**Meet the editor**

Dinesh Sriramulu graduated from the Technical University of Braunschweig, Germany, with his doctorate degree in Medical Microbiology. He started his research career at the Helmholtz Centre for Infection Research, Braunschweig, Germany, in collaboration with the Karolinska Institutet, Stockholm, Sweden. His area of expertise is on the adaptation of bacteria toward diverse

niches, ranging from the human lung to the cattle rumen. He also worked on tumor microenvironment in the case of esophageal and breast cancers. He continued his research work at various reputed institutions worldwide: University College Cork, Ireland; University of Medicine and Dentistry of New Jersey, USA; Food and Drug Administration, Rockville, USA; University of Southern California, Los Angeles, USA; University of Trento, Italy; and University of Cape Town, South Africa. He has published his research findings in various international peer-reviewed journals and presented his works at international conferences. He has been serving as an editorial board member, a peer reviewer, and an expert referee for scientific journals and research funding agencies.

## Contents

#### **Preface XI**


## Preface

The Open Access Initiative has been making its stride toward liberal dissemination of scien‐ tific knowledge to the world community through unrestricted access to full-text articles, chapters, and other contents. Since the inception of virtual media science publishing has been adopting a constant fast-track update of information as and when it occurs. In this line, InTech has been making its footprint all along by identifying and openly involving eminent scientists worldwide. The book, *Progress in Understanding Cystic Fibrosis*, is the successor of *Cystic Fibrosis – Renewed Hopes Through Research*.

Cystic fibrosis (CF), also known as mucoviscidosis, is an autosomal recessive multisystem genetic disorder that occurs predominantly among Caucasians. Though found highest among Irish population, the incidence of CF is on the rise among other populations includ‐ ing the least-affected ones. The interdisciplinary approach toward better understanding the CF condition and the development of sensitive early diagnostic methods have contributed toward efficient diagnosis, treatment, and management of the disease. The CF condition is characterized by abnormal transport of chloride and sodium across the epithelium that leads to thickening of secretions especially in the lungs, pancreas, liver, and intestine. The complex nature of this disease involving multiple organs and subsequent secondary infec‐ tions by microbes is the basis for mortality in CF population. Decades of research by scien‐ tists worldwide has narrowed down the cause of CF to a single target gene. But the complexity of the disease is the most challenging impediment to finding a single-shot cure. This book is a simple collection of chapters on CF-related cellular biochemistry, diabetes, microbiome, and immunotherapy that highlight the progress in CF research. From the infor‐ mation contained in the chapters of this book, it is obvious that only with the help of inter‐ disciplinary research, better understanding and management of the CF disease condition would be possible and this approach has certainly increased the level of life expectancy among CF patients. In addition, a cohort- or patient-specific treatment strategy supported by intense bench-to-bedside research flow seems to be a feasible option to reduce morbidity and mortality in CF population.

I thank InTech for appointing me as the editor of this book and for providing me the oppor‐ tunity to contribute to the scientific community. I thank the authors of the chapters for their valuable contributions. I thank Helmholtz Centre for Infection Research, Braunschweig, Germany, which served as the knowledge center for me to gain expertise in this field.

> **Dinesh Sriramulu** Division of Cell and Immune Biology Helmholtz Centre for Infection Research Braunschweig, Germany

#### **CFTR Involvement in Cell Migration and Epithelial Restitution CFTR Involvement in Cell Migration and Epithelial Restitution**

Scott M. O'Grady Scott M. O'Grady

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66309

#### **Abstract**

Over the past decade, research has shown that *c*ystic *f*ibrosis *t*ransmembrane conductance *r*egulator (CFTR) plays an important role in epithelial cell migration and wound healing. Experiments with airway epithelium, ovarian epithelial cells, placental epithelium and epidermal keratinocytes demonstrated that CFTR function is necessary to achieve maximum migration rates during restitution and in certain cancer cells, CFTR activity contributes to tumor cell invasion. Multiple mechanisms appear to underlie the motility‐promoting actions of CFTR, and although many details remain to be established, our present understanding indicates that processes such as electrotaxis (galvanotaxis), integrin‐mediated cell adhesion and lamellipodia protru‐ sion are dependent on normal CFTR function. In this chapter, the role of CFTR in epithelial cell migration and its implications in cystic fibrosis (CF) will be reviewed with emphasis on the underlying mechanisms that may explain observations made in various epithelial tissues, particularly in airways. Ultimately, a better understanding of CFTR involvement in epithelial repair may lead to new therapeutic approaches to improve barrier function and reduce airway infection and inflammation associated with CF.

**Keywords:** cystic fibrosis, CFTR, wound healing, collective migration, barrier func‐ tion, inflammation

## **1. Introduction**

#### **1.1. Ion channels and cell motility**

The role of ion channels and membrane transporters in cell migration has been the subject of severalrecentreviews [1–4], soonlya fewexampleswillbehighlightedinthis sectiontoprovide

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the reader with an appreciation of their importance in cell motility. Ion channels and other membrane transport pathways participate in multiple housekeeping functions within cells that include regulation of membrane potential, intracellular [Ca2+], cytoskeletal assembly, integrin‐ mediated signaling, cell volume regulation, as well as the maintenance of intracellular and extracellularpH.Eachofthesehousekeepingfunctionscaninfluencecellmigration.Forinstance, changes in ion channel activity produces changes in membrane potential that can facilitate uptake of Ca2+ from the extracellular media. A recent example involves the slow calcium wave that develops approximately 1 h after wounding of corneal endothelial cells [5]. The rise in intracellular[Ca2+]isassociatedwithplasmamembranedepolarizationof cellsalongthemargin of the wound and serves to increase the rate of cell migration. This depolarization has been attributed to increased expression and activity of epithelial Na+ channels (ENaC) within cells that boarder the wound, resulting in elevated intracellular [Na+ ]. The combined effect of depolarizationandincreasedNa+ loaddrivesNa+ /Ca2+ exchange (NCX) activityinreversemode to produce Ca2+ uptake, which propagates from the border of the wound into the epithelium. There may also be an additional role for transient receptor potential (TRP) channels in this process since inhibition of NCX activity does not completely block Ca2+ uptake, whereas inhibition of both NCX and TRP activity abolishes the increase in intracellular [Ca2+].

Membrane hyperpolarization can also stimulate Ca2+ uptake and enhance the rate of cell migration. Differentiated intestinal epithelial cells with increased expression of voltage‐gated K+ channels (Kv1.1/Kv1.5) exhibit membrane hyperpolarization and increased intracellular [Ca2+] as a result of a greater driving force for electrogenic Ca2+ uptake across the plasma membrane [6]. The elevation in intracellular[Ca2+] was shown to augment formation of myosin II containing stress fibers necessary for efficient cell migration. Similarly, ionotropic P2X7 receptors have also been shown to contribute to changes in intracellular [Ca2+] and cell migration. During injury of corneal epithelial cells, P2X7 receptors redistribute to the leading edge of cells that border the wound [7]. Adenosine triphosphate (ATP) is released from the damaged cells leading to activation of these receptors and subsequent uptake of Ca2+ from the extracellular solution. The increase in intracellular [Ca2+] induces actin cytoskeletal rearrange‐ ments that facilitate the formation of branched dendritic networks of actin within lamellipodia, promoting the dynamic regulation of focal adhesions within cells at the wound margin.

ATP release and P2X7 receptor activation have also been shown to be initiated in response to ligand‐activated αVβ3 integrin and syndecan‐4 engagement leading to increased formation of focal adhesions and an enhanced rate of migration in astrocytes [8]. The mechanism of ATP release involved activation of PI3K, PLCγ and IP3 receptors following integrin activation. This resulted in opening of Cx43/Panx‐1 hemichannels in the plasma membrane, facilitating ATP release, transactivation of P2X7 receptors and ultimately, an increase in intracellular [Ca2+]. Furthermore, enhanced expression of both α6β4 integrin and TRPV1 receptors at the leading edge of keratinocytes after wounding has also been linked to increases in intracellular [Ca2+]. Evidence appears to support a model where TRPV1‐mediated increases in intracellular [Ca2+] trigger the activation of transcription factors such as nuclear factor of activated T cells (NFAT) and cAMP response element binding protein (CREB) to stimulate expression of β4 integrins in cells at the margin of the wound leading to an increase in directional migration [9]. Direct coupling between the β1 integrin and KCa3.1 channel expression has been demonstrated in alveolar type II cells grown on a fibronectin matrix and inhibition of channel activity was shown to decrease the rate of migration [10]. This inhibitory effect may be due in part to reducing Ca2+ uptake through TRPC4 channels which were also shown to participate in migration during wound repair. TRP channel‐associated Ca2+ uptake has also been shown to be stimulated by mechanical stretch of the plasma membrane associated with tension and cell shape changes occurring during migration. A specific example involves activation of TRPM7 which mediates transient and highly localized increases in intracellular [Ca2+] known as Ca2+ flickers that take place within lamellipodia in response to mechanical forces linked to contrac‐ tion [11]. This initial Ca2+ response is amplified by localized Ca2+‐induced Ca2+ release from internal stores leading to transactivation of protein kinase A (PKA) through stimulation of Ca2+‐sensitive adenylyl cyclases. PKA is known to have multiple cell migration‐associated targets including components of the cytoskeleton and the focal adhesion proteome that can have both positive and negative effects on migration depending on intracellular localization.

An interesting example of enhanced cell migration linked to K+ channel regulation has been reported in glioblastoma cells [12]. In astrocytes and oligodendrocytes from normal brain tissue, the α9β1 integrin is not expressed; however, expression has been shown to increase with glioma grade and appears to be critical for sustaining increased migration rates following exposure to urokinase receptor (uPAR), agonists. A unique feature of the α9 subunit is that its cytoplasmic domain specifically interacts with spermidine/spermine‐N‐acetyl transferase (SSAT), which catalyzes the breakdown of higher‐order polyamines (spermidine and sper‐ mine) to putrescine. Spermidine and spermine are known to regulate the rectification prop‐ erties of Kir channels by binding to negatively charged residues within the channel pore, significantly reducing K+ efflux from the cell. In contrast, putrescine is a much less effective blocker of outward K+ current in Kir channels. In glioma cells, the α9 subunit colocalizes with Kir4.2 and silencing of the channel inhibits uPAR‐enhanced cell migration. A proposed mechanism to explain the increase in migration rate involves activation of SSAT in response to uPAR‐dependent α9β1 integrin activation, which produces a localized decrease in the [spermidine/spermine] ratio ultimately leading to reduced rectification, increased K+ efflux and membrane hyperpolarization.

#### **1.2. Airway inflammation and epithelial damage**

the reader with an appreciation of their importance in cell motility. Ion channels and other membrane transport pathways participate in multiple housekeeping functions within cells that include regulation of membrane potential, intracellular [Ca2+], cytoskeletal assembly, integrin‐ mediated signaling, cell volume regulation, as well as the maintenance of intracellular and extracellularpH.Eachofthesehousekeepingfunctionscaninfluencecellmigration.Forinstance, changes in ion channel activity produces changes in membrane potential that can facilitate uptake of Ca2+ from the extracellular media. A recent example involves the slow calcium wave that develops approximately 1 h after wounding of corneal endothelial cells [5]. The rise in intracellular[Ca2+]isassociatedwithplasmamembranedepolarizationof cellsalongthemargin of the wound and serves to increase the rate of cell migration. This depolarization has been

to produce Ca2+ uptake, which propagates from the border of the wound into the epithelium. There may also be an additional role for transient receptor potential (TRP) channels in this process since inhibition of NCX activity does not completely block Ca2+ uptake, whereas

Membrane hyperpolarization can also stimulate Ca2+ uptake and enhance the rate of cell migration. Differentiated intestinal epithelial cells with increased expression of voltage‐gated K+ channels (Kv1.1/Kv1.5) exhibit membrane hyperpolarization and increased intracellular [Ca2+] as a result of a greater driving force for electrogenic Ca2+ uptake across the plasma membrane [6]. The elevation in intracellular[Ca2+] was shown to augment formation of myosin II containing stress fibers necessary for efficient cell migration. Similarly, ionotropic P2X7 receptors have also been shown to contribute to changes in intracellular [Ca2+] and cell migration. During injury of corneal epithelial cells, P2X7 receptors redistribute to the leading edge of cells that border the wound [7]. Adenosine triphosphate (ATP) is released from the damaged cells leading to activation of these receptors and subsequent uptake of Ca2+ from the extracellular solution. The increase in intracellular [Ca2+] induces actin cytoskeletal rearrange‐ ments that facilitate the formation of branched dendritic networks of actin within lamellipodia, promoting the dynamic regulation of focal adhesions within cells at the wound margin.

ATP release and P2X7 receptor activation have also been shown to be initiated in response to ligand‐activated αVβ3 integrin and syndecan‐4 engagement leading to increased formation of focal adhesions and an enhanced rate of migration in astrocytes [8]. The mechanism of ATP release involved activation of PI3K, PLCγ and IP3 receptors following integrin activation. This resulted in opening of Cx43/Panx‐1 hemichannels in the plasma membrane, facilitating ATP release, transactivation of P2X7 receptors and ultimately, an increase in intracellular [Ca2+]. Furthermore, enhanced expression of both α6β4 integrin and TRPV1 receptors at the leading edge of keratinocytes after wounding has also been linked to increases in intracellular [Ca2+]. Evidence appears to support a model where TRPV1‐mediated increases in intracellular [Ca2+] trigger the activation of transcription factors such as nuclear factor of activated T cells (NFAT) and cAMP response element binding protein (CREB) to stimulate expression of β4 integrins in cells at the margin of the wound leading to an increase in directional migration [9]. Direct

inhibition of both NCX and TRP activity abolishes the increase in intracellular [Ca2+].

channels (ENaC) within cells

/Ca2+ exchange (NCX) activityinreversemode

]. The combined effect of

attributed to increased expression and activity of epithelial Na+

depolarizationandincreasedNa+ loaddrivesNa+

2 Progress in Understanding Cystic Fibrosis

that boarder the wound, resulting in elevated intracellular [Na+

Loss of CFTR function in the airways of CF patients leads to reduced anion secretion, en‐ hanced Na+ absorption and a decrease in the depth of airway surface liquid that ultimately impairs mucociliary clearance and the removal of pathogens from the lungs [13–15]. Reduced pathogen clearance facilitates infection that induces neutrophilic inflammation, leading to progressive epithelial damage within the conducting airways [16–19]. Over time, a recurrent cycle of intense inflammation, epithelial injury and airway remodeling produce irrevocable damage that dramatically compromises lung function [20–22]. Mounting evidence from in vitro studies and animal models of CF indicate that CFTR malfunction appears to alter the innate immune response of the airways leading to increased release of proinflammatory mediators evoking an amplified, yet less effective inflammatory reaction that is unable to eliminate airway pathogens [17, 18]. In some cases, elevated cytokine levels, neutrophil infiltration and neutrophil elastase (NE) concentrations within the bronchial alveolar lavage (BAL) fluid have been reported in infants without signs of infection, although other studies support the concept that infection is necessary to initiate inflammation [17, 23–25]. Neutrophils represent the major inflammatory leukocyte recruited into CF airways where they release a variety of mediators including oxidants and proteases such as neutrophil elastase (NE), which possess bacteriocidal properties [26, 27]. Moreover, NE catalytic activity is also known to damage the epithelium and reduce structural integrity of the airways leading to bronchiectasis and deteriorating lung function [28–31]. Furthermore, the airways of CF patients encounter various reactive oxygen species (ROS) derived from bacterial pathogens or from the environ‐ ment [32]. ROS production can exceed the endogenous oxidative defense capacity of the airways leading to oxidative stress and additional injury. In adults, the concentration of reduced glutathione (GSH), a major ROS scavenger present in the airway surface liquid, is significantly reduced in CF patients [33, 34]. This condition may be directly related to the loss of CFTR function since the channel is known to transport GSH in addition to anions in normal airways [35].

Decreases in CFTR channel activity also result in acidification of airway surface liquid coupled to an increase in intracellular pH, which reduces antimicrobial function of the airway surface liquid, promoting bacterial infection [36–41]. Intracellular alkalinization also appears to enhance the accumulation of ceramide, a metabolite of sphingomyelin, within lysosomes [42–44]. Ceramide is thought to amplify the inflammatory response by triggering tumor necrosis factor (TNF)α signaling pathways involving mitogen‐activated protein kinases (MAPK), IκB‐kinase degradation [an inhibitory regulator of necrosis factor (NF)‐κB] and NF‐kB nuclear localization [45–47]. Additionally, for class II CFTR mutations, the accumulation of misfolded CFTR protein within the endoplasmic reticulum (ER) induces stress and stimulates what is known as an unfolded protein response, which involves activation of signaling pathways that mitigate ER stress [48–51]. For the most common class II mutation, retention of misfolded ΔF508 CFTR within the ER causes an unfolded protein response that stimulates inflammation by activating NF‐κB and inducing cytokine secretion that can result in apoptosis.

#### **1.3. Evidence for defective epithelial regeneration in CF**

In an earlier investigation, a humanized airway xenograph model was created by inoculation of CF and non‐CF airway epithelial cells onto epithelium‐deleted rat trachea that was then subcutaneously implanted into nude mice over a period from 4 to 35 days [52]. This model was then used to investigate the process of reepithelialization following injury and to deter‐ mine if remodeling of CF epithelium is a consequence of defective epithelial regeneration independent of infection. The results showed that CF epithelial cells exhibited enhanced proliferation along with continuous expression of IL‐8, matrix metalloproteinases (MMP7, MMP9) and tissue inhibitor of metalloproteinase (TIMP)‐1. Moreover, regeneration was delayed and final restitution resulted in a remodeled epithelium that appeared to be a product of aberrant regeneration unrelated to bacterial contamination. A relationship between abnormal regeneration and loss of CFTR function was not identified in this study, although it was concluded that it might be a consequence of altered MMP/TIMP/IL‐8 expression observed in CF epithelium. In a subsequent study, wound healing experiments using immortalized normal (NuLi‐1 cells) and CF (CuFi‐1 cells) human airway epithelial cells revealed that CuFi‐ 1 cell migration was significantly delayed relative to NuLi‐1 cells [53, 54]. This difference in migration activity was attributed to defective epidermal growth factor (EGF)/epidermal growth factor receptors (EGFR) signaling and reduced K+ channel expression. Interestingly, no significant effect on migration was reported in the presence of the CFTR inhibitor, CFTRinh‐ 172 [53]. In subsequent investigations described below, loss of CFTR function was shown to directly contribute to delayed epithelial repair in CF airways and that expression of normal CFTR augments epithelial restitution.

## **2. Anion channels, cell migration and epithelial restitution**

#### **2.1. Volume‐sensitive anion channels in cell migration and invasion**

eliminate airway pathogens [17, 18]. In some cases, elevated cytokine levels, neutrophil infiltration and neutrophil elastase (NE) concentrations within the bronchial alveolar lavage (BAL) fluid have been reported in infants without signs of infection, although other studies support the concept that infection is necessary to initiate inflammation [17, 23–25]. Neutrophils represent the major inflammatory leukocyte recruited into CF airways where they release a variety of mediators including oxidants and proteases such as neutrophil elastase (NE), which possess bacteriocidal properties [26, 27]. Moreover, NE catalytic activity is also known to damage the epithelium and reduce structural integrity of the airways leading to bronchiectasis and deteriorating lung function [28–31]. Furthermore, the airways of CF patients encounter various reactive oxygen species (ROS) derived from bacterial pathogens or from the environ‐ ment [32]. ROS production can exceed the endogenous oxidative defense capacity of the airways leading to oxidative stress and additional injury. In adults, the concentration of reduced glutathione (GSH), a major ROS scavenger present in the airway surface liquid, is significantly reduced in CF patients [33, 34]. This condition may be directly related to the loss of CFTR function since the channel is known to transport GSH in addition to anions in normal

Decreases in CFTR channel activity also result in acidification of airway surface liquid coupled to an increase in intracellular pH, which reduces antimicrobial function of the airway surface liquid, promoting bacterial infection [36–41]. Intracellular alkalinization also appears to enhance the accumulation of ceramide, a metabolite of sphingomyelin, within lysosomes [42–44]. Ceramide is thought to amplify the inflammatory response by triggering tumor necrosis factor (TNF)α signaling pathways involving mitogen‐activated protein kinases (MAPK), IκB‐kinase degradation [an inhibitory regulator of necrosis factor (NF)‐κB] and NF‐kB nuclear localization [45–47]. Additionally, for class II CFTR mutations, the accumulation of misfolded CFTR protein within the endoplasmic reticulum (ER) induces stress and stimulates what is known as an unfolded protein response, which involves activation of signaling pathways that mitigate ER stress [48–51]. For the most common class II mutation, retention of misfolded ΔF508 CFTR within the ER causes an unfolded protein response that stimulates inflammation by activating NF‐κB and inducing cytokine secretion

In an earlier investigation, a humanized airway xenograph model was created by inoculation of CF and non‐CF airway epithelial cells onto epithelium‐deleted rat trachea that was then subcutaneously implanted into nude mice over a period from 4 to 35 days [52]. This model was then used to investigate the process of reepithelialization following injury and to deter‐ mine if remodeling of CF epithelium is a consequence of defective epithelial regeneration independent of infection. The results showed that CF epithelial cells exhibited enhanced proliferation along with continuous expression of IL‐8, matrix metalloproteinases (MMP7, MMP9) and tissue inhibitor of metalloproteinase (TIMP)‐1. Moreover, regeneration was delayed and final restitution resulted in a remodeled epithelium that appeared to be a product of aberrant regeneration unrelated to bacterial contamination. A relationship between

airways [35].

4 Progress in Understanding Cystic Fibrosis

that can result in apoptosis.

**1.3. Evidence for defective epithelial regeneration in CF**

Earlier electrophysiological studies of human glioma cells showed that they express voltage‐ sensitive Cl‐ channels that were blocked by chlorotoxin (Ctx), a peptide isolated from scorpion venom as well as tamoxifen, an estrogen receptor modulator [55–57]. Furthermore, hypotonic solutions were also shown to activate tamoxifen and 5‐nitro‐2‐(3‐phenylpropylamino)‐ benzoate (NPPB)‐sensitive, outwardly rectifying Cl‐ currents carried by channels that were shown to contribute to the resting Cl‐ conductance under isotonic conditions [55]. Treatment with either Ctx or NPPB inhibited glioma cell migration and invasiveness in transwell migration assays. Similarly, osmotically activated cell swelling and regulatory volume decrease (RVD) were also blocked by Ctx and tamoxifin indicating a role in the regulation of cell volume that contributes to migration and tumor cell invasion [56]. Simultaneous time lapse imaging and patch clamp recording of glioma cells demonstrated detectable changes in cell shape and movement that was associated with activation of volume‐sensitive Cl‐ currents. Changes in cell shape and motility were attributed to Cl‐ efflux coupled to K+ and water movement across the plasma membrane resulting in cell shrinkage, which appeared to be localized at the leading edge of the cell. Consequently, cell flattening at the leading edge was proposed to facilitate protrusion through restricted extracellular spaces required for tumor cell invasion [58].

