Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases

*Stephan F. van Eeden and Don D. Sin*

#### **Abstract**

Macrophages are key immune cells, where they play a pivotal role in host defense and tissue homeostasis. The lungs have two major subsets, alveolar macrophages (AMs) found in airspaces and interstitial macrophages (IMs) found in lung tissues. Lung macrophages (LM) are highly heterogeneous and have high levels of plasticity. A longlasting population of LM with self-renewal ability populate the lung during embryogenesis and monocyte-derived macrophages recruited during infection, inflammation, or tissue repair, which are more short lived. AMs have been the main focus of research due in part to their abundance, accessibility, and ease of isolation compared with IMs. With advances in multichannel flow cytometry and single-cell sequencing, the importance of IMs has been recently appreciated. LM's functions in the lungs include maintenance of homoeostasis, immune surveillance, removal of cellular debris, tissue repair, clearance of pathogens, and the resolution of inflammation. They also activate the adaptive immune response by functioning as antigen-presenting cells. LMs are pivotal in the pathogenesis of acute and chronic inflammatory lung conditions including lung cancer. This chapter will discuss the ontology, phenotypic heterogeneity, and functions of LM's and how these characteristics orchestrate and impact common acute and chronic lung conditions.

**Keywords:** alveolar macrophages, interstitial macrophages, macrophage phenotypes, lung infections, asthma, lung cancer, lung fibrosis

#### **1. Introduction**

Macrophages are immune effector cells that are present in most organs and tissues, are highly phagocytic in nature and produce large amounts of a wide variety of mediators. They are either resident in tissues or are recruited and as part of the innate immune effector system, are activated to mount an appropriate immune response to neutralize harmful insults [1].

The lungs are continuously challenged by a variety of foreign inhaled substances which include allergens, microbial pathogens, chemicals, particulates matter and

noxious gasses. These insults require an exquisite capacity to appropriately calibrate inflammatory responses in the airways and lung tissues to maintain physiologic homeostasis (e.g., gas exchange). The lung macrophages have all the properties to orchestrate and calibrate such inflammatory responses given their location in the lungs, their large abundance in tissue and their high degree of functional plasticity and ability to communicate with neighboring cells. Research over the past three decades has shed light on the origins of lung macrophages, their ability to adapt to the local microenvironment, their plasticity, and their functional responses to maintain tissue homeostasis. In addition, their pivotal role in orchestrating the innate immune response and activating adaptive immunity when airways are challenged with pathogenic insults has also been elucidated [1, 2].

#### **1.1 Macrophage populations in the lung**

Two well-studied populations of lung macrophages have been defined: (1) alveolar macrophages (AMs), which are predominantly located on alveolar epithelial surfaces and can be harvested from the lungs by bronchoalveolar lavage (BAL) and (2) interstitial macrophages (IMs), which are located within alveolar walls or interstitial lung tissue and can be harvested directly from lung tissue specimens through biopsy or surgical resections [3]. Two less well-defined populations of macrophages are airway macrophages, which are found on mucosal surfaces of the airways and intravascular macrophages, which reside on capillary blood vessel walls. Limited data suggest that airway macrophages are phenotypically and functionally very similar to AMs and may represent AMs that have migrated up the tracheobronchial tree [4]. Airway macrophages are usually grouped with AMs because of their phenotypic similarity and their ability to be captured by BAL. The intravascular macrophages are located on the inner side of capillaries, suggesting that they fight against blood-borne pathogens. Limited (older) data suggest that their function is comparable to that of AMs and that they may reflect an intermediate stage of differentiation between blood monocytes and AMs [5, 6].