Experiments with murine primary microglial cells or a microglial (BV‐2) cell line demonstrated that exposure to hypotonic saline or an elevated extracellular [K+ ] produced localized swelling and protrusion of lamellipodia at the leading edge of these cells [59]. Blockade of volume‐ activated Cl‐ channels or inhibition of K‐Cl co‐transporters (KCC) effectively inhibited lamellipodia formation. The migratory response induced by localized increases in extracellular [K+ ] may likely result from cell death caused by injury. Ischemia for example, has been shown to increase extracellular [K+ ] by more than 20 fold [60]. Such increases in [K+ ] would provide a favorable driving force for KCl uptake by KCC leading to cell swelling and produce mem‐ brane depolarization. This would establish conditions for electrogenic Cl‐ influx through volume‐activated anion channels which also contributes to localized swelling. Furthermore, signaling proteins such as the chemokine ligand CCL21 is released by damaged neurons and is known to induce a chemotaxis response in microglia which is inhibited by Cl‐ channel blockers [61]. This response was not dependent on activation of the canonical CCL21 receptor CCR7, but instead was shown to stimulate CXCR3 receptors. Short‐duration exposure (30 s) to CCL21 or the selective CXCR3 ligand CXCL10 in either brain slice preparations or microglial cells in culture produced a sustained increase in Cl‐ channel activation that appears to represent an initial trigger for stimulating directed cell migration in response to neuronal injury.

#### **2.2. CFTR and epithelial wound repair**

The first direct evidence of a role for CFTR in cell migration was obtained from studies of airway epithelial cells [62]. Experiments using Calu‐3 cells, a human airway adenocarcinoma cell line and normal human bronchial epithelial cells revealed that inhibition of CFTR channel activity with the selective CFTR blocker, CFTRinh‐172 or silencing CFTR expression by RNAi significantly slowed cell migration and epithelial restitution (see **Figure 1**). Moreover, CFTR channel inhibition or silencing also reduced the extent of lamellipodia protrusion during migration. These results demonstrated that the ion transport activity of CFTR was necessary for airway epithelial cells to achieve a maximum rate of migration during wound closure and that lamellipodia protrusion was at least one aspect of the migration process that was affected by the loss of CFTR function. Following publication of this initial investigation, Sun et al. (2011) showed that epithelial wound repair in a tracheal preparation from rhesus monkeys was delayed following treatment with CFTRinh‐172 [63]. Experiments employing the use of a noninvasive vibrating probe demonstrated that inhibition of CFTR activity inhibited the spontaneous outward current induced by wounding and that treatment with aminophylline, a phosphodiesterase inhibitor and CFTR activator, stimulated this outward current. These results suggested that CFTR activity contributes to the wound current that serves as a guidance cue for directed migration and that inhibition of CFTR activity disrupts the process of electrotaxis, thus delaying wound closure. Further support for the importance of CFTR in airway cell migration and epithelial restitution was provided by a set of rescue experiments involving (i) expression of wild‐type CFTR into CF airway epithelial cell lines to restore the normal rate of wound closure and (ii) treatment with VRT‐325, a CFTR corrector molecule that facilitates apical membrane localization of CFTR with the ΔF508 mutation in CFBE‐ΔF508 cells and in primary bronchial epithelial cells obtained from CF patients [64].

Involvement of CFTR in cell migration has also been observed in other epithelial cell types besides airways. For example, in human trophoblast (BeWo) cells, CFTR activation with forskolin increased cell migration into the wound and subsequent addition of CFTRinh‐172 significantly inhibited the response to forskolin [65]. Poor trophoblast migration/invasiveness and associated spiral artery remodeling represent early recognizable pathologies that underlie preeclampsia and previous studies demonstrated that CFTR expression is reduced in pree‐ clamptic placentas [66]. Thus, changes in CFTR function not only appear to have consequences on placental ion and fluid transport but may also contribute to altered trophoblast invasion in preeclampsia. Another example based on experiments with human ovarian carcinoma cells showed that CFTR silencing by RNAi significantly reduced cell migration and invasion under in vitro conditions and that the tumorigenic potential of these cells in vivo was suppressed compared to controls [67]. This result was consistent with the observation that CFTR expres‐ sion in ovarian cancer was higher relative to normal ovarian epithelial cells or benign ovarian tumors and that enhanced CFTR expression was associated with advanced International Federation of Gynecology and Obstetrics (FIGO) staging and poor histopathology grade. Lastly, CFTR was also shown to play a role in cutaneous wound healing, where ΔF508*cftr*‐*/*‐ mice that lack plasma membrane localization and normal CFTR channel function exhibited delayed wound closure compared to wild‐type mice [68].

volume‐activated anion channels which also contributes to localized swelling. Furthermore, signaling proteins such as the chemokine ligand CCL21 is released by damaged neurons and is known to induce a chemotaxis response in microglia which is inhibited by Cl‐ channel blockers [61]. This response was not dependent on activation of the canonical CCL21 receptor CCR7, but instead was shown to stimulate CXCR3 receptors. Short‐duration exposure (30 s) to CCL21 or the selective CXCR3 ligand CXCL10 in either brain slice preparations or microglial

an initial trigger for stimulating directed cell migration in response to neuronal injury.

and in primary bronchial epithelial cells obtained from CF patients [64].

Involvement of CFTR in cell migration has also been observed in other epithelial cell types besides airways. For example, in human trophoblast (BeWo) cells, CFTR activation with forskolin increased cell migration into the wound and subsequent addition of CFTRinh‐172 significantly inhibited the response to forskolin [65]. Poor trophoblast migration/invasiveness and associated spiral artery remodeling represent early recognizable pathologies that underlie preeclampsia and previous studies demonstrated that CFTR expression is reduced in pree‐ clamptic placentas [66]. Thus, changes in CFTR function not only appear to have consequences on placental ion and fluid transport but may also contribute to altered trophoblast invasion in preeclampsia. Another example based on experiments with human ovarian carcinoma cells

The first direct evidence of a role for CFTR in cell migration was obtained from studies of airway epithelial cells [62]. Experiments using Calu‐3 cells, a human airway adenocarcinoma cell line and normal human bronchial epithelial cells revealed that inhibition of CFTR channel activity with the selective CFTR blocker, CFTRinh‐172 or silencing CFTR expression by RNAi significantly slowed cell migration and epithelial restitution (see **Figure 1**). Moreover, CFTR channel inhibition or silencing also reduced the extent of lamellipodia protrusion during migration. These results demonstrated that the ion transport activity of CFTR was necessary for airway epithelial cells to achieve a maximum rate of migration during wound closure and that lamellipodia protrusion was at least one aspect of the migration process that was affected by the loss of CFTR function. Following publication of this initial investigation, Sun et al. (2011) showed that epithelial wound repair in a tracheal preparation from rhesus monkeys was delayed following treatment with CFTRinh‐172 [63]. Experiments employing the use of a noninvasive vibrating probe demonstrated that inhibition of CFTR activity inhibited the spontaneous outward current induced by wounding and that treatment with aminophylline, a phosphodiesterase inhibitor and CFTR activator, stimulated this outward current. These results suggested that CFTR activity contributes to the wound current that serves as a guidance cue for directed migration and that inhibition of CFTR activity disrupts the process of electrotaxis, thus delaying wound closure. Further support for the importance of CFTR in airway cell migration and epithelial restitution was provided by a set of rescue experiments involving (i) expression of wild‐type CFTR into CF airway epithelial cell lines to restore the normal rate of wound closure and (ii) treatment with VRT‐325, a CFTR corrector molecule that facilitates apical membrane localization of CFTR with the ΔF508 mutation in CFBE‐ΔF508 cells

channel activation that appears to represent

cells in culture produced a sustained increase in Cl‐

**2.2. CFTR and epithelial wound repair**

6 Progress in Understanding Cystic Fibrosis

**Figure 1.** Inhibition of CFTR channel activity or silencing expression by RNAi delay airway epithelial restitution. (**A**)– (**C**) Impedance‐sensing arrays were used to track the process of Calu‐3 cell migration over the surface of a 250 μm di‐ ameter electrode following wounding by electroporation. Images show the extent of Calu‐3 cell confluence at three time points (0, 120 and 300 min). (**D)** Normalized impedance (*Z*/*Z*max) measurements as a function of time for Calu‐3 cells expressing shRNAs designed to selectively target CFTR (shCFTR cells) or have an altered sequence that no longer recognizes CFTR mRNA (shALTR cells). Note that as cells reach confluence on the electrode surface, the normalized impedance value approaches 1, which indicates complete epithelial restitution. For these experiments, shALTR cells were used as controls where the black line represents the mean *Z*/*Z*max values and the shaded grey area corresponds to the SEM (*n* = 8). The blue line (mean) and light‐blue‐shaded area (SEM) shows the effects of silencing CFTR on wound closure, where the slope provides a measure of the average rate of cell migration into the wound (*n* = 8). Finally, the red line (mean) and pink‐shaded area (SEM) are the results from shALTR cells treated with 20 μM CFTRinh‐172, a selective inhibitor of CFTR channel activity, throughout the process of restitution (*n* = 8). Images were adapted from Ref. [114].

Exceptions to the migration‐promoting actions of CFTR can be found in studies of non‐small cell lung cancer (NSCLC) cells and human keratinocytes [69]. Experiments with NSCLC cells showed reduced CFTR expression which correlated with an advanced stage of the cancer, lymph node metastasis and enhanced malignant behavior which manifested as an increase in epithelial‐mesenchymal transition, invasion and migration. In contrast, overexpression of CFTR reduced cancer progression and metastasis, supporting the observation that in some types of cancer, CFTR appears to function as a tumor suppressor. Similarly, CFTR silencing by RNAi in human keratinocytes was shown to promote cell migration and inhibit differentiation, whereas overexpression inhibited migration and stimulated differentiation [68]. The effects of manipulating CFTR expression on migration appeared to be related to its role in the formation of epithelial junctions since silencing the channel downregulated adhesion molecule (E‐ cadherin, ZO‐1 and β‐catenin) expression and intercellular junction formation while overex‐ pression promoted junction formation.

#### **2.3. ANO1, cell migration and cystic fibrosis**

TMEM16A/ANO1 is one of the 10 known members of the anoctamin family (TMEM16A‐K) of proteins, some of which function as anion channels. Certain members of this family, such as ANO1, ANO2 and ANO6, can be activated by increases in intracellular [Ca2+] and are classified as Ca2+‐activated chloride channels (CaCCs) [70–72]. CaCCs exhibit voltage dependence, outward rectification and are perhaps best known for their role in Ca2+‐dependent Cl‐ secretion in various epithelial tissues. Compounds including T16Ainh‐A01, CaCCinh‐A01 and NS3728 block channel activity to varying degrees depending on cell type [73]. Prior to the discovery of its anion channel activity, ANO1 was regarded as either a tumor cell marker or as an oncogene in human cancers with poor prognosis [74, 75].

In prostate cancer (LNCaP and PC‐3) cells, ANO1 is highly expressed and these cells exhibit large CaCC currents in response to increases in cytosolic [Ca+ ] [76]. Silencing ANO1 by RNAi in PC‐3 cells significantly inhibited cell proliferation and migration/invasion. Studies using Ehrlich Lettre ascites (ELA) cells revealed that they express both ANO1 and ANO6 [71]. Interestingly, silencing ANO1 expression was shown to alter directionality of ELA migration while knockdown of ANO6 was shown to cause a ∼40% decrease in the overall rate of migration. Although the mechanism responsible for ANO1‐dependent control of directionality is not understood, it is likely that some contribution to outward current associated with wounding may be important in electrotaxis. Various pancreatic ductal adenocarcinoma cells have also been shown to have increased expression of ANO1 and enhanced CaCC activity. Knockdown of ANO1 or inhibition by CaCC blockers including CaCCinh‐A01, and NS3728 delay migration in BxPC‐2 cells, however, T16Ainh‐A01 exhibited no effect [77]. The authors speculated that activation of ANO1 was important for cell volume changes necessary to control cell shape and that the channel may serve as a potential target for reducing the metastatic potential of pancreatic tumor cells.

Investigations of bronchial epithelial cell repair in cystic fibrosis (CF) demonstrated that the expression of ANO1 and CaCC channel activity were significantly reduced in CF cells compared to bronchial epithelial cells from normal subjects [78]. Consequently, epithelial restitution in wound healing assays was delayed in CF cells relative to non‐CF cells. Moreover silencing ANO1 expression in non‐CF cells reduced the rate of migration, whereas overex‐ pression of ANO1 in CF cells partially restored cell motility, although complete recovery was not achieved. To establish whether ANO1 channel function was necessary for supporting cell migration, primary non‐CF cells were treated with T16Ainh‐A01 which produced a significant delay in wound closure. These findings indicate that reduced rates of cell migration in bronchial epithelial cells from CF patients may be attributed to an overall decrease in apical membrane Cl‐ conductance resulting from loss of both CFTR and ANO1 anion channel activity.

## **3. Mechanisms of CFTR‐dependent cell migration and epithelial repair**

Although the molecular mechanisms underlying the contribution of CFTR to the processes of cell migration and epithelial restitution remain to be fully characterized, the data collected so far have identified three important aspects of migration that merit further investigation. These include the process of lamelliopdia protrusion, electrotaxis and the dynamics of integrin‐ mediated adhesion, each of which are discussed in more detail below.

#### **3.1. Lamellipodia protrusion**

Exceptions to the migration‐promoting actions of CFTR can be found in studies of non‐small cell lung cancer (NSCLC) cells and human keratinocytes [69]. Experiments with NSCLC cells showed reduced CFTR expression which correlated with an advanced stage of the cancer, lymph node metastasis and enhanced malignant behavior which manifested as an increase in epithelial‐mesenchymal transition, invasion and migration. In contrast, overexpression of CFTR reduced cancer progression and metastasis, supporting the observation that in some types of cancer, CFTR appears to function as a tumor suppressor. Similarly, CFTR silencing by RNAi in human keratinocytes was shown to promote cell migration and inhibit differentiation, whereas overexpression inhibited migration and stimulated differentiation [68]. The effects of manipulating CFTR expression on migration appeared to be related to its role in the formation of epithelial junctions since silencing the channel downregulated adhesion molecule (E‐ cadherin, ZO‐1 and β‐catenin) expression and intercellular junction formation while overex‐

TMEM16A/ANO1 is one of the 10 known members of the anoctamin family (TMEM16A‐K) of proteins, some of which function as anion channels. Certain members of this family, such as ANO1, ANO2 and ANO6, can be activated by increases in intracellular [Ca2+] and are classified as Ca2+‐activated chloride channels (CaCCs) [70–72]. CaCCs exhibit voltage dependence, outward rectification and are perhaps best known for their role in Ca2+‐dependent Cl‐ secretion in various epithelial tissues. Compounds including T16Ainh‐A01, CaCCinh‐A01 and NS3728 block channel activity to varying degrees depending on cell type [73]. Prior to the discovery of its anion channel activity, ANO1 was regarded as either a tumor cell marker or as an

In prostate cancer (LNCaP and PC‐3) cells, ANO1 is highly expressed and these cells exhibit

in PC‐3 cells significantly inhibited cell proliferation and migration/invasion. Studies using Ehrlich Lettre ascites (ELA) cells revealed that they express both ANO1 and ANO6 [71]. Interestingly, silencing ANO1 expression was shown to alter directionality of ELA migration while knockdown of ANO6 was shown to cause a ∼40% decrease in the overall rate of migration. Although the mechanism responsible for ANO1‐dependent control of directionality is not understood, it is likely that some contribution to outward current associated with wounding may be important in electrotaxis. Various pancreatic ductal adenocarcinoma cells have also been shown to have increased expression of ANO1 and enhanced CaCC activity. Knockdown of ANO1 or inhibition by CaCC blockers including CaCCinh‐A01, and NS3728 delay migration in BxPC‐2 cells, however, T16Ainh‐A01 exhibited no effect [77]. The authors speculated that activation of ANO1 was important for cell volume changes necessary to control cell shape and that the channel may serve as a potential target for reducing the metastatic

Investigations of bronchial epithelial cell repair in cystic fibrosis (CF) demonstrated that the expression of ANO1 and CaCC channel activity were significantly reduced in CF cells compared to bronchial epithelial cells from normal subjects [78]. Consequently, epithelial

] [76]. Silencing ANO1 by RNAi

pression promoted junction formation.

8 Progress in Understanding Cystic Fibrosis

potential of pancreatic tumor cells.

**2.3. ANO1, cell migration and cystic fibrosis**

oncogene in human cancers with poor prognosis [74, 75].

large CaCC currents in response to increases in cytosolic [Ca+

Lamellipodia are actin‐containing, sheet‐like structures that protrude from the leading edge of migrating cells [79]. They are capable of sensing environmental cues and are necessary for sustained directional migration. A key force contributing to the protrusion of lamellipodia is provided by the extension of actin filaments at the leading edge of the cell. Within lamellipodia, actin forms networks of branched filaments with highest density near the membrane at the leading edge, where the barbed (positive) ends of the filaments are directed toward the plasma membrane to form brush‐like assemblies [80, 81]. Elongation occurs primarily at junctions formed by a multiprotein structure known as the Arp2/3 complex, which functions as a nucleation site for new actin monomers to attach to the sides of existing actin polymers to create a branched arrangement of fibers [82]. As these monomers add to the growing meshwork at the barbed end, cleavage and dissociation of monomers takes place at the pointed (minus) end of filaments located in the more proximal regions of the lamellipodium. ATPase activity associated with actin filaments facilitates accumulation of ADP‐actin at the pointed ends as filament disassembly takes place. This dynamic process of simultaneous actin monomer addition to the barbed end and dissociation at the pointed end of the filament is known as treadmilling and is controlled by several actin‐regulatory proteins [82, 83] as well as intracel‐ lular pH [84, 85]. Previous studies have shown that during polarization along the axis of movement, a redistribution of the Na+ ‐H+ exchanger (NHE1) occurs, which localizes toward the leading edge of the cell. Redistribution of NHE1 results in the development of a steady‐ state pH gradient extending from the front of the cell, which becomes more alkaline, to the rear, developing a more acidic pH relative to the leading edge [86–88]. A key regulator of polarization is Cdc42, a small guanosine‐5'‐triphosphatase (GTP)ase that accumulates at the leading edge where it stimulates actin polymerization. Cdc42 activation is pH sensitive, requiring NHE1 activation and proton efflux to produce localized alkalinization of the cytoplasm to enhance its activity [86]. Moreover, alkalinization also promotes F‐actin cleavage by cofilin, an actin binding protein that facilitates treadmilling by causing depolymerization at the pointed ends of actin filaments [87]. Other acid extruding or base loading transport mechanisms could potentially contribute to this alkalinization process, including CFTR and its ability to conduct bicarbonate ions, provided that a favorable electrochemical driving force exists.

As previously mentioned localized osmotic swelling can also contribute to the force that powers lamellipodia protrusion [59]. Solute uptake serves as a driving force for fluid uptake into the cell, often involving electroneutral transporters that couple cation uptake with Cl‐ transport (e.g. KCC or NKCC cotransporters). It is also possible that if the plasma membrane is depolarized to a voltage that is more positive than the reversal potential of anion channels such as CFTR or ANO1, then the inwardly directed Cl‐ concentration gradient would facilitate influx, setting up a favorable osmotic gradient for fluid uptake and lamellipodia protrusion. Whether Cl‐ influx or efflux is occurring at the leading edge may not be predicable, since this would depend on the activity of multiple electrogenic transport pathways or conditions associated with injury. It is worth emphasizing that depending on the electrochemical gradient for Cl‐ , CFTR could contribute instead to retraction taking place at the rear of the cell. In this case, efflux of Cl‐ , perhaps coordinated with K+ channel activity, would enable localized solute and fluid exit at the trailing edge of the cell, promoting forward movement [3].

#### **3.2. Electrotaxis**

Epithelia engaged in active electrolyte transport typically generate spontaneous transepithelial potentials (TEP) that provide an electrical driving force for paracellular ion movement across the epithelium [89]. Following wounding, the TEP at the site of the wound collapses as laterally oriented electric fields develop with the cathode (negative pole) located at the center of the wound. In dermal, corneal and airway epithelia, for example, outward current can be detected using a noninvasive technique that employs a self‐referencing vibrating probe [90]. Many epithelial cell types respond to wound‐induced electric fields by migrating toward the cathode although some cell types exhibit anodal migration in response to electric field stimulation [91, 92]. In fact, changing the polarity of the field will reverse the direction of migration. In experiments with primate tracheobronchial epithelial cells, an applied electric field with a threshold intensity of 23 mV/mm was effective at stimulating migration with a displacement speed that increased with field strength [63]. The displacement speed reflected greater migration efficiency and in the case of tracheobronchial epithelial cells, the increase in speed primarily resulted from improved directionality, which was quantitatively expressed using a directedness parameter for the migrating cells. Directedness was expressed as the angle (*θ*) that individual cells moved relative to the electric field vector, where cosine *θ* was defined as the directedness value. Cells moving randomly in the absence of an electric field have an average directedness value near zero, whereas those that move entirely along the electric field lines toward the cathode have a value approaching 1. Experiments with tracheobronchial epithelial cells showed that directedness increased with increasing field strength such that when the voltage achieved 90 mV/mm, a number of the cells migrated directly toward the cathode. The finding that inhibition of CFTR reduces the electric field evoked by wounding and that CFTR inhibition or silencing decreases lamellipodia protrusion [62], strongly suggests that CFTR activity plays an important role in sustaining directed migration in airway epithelial cells.

The cellular mechanisms underlying the increase in directed migration induced by wound‐ evoked electric fields are complex and cell type dependent. Studies of corneal epithelial cells and keratinocytes, for instance, revealed changes in the localization of epidermal growth factor receptors (EGFR) toward the cathode‐facing borders of migrating cells, and in at least one study, EGFRs appeared to be activated by the electric field independently of ligand binding to the receptor [93–97]. Moreover, inhibition of EGFR‐MAPK signaling was shown to alter the actin cytoskeleton at the leading edge and diminish directed migration of epithelial cells, demonstrating an important role for EGFR in detecting and initiating the epithelial response to electric field stimulation [96]. Additionally, electric fields can redistribute and activate PI3K/ Akt signaling in a polarized manner at the cathode‐oriented leading edge of the cell [98]. When PI3K is activated, membrane protrusion and lamellipodia formation is initiated at that site, facilitating directed migration toward the cathode. Pharmacological inhibition of PI3K activity or selective disruption of the PI3K‐γ isoform has been shown to block electrotaxis in wound healing assays and organ cultures [98, 99]. Furthermore, in keratinocytes, deletion of phosphatase and tensin homolog (PTEN), a phosphatase that functions as a negative regulator of PI3K, resulted in increased Akt phosphorylation and enhanced electrotaxis. Other kinases linked to the control of cell motility such as extracellular signal‐regulated kinase (ERK) have also been shown to be involved in electric field‐evoked migration [97, 100, 101]. In experiments with glioma and fibrosarcoma cells, electric field stimulation induced NADPH oxidase activation, resulting in the production of reactive oxygen species (ROS) [100]. Intracellular accumulation of ROS stimulates ERK phosphorylation/activation which leads to reorganization of the cytoskeleton and an increase in directed migration [101]. Based on these observations, it appears likely that further studies will uncover additional molecular targets and signaling pathways involved in the detection and regulation of directionality by injury‐ induced electric fields.

#### **3.3. Dynamics of integrin‐mediated adhesion**

cytoplasm to enhance its activity [86]. Moreover, alkalinization also promotes F‐actin cleavage by cofilin, an actin binding protein that facilitates treadmilling by causing depolymerization at the pointed ends of actin filaments [87]. Other acid extruding or base loading transport mechanisms could potentially contribute to this alkalinization process, including CFTR and its ability to conduct bicarbonate ions, provided that a favorable electrochemical driving force

As previously mentioned localized osmotic swelling can also contribute to the force that powers lamellipodia protrusion [59]. Solute uptake serves as a driving force for fluid uptake into the cell, often involving electroneutral transporters that couple cation uptake with Cl‐ transport (e.g. KCC or NKCC cotransporters). It is also possible that if the plasma membrane is depolarized to a voltage that is more positive than the reversal potential of anion channels

influx, setting up a favorable osmotic gradient for fluid uptake and lamellipodia protrusion. Whether Cl‐ influx or efflux is occurring at the leading edge may not be predicable, since this would depend on the activity of multiple electrogenic transport pathways or conditions associated with injury. It is worth emphasizing that depending on the electrochemical gradient

Epithelia engaged in active electrolyte transport typically generate spontaneous transepithelial potentials (TEP) that provide an electrical driving force for paracellular ion movement across the epithelium [89]. Following wounding, the TEP at the site of the wound collapses as laterally oriented electric fields develop with the cathode (negative pole) located at the center of the wound. In dermal, corneal and airway epithelia, for example, outward current can be detected using a noninvasive technique that employs a self‐referencing vibrating probe [90]. Many epithelial cell types respond to wound‐induced electric fields by migrating toward the cathode although some cell types exhibit anodal migration in response to electric field stimulation [91, 92]. In fact, changing the polarity of the field will reverse the direction of migration. In experiments with primate tracheobronchial epithelial cells, an applied electric field with a threshold intensity of 23 mV/mm was effective at stimulating migration with a displacement speed that increased with field strength [63]. The displacement speed reflected greater migration efficiency and in the case of tracheobronchial epithelial cells, the increase in speed primarily resulted from improved directionality, which was quantitatively expressed using a directedness parameter for the migrating cells. Directedness was expressed as the angle (*θ*) that individual cells moved relative to the electric field vector, where cosine *θ* was defined as the directedness value. Cells moving randomly in the absence of an electric field have an average directedness value near zero, whereas those that move entirely along the electric field lines toward the cathode have a value approaching 1. Experiments with tracheobronchial epithelial cells showed that directedness increased with increasing field strength such that when the voltage achieved 90 mV/mm, a number of the cells migrated directly toward the

, CFTR could contribute instead to retraction taking place at the rear of the cell. In this

concentration gradient would facilitate

channel activity, would enable localized solute

such as CFTR or ANO1, then the inwardly directed Cl‐

, perhaps coordinated with K+

and fluid exit at the trailing edge of the cell, promoting forward movement [3].

exists.

10 Progress in Understanding Cystic Fibrosis

for Cl‐

case, efflux of Cl‐

**3.2. Electrotaxis**

A recent study examining the consequences of CFTR silencing on cell migration and epithelial repair in human airway (Calu‐3) epithelial cells demonstrated a ∼60% reduction in GM1 ganglioside content within the plasma membrane of CFTR deficient cells compared to controls [102, 103], which was restored following expression of wild‐type CFTR. Similarly, treatment of cells with the selective CFTR blocker, CFTRinh‐172, also produced comparable reductions in GM1 content in cells expressing wild‐type CFTR. These observations were consistent with earlier studies showing reduced levels of sialylated gangliosides in cells expressing CFTR with the ΔF508 mutation [104, 105]. Furthermore, previous investigations have also shown that gangliosides are capable of regulating integrin signaling and cell migration [106, 107]. Experiments with Calu‐3 cells revealed that CFTR knockdown did not directly affect β1 integrin surface expression; however, the level of activated β1 integrin was significantly lower than observed in CFTR expressing control cells [102]. β1‐integrin activation could be completely recovered by incubating CFTR deficient cells with exogenous GM1, but not with GM3 gangliosides, confirming that integrin activation was dependent on GM1 and CFTR expression. Reduced β1 integrin phosphorylation was associated with lower levels of focal adhesion kinase (FAK) and Crk‐associated substrate (CAS) phosphorylation which was

**Figure 2.** Colocalization of CFTR and the β2‐adrenergic receptor (β2‐AR) in the apical membrane of Calu‐3 cells and in cilia of differentiated primary normal human bronchial epithelial (NHBE) cells grown under air‐liquid interface condi‐ tions. (**A**) Antibody labeling of the β2‐AR, CFTR and merged images (where yellow represents colocalization) collected from wild‐type Calu‐3 cells (wt Calu‐3), Calu‐3 cells expressing shRNA that does not recognize CFTR (shALTR) and CFTR‐deficient cells were CFTR expression was silenced by RNAi (shCFTR). (**B**) Labeling of the β2‐AR and CFTR with‐ in the cilia of differentiated primary NHBE cells. Yellow‐orange represents colocalization of the receptor and channel. (**C**) Cross section of differentiated, pseudostratified primary NHBE cells showing colocalization of the β2‐AR and CFTR within the cilia. Note the layering of nuclei reflecting pseudostratification. Images were adapted from Refs. [114] and [115].

also restored by incubation with exogenous GM1 ganglioside. A possible explanation for the reduction in β1 activation may be related to loss of localization to specific membrane micro‐ domains that function as integrin signaling platforms. This would be consistent with GM1 localization within lipid raft domains where it is known to associate with CFTR [108, 109]. Moreover, recovery of FAK and CAS phosphorylation along with β1 integrin activation with GM1 repletion also produced partial restoration of cell migration, suggesting that reduced integrin engagement with the extracellular matrix, presumably at the leading edge of the cell, accounts for at least part of the effect that loss of CFTR expression or inhibition of channel activity has on epithelial restitution.

significantly lower than observed in CFTR expressing control cells [102]. β1‐integrin activation could be completely recovered by incubating CFTR deficient cells with exogenous GM1, but not with GM3 gangliosides, confirming that integrin activation was dependent on GM1 and CFTR expression. Reduced β1 integrin phosphorylation was associated with lower levels of focal adhesion kinase (FAK) and Crk‐associated substrate (CAS) phosphorylation which was

12 Progress in Understanding Cystic Fibrosis

**Figure 2.** Colocalization of CFTR and the β2‐adrenergic receptor (β2‐AR) in the apical membrane of Calu‐3 cells and in cilia of differentiated primary normal human bronchial epithelial (NHBE) cells grown under air‐liquid interface condi‐ tions. (**A**) Antibody labeling of the β2‐AR, CFTR and merged images (where yellow represents colocalization) collected from wild‐type Calu‐3 cells (wt Calu‐3), Calu‐3 cells expressing shRNA that does not recognize CFTR (shALTR) and CFTR‐deficient cells were CFTR expression was silenced by RNAi (shCFTR). (**B**) Labeling of the β2‐AR and CFTR with‐ in the cilia of differentiated primary NHBE cells. Yellow‐orange represents colocalization of the receptor and channel. (**C**) Cross section of differentiated, pseudostratified primary NHBE cells showing colocalization of the β2‐AR and CFTR within the cilia. Note the layering of nuclei reflecting pseudostratification. Images were adapted from Refs. [114] and

[115].

More recently, stimulation of β2‐adrenergic receptors (β2‐AR) expressed on the apical mem‐ brane of normal human bronchial epithelial cells and Calu‐3 cells caused a significant delay in cell migration and wound closure. This effect could be reproduced using carvedilol, a β2‐ AR agonist that functions as a bias ligand to activate cAMP‐independent, β‐arrestin‐dependent signaling cascades [110]. The inhibitory effects of β2‐AR agonists could be blocked if cells were pretreated with an inhibitor of PP2A phosphatase, indicating that PP2A activation was a critical step in regulating cell migration [111, 112]. Interestingly, in airway epithelial cells, β2‐ARs form a tightly coupled signaling complex with CFTR in the apical membrane (see **Figure 2**) such that receptor activation by endogenous ligands, such as epinephrine, or by selective β2‐AR agonists, like salbutamol, stimulate CFTR channel activity [113]. The reduced rate of migration following β2‐AR activation was associated with a reduction in lamellipodia protrusion, similar in magnitude to the effect produced by CFTR channel inhibition with CFTRinh‐172 or silencing by RNAi. Furthermore, β2‐AR agonists, including carvedilol, decreased β1‐integrin activation, and in CFTR‐deficient Calu‐3 cells, β2‐AR activation had no effect on cell migration [114]. These findings suggested a model where exposure to β2‐AR agonists stimulates PP2A phosphatase

**Figure 3.** Summary of proposed interactions and pathways accounting for the decrease in cell migration and epithelial repair associated with loss of CFTR function and β2‐AR activation in airway epithelial cells. This model was adapted from Ref. [114].

activity to produce dephosphorylation of multiple proteins involved in the control of cell motility (see **Figure 3**). Moreover, CFTR inhibition or silencing and β2‐AR stimulation appear to converge on a common control point involving the activation of β1 integrin, which is thought to be the reason why CFTR silencing and β2‐AR activation do not produce additive effects on cell migration.

## **4. Conclusions**

This chapter has focused on the impact of CFTR dysfunction on cell migration and epithelial repair that has direct relevance to airway barrier function. This role for CFTR constitutes an important intrinsic deficit of the CF epithelium that contributes to disease progression. Other intrinsic deficits such as those linked to altered innate immune function appear to underlie abnormal regeneration and remodeling of the CF epithelium that occurs in the absence of infection. Delayed wound repair exacerbates intrinsic inflammation by providing opportuni‐ ties for pathogen access to the airway submucosa, augmenting inflammation and tissue damage. Furthermore, dysregulation of the repair process establishes a chronic cycle of injury and inadequate restitution that intensifies remodeling, ultimately leading to deterioration of lung function. Further investigation is required to more clearly understand the molecular and cellular mechanisms by which CFTR expression and function affect cell motility. Results from these studies should aid in identifying pathways that could be targeted for development of novel pharmacotherapies to reduce airway infection and inflammation in CF.

## **Author details**

Scott M. O'Grady

Address all correspondence to: ograd001@umn.edu

Departments of Animal Science, Integrative Biology and Physiology, University of Minnesota, St. Paul, MN, USA

## **References**


[3] Schwab A, Fabian A, Hanley PJ, Stock C. Role of ion channels and transporters in cell migration. Physiol Rev. 2012; 92:1865–913.

activity to produce dephosphorylation of multiple proteins involved in the control of cell motility (see **Figure 3**). Moreover, CFTR inhibition or silencing and β2‐AR stimulation appear to converge on a common control point involving the activation of β1 integrin, which is thought to be the reason why CFTR silencing and β2‐AR activation do not produce additive effects on

This chapter has focused on the impact of CFTR dysfunction on cell migration and epithelial repair that has direct relevance to airway barrier function. This role for CFTR constitutes an important intrinsic deficit of the CF epithelium that contributes to disease progression. Other intrinsic deficits such as those linked to altered innate immune function appear to underlie abnormal regeneration and remodeling of the CF epithelium that occurs in the absence of infection. Delayed wound repair exacerbates intrinsic inflammation by providing opportuni‐ ties for pathogen access to the airway submucosa, augmenting inflammation and tissue damage. Furthermore, dysregulation of the repair process establishes a chronic cycle of injury and inadequate restitution that intensifies remodeling, ultimately leading to deterioration of lung function. Further investigation is required to more clearly understand the molecular and cellular mechanisms by which CFTR expression and function affect cell motility. Results from these studies should aid in identifying pathways that could be targeted for development of

Departments of Animal Science, Integrative Biology and Physiology, University of Minnesota,

[1] Stock C, Ludwig FT, Hanley PJ, Schwab A. Roles of ion transport in control of cell

[2] Schwab A, Stock C. Ion channels and transporters in tumour cell migration and

novel pharmacotherapies to reduce airway infection and inflammation in CF.

Address all correspondence to: ograd001@umn.edu

motility. Compr Physiol. 2013; 3:59–119.

invasion. Philos Trans R Soc Lond B Biol Sci. 2014; 369:1–8.

cell migration.

14 Progress in Understanding Cystic Fibrosis

**4. Conclusions**

**Author details**

Scott M. O'Grady

St. Paul, MN, USA

**References**


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## **Chapter 2**

## **Cystic Fibrosis–Related Diabetes**

Bernadette Prentice, Shihab Hameed, Chee Y. Ooi,

Charles F. Verge and John Widger

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66452

#### **Abstract**

Cystic fibrosis–related diabetes (CFRD) results in significant morbidity and mortality for patients with cystic fibrosis (CF). It is the endpoint of a spectrum of progressive insulin deficiency with resulting abnormalities of glucose tolerance. The consequence of glycaemic abnormalities in CF is poorer nutritional status, an increase in respiratory exacerbations with decline in lung function and ultimately greater morbidity and mortality. CFRD can be diagnosed by the standard oral glucose tolerance test (OGTT) usually performed from 10 years of age. However, this may miss early glycaemic abnormalities which appear to be clinically important. Early recognition of CFRD and treatment have been shown to improve outcomes in CF. Novel diagnostic methods such as 30-min sampled OGTT and continuous glucose monitoring (CGM) may prove to be useful in screening for this disorder and in the early identification of glycaemic abnormalities.

**Keywords:** cystic fibrosis–related diabetes, glucose, insulin, abnormal glucose tolerance, indeterminate glycaemia, impaired glucose tolerance, oral glucose tolerance test, continuous glucose monitoring

#### **1. Introduction**

Cystic fibrosis (CF) is the most common life-limiting autosomal recessive genetic condition seen in the Caucasian population, affecting approximately 1/2500 live births in Australia [1]. It is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, located on the long arm of chromosome 7 [1] and expressed in the epithelial cells of lungs, pancreas and sweat glands and other organs. Cystic fibrosis–related diabetes (CFRD) is one of the most important complications of the disease as it is known to have a significant impact on morbidity and mortality [2]. Patients with CF ultimately die from recurrent respiratory tract infections

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and respiratory failure which may be hastened by abnormalities of glucose tolerance affecting respiratory function and nutrition.

## **2. Pathophysiology**

The pathophysiology of CFRD is likely multifactorial and complex. Historically CFRD was thought to be the result of progressive pancreatic destruction by secretions of the exocrine pancreas, pancreatic autodigestion and replacement with nonfunctioning fatty tissue, amyloid deposits or fibrotic tissue [3, 4]. This theory was supported by ultrasound findings in patients with CF of an "echogenic" and atrophied pancreas which progresses with age. MRI has also been used to study the pancreas of patients with CF. Sequeiros et al. attempted to determine the pancreatic volume of patients with CF using MRI and compare with Type 1 diabetic patients and controls. In over 70% of patients with CF, the pancreas could not be visualised and this was irrespective of glycaemic status [5]. Pancreatic tissue on autopsies of patients with CF has also noted to have fewer islet cells and replacement with fibrotic tissue. Histologically, patients with CFRD have a relative decrease in the number of islet cells and insulin-containing cells within the islets, relative to the non-CFRD cohort [4, 6].

However, recent information supports the theory that destruction of the physical pancreas does not entirely explain the glycaemic abnormalities in patients with CF. Insulin deficiency has been shown to occur in young children and infants with CF [7], and even infants have been reported to have CFRD [8]. This has also been demonstrated in animal models of CF. In both the pig and ferret CF models, the animals demonstrate abnormal insulin secretion from birth, suggesting that CFTR may play a more direct role in insulin secretion [9, 10]. In the pig model, newborn pigs were noted to develop hyperglycaemia even when there was no significant islet cell destruction [10]. Recent studies of the CFTR potentiator ivacaftor (Kalydeco™), which improves gating defects and thus should not have any impact on fatty or fibrotic tissue, have demonstrated an improvement in glucose abnormalities [11, 12]. This suggests that the intrinsic abnormality in the CFTR protein may play a role in glycaemic control in CF.

The timeframe during which patients with CF develop glycaemic abnormalities and CFRD has significant variability, and the specific CFTR class abnormality does not entirely account for this unpredictability. Non-CFTR genetic modifiers may play a key role in determining this risk. Patients with CF who have a family history of Type 2 diabetes are known to have an increased risk of CFRD [13, 14]. Polymorphisms in *TCF7L2*, a "susceptibility gene" for Type 2 diabetes, are more common in patients with CFRD. The pathophysiology also appears to have similarities. Couce et al. noted that islet cell amyloidosis, which is characteristic of pancreatic histology of patients with Type 2 Diabetes mellitus, is also present in CF patients with CFRD and "borderline diabetes" but not in nondiabetic CF patients or controls [3]. Other genetic modifiers have been shown to modify CF phenotypes, including *SLC26A9* which has been demonstrated to be more common in patients who develop CFRD [15].

In normal insulin physiology, insulin secretion occurs in two phases—the first phase results from exocytosis of preformed insulin granules which is the result of a voltage-dependent calcium channel being triggered by blood glucose elevations [16–18]. The second phase requires maturation of insulin granules and lasts minutes to hours [19, 20]. Oral glucose ingestion results in a limited and delayed first-phase insulin peak when compared with intravenous administration [21, 22]. Overall, the amount of insulin secreted appears to be amplified when glucose is given orally, rather than intravenously. Incretins (glucagon-like peptide and gastric inhibitory peptide) are secreted from neuroendocrine cells of the gastrointestinal system and increase insulin secretion and decrease glucagon secretion. The secretion of incretins is hypothesised to be the result of the action of oral glucose within the gastrointestinal tract [21]. The role of incretins in CFRD has not yet been fully elucidated, and it is unclear whether or not patients with CFRD have abnormal levels of incretins. However, the diet of CF patients may play a role in the development of CFRD. In patients with Type 2 diabetes mellitus (DM), those prescribed orlistat, a lipase inhibitor, had diminished fat digestion which resulted in greater postprandial hyperglycaemia [23]. In a randomised crossover trial, Perano et al. demonstrated that adolescent patients with CF, who did not take appropriate pancreatic enzyme supplementation, experienced amplified postprandial hyperglycaemia [24]. Barrio postulates in her review that inadequate enzyme supplementation in patients with CF results in fat malabsorption, which may hasten gastric emptying, thereby inhibiting the normal augmentation of insulin response by the neuroendocrine cells [25]. Exogenous incretin therapy has proven beneficial in patients with Type 2 DM, but it has also been associated with weight loss in this cohort, an undesirable outcome for patients with CF [26]. Hyperglycaemia is known to promote beta-cell apoptosis, and as such, postprandial hyperglycaemia from dysfunctional incretin secretion in CF may potentiate the glycaemic abnormalities demonstrated and hasten the progression to CFRD.

and respiratory failure which may be hastened by abnormalities of glucose tolerance affecting

The pathophysiology of CFRD is likely multifactorial and complex. Historically CFRD was thought to be the result of progressive pancreatic destruction by secretions of the exocrine pancreas, pancreatic autodigestion and replacement with nonfunctioning fatty tissue, amyloid deposits or fibrotic tissue [3, 4]. This theory was supported by ultrasound findings in patients with CF of an "echogenic" and atrophied pancreas which progresses with age. MRI has also been used to study the pancreas of patients with CF. Sequeiros et al. attempted to determine the pancreatic volume of patients with CF using MRI and compare with Type 1 diabetic patients and controls. In over 70% of patients with CF, the pancreas could not be visualised and this was irrespective of glycaemic status [5]. Pancreatic tissue on autopsies of patients with CF has also noted to have fewer islet cells and replacement with fibrotic tissue. Histologically, patients with CFRD have a relative decrease in the number of islet cells and

However, recent information supports the theory that destruction of the physical pancreas does not entirely explain the glycaemic abnormalities in patients with CF. Insulin deficiency has been shown to occur in young children and infants with CF [7], and even infants have been reported to have CFRD [8]. This has also been demonstrated in animal models of CF. In both the pig and ferret CF models, the animals demonstrate abnormal insulin secretion from birth, suggesting that CFTR may play a more direct role in insulin secretion [9, 10]. In the pig model, newborn pigs were noted to develop hyperglycaemia even when there was no significant islet cell destruction [10]. Recent studies of the CFTR potentiator ivacaftor (Kalydeco™), which improves gating defects and thus should not have any impact on fatty or fibrotic tissue, have demonstrated an improvement in glucose abnormalities [11, 12]. This suggests that the

insulin-containing cells within the islets, relative to the non-CFRD cohort [4, 6].

intrinsic abnormality in the CFTR protein may play a role in glycaemic control in CF.

demonstrated to be more common in patients who develop CFRD [15].

The timeframe during which patients with CF develop glycaemic abnormalities and CFRD has significant variability, and the specific CFTR class abnormality does not entirely account for this unpredictability. Non-CFTR genetic modifiers may play a key role in determining this risk. Patients with CF who have a family history of Type 2 diabetes are known to have an increased risk of CFRD [13, 14]. Polymorphisms in *TCF7L2*, a "susceptibility gene" for Type 2 diabetes, are more common in patients with CFRD. The pathophysiology also appears to have similarities. Couce et al. noted that islet cell amyloidosis, which is characteristic of pancreatic histology of patients with Type 2 Diabetes mellitus, is also present in CF patients with CFRD and "borderline diabetes" but not in nondiabetic CF patients or controls [3]. Other genetic modifiers have been shown to modify CF phenotypes, including *SLC26A9* which has been

In normal insulin physiology, insulin secretion occurs in two phases—the first phase results from exocytosis of preformed insulin granules which is the result of a voltage-dependent

respiratory function and nutrition.

26 Progress in Understanding Cystic Fibrosis

**2. Pathophysiology**

CFRD is distinct from both Type 1 and Type 2 diabetes. CFRD is not an autoimmune condition like Type 1 DM and is not associated with autoantibodies found in Type 1 DM. Moreover, Type 2 DM is primarily a disorder of insulin resistance, whereas glucose abnormalities in CF are primarily the result of insulin deficiency, which is present even in CF patients with normal glucose tolerance on oral glucose tolerance test (OGTT) [7]. One of the features of CFRD that differentiates it from other forms of diabetes is the variation in glucose tolerance demonstrated over time [27]. Although abnormalities of glucose tolerance are known to progress and the complications of diabetes increase in the degree of abnormal glycaemia, some patients with the diagnosis of CFRD will have OGTT results that normalise [27]. The role of insulin resistance has been less well defined although there is emerging evidence of its importance. Ahmad et al. illustrated that patients with CF actually had an increase in peripheral insulin sensitivity compared to healthy controls matched for age and body mass index. They concluded that this increase in peripheral sensitivity in CF patients was a metabolic compensation for insulin deficiency [28]. Moran et al. replicated these findings in exocrine-insufficient CF patients without diabetes. However, once CFRD had developed, there was an increase in peripheral insulin resistance [29]. The mechanism by which this may occur could be the result of a downregulation of GLUT-4 insulin-sensitive channels secondary to chronic hyperglycaemia [30] ("glucose toxicity"). Insulin resistance is also thought to vary over time which could explain the variability of glucose tolerance seen in patients with CF, including a normalisation of previously abnormal glucose tolerance on OGTT. It is often cited that glucose abnormalities worsen during pulmonary exacerbations (due to cytokine and stress hormone release), but the data to support this suggestion is limited and was not found in the study by Widger et al. [31]. This group performed OGTT in patients with a pulmonary exacerbation and then repeated the OGTT when well. Although the sample size was small, 8/9 patients remained within their glycaemic category even when recovered from their pulmonary exacerbation. However insulin resistance is known to increase during periods of corticosteroid usage, overnight feeds [32, 33], pregnancy and during puberty [34–36]. In the latter case, insulin resistance is thought to increase as a result of a physiological elevation in growth hormone [34], and this may account for the increased detection of CFRD in this age group.