It was previously thought that resident lung macrophages originate primarily from blood monocytes, which are produced and released from the bone marrow [7, 8]. The last decade this paradigm has been turned on its head as research has shown that most resident macrophages in different tissues throughout the body, including lung macrophages, arise predominantly from embryonic precursors. These macrophages are produced before birth, become colonized in the lungs in the prenatal period and are maintained throughout life by local proliferation [9–11]. Resident alveolar macrophages originate as erythromyeloid progenitors (EMPs) in the yolk sac on embryonic day (E) 8.5. EMPs colonize the fetal liver by E10.5, and then give rise to fetal monocytes, which migrate to the lungs by E12.5 [12]. The maturation of fetal monocytes to alveolar macrophages occurs in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF) and is fully completed by the third postnatal day (**Figure 1**).

These resident lung macrophages are the primary "janitors" of the lung, protecting the lungs from inhaled environmental insults. In contrast to resident alveolar macrophages, recruited monocytes, which enter airspaces 24-72 hrs after the onset of an inflammatory stimulus in the lung, differentiate into macrophages in the tissues where they initially take on a pro-inflammatory phenotype (by promoting inflammation) and later assumes a regulatory role by suppressing the inflammatory process. Classically, the initial phenotype has been described as M1 macrophages and the regulatory phenotype as M2 macrophages.

*Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

#### **Figure 1.**

*The origin of alveolar (AM) and interstitial (IM) macrophages in mice. Resident AMs, derived from the embryo (yolk sac and/orfetal liver), are capable of self-replicating during homeostasis and when challenge in lung. IMs are derived from bone marrow monocytes and there are three unique IMs in murine lung in homeostasis: IM1, IM2 and IM3 (Gibbings et al.). During steady state, the maintenance of AM pool does not need the contribution of bone marrow -derived monocytes, but in the circumstance of inflammation, monocytes are strongly recruited to areas of inflammatory alveoli and differentiated into recruited monocyte-derived AMs. IM3 are originated from BM-derived monocytes and play different roles in diseases such as pulmonary fibrosis (Chakarov et al.).*

While M1 macrophages promote inflammation, M2 macrophages induce efferocytosis (phagocytosis of apoptotic and dead cells) to enable inflammatory clearance, and secrete anti-inflammatory cytokines such as IL-10 and soluble (decoy) IL-1R [13].

Studies in animal models have shown that resident AMs can be replaced by IMs or be derived from circulating monocytes [8, 14, 15], underlining the adaptability of macrophage kinetics and function. Schneider and co-workers have shown that the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-) is essential for the differentiation of AMs and that PPAR- deletion in the myeloid lineage cells prevents the formation of mature AMs, resulting in impaired microbial clearance and development of a clinical condition called pulmonary alveolar proteinosis, which is characterized by excess surfactant accumulation in lungs [16]. PPAR- expression requires GM-CSF, which is produced largely by type II alveolar epithelial cells. A lack of GM-CSF leads to a loss of mature AMs, accumulation of

surfactant and ultimately alveolar proteinosis [9]. Recently the interesting concept of innate immune memory resulting from resetting of the cell's epigenetic program and functional state after infection, leading to enhanced protection against a secondary infection in the ensuing weeks to months [17]. These "memory AMs" have been shown to originate from resident AMs following mild infection and are characterized by a high level of MHCII molecules and host defense-ready genes, and enhanced production of neutrophil attracting chemokines and interferon-gamma (IFN-γ) derived from effector T cells, which are primed to respond during infection [18].

Interstitial macrophages are less well defined mostly because access to pure populations are difficult in humans. Studies in laboratory animals suggest that there are at least 3 different populations of IM: IM1, IM2 and IM3. Expression of CD11c and MHCII on IMs at a steady state classifies these population into three subsets, IM1 (CD11cloMHCIIlo), IM2 (CD11cloMHCIIhi), and IM3 (CD11chiMHCIIhi) in mice [19]. At least two of these three populations have been identified in humans [20, 21]. Whether these IMs are resident, long lived cells possessing self-renewal properties similar to resident AMs, or represent derived cells from blood monocytes is still unclear.