Chronic inflammation may play a key role in the development of glucose abnormalities in CF. Bismuth et al. demonstrated in their cohort of patients with CF that the erythrocyte sedimentation rate (ESR), a marker of inflammation, positively correlated with HbA1c and the area under the curve (AUC) for glucose in patients undergoing OGTT [37]. Significant and ongoing oxidative stress is one mechanism hypothesised to result in an inflammatory state and beta-cell apoptosis [38, 39]. One review postulated that the imbalance in inflammatory T-cell lymphocytes known to play a role in the development of other forms of diabetes may contribute to lung inflammation and thereby a chronic inflammatory states resulting in glucose abnormalities [40]. T-helper 17 (Th-17) lymphocyte cells secrete a pro-inflammatory cytokine-IL-17 known to be involve in pulmonary inflammation in CF and is known to be present in higher levels compared to controls in patients with Type 2 diabetes. Furthermore, studies also suggest that IL-17 may play an important role in the development of Type 1 diabetes [41] and may contribute to β-cell destruction. It has also been postulated that cytokines such as TNF-α may act directly on the insulin receptor by inducing insulin resistance, thereby inhibiting the potential action of insulin [42].

The pathophysiology of CFRD is likely to be multifactorial but ultimately resulting from progressive insulin deficiency secondary to islet cell destruction and defective beta-cell secretion, combined with stressors that intermittently increase insulin resistance resulting in a further deterioration of glycaemic status. Certain patients may be more at risk if non-CFTR genetic modifiers are present [13, 14], and perhaps these patients are unable to compensate for the degree of histological pancreatic destruction and defective beta-cell functioning.

## **3. Epidemiology**

### **3.1. Prevalence of glycaemic abnormalities in CF**

CFRD is known to occur in up to 50% of patients with CF by the age of 30 years [43] and the prevalence increases with age. CFRD can occur in young children with CF but is rare [8]. Recent studies suggest that CFRD affects approximately 9% in the 5–9 year age group [44] and a smaller proportion of children under 5 may also meet the CFRD diagnostic criteria. Yi et al. recently reported a series that suggested 5% of their cohort between 6 months and 5 years had CFRD [45]. Although a small proportion of young children have CFRD, the average age of onset is 20 years [46]. CFRD occurs more commonly in females with a prevalence of 17% in young female adults compared with 12% in males previously described [47].

Children with CF are known to be insulin deficient from birth. Milner et al. demonstrated that children with CF in the first year of life had lower insulin levels than controls [7]. Insulin deficiency will progress over time and results in a gradual deterioration of glucose tolerance. As such, impaired glucose tolerance is much more common than CFRD and can affect up to 41% of children in the 6–9 year age group [48], compared with only 10% of this group being classified as CFRD. The risk of early CFRD is much higher in children with abnormal glucose tolerance on OGTT [48].

#### **3.2. Screening**

to cytokine and stress hormone release), but the data to support this suggestion is limited and was not found in the study by Widger et al. [31]. This group performed OGTT in patients with a pulmonary exacerbation and then repeated the OGTT when well. Although the sample size was small, 8/9 patients remained within their glycaemic category even when recovered from their pulmonary exacerbation. However insulin resistance is known to increase during periods of corticosteroid usage, overnight feeds [32, 33], pregnancy and during puberty [34–36]. In the latter case, insulin resistance is thought to increase as a result of a physiological elevation in growth hormone [34], and this may account for the increased detection of CFRD in this age

Chronic inflammation may play a key role in the development of glucose abnormalities in CF. Bismuth et al. demonstrated in their cohort of patients with CF that the erythrocyte sedimentation rate (ESR), a marker of inflammation, positively correlated with HbA1c and the area under the curve (AUC) for glucose in patients undergoing OGTT [37]. Significant and ongoing oxidative stress is one mechanism hypothesised to result in an inflammatory state and beta-cell apoptosis [38, 39]. One review postulated that the imbalance in inflammatory T-cell lymphocytes known to play a role in the development of other forms of diabetes may contribute to lung inflammation and thereby a chronic inflammatory states resulting in glucose abnormalities [40]. T-helper 17 (Th-17) lymphocyte cells secrete a pro-inflammatory cytokine-IL-17 known to be involve in pulmonary inflammation in CF and is known to be present in higher levels compared to controls in patients with Type 2 diabetes. Furthermore, studies also suggest that IL-17 may play an important role in the development of Type 1 diabetes [41] and may contribute to β-cell destruction. It has also been postulated that cytokines such as TNF-α may act directly on the insulin receptor by

inducing insulin resistance, thereby inhibiting the potential action of insulin [42].

degree of histological pancreatic destruction and defective beta-cell functioning.

young female adults compared with 12% in males previously described [47].

The pathophysiology of CFRD is likely to be multifactorial but ultimately resulting from progressive insulin deficiency secondary to islet cell destruction and defective beta-cell secretion, combined with stressors that intermittently increase insulin resistance resulting in a further deterioration of glycaemic status. Certain patients may be more at risk if non-CFTR genetic modifiers are present [13, 14], and perhaps these patients are unable to compensate for the

CFRD is known to occur in up to 50% of patients with CF by the age of 30 years [43] and the prevalence increases with age. CFRD can occur in young children with CF but is rare [8]. Recent studies suggest that CFRD affects approximately 9% in the 5–9 year age group [44] and a smaller proportion of children under 5 may also meet the CFRD diagnostic criteria. Yi et al. recently reported a series that suggested 5% of their cohort between 6 months and 5 years had CFRD [45]. Although a small proportion of young children have CFRD, the average age of onset is 20 years [46]. CFRD occurs more commonly in females with a prevalence of 17% in

group.

28 Progress in Understanding Cystic Fibrosis

**3. Epidemiology**

**3.1. Prevalence of glycaemic abnormalities in CF**

The prevalence of identified CFRD has been shown to increase after the introduction of screening [49]. Unlike Type 1 or Type 2 diabetes which are often symptomatic, CFRD does not often present with symptoms of hyperglycaemia although this can occur in approximately one third of patients. Symptoms can include polyuria and polydipsia, but CFRD is more likely to present insidiously with the catabolic complications of insulin deficiency such as nutritional deterioration or decline in pulmonary function. When routine screening was introduced in Australia, the incidence of CFRD increased from 2.0 to 22.1 per 1000 person years between 2000 and 2008, which represents a tenfold increase [50]. A decline in the age of diagnosis has also been demonstrated after the introduction of routine screening; Noronha et al. reported a reduction in the mean age of diagnosis from 22.3 years to 13.5 years [49]. Routine screening from at least 10 years of age with an OGTT is recommended by most guidelines [51, 52].

#### **3.3. Risk factors for CFRD**

The risk factors for the development of CFRD are closely linked to the specific CFTR genotype and the severity of the CFTR protein dysfunction [53]. CFTR mutations are classified according to the resulting functional deficit [54]. Class 1 and class 2 mutations result in the total or partial absence of CFTR protein at the surface membrane due to defective/non-functional protein (Class 1, e.g. stop codon mutations) or due to defective transfer of the protein to the cell membrane, i.e. defective "trafficking" (Class 2, e.g. F508) [25]. Classes 3, 4, 5 and 6 have irregularities in regulation, conductance, prevalence and stability of CFTR at the membrane [55]. Of the latter, 4 classes, all except class 3, which is known as a gating mutation, have partial function. Those classes with no action have a more severe phenotype and are associated with a greater risk of CFRD, such as homozygous F508 patients [46].

CFRD generally occurs in patients with pancreatic insufficiency. There have been reports of CFRD in patients who are pancreatic sufficient, but the diagnostic criteria for exocrine pancreatic function do not appear to be robust [47]. Some of these patients were classified as pancreatic sufficient because they were not taking replacement enzymes, but had not undergone any formal diagnostic testing such as faecal elastase or 3-day fat stool sampling. More recent studies have demonstrated that the degree of pancreatic exocrine function appears to correlate with the development of CFRD. Soave et al. demonstrated a causal relationship between the level of serum trypsinogen on the newborn screen (a marker of exocrine pancreatic function used to diagnose CF) and the development of CFRD over time [15]. Trypsinogen is an inactive pancreatic enzyme precursor required for protein digestion and absorption. It is converted to trypsin when secreted into the small intestine, but this process is inhibited in CF and results in an elevated serum trypsinogen. A significant elevation in the blood levels of immunoreactive trypsinogen (IRT) on newborn screening is used to identify neonates with CF. The IRT level is known to decline rapidly over Time with ongoing pancreatic destruction. Soave et al. postulated that patients with CF who had more significant pancreatic disease at birth would have IRT levels that had already started to decline and would be relatively lower than the rest of the CF cohort [15]. They also demonstrated that those children with relatively low IRT amongst the CF cohort had an increased risk of CFRD, thus confirming the relationship between exocrine pancreatic function and endocrine disease.

The presence of CF liver disease appears to be a significant risk factor in the development of CFRD. Leung et al. examined over 700 liver ultrasounds of patients with CF and found that patients with the features of heterogenous or cirrhotic liver disease on ultrasound were more likely to have abnormalities of glucose tolerance, including CFRD, than those with normal liver ultrasounds [56]. The relationship between liver disease and CFRD remains unclear. It could be a result of the more severe genotypes causing CFRD also increasing the risk of liver disease, or it could be the result of a non-CFTR genetic modifier.

Abnormal glucose tolerance is a known risk factor for progression to CFRD. CF patients with glucose abnormalities are up to 11 times more likely to develop early CFRD than other 6–9-year-old patients [48].

## **4. The clinical impact of glucose abnormalities in CF**

Glucose abnormalities in CF are associated with significantly increased morbidity and mortality [2]. Prior to the introduction of routine screening for CFRD, less than 25% of CFRD patients survived to age 30, compared with 60% of patients without diabetes [57]. When Moran et al. examined female CF cohorts with and without CFRD in the 1990s and compared them with cohorts after the introduction of routine CFRD screening, mortality rates had halved: 6.9 per 100 patients years in patients with CFRD versus 3.2 per 100 patient years in CF without diabetes, with similar results seen in men were reported [58]. Although mortality rates for patients with CFRD have seen a marked improvement, a significant difference between CF patients with and without diabetes persists [59].

CFRD leads to a significant increase in respiratory exacerbations, increased infection with CF pathogens [60] (including *Pseudomonas aeruginosa*) and poorer lung function [57, 61]. The mechanism by which insulin deficiency resulting in CFRD has such a negative impact on lung function in CF is probably multifactorial. The hyperglycaemic environment is said to create a "pro-inflammatory" environment, optimal for bacterial growth and colonisation that allows CF pathogens to thrive [52]. In vitro studies have demonstrated an amplification of bacterial growth, in particular *Staphylococcus aureus* and *P*. *aeruginosa* with increasing glucose concentrations [62], and this evidence supports the hypothesis of glycaemic abnormalities playing a significant and direct role in the infection and colonisation of patients with CF.

Blood glucose levels >8 mmol/L correlate with increased airway glucose levels in patients with CF [62]. In non-CF patients, elevated airway glucose has been demonstrated to be a risk factor for respiratory infections, including MRSA (based on studies in patients intubated due to critical illness in the intensive care unit [63]). When Brennan et al. examined the airway glucose of patients with CF, they demonstrated that even patients with normal glucose tolerance on OGTT had glucose in their airway for longer periods of time than the control population. The duration of time spent with airway glucose levels >8 mmol/L correlated with the degree of glucose abnormality [62]. The level at which glucose appears in the airway is much lower than the 2-h OGTT glycaemic threshold for CFRD and also appears to be very close to the level of blood glucose level (BGL) which correlates with significant nutritional and respiratory decline [64].

converted to trypsin when secreted into the small intestine, but this process is inhibited in CF and results in an elevated serum trypsinogen. A significant elevation in the blood levels of immunoreactive trypsinogen (IRT) on newborn screening is used to identify neonates with CF. The IRT level is known to decline rapidly over Time with ongoing pancreatic destruction. Soave et al. postulated that patients with CF who had more significant pancreatic disease at birth would have IRT levels that had already started to decline and would be relatively lower than the rest of the CF cohort [15]. They also demonstrated that those children with relatively low IRT amongst the CF cohort had an increased risk of CFRD, thus confirming the relation-

The presence of CF liver disease appears to be a significant risk factor in the development of CFRD. Leung et al. examined over 700 liver ultrasounds of patients with CF and found that patients with the features of heterogenous or cirrhotic liver disease on ultrasound were more likely to have abnormalities of glucose tolerance, including CFRD, than those with normal liver ultrasounds [56]. The relationship between liver disease and CFRD remains unclear. It could be a result of the more severe genotypes causing CFRD also increasing the risk of liver

Abnormal glucose tolerance is a known risk factor for progression to CFRD. CF patients with glucose abnormalities are up to 11 times more likely to develop early CFRD than other

Glucose abnormalities in CF are associated with significantly increased morbidity and mortality [2]. Prior to the introduction of routine screening for CFRD, less than 25% of CFRD patients survived to age 30, compared with 60% of patients without diabetes [57]. When Moran et al. examined female CF cohorts with and without CFRD in the 1990s and compared them with cohorts after the introduction of routine CFRD screening, mortality rates had halved: 6.9 per 100 patients years in patients with CFRD versus 3.2 per 100 patient years in CF without diabetes, with similar results seen in men were reported [58]. Although mortality rates for patients with CFRD have seen a marked improvement, a significant difference between CF patients

CFRD leads to a significant increase in respiratory exacerbations, increased infection with CF pathogens [60] (including *Pseudomonas aeruginosa*) and poorer lung function [57, 61]. The mechanism by which insulin deficiency resulting in CFRD has such a negative impact on lung function in CF is probably multifactorial. The hyperglycaemic environment is said to create a "pro-inflammatory" environment, optimal for bacterial growth and colonisation that allows CF pathogens to thrive [52]. In vitro studies have demonstrated an amplification of bacterial growth, in particular *Staphylococcus aureus* and *P*. *aeruginosa* with increasing glucose concentrations [62], and this evidence supports the hypothesis of glycaemic abnormalities playing a significant and direct role in the infection and colonisation of patients

ship between exocrine pancreatic function and endocrine disease.

disease, or it could be the result of a non-CFTR genetic modifier.

**4. The clinical impact of glucose abnormalities in CF**

6–9-year-old patients [48].

30 Progress in Understanding Cystic Fibrosis

with and without diabetes persists [59].

with CF.

Respiratory tract infections may not entirely account for the deterioration in lung function seen in patients with CF. Patients with diabetes mellitus from other causes have also been demonstrated to have poorer lung function than matched controls, even in the absence of respiratory disease [65, 66]. It is unclear whether this is a direct result of glucose in the airways or an indirect result of inflammation from relative insulin deficiency.

Nutrition in CF has a direct correlation with survival [67], and insulin, an anabolic hormone, plays an integral role in maintaining weight and building muscle [18]. When CF patients are insulin deficient, this manifests as poorer nutritional status. Multiple studies have demonstrated the impact of CFRD and insulin deficiency on nutrition and growth [37]. The data of over 8000 CF patients on the epidemiologic study of cystic fibrosis (ESCF) was analysed in 2005 and confirmed a greater impairment in nutrition in the CFRD group when compared with the nondiabetic group [47]. The CFRD cohort had statistically lower height for age percentiles, weight for age percentiles and BMI (p < 0.001 for all three parameters). A statistically significant difference in body weight and BMI has also been demonstrated in the "prediabetic" CF patients when compared with CF patients with normal glucose tolerance [61]. This decline was detected by Lanng et al. in some patients 4 years prior to the diagnosis of CFRD being. Given the insidious nature of glycaemic abnormalities and the inherent difficulties with nutrition in patients with CF, particularly those with exocrine pancreatic insufficiency, the impact of insulin deficiency is often not recognised until CFRD is diagnosed on routine screening.

## **5. The spectrum of glucose abnormalities in CF**

Insulin deficiency is progressive and results in a deterioration of glucose tolerance over time. CFRD lies at the end of a spectrum of glucose abnormalities. Glycaemic categories in CF are determined based on the results of the oral glucose tolerance test (OGTT) [51]. To perform an OGTT, a glucose load of 1.75 g/kg (maximum 75 g) is consumed after fasting. Classically the blood glucose level (BGL) is measured at 0 and 120 min [68]. Additional information about glucose tolerance is gained by also checking the BGL at 30 min, 60 min and 90 min, i.e. a 30-min sampled OGTT [64].

The diagnosis of CFRD is made based on the American Diabetic Association (ADA) criteria [51] (see **Table 1**). CFRD is diagnosed when the 2-h OGTT level is ≥ 11.1 mmol/L and can


**Table 1.** Classification of abnormalities of glucose tolerance in cystic fibrosis on OGTT.

occur with or without fasting hyperglycaemia (fasting BGL ≥ 7.0 mmol/L is defined as fasting hyperglycaemia). Fasting hyperglycaemia can also be considered diagnostic of CFRD, if still abnormal when repeated. One fasting BGL ≥ 7.0 mmol/L and another non-fasting level ≥ 11.1 mmol/L can also make a diagnosis of CFRD. If a patient is sick and glycaemic abnormalities persist for two days, then the diagnosis can also be made. Most guidelines recommend the OGTT/BGL is repeated before the diagnosis is confirmed. Some guidelines subclassify CFRD based on the fasting BGL, but this distinction does not alter management, as insulin treatment is recommended for those with and without fasting hyperglycaemia.

Additional criteria have been published to subclassify the patients into glycaemic categories based on 30-min samples (see **Table 1**) [1]. Patients with normal glucose tolerance have fasting BGL <7.0 mmol/L and 2-h level <7.8 mmol/L. Indeterminate glycaemia (INDET) is defined as normal fasting and 2-h levels with a midpoint level ≥ 11.1 mmol/L. Impaired glucose tolerance (IGT) is defined by a 2-h level <11.1 mmol/L but ≥7.8 mmol/L.

Children with abnormal glucose tolerance and CF may fluctuate between glycaemic categories because of increasing insulin requirements at times of illness or because of variable levels of resistance. In one study 18% of CF patients with abnormal glucose tolerance had glycaemic abnormalities that improved over time. Twenty-two percent of patients had a deterioration in their glucose tolerance [27]. This variability was replicated by Lanng et al. who saw a normalisation of the patient OGTT in 58% of adult patients with CF when followed up after 5 years [69]. Yi et al. examined glucose tolerance in young children (<6 years) and found that some of these children with abnormal glucose tolerance that normalised, including those that met CFRD criteria [45]. This variability adds to the difficulty seen in managing patients with CFRD, particularly younger children.

## **6. Issues with the OGTT in CFRD**

The OGTT was not designed to diagnose diabetes in the CF population. The test was designed to determine the treatment threshold for Pima Native American population with Type 2 diabetes based on their risk of developing microvascular complications [70]. Although microvascular diabetes complications can occur in CF, the major concern for CFRD is its impact on nutrition and lung function. Complications from chronic intermediate hyperglycaemia may also result in microvascular disease prior to the patient meeting the criteria for CFRD [71]. More practical goals would include an initiation of treatment at a time that would alleviate significant respiratory morbidity such as recurrent infections and respiratory function decline. The drop in nutritional status and weight, or poor growth in younger children because of insulin deficiency catabolism, would be a more relevant CF-specific outcome to guide diagnostic targets.

The decrease in lung function and nutrition seen in CFRD actually precedes the diagnosis by several years and is often insidious. Lanng et al. noted that a decline was present up to four years prior to the OGTT 2-h criteria being met [61]. Furthermore, insulin therapy has been demonstrated to reverse some of the nutritional decline seen in patients with abnormal glycaemia [72, 73]. However, once patients meet the criteria for CFRD, recovery of lung function is not always possible. Widger et al. postulate that by waiting until the patient meets the CFRD criteria to start insulin, the conceded progression from abnormal glucose tolerance to CFRD allows irreversible structural remodelling of the lungs that cannot be corrected with insulin therapy [74].

occur with or without fasting hyperglycaemia (fasting BGL ≥ 7.0 mmol/L is defined as fasting hyperglycaemia). Fasting hyperglycaemia can also be considered diagnostic of CFRD, if still abnormal when repeated. One fasting BGL ≥ 7.0 mmol/L and another non-fasting level ≥ 11.1 mmol/L can also make a diagnosis of CFRD. If a patient is sick and glycaemic abnormalities persist for two days, then the diagnosis can also be made. Most guidelines recommend the OGTT/BGL is repeated before the diagnosis is confirmed. Some guidelines subclassify CFRD based on the fasting BGL, but this distinction does not alter management, as insulin treatment is recommended for those with and without fasting hyperglycaemia.

<7 mmol/L ≥11.1 mmol/L <7.8 mmol/L

<7 mmol/L ≥11.1 mmol/L

≥7 mmol/L ≥11.1 mmol/L

<7 mmol/L ≥7.8 and < 11.1 mmol/L

**Category Fasting level Midpoint peak (1 h) 2-h plasma level** Normal glucose tolerance <7 mmol/L <11.1 mmol/L <7.8 mmol/L

Additional criteria have been published to subclassify the patients into glycaemic categories based on 30-min samples (see **Table 1**) [1]. Patients with normal glucose tolerance have fasting BGL <7.0 mmol/L and 2-h level <7.8 mmol/L. Indeterminate glycaemia (INDET) is defined as normal fasting and 2-h levels with a midpoint level ≥ 11.1 mmol/L. Impaired glucose tolerance

Children with abnormal glucose tolerance and CF may fluctuate between glycaemic categories because of increasing insulin requirements at times of illness or because of variable levels of resistance. In one study 18% of CF patients with abnormal glucose tolerance had glycaemic abnormalities that improved over time. Twenty-two percent of patients had a deterioration in their glucose tolerance [27]. This variability was replicated by Lanng et al. who saw a normalisation of the patient OGTT in 58% of adult patients with CF when followed up after 5 years [69]. Yi et al. examined glucose tolerance in young children (<6 years) and found that some of these children with abnormal glucose tolerance that normalised, including those that met CFRD criteria [45]. This variability adds to the difficulty seen in managing patients with

The OGTT was not designed to diagnose diabetes in the CF population. The test was designed to determine the treatment threshold for Pima Native American population with Type 2 diabetes based on their risk of developing microvascular complications [70].

(IGT) is defined by a 2-h level <11.1 mmol/L but ≥7.8 mmol/L.

**Table 1.** Classification of abnormalities of glucose tolerance in cystic fibrosis on OGTT.

CFRD, particularly younger children.

Indeterminate glycaemia

32 Progress in Understanding Cystic Fibrosis

Impaired glucose tolerance

CFRD without fasting hyperglycaemia

CFRD with fasting hyperglycaemia

(INDET)

(IGT)

**6. Issues with the OGTT in CFRD**

Further evidence for insulin therapy at an earlier stage of the glycaemic spectrum is warranted, and initial data has highlighted which patients may benefit most. Schmid et al. demonstrated that in 1000 patients with CF, patients with midpoint level ≥11.1 mmol/L (INDET) were predictive for later development of CFRD [75]. Brodsky et al. were able to establish that the 1-h level on the OGTT correlated with poorer lung function [76]. They examined 101 patients with CF and these patients with higher 1-h levels had poorer respiratory status even when corrected for nutritional status. The 2-h "diagnostic" level in this group did not correlate with BMI or lung function. The findings of Coriati et al. [77] confirm that waiting for the 2-h BGL to be diagnostic of CFRD may be too late. Their cohort of patients with indeterminate glycaemia already had significant loss of lung function, equivalent to the lung function of patients with newly diagnosed CFRD. The criteria to start insulin in the future may be determined by the patient's own risk of developing CFRD or by early clinical signals in lung function and intermediate glucose abnormalities.

Hameed et al. used a 30-min sampled OGTT and found that a peak BGL ≥ 8.2 mmol/L was reliably predictive of a decline in lung function and nutrition in the preceding year [64]. Based


**Table 2.** Proposed new staging criteria for insulin deficiency and early glucose abnormalities in CF, based on the OGTT with samples every 30 min.

on these results, this group proposed a new staging criteria to identify insulin deficiency and early glucose abnormalities in patients with CF (see **Table 2**) [78]. Cystic fibrosis insulin deficiencies (CFID) 4 and 3 correspond to existing CFRD categories with and without fasting hyperglycaemia, respectively. CFID 1 and 2 are earlier stages of insulin deficiency that are distinct from impaired glucose tolerance (IGT) because they are based on the peak glucose level and have 2-h levels < 11.1. CFID 1 is defined by a midpoint peak glucose level ≥8.2 mmol/L, and CFID2 has a midpoint glucose peak ≥11.1 mmol/L.

## **7. Continuous glucose monitoring in CF**

Continuous glucose monitoring (CGM) has been used for several years in the management of Type 1 diabetes although it is not licenced for use as a diagnostic device. CGM uses a small probe inserted into the subcutaneous space where it measures interstitial glucose levels. Inserting the device is a relatively simple procedure that can be done within a few minutes in a clinic environment. It is easy to remove at home by the patient or carer, without any specific medical training. The device averages the glucose readings every five minutes and can be worn for several days whilst the patient continues to participate in normal activities and consumes their normal diet. The CGM device has been validated in CF and non-CF populations and shown to correlate with plasma glucose measurements [79, 80]. When compared with OGTT, CGM appears to be reproducible and a reliable assessment of glycaemic abnormalities. When used in Type 1 diabetes, Bergenstal was able to demonstrate that children and adults on insulin pumps had improved glycaemic control, as measured by HbA1c than those who did not use CGM [81].

CGM may be particularly useful in managing cystic fibrosis. CF patients frequently demonstrate early postprandial hyperglycaemia [79, 82, 83], reflected by elevations in readings on a 30-min sampled OGTT in the setting of a normal 2-h level. This intermittent postprandial hyperglycaemia may be reflected in the poor correlation of HbA1c (glycated haemoglobin) with early glycaemic abnormalities in CF. HbA1c represents an index of the average of blood glucose concentrations in the preceding 2–3-month period, and the result is influenced by the half-life of the red cells [84]. When measured in CF, it is a poor indicator of glycaemic abnormalities as it is often still normal by the time a diagnosis of CFRD has been made. The poor sensitivity of the test may result from the intermittent nature of hyperglycaemia in patients with CF, which is not revealed in the HbA1c level when the glucose levels are "averaged", as well as increased red cell turnover in CF.

CGM provides a useful tool to guide insulin treatment once the diagnosis of CFRD has been made [79], but it may also offer a potential opportunity to capture the moments of postprandial hyperglycaemia in CF in the screening and diagnostic phase. In CF patients with normal glucose tolerance on OGTT, abnormalities on CGM have been detected [79, 82, 83]. This could reflect the fact that patients with CF undergo a period of fasting prior to their glucose load in the OGTT which will only measure two values. When a CGM is worn, patients can be at home and may consume their normal CF diet including a carbohydrate load that may exceed the glucose level consumed during an OGTT. In the same way that HbA1c may not reflect a true picture of glycaemic abnormalities in CF, so too may the OGTT underestimate the hyperglycaemia in these patients, particularly in the early phase of glucose abnormalities.

CGM may be a useful device in predicting which children with CF will develop glycaemic abnormalities. Schiaffini et al. performed OGTT and CGM on children with CF and then repeated the OGTT after 2 years. Children who had diabetic excursions on CGM at baseline, even those with normal glucose tolerance on OGTT, developed impaired glucose tolerance or CFRD when the OGTT was repeated 2 years later [83]. Initial data on CGM does appear to suggest that this tool may be useful in identifying clinically significant glucose abnormalities in CF. Leclercq et al. demonstrated, in a CF population with normal OGTT, that patients who recorded glucose levels in the diabetic range (≥11.1 mmol/L) on CGM had poorer lung function and greater colonisation with CF respiratory pathogens such as *P*. *aeruginosa* [85].

Glycaemic abnormalities are known to have a significant impact on nutrition in patients with CF. CGM may provide an opportunity to highlight which children are at risk of nutritional decline secondary to abnormities of glucose tolerance as described in the study by Hameed et al. [64]. In this study of 25 children with CF undergoing CGM, if ≥ 4.5% of the study duration was spent with an interstitial glucose reading >7.8 mmol/L, this was predictive of a decline in weight standard deviation score. This CGM criterion had a sensitivity of 89% and a specificity of 86% in detecting this nutritional decline. CGM abnormalities do appear to be clinically significant, but there are not studies as yet demonstrating a benefit from treatment based on CGM recordings in CF, and the device is not yet licenced to make a diagnosis of diabetes.

## **8. Management of CFRD**

The main aim of CFRD treatment is to correct the hyperglycaemia and its downstream effects on respiratory function and infections, in addition to reversing significant protein catabolism secondary to insulin deficiency. Optimal management has been shown to improve lung function and morbidity [72]. Although a drop in mortality from late CFRD diagnosis has been seen, the risk of early mortality is still higher in this population. The mainstay of treatment is exogenous insulin therapy, but studies are underway examining the benefits of dietary changes and the use of oral hypoglycaemic agents in CF.

#### **8.1. Insulin**

on these results, this group proposed a new staging criteria to identify insulin deficiency and early glucose abnormalities in patients with CF (see **Table 2**) [78]. Cystic fibrosis insulin deficiencies (CFID) 4 and 3 correspond to existing CFRD categories with and without fasting hyperglycaemia, respectively. CFID 1 and 2 are earlier stages of insulin deficiency that are distinct from impaired glucose tolerance (IGT) because they are based on the peak glucose level and have 2-h levels < 11.1. CFID 1 is defined by a midpoint peak glucose level ≥8.2 mmol/L,

Continuous glucose monitoring (CGM) has been used for several years in the management of Type 1 diabetes although it is not licenced for use as a diagnostic device. CGM uses a small probe inserted into the subcutaneous space where it measures interstitial glucose levels. Inserting the device is a relatively simple procedure that can be done within a few minutes in a clinic environment. It is easy to remove at home by the patient or carer, without any specific medical training. The device averages the glucose readings every five minutes and can be worn for several days whilst the patient continues to participate in normal activities and consumes their normal diet. The CGM device has been validated in CF and non-CF populations and shown to correlate with plasma glucose measurements [79, 80]. When compared with OGTT, CGM appears to be reproducible and a reliable assessment of glycaemic abnormalities. When used in Type 1 diabetes, Bergenstal was able to demonstrate that children and adults on insulin pumps had improved glycaemic control, as measured by HbA1c than those who did not use

CGM may be particularly useful in managing cystic fibrosis. CF patients frequently demonstrate early postprandial hyperglycaemia [79, 82, 83], reflected by elevations in readings on a 30-min sampled OGTT in the setting of a normal 2-h level. This intermittent postprandial hyperglycaemia may be reflected in the poor correlation of HbA1c (glycated haemoglobin) with early glycaemic abnormalities in CF. HbA1c represents an index of the average of blood glucose concentrations in the preceding 2–3-month period, and the result is influenced by the half-life of the red cells [84]. When measured in CF, it is a poor indicator of glycaemic abnormalities as it is often still normal by the time a diagnosis of CFRD has been made. The poor sensitivity of the test may result from the intermittent nature of hyperglycaemia in patients with CF, which is not revealed in the HbA1c level when the glucose levels are "averaged", as

CGM provides a useful tool to guide insulin treatment once the diagnosis of CFRD has been made [79], but it may also offer a potential opportunity to capture the moments of postprandial hyperglycaemia in CF in the screening and diagnostic phase. In CF patients with normal glucose tolerance on OGTT, abnormalities on CGM have been detected [79, 82, 83]. This could reflect the fact that patients with CF undergo a period of fasting prior to their glucose load in the OGTT which will only measure two values. When a CGM is worn, patients can be at home and may consume their normal CF diet including a carbohydrate load that may exceed the glucose level consumed during an OGTT. In the same way that HbA1c may not reflect a true

and CFID2 has a midpoint glucose peak ≥11.1 mmol/L.

**7. Continuous glucose monitoring in CF**

34 Progress in Understanding Cystic Fibrosis

well as increased red cell turnover in CF.

CGM [81].

Insulin plays a major role in the management of CFRD. Insulin replacement by subcutaneous injection in CFRD has been shown to improve lung function and reduce pulmonary exacerbation frequency [86]. It has also been shown to benefit the nutritional status of the patient, with an improvement in growth seen in children with CF [73]. Recent studies have also demonstrated that insulin therapy in the prediabetic phase may also play a valuable role in the management of patients with CF. Hameed et al. were able to replicate previous studies demonstrating a benefit of insulin therapy on lung function and nutrition in patients with CF and revealed an improvement in weight standard deviation score (p = 0.003) and lung function (FEV1 improvement p = 0.004) with once daily insulin injections (detemir, Levemir™) [73].

Insulin is given via subcutaneous injection. Unlike Type 1 diabetes, a once daily dose of long-acting insulin may be all that is required to demonstrate a benefit for this population [73]. Insulin doses vary with each patient, but because of the important anabolic role insulin plays in growth and nutrition, the highest tolerated dose without hypoglycaemia or other side effects is generally recommended [52] (taking into account patient-specific factors such as ability to recognise hypoglycaemic symptoms). The dose prescribed may vary over time with increasing requirements during times of relative increase in insulin resistance such as with glucocorticoid use or during periods of growth and pregnancy. Given the progressive nature of insulin deficiency in CF, increasing requirements may be seen over time, particularly in the paediatric population with CF that have age- and weight-based doses.

Insulin pumps that continuously deliver a small amount of insulin into the subcutaneous space have been used in patients with CFRD [87] although the uptake in CF has been poor when compared with other forms of diabetes. When wearing a pump, patients are currently required to undertake much more intensive finger-prick blood glucose testing than that required with a once daily insulin injection. This may prove to be too onerous for patients with CFRD who already have a significant treatment burden with multiple oral and nebulised medications and physiotherapy. Future insulin pump devices may include closed loop systems, in which interstitial glucose levels measured by CGM calibrate the rate and amount of insulin secreted by the pump [88]. These devices are currently under investigation for Type 1 DM, but there are no data published about their use in CFRD to date.

#### **8.2. Nutrition**

Nutritional education and support are of utmost importance for patients with a diagnosis of CFRD. Children with CF require a higher caloric intake (may need up to 200% of usual recommendations [89]) to achieve optimal nutritional and growth targets. If nutritional targets are not met, there may be significant consequences as a lower BMI has been associated with increased mortality in CF [67]. These additional calories are best taken from fat and protein-based meals, but a significant proportion is taken from carbohydrates [90]. Patients with abnormalities of glucose tolerance and CFRD will be required to recognise carbohydrates in their diet, as the carbohydrate load will affect the glucose level and the resulting insulin requirements. This is usually done by educating the family and patient about carbohydrate-insulin ratios.

There are very limited data regarding the dietary management of CFRD. This is of particular significance given that hyperglycaemia has been demonstrated to worsen glycaemic abnormalities in CF, possibly by potentiating beta-cell apoptosis. As such, glycaemic control in CFRD needs to be tight, and diets that perpetuate postprandial hyperglycaemia may have a negative impact on glycaemic abnormalities in CF and increase insulin requirements. A low glycaemic diet is often recommended in Type 1 and Type 2 diabetes to optimise control of hyperglycaemia and has been shown to decrease insulin requirements and improve glucose homeostasis, without having a significant impact on quality of life for these patients. Whereas weight loss due to change in diet may be beneficial in Type 2 DM, this may have serious negative consequence in CF. There is not enough information in the literature to recommend any dietary changes that might improve glycaemic control or prevent or delay progression to CFRD if instituted at an earlier stage.

#### **8.3. Oral hypoglycaemic agents**

Insulin is given via subcutaneous injection. Unlike Type 1 diabetes, a once daily dose of long-acting insulin may be all that is required to demonstrate a benefit for this population [73]. Insulin doses vary with each patient, but because of the important anabolic role insulin plays in growth and nutrition, the highest tolerated dose without hypoglycaemia or other side effects is generally recommended [52] (taking into account patient-specific factors such as ability to recognise hypoglycaemic symptoms). The dose prescribed may vary over time with increasing requirements during times of relative increase in insulin resistance such as with glucocorticoid use or during periods of growth and pregnancy. Given the progressive nature of insulin deficiency in CF, increasing requirements may be seen over time, particu-

larly in the paediatric population with CF that have age- and weight-based doses.

**8.2. Nutrition**

36 Progress in Understanding Cystic Fibrosis

Insulin pumps that continuously deliver a small amount of insulin into the subcutaneous space have been used in patients with CFRD [87] although the uptake in CF has been poor when compared with other forms of diabetes. When wearing a pump, patients are currently required to undertake much more intensive finger-prick blood glucose testing than that required with a once daily insulin injection. This may prove to be too onerous for patients with CFRD who already have a significant treatment burden with multiple oral and nebulised medications and physiotherapy. Future insulin pump devices may include closed loop systems, in which interstitial glucose levels measured by CGM calibrate the rate and amount of insulin secreted by the pump [88]. These devices are currently under investigation for Type 1 DM, but there are no data published about their use in CFRD to date.

Nutritional education and support are of utmost importance for patients with a diagnosis of CFRD. Children with CF require a higher caloric intake (may need up to 200% of usual recommendations [89]) to achieve optimal nutritional and growth targets. If nutritional targets are not met, there may be significant consequences as a lower BMI has been associated with increased mortality in CF [67]. These additional calories are best taken from fat and protein-based meals, but a significant proportion is taken from carbohydrates [90]. Patients with abnormalities of glucose tolerance and CFRD will be required to recognise carbohydrates in their diet, as the carbohydrate load will affect the glucose level and the resulting insulin requirements. This is

usually done by educating the family and patient about carbohydrate-insulin ratios.

There are very limited data regarding the dietary management of CFRD. This is of particular significance given that hyperglycaemia has been demonstrated to worsen glycaemic abnormalities in CF, possibly by potentiating beta-cell apoptosis. As such, glycaemic control in CFRD needs to be tight, and diets that perpetuate postprandial hyperglycaemia may have a negative impact on glycaemic abnormalities in CF and increase insulin requirements. A low glycaemic diet is often recommended in Type 1 and Type 2 diabetes to optimise control of hyperglycaemia and has been shown to decrease insulin requirements and improve glucose homeostasis, without having a significant impact on quality of life for these patients. Whereas weight loss due to change in diet may be beneficial in Type 2 DM, this may have serious negative consequence in CF. There is not enough information in the literature to recommend any dietary changes that might improve glycaemic control or prevent or delay progression to CFRD if instituted at an earlier stage.

Oral agents do not play a role in the management of patients with CFRD. Many agents target insulin resistance (e.g. metformin), which is not a major feature in the early glycaemic abnormalities of CF where insulin deficiency plays the key role and as such will not be of significant benefit to CF patients. Significant side effects from oral hypoglycaemic agents such as hepatotoxicity are a serious complication for the CF population where a significant proportion may develop CF liver disease [91]. Insulin therapy in states of insulin deficiency such as Type 1 diabetes has been shown to preserve insulin secretion and "rest" the residual beta cells. Conversely, agents that stimulate insulin secretion may potentially hasten beta-cell loss. For example, agents such as repaglinide may be useful in the short term but ultimately have a negative long-term impact.

#### **8.4. The role of potentiators in CFRD**

Evidence for the use of potentiators in CFRD is limited, but a few pilot studies have been published that suggest a benefit on glucose homeostasis in CF. In a single pair of CF siblings with abnormal glucose tolerance (one with CFRD) and gating mutations, a reduction in the glucose AUC and an improvement in the insulin secretion profile was demonstrated after the introduction of ivacaftor (Kalydeco™). Bellin et al. also demonstrated improvements in glucose homeostasis after the introduction of ivacaftor. In this group of five CF patients with glucose abnormalities, four of five demonstrated improvements in insulin secretion. The patient whose insulin secretion did not improve had long-standing CFRD, whereas the others had earlier glycaemic abnormalities. Theoretically, the patient with long-standing CFRD could already have undergone such significant pancreatic destruction that the abnormalities of glucose tolerance could not be corrected at the level of the CFTR.

## **9. Complications of CFRD**

Long-standing hyperglycaemia and insulin deficiency will result in an increase in respiratory exacerbations and morbidity and poorer nutrition. It will also result in complications from chronic hyperglycaemia seen in other forms of diabetes. Historically the life-limiting nature of CF and in particularly those with CFRD meant that CF patients were unlikely to live long enough to develop end-organ dysfunction from the macrovascular and microvascular complications seen in other forms of diabetes. With an improvement in life expectancy, these long-term issues need to be addressed, and routine screening needs to be a part of CF clinical care. This will include examination for neuropathy and retinopathy and urine screening for microalbuminuria. In one study, 10 years after the diagnosis of CFRD has been made, subjects with fasting glycaemia demonstrated rates of microalbuminuria of approximately 14%, retinopathy 16%, neuropathy 55% and autonomic gastropathy 50% [51]. Gilchrist et al. reported retinopathy in three patients with abnormal glucose tolerance but not meeting criteria for CFRD [71] which further supports the proposition that the OGTT may not be the ideal test for significant glycaemic abnormalities in patients with CF.

## **10. Conclusion**

Cystic fibrosis–related diabetes continues to pose a significant risk of increased morbidity and mortality to the CF population. However, CFRD lies at the endpoint of spectrum of glucose abnormalities, and increasing evidence implies that earlier glycaemic abnormalities may also be clinically significant. The standard OGTT does not appear to be sensitive enough to pick up early, clinically significant abnormalities of glucose tolerance secondary to insulin deficiency and the dysregulation of insulin secretion detected in CF patients. Hyperglycaemia in CF affects lung function, risk of respiratory pathogens, nutrition and growth in young children, and treating teams need to be proactive in the screening and diagnosis of glycaemic abnormalities that may be insidious and potentially irreversible if recognised late. Early recognition of hyperglycaemia in CF is required to prevent significant morbidity. Novel techniques such as continuous glucose monitoring may play a role in screening and early identification of at risk patients, as they have been shown to be predictive of significant glucose abnormalities in the future such as CFRD, but there is not enough evidence as yet to recommend their routine use in diagnosis. Future directions may include the use of potentiators and correctors in CF which appear to have potential to correct abnormalities of glucose tolerance but may be limited if instituted late and once significant pancreatic destruction has occurred.

## **Acknowledgements**

SH and CFV are grateful for funding assistance from the National Health and Medical Research Council of Australia, Australasian Cystic Fibrosis Research Trust, Regional Diabetes Support Scheme, Sydney Children's Hospital Foundation and Australasian Paediatric Endocrine Care Grant from Pfizer and for industry support from Novo Nordisk, Medtronic and Abbott Diagnostics. BP has been awarded a scholarship from the Thoracic Society of Australia and New Zealand and Vertex.

## **Author details**

Bernadette Prentice1,2\*, Shihab Hameed2,3, Chee Y. Ooi2,4, Charles F. Verge2,3 and John Widger1,2

\*Address all correspondence to: bernadette.prentice@health.nsw.gov.au

1 Department of Respiratory Medicine, Sydney Children's Hospital, Randwick, NSW, Australia

2 Discipline of Paediatrics, School of Women's and Children's Health, The University of New South Wales, Sydney, NSW, Australia

3 Department of Endocrinology, Sydney Children's Hospital, Randwick, NSW, Australia

4 Department of Gastroenterology, Sydney Children's Hospital, Randwick, NSW, Australia

## **References**

**10. Conclusion**

38 Progress in Understanding Cystic Fibrosis

significant pancreatic destruction has occurred.

**Acknowledgements**

New Zealand and Vertex.

South Wales, Sydney, NSW, Australia

**Author details**

Cystic fibrosis–related diabetes continues to pose a significant risk of increased morbidity and mortality to the CF population. However, CFRD lies at the endpoint of spectrum of glucose abnormalities, and increasing evidence implies that earlier glycaemic abnormalities may also be clinically significant. The standard OGTT does not appear to be sensitive enough to pick up early, clinically significant abnormalities of glucose tolerance secondary to insulin deficiency and the dysregulation of insulin secretion detected in CF patients. Hyperglycaemia in CF affects lung function, risk of respiratory pathogens, nutrition and growth in young children, and treating teams need to be proactive in the screening and diagnosis of glycaemic abnormalities that may be insidious and potentially irreversible if recognised late. Early recognition of hyperglycaemia in CF is required to prevent significant morbidity. Novel techniques such as continuous glucose monitoring may play a role in screening and early identification of at risk patients, as they have been shown to be predictive of significant glucose abnormalities in the future such as CFRD, but there is not enough evidence as yet to recommend their routine use in diagnosis. Future directions may include the use of potentiators and correctors in CF which appear to have potential to correct abnormalities of glucose tolerance but may be limited if instituted late and once

SH and CFV are grateful for funding assistance from the National Health and Medical Research Council of Australia, Australasian Cystic Fibrosis Research Trust, Regional Diabetes Support Scheme, Sydney Children's Hospital Foundation and Australasian Paediatric Endocrine Care Grant from Pfizer and for industry support from Novo Nordisk, Medtronic and Abbott Diagnostics. BP has been awarded a scholarship from the Thoracic Society of Australia and

Bernadette Prentice1,2\*, Shihab Hameed2,3, Chee Y. Ooi2,4, Charles F. Verge2,3 and John Widger1,2

1 Department of Respiratory Medicine, Sydney Children's Hospital, Randwick, NSW, Australia 2 Discipline of Paediatrics, School of Women's and Children's Health, The University of New

3 Department of Endocrinology, Sydney Children's Hospital, Randwick, NSW, Australia

4 Department of Gastroenterology, Sydney Children's Hospital, Randwick, NSW, Australia

\*Address all correspondence to: bernadette.prentice@health.nsw.gov.au


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42 Progress in Understanding Cystic Fibrosis

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## **The Cystic Fibrosis Airway Microbiome and Pathogens**

Ibrahim A. Janahi and Abdul Rehman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67846

#### **Abstract**

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44 Progress in Understanding Cystic Fibrosis

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Cystic fibrosis (CF) is an autosomal recessive genetic disorder resulting from geneticdefects in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR dysfunction in patients with CF leads to a number of pleiotropic manifestations with the prime pathology being mucus plugging in the airways and paranasal sinuses. Patients with CF are prone to polymicrobial infections and the airway microbiome in such patients changes continuously and evolves over time. The composition of the airway microbiome in CF patients is dependent on a number of factors including geographic variation, type of genetic mutation (e.g., ΔF508), antibiotic exposures, and chronic infection with certain pathogenic bacteria (e.g., *Pseudomonas aeruginosa*). Proteomic and genomic approaches to understanding the microbiome of patients with CF have provided new insights into the pathogenesis of this disease. High‐throughput pyrosequencing, Sanger sequencing, and phylogenetic microarray analysis have enabled the recognition of multiple lineages and clonal populations of a single bacterial species within the same patient. This provides a unique opportunity to explore novel therapeutic approaches to this disease (for instance, use of probiotics and environmental manipulation) and potentially translate them into bedside clinical interventions.