Macrophages in the lungs have a range of function, including maintenance of homeostasis, immune surveillance, repair, removal of cellular debris and surfactant clearance, and elimination of microbes, allergens and particulate matter, and resolution of inflammation. These distinct functions are linked to the different subsets of macrophages, ontogeny, and location of the macrophages and influence of the local microenvironment.

#### **2. Macrophage phenotypes**

Similar to T helper 1/2 (Th1/Th2) cells, macrophages are categorized as either classically activated M1 macrophages or alternatively activated M2 macrophages. The classical or M1 macrophages are activated by microbial products and/or IFN-γ. MI cells are pro-inflammatory and possess anti-microbial functions, and ability destroy tumor cells [22]. Signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor (IRF)3, IRF5, and nuclear factor-κβ (NF-κβ) are key molecules that become activated in M1 macrophages to generate pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), Interleukin-1 (IL-1), IL- 12 and IL- 23, [23], nitric oxide (NO), and reactive oxygen intermediates (ROI). They also promote increased expression of major histocompatibility complex (MHC) molecules (**Figure 2**). Over the inflammatory period, the microenvironment changes and Th2 cytokines, including IL- 4 and IL- 13, stimulate monocytes or macrophages to transform into a M2 phenotype. M2 macrophages promote resolution of the inflammatory process and stimulate wound healing, favoring a milieu of angiogenesis and tissue remodeling. IL-10, glucocorticoid hormones and IL-1R may also induce M2 macrophage polarization.

The M2 macrophage phenotype is not homogenous and consists of several subtypes including M2a, M2b, M2c and M2d (**Figure 3**). The M2a macrophages are produced by exposure to cytokine such as IL-4 or IL-13. They can also be induced by fungal and helminth infections. They are characterized by expression of high levels of the mannose receptor (CD 206), CD209, IL-4R and FcεR, secreting transforming growth factor β1 (TGF-β1) and insulin-like growth factor. Functionally M2a macrophages contribute towards wound healing and tissue repair [24]. In contrast,

*Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

#### **Figure 2.**

*Paradigm of macrophage phenotypes. Naïve macrophages are activated to either a M1 phenotype (classical activation) or to M2 phenotype (alternatively activated) dependent on their microenvironment milieau. Macrophages are polarized to an M1 (or T-helper type 1 cell [Th1]) phenotype in response to IFN-g, granulocyte–macrophage colony–stimulating factor (GM-CSF), and bacterial products (e.g., LPS). They are characterized by a high production of the proinflammatory cytokines IL-12 and IL-23, and with high expression of major histocompatibility complex class II (MHCII) molecules and coactivation proteins (CD80 and CD86) are considered excellent antigen-presenting cells that can activate an adaptive immune response and fight foreign insults through the production of toxic intermediates such as reactive oxygen intermediates [ROI]). Macrophages are polarized to an M2 (or Th2) phenotype in response to macrophage colony–stimulating factor (M-CSF), IL-3 and IL-10 and antiinflammatoryfactors. They produce a variety of antiinflammatorycytokines, immunosuppressive mediators, and matrix-degrading enzymes, and promote angiogenesis and are involved in tissue remodeling and repair.*

#### **Figure 3.**

*Different M2 macrophage phenotypes are generated by different microenvironments and stimuli. The functional responses of the four different M2 sub-phenotypes are determine by them producing different mediators.*

M2b macrophages are induced by immune complexes, IL-1β and lipopolysaccharide (LPS) exposure, and secrete abundance of IL-10, IL-1β, IL-6 and TNF-α, that have predominantly anti-inflammatory properties [25]. The M2c macrophages are induced cytokines such as IL-10, TGF-β and glucocorticoids and have increased expression of receptor for advance glycation and end products (RAGE), CD163 and CD206. M2c macrophages are thought to be involved in immunosuppression, tissue repair and matrix remodeling [26, 27]. Lastly, M2d macrophages, are activated by leukocyte inhibitory factor, toll like receptor (TLR) ligands and adenosine. They express low levels of CD206, and produce large amounts of IL-10, TGF-β and vascular endothelial growth factor (VEGF) which facilitate immunosuppression and angiogenesis [28].