**Keywords:** cystic fibrosis, microbiome, dysbiosis, *Pseudomonas aeruginosa*, burkholderia cenocepacia

## **1. Introduction**

Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene [1]. CF is most prevalent in the Caucasian population and is a common life‐limiting disease [2]. CFTR is expressed on the apical surface of epithelial cells of the respiratory, gastrointestinal, pancreatic and reproductive tracts, and sweat glands [3]. The prime function of CFTR ion channel is to transport chloride ions across epithelial surfaces in order to maintain the osmotic gradient. Chloride ions are actively

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

pumped out into the luminal side of the gastrointestinal and respiratory tracts, which decrease water potential on the luminal side. Subsequently, water molecules move from a higher osmotic potential to a lower osmotic potential (down the osmotic gradient) and combine with mucin glycoproteins to keep them adequately hydrated. This in turn helps to maintain the thin consistency of the mucus layer, which is essential for optimal mucociliary function [4]. Thick and viscid mucus caused by a defect in chloride‐conducting transmembrane channel results in stagnation of mucus. Moreover, CFTR channel also plays an important role in regulating the transepithelial transport of sodium and bicarbonate ions [5]. Defective CFTR functioning leads to an increase in pH of the mucus layer, which compromises the innate immune system and promotes inflammation. Defects in innate immunity and chronic inflammation predispose patients to recurrent pulmonary infections, which result in permanent lung damage—the prime cause of morbidity and mortality [6]. Pulmonary system is not the only organ‐system affected in CF; endocrine, gastrointestinal, and reproductive systems are also involved in this multisystem disorder [3].

The human microbiome project aims to identify and characterize microbial flora of healthy and diseased individuals [7]. Understanding the role of infectious pathogens in the pathogenesis of CF in general and pulmonary exacerbations and lung damage in particular has enabled the scientific community to devise new treatment modalities for CF patients, which can potentially improve outcomes and survival in such patients. In patients with CF, different bacteria inhabit different parts of the lung at various stages of the disease and persistent inflammation in the lungs can change and modify the composition of the microbiome [8]. For instance, methicillin‐sensitive *Staphylococcus aureus* (MSSA) and *Hemophilus influenzae* are common pathogens early in life of such patients [9]. As the disease progresses, more virulent pathogens—such as *Pseudomonas aeruginosa* and methicillin‐resistant *S. aureus* (MRSA)—invade the lung and cause pulmonary damage [10]. By understanding the evolution of the CF microbiome, we can gain further insights into the natural course of CF. This in turn can have important implications for developing interventions that can halt or reverse the course of progressive pulmonary damage and prolong survival and quality of life in CF patients [11]. In the following pages, we discuss the CF microbiome, its evolution and heterogeneity in CF patients, interaction between different bacteria within the CF lung and the factors that potentially affect the CF microbiome.

## **2. The microbiome**

As mentioned previously, the human microbiome project aims to identify and characterize microbial flora of healthy and diseased individuals [7]. There is a diversity of microbes in every single human being i.e., diversity being defined as the number and distribution of a particular type of organism in a body habitat. Every human has particular and distinct microbes; dysbiosis (alteration in composition and balance) of these microbes is now thought to underlie the pathogenesis of many diseases, such as inflammatory bowel disease, *Clostridium difficile* (CD) colitis, bacterial vaginosis, obesity, and CF [12]. The human microbiome plays a very important role in human biology, defense mechanisms, metabolic processes (such as digestion, absorption, and assimilation) and even pathogenesis of acute and chronic diseases [13]. For instance, CD colitis is a disease that arises as a consequence of interaction of bacterial virulence factors, host immune mechanism and the intestinal microbiome [14]. Research studies have shown that variability in the innate host response may also impact upon the severity of CD colitis, and this variation may be accounted for by alterations in the gut microbiota [15]. Based on improved understanding of the pathogenesis of CD colitis, fecal microbiota transplantation (FMT) and other novel types of bacteriotherapy have become potentially effective treatment options for this deadly disease [16].

pumped out into the luminal side of the gastrointestinal and respiratory tracts, which decrease water potential on the luminal side. Subsequently, water molecules move from a higher osmotic potential to a lower osmotic potential (down the osmotic gradient) and combine with mucin glycoproteins to keep them adequately hydrated. This in turn helps to maintain the thin consistency of the mucus layer, which is essential for optimal mucociliary function [4]. Thick and viscid mucus caused by a defect in chloride‐conducting transmembrane channel results in stagnation of mucus. Moreover, CFTR channel also plays an important role in regulating the transepithelial transport of sodium and bicarbonate ions [5]. Defective CFTR functioning leads to an increase in pH of the mucus layer, which compromises the innate immune system and promotes inflammation. Defects in innate immunity and chronic inflammation predispose patients to recurrent pulmonary infections, which result in permanent lung damage—the prime cause of morbidity and mortality [6]. Pulmonary system is not the only organ‐system affected in CF; endocrine, gastrointestinal, and reproductive systems are also involved in this

The human microbiome project aims to identify and characterize microbial flora of healthy and diseased individuals [7]. Understanding the role of infectious pathogens in the pathogenesis of CF in general and pulmonary exacerbations and lung damage in particular has enabled the scientific community to devise new treatment modalities for CF patients, which can potentially improve outcomes and survival in such patients. In patients with CF, different bacteria inhabit different parts of the lung at various stages of the disease and persistent inflammation in the lungs can change and modify the composition of the microbiome [8]. For instance, methicillin‐sensitive *Staphylococcus aureus* (MSSA) and *Hemophilus influenzae* are common pathogens early in life of such patients [9]. As the disease progresses, more virulent pathogens—such as *Pseudomonas aeruginosa* and methicillin‐resistant *S. aureus* (MRSA)—invade the lung and cause pulmonary damage [10]. By understanding the evolution of the CF microbiome, we can gain further insights into the natural course of CF. This in turn can have important implications for developing interventions that can halt or reverse the course of progressive pulmonary damage and prolong survival and quality of life in CF patients [11]. In the following pages, we discuss the CF microbiome, its evolution and heterogeneity in CF patients, interaction between different bacteria within the CF lung and the factors that poten-

As mentioned previously, the human microbiome project aims to identify and characterize microbial flora of healthy and diseased individuals [7]. There is a diversity of microbes in every single human being i.e., diversity being defined as the number and distribution of a particular type of organism in a body habitat. Every human has particular and distinct microbes; dysbiosis (alteration in composition and balance) of these microbes is now thought to underlie the pathogenesis of many diseases, such as inflammatory bowel disease, *Clostridium difficile* (CD) colitis, bacterial vaginosis, obesity, and CF [12]. The human microbiome plays a very important role in human biology, defense mechanisms, metabolic processes (such as

multisystem disorder [3].

46 Progress in Understanding Cystic Fibrosis

tially affect the CF microbiome.

**2. The microbiome**

Another example of a disease where microbiota plays a major role in pathogenesis is Crohn's disease. The exact cause of Crohn's disease is unknown; however, evidence suggests that microbiota contribute to the underlying pathology and disease development [17]. No single bacterium has been convincingly shown to contribute to the overall pathogenesis of Crohn's disease. Instead, dysbiosis (bacterial imbalance) is more widely accepted as a leading factor in the disrupted host immune system cross‐talk that results in subsequent intestinal inflammation [18]. Depletion of symbiont (beneficial) microbes (including Firmicutes, Bifidobacteriaceae, and Clostridia) in conjunction with an increase in pathobiont (harmful) microbes (such as Bacteroidetes and Enterobacteriaceae) is a striking feature observed in Crohn's disease. No single factor has been definitely identified as driving this dysbiosis; instead, a host of environmental factors—such as the diet, antibiotic exposures and possible early life infections—in the presence of underlying genetic susceptibilities may contribute to the overall pathogenesis of Crohn's disease [17].

In CF patients, composition of the microbiome of pulmonary and gastrointestinal tracts changes over time, presumably as a consequence of inflammation [19]. Most research studies have demonstrated the influence of inflammation in negatively selecting against potential pathogens. Moreover, some bacterial species may also have the ability to exploit inflammatory byproducts for their benefit, which may promote their natural selection in inflamed habitats [20]. Reactive nitrogen species produced during inflammatory responses can be exploited by pathogens for their growth. Moreover, inflammatory mediators can provide an environment for some bacteria to grow and use these inflammatory mediators for their survival [21]. Examples of such bacteria include *Escherichia coli* and *P. aeruginosa* in the gastrointestinal and respiratory tracts of CF patients, respectively. *P. aeruginosa* uses nitric oxide produced in the process of inflammation for its anaerobic respiration and promotes its growth in inflammatory environments. Likewise, *E. coli* uses increased nitrate in the environment for its anaerobic respiration and enhances its growth in the inflamed gut of CF patients [19].

## **3. Heterogeneity of the CF airway microbiome**

Due to defects in innate immunity, CF patients are prone to polymicrobial infections and their airway microbiome changes continuously and evolves over time. The primary cause of death in CF patients is respiratory failure due to persistent and recurrent pulmonary infections with different pathogenic organisms [22]. Over the past decade, the median survival for such patients stands at 37 years despite increases in life expectancy [23]. MSSA and *H. influenzae* are one of the most common pathogens cultured from sputum samples of affected children. *P. aeruginosa* has been associated with increased morbidity as most strains of this organism are multidrug resistant. Infections with bacteria of the Bukholderia cepacia complex (BCC) are associated with a worse prognosis [24]. Likewise, other multidrug resistant organisms, such as *Achromobacter xylosoxidans* and *Stenotrophomonas maltophilia*, can also be isolated from CF patients with end‐stage pulmonary disease [25]. Nontuberculous mycobacterium (NTM) has also been identified as emerging causes of infections in patients with CF and their incidence may have been underestimated in the past [26]. More recently, research studies have shown that when sputum samples obtained from adults with CF are cultured, a significantly high density of anaerobic bacteria can be isolated—the most common of which are *Streptococcus milleri*, *Prevotella* spp., *Actinomyces*, and *Veillonella* [27].

Microbes of the lower airways in all humans exist in a dynamic state. Literature published on microbiome of CF patients has shown a complex and dynamic interaction between different organisms in the airways of such patients [28]. Organisms within a single patient are genetically and phenotypically diverse and heterogeneity is detectable even in different parts of the same lung. Over a period of time, community diversity of bacteria declines in CF patients as pulmonary function declines and lung disease progressively worsens. Studies have shown that diversity of microbial communities correlates positively with pulmonary function and outcome [29]. Such diversity was previously unrecognized as most studies relied solely on culture‐based methods of culturing bacteria. However, novel state‐of‐the‐art molecular techniques (such as Sanger sequencing of clone libraries, terminal restriction fragment length polymorphism [RFLP] analysis and microarray hybridization) have enabled the detection of subtle molecular diversity among seemingly similar bacterial species [30]. This diversity may be influenced by a number of factors including the patient's age, sex, type of CFTR mutation, antibiotic exposures, environmental factors, and extent and severity of lung disease. In a study by Zhao et al., sputum samples were collected from six CF patients over a period of 10 years. Of a total of 126 sputum samples, 662 operational taxonomic units (OTU) were identified and each patient had 5–114 different OTUs [29]. Similarly, in another observational study, sputum samples of patients with acute infective exacerbation of non‐CF related bronchiectasis were collected. Sputum cultures from each patient contained large quantities of multiple bacterial species with a single predominant pathogenic species [31]. In one study, polymerase chain reaction (PCR)‐temporal temperature gel electrophoresis (PCR‐TTGE) was used to evaluate intraspecific and intragenomic 16S rDNA variability among commonly isolated respiratory pathogens from CF patients [32]. Significant discordance in intraspecific and intragenomic variability was noted among different bacterial species with *H. influenzae* displaying the highest level of intraspecific variability.

#### **4. Composition of the CF microbiome and its determinants**

The composition of the airway microbiome in CF patients is dependent on a number of factors including geographic variation (more common in white population), type of genetic mutation (e.g., ΔF508), antibiotic exposures, and chronic infection with certain pathogenic bacteria (e.g., *P. aeruginosa*) [8]. Fetal lungs are sterile, just like fetal gastrointestinal tract, but they soon become colonized after birth. Fetal skin becomes colonized with microbes present in maternal reproductive and gastrointestinal tracts and lungs become colonized from gut flora of the child [33]. The common phyla found in healthy lungs include Bacteroides, Firmicutes, and Proteobacterium. Other genera include Prevotella, Veillonella, Streptococcus and Pseudomonas [34]. Many techniques have been used for the detection of microbes in CF patients. Some of these techniques include terminal RFLP profiling, microarray analysis, clone library sequencing, and pyrosequencing. The most frequently used samples from CF patients for analysis are expectorated sputum, tracheal aspirates, bronchial washings, and bronchoalveolar lavage (BAL).

patients stands at 37 years despite increases in life expectancy [23]. MSSA and *H. influenzae* are one of the most common pathogens cultured from sputum samples of affected children. *P. aeruginosa* has been associated with increased morbidity as most strains of this organism are multidrug resistant. Infections with bacteria of the Bukholderia cepacia complex (BCC) are associated with a worse prognosis [24]. Likewise, other multidrug resistant organisms, such as *Achromobacter xylosoxidans* and *Stenotrophomonas maltophilia*, can also be isolated from CF patients with end‐stage pulmonary disease [25]. Nontuberculous mycobacterium (NTM) has also been identified as emerging causes of infections in patients with CF and their incidence may have been underestimated in the past [26]. More recently, research studies have shown that when sputum samples obtained from adults with CF are cultured, a significantly high density of anaerobic bacteria can be isolated—the most common of which are *Streptococcus* 

Microbes of the lower airways in all humans exist in a dynamic state. Literature published on microbiome of CF patients has shown a complex and dynamic interaction between different organisms in the airways of such patients [28]. Organisms within a single patient are genetically and phenotypically diverse and heterogeneity is detectable even in different parts of the same lung. Over a period of time, community diversity of bacteria declines in CF patients as pulmonary function declines and lung disease progressively worsens. Studies have shown that diversity of microbial communities correlates positively with pulmonary function and outcome [29]. Such diversity was previously unrecognized as most studies relied solely on culture‐based methods of culturing bacteria. However, novel state‐of‐the‐art molecular techniques (such as Sanger sequencing of clone libraries, terminal restriction fragment length polymorphism [RFLP] analysis and microarray hybridization) have enabled the detection of subtle molecular diversity among seemingly similar bacterial species [30]. This diversity may be influenced by a number of factors including the patient's age, sex, type of CFTR mutation, antibiotic exposures, environmental factors, and extent and severity of lung disease. In a study by Zhao et al., sputum samples were collected from six CF patients over a period of 10 years. Of a total of 126 sputum samples, 662 operational taxonomic units (OTU) were identified and each patient had 5–114 different OTUs [29]. Similarly, in another observational study, sputum samples of patients with acute infective exacerbation of non‐CF related bronchiectasis were collected. Sputum cultures from each patient contained large quantities of multiple bacterial species with a single predominant pathogenic species [31]. In one study, polymerase chain reaction (PCR)‐temporal temperature gel electrophoresis (PCR‐TTGE) was used to evaluate intraspecific and intragenomic 16S rDNA variability among commonly isolated respiratory pathogens from CF patients [32]. Significant discordance in intraspecific and intragenomic variability was noted among different bacterial species with *H. influenzae* displaying the high-

*milleri*, *Prevotella* spp., *Actinomyces*, and *Veillonella* [27].

48 Progress in Understanding Cystic Fibrosis

est level of intraspecific variability.

**4. Composition of the CF microbiome and its determinants**

The composition of the airway microbiome in CF patients is dependent on a number of factors including geographic variation (more common in white population), type of genetic The microbiome in patients with CF evolves as patients grow older, and this is a consequence of the wide adaptability of pathogenic bacteria. Clustering of phylogenetically similar bacterial communities and loss of the architectural diversity of the airway microbiome is a key feature of late‐stage CF airway disease. Moreover, the type of bacterial species predominating at a particular age group is also of immense importance. In one study, phylogenetic diversity of CF airway microbiota in patients of different age groups was studied using microarray analysis [35]. *S. aureus* was detected in 65% of sputum samples and was more common in the pediatric population (72% of the pediatric sample). *Pseudomonas* spp. was found in 73% of samples and were most common in adults (91% of the adult sample). In the same study, older CF patients had reduced airway bacterial diversity and aggregation of relatively similar organisms; this process occurred in conjunction with a progressive decline in pulmonary function. *H. influenzae* was most prevalent in the pediatric population when the bacterial diversity was highest. Conversely, *P. aeruginosa* was most common in older individuals with a lower level of bacterial diversity. Likewise, members of the Mycobacteriaceae family and obligate intracellular pathogens (such as Chlamydia and Mycoplama spp.) were more prevalent in younger CF patients. Certain known or potential pathogens of CF patients, such as members of the Burkholderiaceae and Thermoactinomycetaceae families, were almost exclusively observed among adult patients.

In another study [29], CF patients with progressive lung disease were noted to have a decrease in bacterial diversity with increasing age, but the total bacterial density remained stable over time. Antibiotic exposures in conjunction with recurrent pulmonary exacerbations were proposed as a possible contributing factor toward this observation. In a study by Tunney et al., several anaerobic species (including a number of Veillonella and Prevotella species) constituted a significant portion of the CF airway microbiota [36]. In a unique study, next generation sequencing was used to study the microorganisms of gastric juice among patients with CF and non‐CF controls [37]. CF gastric juice was noted to have an abundance of Pseudomonas spp. and a relative paucity of normal gut bacteria (such as Bacteroides and Faecalibacterium), which was in contrast with normal gastric juice samples. These results suggest that CF patients possess a unique aerodigestive microbiome that is inter‐related. This explanation seems plausible as the factors that influence the airway microbiome (for instance, antibiotic exposures) are also likely to influence the microbiota of gut and other organ‐systems of the body [38].

In patients with CF, different bacterial colony morphotypes can be isolated from a single sputum sample. There is some evidence to suggest that these different morphotypes arise from a single bacterial strain [39]. Microbes in the lungs of CF patients are capable of constantly adapting to selection pressures. Some of the mechanisms that enable the evolution of microbes include motility, type III secretion systems, lipopolysaccharide, plasmids (encoding for antibiotic resistance), biofilm formation, small colony variants, quorum sensing, and hypermutability. As a consequence of these mechanisms, different phenotypes arise from a single bacterial species and, over time, a single bacterial strain with dominating features may evolve [40]. Given that different bacterial strains have differing capacities to evolve, multiple lineages of bacterial colonies evolve and coexist [41]. Some studies have shown that complexity of bacterial communities inversely correlates with patient age, antibiotic exposures, and presence of *P. aeruginosa* [42]. In one study, heterozygosity for the ΔF508 mutation and presence of mutations other than the ΔF508 was associated with relative preservation of airway bacterial diversity over time [35]. This shows that apart from environmental exposures (such as antibiotic pressures), patients' genotype (type of mutation) also plays an important role in determining the composition of the CF airway microbiome. In terms of environmental exposures, antibiotic use has been shown to be the prime factor that adversely affects microbial diversity among CF patients [29]. Loss of bacterial diversity (under the selection pressure of antibiotics) has been associated with an increased risk of pneumonia in mechanically ventilated patients colonized with *P. aeruginosa* [43]. Smith et al. studied this further by performing whole genomic analysis of a single species of *P. aeruginosa* isolated from a patient with CF. Whole genomic sequencing was repeated multiple times during the course of the patient's illness, which enabled the detection of an overwhelming number of mutations. Based on these analyses, it was found that the strain of *P. aeruginosa* that inhabits patients with advanced CF differs significantly from wild‐type *P. aeruginosa* [40].

The interaction among different bacterial colonies has also become a subject of intense research and genomic and proteomic approaches are currently being used to understand their interrelationships. In an experimental study, production of 4‐hydroxy‐2‐heptylquinoline‐N‐oxide (HQNO) by a strain of *P. aeruginosa* enhanced the aminoglycoside resistance of *S. aureus* [44]. This study provided some evidence of how bacterial interspecies interaction can alter the airway microbiome by selecting for resistant strains of a bacterial species. Previous studies have shown that HQNO is detectable in the sputum of infected CF patients. Therefore, an interaction between *P. aeruginosa* and *S. aureus* may account for the increased incidence of small colony variant (SCV) of *S. aureus* species in CF patients with advanced lung disease.

In the recent literature, an increasing number of unusual microbes have been reported as the cause of infective exacerbations of CF. Such bacteria include multidrug resistant pathogens like *S. maltophilia*, multidrug resistant *P. aeruginosa*, MRSA, *Burkholderia cenocepacia* and even NTM [45]. The emergence of such bacteria as members of the CF airway microbiome can have important implications for management and prognosis for patients. For instance, studies have shown that in CF patients with an acute exacerbation, there is discordance between the results of microbial sensitivity testing and response to antibacterial therapy [46]. Polymicrobial infections and presence of fastidious organisms may account for this observation. Moreover, such pathogenic bacteria can interact with other less virulent bacterial species and lead to architectural distortion of the entire CF microbiome. In the following lines, we discuss common members of the CF airway microbiome, some of which are commonly implicated in infective exacerbations.

#### **4.1. Methicillin‐sensitive** *Staphylococcus aureus*

In patients with CF, different bacterial colony morphotypes can be isolated from a single sputum sample. There is some evidence to suggest that these different morphotypes arise from a single bacterial strain [39]. Microbes in the lungs of CF patients are capable of constantly adapting to selection pressures. Some of the mechanisms that enable the evolution of microbes include motility, type III secretion systems, lipopolysaccharide, plasmids (encoding for antibiotic resistance), biofilm formation, small colony variants, quorum sensing, and hypermutability. As a consequence of these mechanisms, different phenotypes arise from a single bacterial species and, over time, a single bacterial strain with dominating features may evolve [40]. Given that different bacterial strains have differing capacities to evolve, multiple lineages of bacterial colonies evolve and coexist [41]. Some studies have shown that complexity of bacterial communities inversely correlates with patient age, antibiotic exposures, and presence of *P. aeruginosa* [42]. In one study, heterozygosity for the ΔF508 mutation and presence of mutations other than the ΔF508 was associated with relative preservation of airway bacterial diversity over time [35]. This shows that apart from environmental exposures (such as antibiotic pressures), patients' genotype (type of mutation) also plays an important role in determining the composition of the CF airway microbiome. In terms of environmental exposures, antibiotic use has been shown to be the prime factor that adversely affects microbial diversity among CF patients [29]. Loss of bacterial diversity (under the selection pressure of antibiotics) has been associated with an increased risk of pneumonia in mechanically ventilated patients colonized with *P. aeruginosa* [43]. Smith et al. studied this further by performing whole genomic analysis of a single species of *P. aeruginosa* isolated from a patient with CF. Whole genomic sequencing was repeated multiple times during the course of the patient's illness, which enabled the detection of an overwhelming number of mutations. Based on these analyses, it was found that the strain of *P. aeruginosa* that inhabits patients with advanced CF

The interaction among different bacterial colonies has also become a subject of intense research and genomic and proteomic approaches are currently being used to understand their interrelationships. In an experimental study, production of 4‐hydroxy‐2‐heptylquinoline‐N‐oxide (HQNO) by a strain of *P. aeruginosa* enhanced the aminoglycoside resistance of *S. aureus* [44]. This study provided some evidence of how bacterial interspecies interaction can alter the airway microbiome by selecting for resistant strains of a bacterial species. Previous studies have shown that HQNO is detectable in the sputum of infected CF patients. Therefore, an interaction between *P. aeruginosa* and *S. aureus* may account for the increased incidence of small colony variant (SCV) of *S. aureus* species in CF patients with advanced lung disease.

In the recent literature, an increasing number of unusual microbes have been reported as the cause of infective exacerbations of CF. Such bacteria include multidrug resistant pathogens like *S. maltophilia*, multidrug resistant *P. aeruginosa*, MRSA, *Burkholderia cenocepacia* and even NTM [45]. The emergence of such bacteria as members of the CF airway microbiome can have important implications for management and prognosis for patients. For instance, studies have shown that in CF patients with an acute exacerbation, there is discordance between the results of microbial sensitivity testing and response to antibacterial therapy [46]. Polymicrobial infections and presence of fastidious organisms may account for this observation. Moreover, such pathogenic bacteria can interact with other less virulent bacterial species and lead to

differs significantly from wild‐type *P. aeruginosa* [40].

50 Progress in Understanding Cystic Fibrosis

*S. aureus* is a common colonizer of the anterior nares of adolescent and adult patients [47]. Among patients with CF, MSSA is one of the most common pathogens isolated from sputum samples obtained for culture and sensitivity testing. In the CF Foundation (CFF) patient registry (Bethesda, Maryland, USA), *S. aureus* was most commonly isolated from children and adolescents accounting for approximately 51% of the total samples. Moreover, the overall prevalence of *S. aureus* has been increasing over the past few decades. Infection with *S. aureus* has been associated with increased bronchial inflammation and decreasing pulmonary function [48]. Moreover, when coinfection with *P. aeruginosa* and MSSA occurs, mortality is increased manifold. Interestingly, studies have shown that MSSA is associated with more severe disease in children as compared to adults.

With the widespread use of antistaphylococcal antibiotics, incidence of Gram‐negative infections among CF patients has increased and MSSA has become less common among adult patients. Overall, the most common cause of chronic lung infections in CF patients is *P. aeruginosa*, an oxidase‐positive Gram‐negative bacillus. Moreover, as CF patients grow older, MRSA becomes a more frequent cause of infective exacerbation than MSSA. Over the past few years, the incidence of MRSA infections has been steadily increasing, owing to increasing use of antistaphylococcal penicillins (such as oxacillin and nafcillin) [49]. More recently, a subtype of *S. aureus* species (viz. small colony variant) has been isolated more frequently from CF patients. The small colony variant of *S. aureus* species is fastidious and slow‐growing, and it has also been associated with rapid decline in pulmonary function. As mentioned previously, selection of small colony variant species is promoted by HQNO—a product synthesized and secreted by *P. aeruginosa* species [44]**.** Increasing use of broad‐spectrum antibiotics that select for multidrug resistant pathogens can explain this distortion in the composition of the airway microbiome in patients with CF.

#### **4.2. Methicillin‐resistant** *Staphylococcus aureus*

*S. aureus* is typically the first bacterial pathogen to invade the pulmonary parenchyma in patients with CF. Chronic infection with this organism can persist in the airways of CF patients for several years. Acquisition of mecA gene mediates methicillin resistance in community‐ acquired MRSA by encoding for a mutated penicillin binding protein‐2A (PBP‐2A) [50]. The prevalence of MRSA has increased substantially over the past several years from an estimated 7.3% in 2001 to 22.6% in the year 2008 and 25.7% in 2012 [10]. This increase in prevalence of MRSA was noticed across CF patients of all age groups with the highest increase being in the adolescent age bracket. This increase in the prevalence of MRSA in CF patients has been directly linked to the increase in overall incidence of community‐acquired MRSA in the general population [51]. In a study by Glikman et al., 22 of 34 (64.7%) MRSA isolates from patients with CF contained the gene SCCmec II—a typical feature of health‐care associated MRSA strains. On the other hand, 9 of 34 (26.5%) MRSA strains harbored the SCCmec IV gene, which characterizes them as community‐acquired MRSA strains. Most patients with community‐acquired MRSA were newly colonized with the strain. Additionally, children with CF were more likely to harbor MRSA isolates that were resistant to clindamycin and ciprofloxacin compared with strains from non‐CF patients [52]. Other studies have reported persistent infections in CF patients with both hospital‐acquired and community‐acquired MRSA strains (including Panton‐Valentine leukocidin‐positive strains) with an overall prevalence of 7.8% [53]. In these studies, persistence was due to presence of different clones over time or identical clones that underwent minor modifications in their toxin content. Moreover, isolation of MRSA from CF patients aged 7–24 years has been associated with an increased severity of the disease. Alarmingly, some of these strains may be vancomycin‐intermediate *S. aureus* (VISA), which implies that treatment with glycopeptides (such as vancomycin) may also be ineffective. Highly virulent strains, such as vancomycin‐resistant *S. aureus* (VRSA), have also been reported to cause necrotizing pneumonia in a small number of CF patients [54]. Persistent infection with virulent strains of *S. aureus* has been associated with a rapid decline in pulmonary function [55]. In a case‐control study, CF patients who were colonized with MRSA had a significantly higher rate of decline in FEV<sup>1</sup> (forced expiratory volume in first second) as compared to those who were not colonized with MRSA [56]. Moreover, MRSA‐infected CF patients have been shown to have longer hospital stays than age‐ and sex‐matched controls [57]. Serious manifestations of MRSA infections have also been described in various reports. Cavitary lesions have been described in two CF patients infected with Panton‐Valentine leukocidin‐positive MRSA strains [54]. This observation was consistent with other reports of serious pulmonary manifestations of community acquired MRSA infection [54, 58]. In a cohort study of longitudinal data, risk of death among CF patients who had at least one culture positive for MRSA was 1.27 times greater than for CF patients in whom MRSA was never detected [55]. In a meta‐analysis of 76 studies, a clear and strong association was noted between exposure to antibiotics and isolation of MRSA [59]. The risk of acquiring MRSA was increased by 1.8‐fold in patients who had taken antibiotics as compared to others. The risk ratios for quinolones, glycopeptides, cephalosporins, and other beta‐lactam antibiotics were 3, 2.9, 2.2, and 1.9, respectively.

#### **4.3. Hemophilus influenzae**

*H. influenzae* is a facultative, anaerobic, Gram‐negative bacillus. In many patients, this organism begins to colonize the upper respiratory tract since infancy. Approximately 20% of infants with CF are colonized by the end of first year of life and the rate is even higher for patients of older ages [60]. By the age of 5–6 years, more than 50% of children are colonized with this bacterium [61]. *H. influenzae* is a common pathogen of chronic lung infections and is frequently implicated in infective exacerbations of CF [62]. In children with CF, about 32% are colonized with this microorganism. However, as these patients grow older and are exposed to a wide range of broad‐spectrum antibiotics, more virulent bacteria inhabit their respiratory tracts. Consequently, in adults with CF, the rate of colonization with *H. influenzae* is reported to be only 10–15%. Having said this, the prevalence of *H. influenzae* has increased from 10.3% in the year 1995 to 16.3% in the year 2008.

Similar to the general population, colonization of the upper respiratory tract of CF patients with *H. influenzae* is quite a dynamic process. Children will typically carry multiple strains of this bacterium simultaneously, whilst adults will be colonized with only one strain [63]; again, this is a natural consequence of the loss of microbial diversity induced by antibiotic selection pressures. Even in most healthy adults, the upper airway is colonized with *H. influenzae*; most strains in such healthy subjects are nontypeable. In particular, the nasopharynx is an area of the respiratory tract that serves as a potential reservoir of this bacterium. Eventually, the organism may spread from the nasopharynx to the lower respiratory tract and cause an infection of the pulmonary parenchyma [64]. Studies have shown that most CF patients are cocolonized with two or more distinct strains of *H. influenzae* [65].

*H. influenzae* is not considered a virulent pathogen in patients with CF. Interestingly, some studies have shown that colonization with *H. influenzae* is associated with a relatively preserved lung function. This is in sharp contrast to other microorganisms like *P. aeruginosa* and MRSA, whose colonization of the pulmonary parenchyma is strongly associated with a rapid decline in lung function [66]. In a prospective study, 27 patients with CF (under the age of 12 years) and 27 matched patients with asthma were followed up for 1 year [67]. The isolation rate of noncapsulated (nontypeable) strains of *H. influenzae* was significantly higher in the CF group as compared to that of the asthma group. During exacerbations, the isolation rate of *H. influenzae* in the CF group was significantly greater than at other times, whereas there was no significant difference in the control group. The distribution of biotypes of *H. influenzae* and *Hemophilus parainfluenzae* was similar in the two groups. In the CF group, biotype I was commonly detected and was associated with infective exacerbations of CF. In contrast, biotype V was more common in the asthma group, although it had no association with the development of infective exacerbations [67].

#### **4.4. Pseudomonas aeruginosa**

MRSA strains. On the other hand, 9 of 34 (26.5%) MRSA strains harbored the SCCmec IV gene, which characterizes them as community‐acquired MRSA strains. Most patients with community‐acquired MRSA were newly colonized with the strain. Additionally, children with CF were more likely to harbor MRSA isolates that were resistant to clindamycin and ciprofloxacin compared with strains from non‐CF patients [52]. Other studies have reported persistent infections in CF patients with both hospital‐acquired and community‐acquired MRSA strains (including Panton‐Valentine leukocidin‐positive strains) with an overall prevalence of 7.8% [53]. In these studies, persistence was due to presence of different clones over time or identical clones that underwent minor modifications in their toxin content. Moreover, isolation of MRSA from CF patients aged 7–24 years has been associated with an increased severity of the disease. Alarmingly, some of these strains may be vancomycin‐intermediate *S. aureus* (VISA), which implies that treatment with glycopeptides (such as vancomycin) may also be ineffective. Highly virulent strains, such as vancomycin‐resistant *S. aureus* (VRSA), have also been reported to cause necrotizing pneumonia in a small number of CF patients [54]. Persistent infection with virulent strains of *S. aureus* has been associated with a rapid decline in pulmonary function [55]. In a case‐control study, CF patients who

were colonized with MRSA had a significantly higher rate of decline in FEV<sup>1</sup>

other beta‐lactam antibiotics were 3, 2.9, 2.2, and 1.9, respectively.

**4.3. Hemophilus influenzae**

52 Progress in Understanding Cystic Fibrosis

year 1995 to 16.3% in the year 2008.

tory volume in first second) as compared to those who were not colonized with MRSA [56]. Moreover, MRSA‐infected CF patients have been shown to have longer hospital stays than age‐ and sex‐matched controls [57]. Serious manifestations of MRSA infections have also been described in various reports. Cavitary lesions have been described in two CF patients infected with Panton‐Valentine leukocidin‐positive MRSA strains [54]. This observation was consistent with other reports of serious pulmonary manifestations of community acquired MRSA infection [54, 58]. In a cohort study of longitudinal data, risk of death among CF patients who had at least one culture positive for MRSA was 1.27 times greater than for CF patients in whom MRSA was never detected [55]. In a meta‐analysis of 76 studies, a clear and strong association was noted between exposure to antibiotics and isolation of MRSA [59]. The risk of acquiring MRSA was increased by 1.8‐fold in patients who had taken antibiotics as compared to others. The risk ratios for quinolones, glycopeptides, cephalosporins, and

*H. influenzae* is a facultative, anaerobic, Gram‐negative bacillus. In many patients, this organism begins to colonize the upper respiratory tract since infancy. Approximately 20% of infants with CF are colonized by the end of first year of life and the rate is even higher for patients of older ages [60]. By the age of 5–6 years, more than 50% of children are colonized with this bacterium [61]. *H. influenzae* is a common pathogen of chronic lung infections and is frequently implicated in infective exacerbations of CF [62]. In children with CF, about 32% are colonized with this microorganism. However, as these patients grow older and are exposed to a wide range of broad‐spectrum antibiotics, more virulent bacteria inhabit their respiratory tracts. Consequently, in adults with CF, the rate of colonization with *H. influenzae* is reported to be only 10–15%. Having said this, the prevalence of *H. influenzae* has increased from 10.3% in the

(forced expira-

*P. aeruginosa* is an obligate aerobic, oxidase‐positive, nonlactose fermenting Gram‐negative rod. *P. aeruginosa* is the most common organism implicated in infective exacerbations in patients with CF. In the CFF patient registry (Bethesda, Maryland, USA), more than half of the patients (52.5%) were reported to be infected with *P. aeruginosa* in 1995. The risk of chronic infection with *P. aeruginosa* increased proportionately with increasing age. Moreover, the incidence of *P. aeruginosa* has been reported to be increasing in infants. Despite changes in the management of patients with CF, the frequency of persistent infection with *P. aeruginosa* has remained relatively stable over time [68]. In a study based on the CFF patient registry, prevalence of colonization with *P. aeruginosa* was 60% in 1995 and 56.1% in 2005 [69]. However, recent data suggest that the prevalence of P. aeruginosa is slowly decreasing over time and has been estimated to be 30.4% in the year 2015 [70].

The main reservoir of *P. aeruginosa* is the environment surrounding CF patients. It has been thought that among siblings with CF, prolonged exposure of young children to their older siblings with CF is a potential risk factor for acquisition of *P. aeruginosa*. A study published in 1991 reported that *P. aeruginosa* may be acquired by patients at CF recreation camps, clinics, and/or rehabilitation centers [71]. Studies on genotypes of *P. aeruginosa* performed using conventional pyocin typing and DNA probe analysis reported that most CF patients harbored a persistent strain of *P. aeruginosa* in their lungs [72]. These studies suggested that cross‐colonization possibly could occur among patients. Another study showed that 59% of CF patients harbored a clonal strain of *P. aeruginosa* and the dominant pulsotype was indistinguishable from nonclonal strains with respect to both colony morphology and resistance patterns [73]. Wolz et al. used DNA probe amplification assays and demonstrated that 46% of CF patients (who were initially uninfected) acquired P. aeruginosa infection at the end of a CF recreation camp [74]. Clear evidence of a cross‐infection among patients attending a CF clinic was published in 2001 [75]. In this study, 22 of 154 patients attending an adult CF clinic were chronically infected with similar isolates (based on pyocin typing and pulsed‐ field gel electrophoresis [PFGE] analysis) of *P. aeruginosa* that shared unusual phenotypic features: lack of motility and pigmentation along with a remarkable resistance to many antibiotics. In another study from a large pediatric CF clinic from Australia, 65 patients (55%) were found to be infected with a similar strain of *P. aeruginosa*. These patients were more likely to have been hospitalized in the preceding 1 year for respiratory exacerbations [76]. On the other hand, a study conducted by Speert et al. in Vancouver (Canada) reported a low rate of transmission of *P. aeruginosa* from one CF patient to the other [77]. In this study, a total of 157 genetic types of *P. aeruginosa* were identified, of which 123 were unique to individual patients. These apparently conflicting findings may be accounted for by the highly adaptable nature of *P. aeruginosa* and its ability to evolve. In a study by Mahenthiralingam et al., different strains of *P. aeruginosa* were studied using genomic fingerprinting and random DNA amplification assays [78]. A total of 385 isolates from 20 patients were grouped into 35 random amplified polymorphic DNA (RAPD) strain types. Secretion of mucoid exopolysaccharide, loss of expression of RpoN‐dependent surface factors and acquisition of serum‐susceptible phenotypes in Pseudomonas were shown to be a specific adaptation to infection, rather than being acquired from a new bacterial strain. This explanation is also in congruence with observations from other studies that found different strains of *P. aeruginosa* in unrelated CF patients and identical or closely related strains among siblings [79]. The presence of distinct strains of *P. aeruginosa* in these studies reflects an absence of nosocomial transmission of organisms at respective CF centers [80]. This may be a consequence of strict hygiene measures and microbiologic surveillance instituted at most CF centers across the world following reports of nosocomial spread [75, 76].

The effects of *P. aeruginosa* infection on the CF lung are deleterious. In one observational study, outcomes of CF children colonized with *P. aeruginosa* were compared with those of noncolonized patients. Children colonized with *P. aeruginosa* had a worse outcome and experienced rapid decline in pulmonary function as measured by FEV<sup>1</sup> and FEF25 (forced expiratory flow at 25% of vital capacity) [81]. In another longitudinal observational study, the temporal relationship between *P. aeruginosa* infection and pulmonary damage (as measured by FEV<sup>1</sup> and Wisconsin additive chest radiograph score) was explored. Acquisition of *P. aeruginosa* was independently associated with a worsening pulmonary status in children with CF [82]. Moreover, in these studies, decline in pulmonary function after colonization with *P. aeruginosa* was observed to be gradual. This decline in pulmonary function associated with *P. aeruginosa* infection is noted across all age groups. In another study, acquisition of mucoid strains of *P. aeruginosa* was associated with an unfavorable prognosis [83]. From a pathologic perspective, *P. aeruginosa* causes repeated airway infections with eventual progression to chronic airway infection. This organism can also lead to necrotizing pneumonia, chronic bronchopneumonia, and chronic parenchymal lung disease. While the aggressive use of antipseudomonal antibiotics has been shown to delay the onset of chronic infection, prevalence rates of *P. aeruginosa* colonization have remained relatively stable over the past two decades [84, 85].

The CF airway provides a pathological milieu and a scaffold for chronic infection with resistant organisms, the most notable of them being *P. aeruginosa*. A number of virulent factors enable this resilient organism to establish it within the CF airways. One such virulence factor—overproduction of alginate slime capsule—characterizes the mucoid type of *P. aeruginosa*, which allows it to adhere firmly to the airway epithelium. Being encoded by the AlgT gene, alginate negatively regulates flagella, fimbriae, and quorum sensing. TTSS (injectosome) positively regulates alginate production indirectly through heat shock, osmotic, and oxidative stress responses [86]. In the inflamed CF airway, polymorphonuclear leukocytes (PMN) lead to the production of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) [87]. Moreover, mutated CF epithelial cells are unable to efflux glutathione (a potent free radical scavenger) and unable to absorb other dietary antioxidants. Production of ROS and RNI by PMN leads to DNA damage, lipid peroxidation and denaturation of proteins. At the same time, RNI and ROS lead to upregulation of alginate production by *P. aeruginosa*. The alginate slime capsule enables the bacterium to adhere firmly to the airway epithelial cells and results in persistence of this organism within the airways. At the same time, other virulence factors produced by *P. aeruginosa* (such as exotoxins) incur progressive pulmonary damage and help it to evade the (already impaired) host immune response. Over time, ROI and RNI lead to loss of microbial diversity and disruption of the airway microbiota. Simultaneously, such an environment favors the survival and selection of *P. aeruginosa* within the CF airway and leads to persistent infection with this organism [88, 89]. Moreover, antibiotic exposures select for multidrug resistant variants of the organism and allow them to predominate and colonize the airways [24, 90]. Alarmingly, recent reports from CF centers across the world have described certain strains of *P. aeruginosa* that exhibit resistance to all clinically relevant classes of antimicrobials ("pan‐resistant" *P. aeruginosa*) [91]. This can explain the worse prognosis associated with this organism in most studies of CF patients.

#### **4.5. Burkholderia cepacia complex**

conventional pyocin typing and DNA probe analysis reported that most CF patients harbored a persistent strain of *P. aeruginosa* in their lungs [72]. These studies suggested that cross‐colonization possibly could occur among patients. Another study showed that 59% of CF patients harbored a clonal strain of *P. aeruginosa* and the dominant pulsotype was indistinguishable from nonclonal strains with respect to both colony morphology and resistance patterns [73]. Wolz et al. used DNA probe amplification assays and demonstrated that 46% of CF patients (who were initially uninfected) acquired P. aeruginosa infection at the end of a CF recreation camp [74]. Clear evidence of a cross‐infection among patients attending a CF clinic was published in 2001 [75]. In this study, 22 of 154 patients attending an adult CF clinic were chronically infected with similar isolates (based on pyocin typing and pulsed‐ field gel electrophoresis [PFGE] analysis) of *P. aeruginosa* that shared unusual phenotypic features: lack of motility and pigmentation along with a remarkable resistance to many antibiotics. In another study from a large pediatric CF clinic from Australia, 65 patients (55%) were found to be infected with a similar strain of *P. aeruginosa*. These patients were more likely to have been hospitalized in the preceding 1 year for respiratory exacerbations [76]. On the other hand, a study conducted by Speert et al. in Vancouver (Canada) reported a low rate of transmission of *P. aeruginosa* from one CF patient to the other [77]. In this study, a total of 157 genetic types of *P. aeruginosa* were identified, of which 123 were unique to individual patients. These apparently conflicting findings may be accounted for by the highly adaptable nature of *P. aeruginosa* and its ability to evolve. In a study by Mahenthiralingam et al., different strains of *P. aeruginosa* were studied using genomic fingerprinting and random DNA amplification assays [78]. A total of 385 isolates from 20 patients were grouped into 35 random amplified polymorphic DNA (RAPD) strain types. Secretion of mucoid exopolysaccharide, loss of expression of RpoN‐dependent surface factors and acquisition of serum‐susceptible phenotypes in Pseudomonas were shown to be a specific adaptation to infection, rather than being acquired from a new bacterial strain. This explanation is also in congruence with observations from other studies that found different strains of *P. aeruginosa* in unrelated CF patients and identical or closely related strains among siblings [79]. The presence of distinct strains of *P. aeruginosa* in these studies reflects an absence of nosocomial transmission of organisms at respective CF centers [80]. This may be a consequence of strict hygiene measures and microbiologic surveillance instituted at most CF centers across the world following

The effects of *P. aeruginosa* infection on the CF lung are deleterious. In one observational study, outcomes of CF children colonized with *P. aeruginosa* were compared with those of noncolonized patients. Children colonized with *P. aeruginosa* had a worse outcome and

expiratory flow at 25% of vital capacity) [81]. In another longitudinal observational study, the temporal relationship between *P. aeruginosa* infection and pulmonary damage (as measured

*P. aeruginosa* was independently associated with a worsening pulmonary status in children with CF [82]. Moreover, in these studies, decline in pulmonary function after colonization with *P. aeruginosa* was observed to be gradual. This decline in pulmonary function associated with *P. aeruginosa* infection is noted across all age groups. In another study, acquisition of mucoid

and Wisconsin additive chest radiograph score) was explored. Acquisition of

and FEF25 (forced

experienced rapid decline in pulmonary function as measured by FEV<sup>1</sup>

reports of nosocomial spread [75, 76].