A unique characteristic of macrophages is their plasticity and their ability to differentiate into several phenotypes and also to de-differentiate. For example, M2 macrophages may revert to M1 macrophages under certain conditions. This switch in polarization is dynamic and induced predominantly by their microenvironment. The distinct M1 and M2 subtypes is an overly simplification of macrophage polarization, for example, 5% of the macrophages in lung cancer specimens express both M1 and M2 markers [29], and mixed macrophage polarization have been described in other lung conditions [29, 30]. With the advent of genetic analysis and specifically single cell RNA sequencing technology, the basic macrophage characterization of M1 and M2 phenotypes has been challenged and may change in the near future [31].

#### **3. Macrophages in lung infections**

Airspace or resident AMs represent the first line of defense and play a central role in protecting the lungs against a range of respiratory pathogens. Yet, pulmonary immune responses need to be contained and refined to avoid excessive tissue damage and safeguard gas exchange. Resident AMs (ResAM) are less responsive than recruited monocyte-derived macrophages in the context of infection in the lung [32]. As resident AMs are predominantly involved in lung tissue homeostasis, resident AMs are generally "anti-inflammatory", which in part is linked to their production of type I interferons (IFNs) [33]. Notably Type I IFNs negatively regulate IL-1 production and positively regulate IL-10 production in monocyte-derived macrophages [34]. AMs have low expression of complement-associated genes such as *C1qa*, *C1qb*, *C1qc*, *C2*, *C4b* and *C3ar1* [19], in contrast to macrophages isolated from other tissues. Epigenetic profiling of AMs during inflammation has shown that while the *C1q* locus is inaccessible in ResAMs, complement genes are highly accessible in inflammatory AMs (InfResAMs) that arise in the AM pool during influenza infection [35]. With human SARS-CoV-2 infection, recruited macrophages were more C1qhi compared with the ResAMs, which were more C1qlo, suggesting that repression of the complement-associated genes in ResAMs is conserved in humans [36]. Importantly, the overt activation of complements has been linked to coronavirus 2019 (COVID-19) severity [37, 38]. It is reasonable to suggest that the sharp drop in ResAMs and their replacement by recruited macrophages expressing high levels of the complementassociated genes during SARS-CoV-2 infection may fuel the complement cascade and actively participate in the COVID-19 pathogenesis. Furthermore, it could be that repression of these complement genes in ResAMs is the result of evolutionary pressure to protect the lungs from complement-mediated collateral damage during infections.

#### *Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

Kinetic studies have shown that bone marrow derived monocytes are recruited into alveolar space within 24 hrs following a focal instillation of *Streptococcus pneumoniae* into the lung [15]. These monocyte derived macrophages become the dominant immune cells in the airspaces during the resolution phase of pneumonia. Bacteria have developed strategies to take advantage of hypo-responsive ResAMs by enhancing their survival and promoting spread [39]. An example of this concept is the relocation of *Mycobacterium tuberculosis*-infected AMs into pulmonary tissues, which is a pivotal preparatory step for bacterial dissemination in the host [40]. Furthermore, following viral infection such as influenza, the resident AM population are depleted and subsequently restored by monocyte recruitment from the bloodstream which differentiate into long-lived InfResAMs and partially replace the resident AM [41, 42]. These newly recruited monocytes replace the resident macrophages and the extend of this replacement is linked to the dose and/or virulence of the pathogen. The reduction of resident macrophages by viral infections renders the host more susceptible to other microbial infections until the resident macrophages recover their numbers via self-renewal [43]. InfResAMs that develop during a viral infection have been shown to differ functionally from the ResAMs that were present before the infection [16, 42]. For example, InfResAMs that developed during influenza infection are more responsive to TLR ligands and subsequent *S. pneumoniae* infection compared with ResAMs. The InfResAMs acquired innate memory qualities via enhanced IL-6 production that provides protection against subsequent bacterial infections such as *S. pneumoniae* infection. Comparing the epigenetic signature of these ResAMs and InfResAMs shows that monocytes recruited by the virus infection have an epigenetic profile similar to tissue residency, but addition also acquire a more inflammatory signature induced by the viral infection. For example, the IL-6 enhancer regions are more accessible in InfResAMs that are recruited during an influenza lung infection compared with ResAMs. This altered epigenetic signature is still evident 1 month after the viral infection but started to alter at 2 months post-infection (**Figure 4**).

Long-term memory cells, which are characterized by altered gene expression, metabolism and antimicrobial responsiveness, have been proposed as a subset of ResAMs [44]. These cells have reduced phagocytic capacity by over-expressing signal regulatory protein α [45]. Similarly, InfResAMs that develop during an infection can acquire innate memory, thus altering their responses to subsequent infections. However, these InfResMacs show increased epigenetic plasticity. Much less is known about the impact of viral infections on resident IMs and their plasticity after an infectious insult. These cells have a significantly shorter half-life compared with ResAMs and robust fate-mapping systems have not been developed for IM.

Our ability to separate lung ResMacs from InfResMacs has shown that InfResMacs are more strongly imprinted across a range of infectious stimuli. This imprinting has the capacity to generate long-lasting innate immune memory cells that alter macrophage function during subsequent challenges. Multiple lung infectious challenges may therefore allow the engraftment of many waves of InfResMacs into lungs. However, over time and in a steady state, InfResMac may lose their unique genetic or epigenetic signature, leading to a loss in memory. Recurrent infections or inflammatory stimuli, on the other hand, could revive the memory of these InfResMacs. Innate memory in InfResMacs may be beneficial in some cases and may confer resistance to subsequent infections, but could also exacerbate tissue inflammation and injury during subsequent infections [46]. This has been proposed as a potential reason for the difference in severity of Covid-19 infection in older versus younger subjects and the generally mild inflammatory response seen in children. Therefore, the duration

#### **Figure 4.**

*Inflammation and/or infection in the lung and subsequent resolution reshape the composition of the pulmonary macrophage pool. A) An infectious episode in the lung results in the resident macrophages (ResMac) send signals for recruitment of monocytes from the blood. B) Monocytes from the blood are recruited into alveolar spaces to help dealing with the infection. C) these monocytes change intotransitional inflammatory macrophages that help clearing the infection. D) some of these transitional macrophages change intoinflammatory resident macrophages (InfResMac), the rest disappear. The InfResMacremain in the airspaces for months and ResMacproliferate to slowly replace them over time.*

of residence in a homeostatic environment such as the lung, becomes a key factor determining lung macrophage biology [47, 48].

#### **4. Macrophages and COPD**

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory lung disease caused by the long-term exposure to toxic particles and gasses. Worldwide, tobacco cigarette smoke is the main culprit, though biomass exposure may be a more important cause of COPD in some parts of the developing world [49]. These exposures elicit a persistent innate and eventually an adaptive immune response in the airways and lung tissues, which is characterized by overproduction of mucus in the central airways, fibrosis and obstruction of small airways and eventual destruction of the lung parenchyma leading to emphysema [50]. There is also evidence for impaired tissue repair responses and altered tissue remodeling that contribute to a progressive disease phenotype [51].