54 Progress in Understanding Cystic Fibrosis

by FEV<sup>1</sup>

More than 60 species belonging to the genus Burkholderia are not pathogenic to humans, but some of the remaining species are implicated in serious infections in CF patients. Using 16S rDNA and recA gene analysis, 17 species of this genus have been grouped together as the Burkholderia cepacia complex (BCC). BCC is a group of virulent pathogens that are frequently implicated in infective exacerbations in CF patients with end‐stage lung disease. Colonization with BCC in CF patients indicates a poor prognosis and has been shown to be associated with a requirement for lung transplantation. This worse prognosis is due to the inherent antibiotic resistance possessed by these organisms and their ability to rapidly spread from patient to patient. In some cases, infection with BCC can lead to the development of cepacia syndrome—a rapid fulminating pneumonia that often leads to bacteremia and sepsis. Given their virulent nature, strict infection control measures are essential to prevent outbreaks of BCC in CF clinics and centers [92]. A report of rapid spread and outbreak of BCC infection was reported in a CF center in Toronto [93]. This center reported the development of cepacia syndrome in many patients, being characterized by rapidly deteriorating pulmonary function, fever, leukocytosis, elevated markers of inflammation, and BCC bacteremia. Furthermore, in another report, cepacia syndrome occurred in approximately 20% of infected patients and had a case fatality rate of 62% [93].

Outside of the BCC group, a few other species of the Burkholderia genus are also implicated in infective exacerbations. These species include *Burkholderia gladioli*, *Burkholderia fungorum*, *Burkholderia multivorans* and *Burkholderia pseudomallei* [94]. Of these, *B. gladioli* now accounts for a significant proportion of Burkholderia infections in CF patients [95]. In the United States, *B. multivorans* and *B. gladioli* together account for more than 50% of Burkholderia infections in CF patients.

Most infected CF patients harbor genotypically distinct strains of the BCC. Strains of Burkholderia spp. that are shared by multiple CF patients are very uncommon. This suggests that most Burkholderia infections in CF patients result from acquisition of strains from the natural environment [92, 96]. In this regard, *B. gladioli* and *B. cepacia* have been described as recognized plant pathogens. In one study, multilocus sequence typing of Burkholderia spp. revealed that more than 20% of CF isolates were identical to strains recovered from the environment [97].

In the CFF patient registry, prevalence of BCC was reported to have declined from 9% in 1985 to 4% in 2005. Incidence of BCC was also found to be reduced from 1.3% in 1995 to 0.8% in 2005 [69]. This has not changed significantly over the past decade as shown by data published in 2016 [70]. Ramette et al. analyzed 285 confirmed isolates of BCC using restriction analysis of recA and identified seven different BCC species in the environment [98]. Healthcare‐associated outbreaks of BCC infections as a consequence of contaminated medical devices and products (such as mouthwashes, ultrasound gels, skin antiseptics, and medications) have been reported previously. While most of these outbreaks have generally involved non‐CF patients, the potential for developing such outbreaks among CF patients remains a hazard [99]. Infection of the respiratory tract with BCC species in CF patients often results in a chronic persistent infection [100]. In most such cases, a single strain of Burkholderia spp. colonizes the respiratory tract.

Infection with BCC species has been associated with a worse prognosis. In one study, CF patients who were infected with *Burkholderia dolosa* had a rapid decline in FEV<sup>1</sup> over time [101]. In another study, patients colonized with *B. cenocepacia* had a worse outcome in terms of body mass index (BMI) and FEV<sup>1</sup> as compared to those colonized with *P. aeruginosa* or *B. multivorans* [102].

#### **4.6. Anaerobic bacteria**

Anaerobic bacteria have been described in the airways of people with healthy lungs and are generally not considered to be pathogenic. In patients with CF, anaerobic bacteria are persistent members of the lower airway community as the anaerobic conditions (and steep oxygen gradients) in the lower airways provide an ideal environment for their growth [88, 103]. However, in the CF lung, anaerobic bacteria can produce virulence factors and damage the lung parenchyma (perhaps as a consequence of impaired innate immunity), which may worsen pulmonary function and exacerbate the inflammatory response. Short‐chain fatty acids produced by anaerobic bacteria can increase production of interleukin‐8 (IL‐8) by upregulating expression of the short‐chain fatty acid receptor GPR41 [104]. Moreover, in the CF microbiome, anaerobic bacteria can interact with other established pathogens and lead to progressive pulmonary damage [105]. Previously, anaerobic bacteria were thought to be an infrequent cause of CF exacerbation; however, with the advent of novel (culture‐independent) microbial detection methods [106–109], anaerobes have been isolated from more frequently. In one study, 23.8% of sputum specimens from CF patients grew more than 105 colony forming units (CFU) per milliliter of anaerobic bacteria [110]. In another study, 15 genera of obligate anaerobes were identified in 91% of CF patients with counts (CFU/ml) being comparable to that of *P. aeruginosa* and *S. aureus* [111]. The most common anaerobes were *Staphylococcus saccharolyticus* and *Peptostreptococcus prevotii*. Some studies suggest that patients with lower aerobic and anaerobic bacterial load have worse pulmonary function and higher levels of inflammatory markers [112]. From a biological standpoint, lower quantity of aerobes and anaerobes may reflect disruption of the CF microbiota. Studies have shown that antibiotic therapy directed against *P. aeruginosa* during acute exacerbations does not affect anaerobes [111]. This observation could be explained by considering the resistance patterns of anaerobes. In 58% of patients, obligate anaerobes detected during acute infective exacerbations were resistant to antibiotics used for treatment. The chief obligate anaerobes in such cases were *Bacteroides* spp., *Porphyromonas* spp., *Prevotella* sp., *Veillonella*, *anaerobic Streptococcus* spp., *Proprionibacterium*, *Actinomyces*, *S. saccharolyticus* and *P. prevotii* [36, 111, 113]. Interestingly, infection with *P. aeruginosa* significantly increases the likelihood of isolating anaerobic bacteria from CF patients [36]. Some of these anaerobic bacteria (such as S. milleri) are now known to be associated with worse clinical outcomes. Furthermore, new anaerobic organisms have been detected for the first time from samples of CF patients. Such bacteria, for instance Gemella and Rothia mucilaginosa, have been found to be associated with dismal pulmonary outcomes. Most such patients are often coinfected with *P. aeruginosa* as well [114, 115].

#### **4.7. Nontuberculous mycobacteria**

possessed by these organisms and their ability to rapidly spread from patient to patient. In some cases, infection with BCC can lead to the development of cepacia syndrome—a rapid fulminating pneumonia that often leads to bacteremia and sepsis. Given their virulent nature, strict infection control measures are essential to prevent outbreaks of BCC in CF clinics and centers [92]. A report of rapid spread and outbreak of BCC infection was reported in a CF center in Toronto [93]. This center reported the development of cepacia syndrome in many patients, being characterized by rapidly deteriorating pulmonary function, fever, leukocytosis, elevated markers of inflammation, and BCC bacteremia. Furthermore, in another report, cepacia syndrome occurred

Outside of the BCC group, a few other species of the Burkholderia genus are also implicated in infective exacerbations. These species include *Burkholderia gladioli*, *Burkholderia fungorum*, *Burkholderia multivorans* and *Burkholderia pseudomallei* [94]. Of these, *B. gladioli* now accounts for a significant proportion of Burkholderia infections in CF patients [95]. In the United States, *B. multivorans* and *B. gladioli* together account for more than 50% of Burkholderia infections in CF patients. Most infected CF patients harbor genotypically distinct strains of the BCC. Strains of Burkholderia spp. that are shared by multiple CF patients are very uncommon. This suggests that most Burkholderia infections in CF patients result from acquisition of strains from the natural environment [92, 96]. In this regard, *B. gladioli* and *B. cepacia* have been described as recognized plant pathogens. In one study, multilocus sequence typing of Burkholderia spp. revealed that more than 20% of CF isolates were identical to strains recovered from the environment [97].

In the CFF patient registry, prevalence of BCC was reported to have declined from 9% in 1985 to 4% in 2005. Incidence of BCC was also found to be reduced from 1.3% in 1995 to 0.8% in 2005 [69]. This has not changed significantly over the past decade as shown by data published in 2016 [70]. Ramette et al. analyzed 285 confirmed isolates of BCC using restriction analysis of recA and identified seven different BCC species in the environment [98]. Healthcare‐associated outbreaks of BCC infections as a consequence of contaminated medical devices and products (such as mouthwashes, ultrasound gels, skin antiseptics, and medications) have been reported previously. While most of these outbreaks have generally involved non‐CF patients, the potential for developing such outbreaks among CF patients remains a hazard [99]. Infection of the respiratory tract with BCC species in CF patients often results in a chronic persistent infection [100]. In most such cases, a single strain of Burkholderia spp. colonizes the respiratory tract.

Infection with BCC species has been associated with a worse prognosis. In one study, CF patients

study, patients colonized with *B. cenocepacia* had a worse outcome in terms of body mass index

Anaerobic bacteria have been described in the airways of people with healthy lungs and are generally not considered to be pathogenic. In patients with CF, anaerobic bacteria are persistent members of the lower airway community as the anaerobic conditions (and steep oxygen gradients) in the lower airways provide an ideal environment for their growth [88, 103].

as compared to those colonized with *P. aeruginosa* or *B. multivorans* [102].

over time [101]. In another

who were infected with *Burkholderia dolosa* had a rapid decline in FEV<sup>1</sup>

(BMI) and FEV<sup>1</sup>

**4.6. Anaerobic bacteria**

56 Progress in Understanding Cystic Fibrosis

in approximately 20% of infected patients and had a case fatality rate of 62% [93].

Traditionally, the frequency of CF patients infected with NTM has been reportedly low. In the CFF patient registry, the prevalence of NTM infections among CF patients has been estimated to be 2.2%. Nevertheless, the prevalence of NTM has been increasing slowly over the past few decades. The prevalence of NTM infection in 1999 among CF patients was 0.85%, which increased to 2.18% in 2008 [116]. More recent data published in 2016 shows that the prevalence of NTM may be as high as 11.9% [70]. The most common NTM species have been reported to be Mycobacterium avium‐intracellulare (MAI) complex and *Mycobacterium abscessus*. Factors associated with a culture positive for NTM are older age, greater FEV<sup>1</sup> , higher frequency of MSSA colonization and lower frequency of *P. aeruginosa* infection [117]. In most patients, unique strains of NTM are detected by molecular typing, which suggests that neither person‐to‐person transmission nor nosocomial acquisition is implicated. In one study, the prevalence of NTM infection among 385 patients in three Parisian centers was 8.1%. *M. abscessus*

was isolated in all age groups. About 4.1% (16/385) of the study cohort met the American Thoracic Society (ATS) criteria for NTM‐related lung disease [118]. In another multicenter study done in Israel [119], prevalence of NTM‐related lung disease (as defined by the 2007 ATS criteria) was 10.8%. This study further suggested that the incidence of NTM infections is increasing over time. Other studies have demonstrated that the incidence of MAI complex infections in CF patients is decreasing with time, while that of *M. abscessus* complex is increasing [120]. Alarmingly, infection with *M. abscessus* complex has been associated with a worse impact on pulmonary function. Some researchers have proposed that eradication of *M. abscessus* complex may provide a significant improvement in terms of pulmonary outcome [121]. However, *M. abscessus* is difficult to manage, commonly affects younger children, and requires prolonged courses of intravenous antibiotics [122].

#### **4.8.** *Stenotrophomonas maltophilia*

*S. maltophilia* is a Gram‐negative bacillus that is commonly implicated in nosocomial infections in non‐CF patients. However, in patients with CF, *S. maltophilia* has been recognized as a cause of acute infective exacerbation. The medical importance of this pathogen is that it is inherently resistant to a wide range of broad‐spectrum antibiotics (most notably carbapenems). The prevalence of infection with this organism has increased from 1 to 4% over a period of 20 years (1985–2005) [68]. In the CFF patient registry, the prevalence of *S. maltophilia* increased from 4.0% in 1996 to 12.4% in 2005 [69]. From 2005 till 2015, the prevalence of *S. maltophilia* seems to have plateaued [70]. *S. maltophilia* infections of the respiratory tract in CF patients tend to be acute and, in most cases, the organism does not persist in the lower airways (although recurrent infections can occur). Most isolates of this organism have been shown to be transmitted from patient‐to‐patient, especially among siblings, or those who are otherwise epidemiologically linked [123]. One‐third of CF patients who experience recurrent infections with *S. maltophilia* harbor more than one strain of the organism [124]. The most important risk factors for acquiring *S. maltophilia* infections are therapy with carbapenems and central venous catheterization [125]. In one study, history of treatment with imipenem was 10 times more frequent among cases (who contracted *S. maltophilia*) than among controls [125]. Furthermore, all fatal infections with *S. maltophilia* occurred in patients who had received imipenem. Based on these results, it is advisable to cover *S. maltophilia* empirically in CF patients who develop super‐infection while receiving imipenem therapy. In a report by Sanyal and Mokaddas [126], most strains of *S. maltophilia* were susceptible to ciprofloxacin and trimethoprim‐sulfamethoxazole. Moreover, some evidence shows that CF patients infected with *S. maltophilia* were more likely to have been hospitalized for many days in the past one year [127]. Other factors associated with *S. maltophilia* acquisition were more than two courses of intravenous antibiotics, isolation of *Aspergillus fumigatus* or *P. aeruginosa* in sputum and oral steroid use [128]. *S. maltophilia* is also more common among CF patients who develop allergic bronchopulmonary aspergillosis (ABPA) [129]. While chronic infection with *S. maltophilia* is infrequent, it can occur in certain patients and requires repeated courses of antibiotics [130]. Chronic infection with *S. maltophilia* confers a threefold higher risk of mortality or the need for lung transplantation [131].

#### **4.9.** *Achromobacter xylosoxidans*

was isolated in all age groups. About 4.1% (16/385) of the study cohort met the American Thoracic Society (ATS) criteria for NTM‐related lung disease [118]. In another multicenter study done in Israel [119], prevalence of NTM‐related lung disease (as defined by the 2007 ATS criteria) was 10.8%. This study further suggested that the incidence of NTM infections is increasing over time. Other studies have demonstrated that the incidence of MAI complex infections in CF patients is decreasing with time, while that of *M. abscessus* complex is increasing [120]. Alarmingly, infection with *M. abscessus* complex has been associated with a worse impact on pulmonary function. Some researchers have proposed that eradication of *M. abscessus* complex may provide a significant improvement in terms of pulmonary outcome [121]. However, *M. abscessus* is difficult to manage, commonly affects younger children, and

*S. maltophilia* is a Gram‐negative bacillus that is commonly implicated in nosocomial infections in non‐CF patients. However, in patients with CF, *S. maltophilia* has been recognized as a cause of acute infective exacerbation. The medical importance of this pathogen is that it is inherently resistant to a wide range of broad‐spectrum antibiotics (most notably carbapenems). The prevalence of infection with this organism has increased from 1 to 4% over a period of 20 years (1985–2005) [68]. In the CFF patient registry, the prevalence of *S. maltophilia* increased from 4.0% in 1996 to 12.4% in 2005 [69]. From 2005 till 2015, the prevalence of *S. maltophilia* seems to have plateaued [70]. *S. maltophilia* infections of the respiratory tract in CF patients tend to be acute and, in most cases, the organism does not persist in the lower airways (although recurrent infections can occur). Most isolates of this organism have been shown to be transmitted from patient‐to‐patient, especially among siblings, or those who are otherwise epidemiologically linked [123]. One‐third of CF patients who experience recurrent infections with *S. maltophilia* harbor more than one strain of the organism [124]. The most important risk factors for acquiring *S. maltophilia* infections are therapy with carbapenems and central venous catheterization [125]. In one study, history of treatment with imipenem was 10 times more frequent among cases (who contracted *S. maltophilia*) than among controls [125]. Furthermore, all fatal infections with *S. maltophilia* occurred in patients who had received imipenem. Based on these results, it is advisable to cover *S. maltophilia* empirically in CF patients who develop super‐infection while receiving imipenem therapy. In a report by Sanyal and Mokaddas [126], most strains of *S. maltophilia* were susceptible to ciprofloxacin and trimethoprim‐sulfamethoxazole. Moreover, some evidence shows that CF patients infected with *S. maltophilia* were more likely to have been hospitalized for many days in the past one year [127]. Other factors associated with *S. maltophilia* acquisition were more than two courses of intravenous antibiotics, isolation of *Aspergillus fumigatus* or *P. aeruginosa* in sputum and oral steroid use [128]. *S. maltophilia* is also more common among CF patients who develop allergic bronchopulmonary aspergillosis (ABPA) [129]. While chronic infection with *S. maltophilia* is infrequent, it can occur in certain patients and requires repeated courses of antibiotics [130]. Chronic infection with *S. maltophilia* confers a threefold higher risk of mortality or the need

requires prolonged courses of intravenous antibiotics [122].

**4.8.** *Stenotrophomonas maltophilia*

58 Progress in Understanding Cystic Fibrosis

for lung transplantation [131].

*A. xylosoxidans* has been recognized as a pathogen and cause of infective exacerbation in patients with CF [132]. In the CFF patient registry, the prevalence of *A. xylosoxidans* infection was 1.9% in 1995 [69]. In 2015, the prevalence had increased almost three‐folds to 6.1% [70]. *A. xylosoxidans* is a ubiquitous organism that occurs widely in natural habitats. This organism is an opportunistic pathogen that affects only immunocompromised patients and those with CF. *A. xylosoxidans* is mostly implicated in nosocomial infections, such as hospital acquired pneumonia, catheter‐associated urinary tract infection, and wound infections. Lung infections with this fastidious organism are difficult to eradicate. Most patients respond to antipseudomonal penicillins (such as piperacillin–tazobactam) and third‐ or fourth‐generation cephalosporins [133]. In one report, two cases of *Achromobacter ruhlandii* developed after indirect contact between CF patients [134]. Another study from a French CF center reported that most isolates of Achromobacter spp. were resistant to fluoroquinolones and carbapenems [135]. In a retrospective study, CF patients who were chronically infected with *A. xylosoxidans* were more likely to have impaired pulmonary function. Additionally, the frequency of hospitalization was higher among such patients than others [136].

## **5. Implications for further research**

Cystic fibrosis is a monogenetic multisystem disorder, but, pulmonary disease is the leading cause of morbidity and mortality. Recurrent pulmonary infections with pathogenic bacteria can lead to progressive pulmonary damage and eventually lead to death. Therefore, understanding the CF airway microbiome has immense importance for understanding the overall pathology of the disease. Disruption of the CF airway microbiome under the influence of environmental factors and antibiotic exposures is a crucial step in the development of end‐stage pulmonary disease in such patients [40]. Colonization of the lower airways with pathogenic bacteria, such as *P. aeruginosa* [82] and *B. cenocepacia* [101], has been associated with end‐stage pulmonary disease.

As the CF airway microbiome evolves under the influence of antibiotic exposures, microbes undergo a number of mutations and changes in their genome [137]. While these genetic mutations are an evolutionary mechanism for microorganisms (for instance, to acquire resistance to antibiotics), they create potential vulnerabilities that may be exploited in unique therapeutic approaches. Traditionally, the approach to management of CF pulmonary exacerbations has been through employment of antibiotics. While antibiotics are useful in the short run, multidrug resistant microbes eventually evolve and become a challenge to tackle. In view of this, novel approaches to the management of CF pulmonary disease have been proposed, which involve manipulating patients' microbial consortia [8]. From a theoretical perspective, such an approach aims to maintain the architecture of the CF airway microbiome and avoids the use of antimicrobials, thereby circumventing the problem of destroying the community structure of a patient's microbiome. Such a novel treatment approach is based on the principles of personalized medicine and aims to tailor treatment according to each patient's individual microbiome [138]. By manipulating and restoring the structure of a patient's airway microbiome, the complex metabolomic profile of the patient's sputum (and other body fluids) can be altered, which may have long‐lasting and pleiotropic consequences [139].

Novel treatment approaches for the treatment of CF patients hold theoretical promise, but their practical applicability and clinical efficacy remains to be established [140]. A recent pilot study compared the use of a probiotic (*Lactobacillus* spp.) versus placebo in pediatric CF patients. Patients receiving the probiotic demonstrated a significant reduction in hospitalization for pulmonary exacerbation and a beneficial effect on the gut in terms of reducing gastrointestinal inflammation [141]. Another clinical trial examined the efficacy of enteric probiotics in reducing the frequency and severity of pulmonary exacerbations in CF patients. Both studies reported that the use of enteric probiotics provided a significant reduction in the frequency of pulmonary exacerbations when compared to the placebo group [142]. Larger randomized controlled studies are needed to more fully evaluate the effect of probiotics on hard clinical endpoints [143]. Other treatment options based on these novel concepts need to be developed further, and they may help to improve the overall outcomes of patients with CF [144].

## **Abbreviations**



## **Author details**

the principles of personalized medicine and aims to tailor treatment according to each patient's individual microbiome [138]. By manipulating and restoring the structure of a patient's airway microbiome, the complex metabolomic profile of the patient's sputum (and other body fluids)

Novel treatment approaches for the treatment of CF patients hold theoretical promise, but their practical applicability and clinical efficacy remains to be established [140]. A recent pilot study compared the use of a probiotic (*Lactobacillus* spp.) versus placebo in pediatric CF patients. Patients receiving the probiotic demonstrated a significant reduction in hospitalization for pulmonary exacerbation and a beneficial effect on the gut in terms of reducing gastrointestinal inflammation [141]. Another clinical trial examined the efficacy of enteric probiotics in reducing the frequency and severity of pulmonary exacerbations in CF patients. Both studies reported that the use of enteric probiotics provided a significant reduction in the frequency of pulmonary exacerbations when compared to the placebo group [142]. Larger randomized controlled studies are needed to more fully evaluate the effect of probiotics on hard clinical endpoints [143]. Other treatment options based on these novel concepts need to be developed further, and they may help to improve the overall outcomes

can be altered, which may have long‐lasting and pleiotropic consequences [139].

of patients with CF [144].

60 Progress in Understanding Cystic Fibrosis

ABPA Allergic bronchopulmonary aspergillosis

CFTR Cystic fibrosis transmembrane conductance regulator

FAFLP Fluorescent amplified fragment length polymorphism

FEF25 Forced expiratory flow at 25% of vital capacity FEV<sup>1</sup> Forced expiratory volume in first second

ATS American Thoracic Society BAL Bronchoalveolar lavage BCC Burkholderia cepacia complex

CFF Cystic Fibrosis Foundation

FMT Fecal microbiota transplantation HQNO 4‐Hydroxy‐2‐heptylquinoline‐N‐oxide

MAI Mycobacterium avium‐intracellulare MRSA Methicillin‐resistant Staphylococcus aureus MSSA Methicillin‐sensitive Staphylococcus aureus

CFU Colony forming units

IL‐8 Interleukin‐8

CD Clostridium difficile CF Cystic fibrosis

**Abbreviations**

Ibrahim A. Janahi<sup>1</sup> \* and Abdul Rehman<sup>2</sup>

\*Address all correspondence to: ijanahi@hamad.qa

1 Pediatric Pulmonology, Department of Pediatrics, Hamad Medical Corporation, Doha, Qatar

2 Internal Medicine Section, Department of Medicine, Hamad Medical Corporation, Doha, Qatar

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