Lung macrophages are key immune effector cells in the pathogenesis of COPD. Airspace macrophages are directly exposed to inhaled antigens, pathogens and noxious particles and gasses, and several studies have shown an increase in their numbers in subjects with COPD compared to controls [52–54]. Cigarette smoking is still the major cause of COPD in developed countries, but biomass exposure is a more important cause of COPD in the developing world [49]. For both of these exposure types, lung macrophages play a pivotal role in processing and clearing these particles from the lungs. Due to the chronicity of these exposures in COPD, functional responses of lung macrophages to these exposures are thought to participate in the development of COPD. There is a significant increase in macrophages in induced sputum and BAL fluid (BALF) samples in COPD patients [55], supporting this notion [54]. In COPD, lung macrophages also secrete large amounts of potential tissue damaging enzymes such as elastase, matrix metalloproteinases (MMPs) MMP-2, MMP-9, MMP-12 and cathepsin S in response to exposure to cigarette smoke, ambient particulate matter or micro-organisms [55, 56]. In addition, continuous exposure to cigarette smoke or

*Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

biomass markedly depletes intracellular anti-oxidants such as glutathione, causing excessive oxidative stress, which suppresses macrophage bacterial phagocytosis and efferocytosis [57]. Therefore, macrophages in COPD generate a more pro-inflammatory milieu that promotes tissue injury. They also demonstrate defective immune surveillance and protective (phagocytic) functions that collectively contribute to the progression of COPD. The majority of acute exacerbations of COPD are triggered by either viral or bacterial respiratory infections that could alter the airway microbiome and cause frequent exacerbations [58], a clinical phenotype that is associated with a poor long-term outcome [51].

In COPD, M1 macrophages demonstrate enhanced pro-inflammatory capacity producing more TNF-α and MMPs [59], leading to increased extracellular matrix (ECM) deposition, elastin breakdown and excessive accumulation of collagen in the lung parenchyma. In contrast, Stout and co-workers [60] showed that M2 macrophages have a lower pro-inflammatory capacity (TNF-α, IL-1β, and IL-6), when stimulated. Morphological studies have shown that macrophages accumulate in areas of persistent inflammation in lung tissues of COPD including airway walls that lead to airway narrowing and obstruction and destructive changes in lung parenchyma [50, 61]. Several studies have shown phenotypic shifts in lung macrophages in COPD airways. Dewhurst and co-workers showed that the total number of macrophages in the airspaces of COPD subjects increased and morphologically became predominantly larger macrophages, which produced fewer pro-inflammatory cytokines and demonstrated reduced phagocytic ability [62]. Berenson and co-workers showed that macrophages from patients with COPD have impaired phagocytosis of respiratory pathogens which strongly correlated with COPD severity (FEV1% predicted) [63]. Studies from our laboratory recently showed that the majority of airspace macrophages in COPD do not express either M1 or M2 markers and that these "non-polarized" macrophages have significantly reduced phagocytic capacity compared to polarized (M1 or M2) macrophages [30]. In this study, airspace macrophages could be divided into 4 distinct groups using surface markers, as either M1, M2, dual positive for M1 & M2 (double polarized) or negative for both M1 & M2 markers (non-polarized). Using the phagocytosis of opsonized *Staphylococcus aureus* as a readout, we showed that the double polarized macrophages had the best phagocytic function while the non-polarized macrophages had the worst (**Figure 5**). These data highlight the importance of macrophage micro-environment that impacts polarization and ultimately function.

The inability of macrophages to polarize may render the airways in COPD more vulnerable for colonization with pathogens and subsequent infection resulting in COPD exacerbation. The presence of large numbers of non-polarized macrophages in COPD may collectively reduce phagocytosis of pathogens and noxious particles, and in certain cases promote a pro-inflammatory milieu that contributes to airway and lung tissue injury and remodeling.

The air spaces have their own unique microbiome shown by next-generation sequencing technologies, such as 16 s RNA gene measurement, and studies in COPD cohorts have shown alterations in this microbiome that vary with the severity of COPD, during and after an acute COPD exacerbations, and with the use of inhaled steroids and/or antimicrobial treatment [64]. Alterations in the lung microbiome may contribute to the pathogenesis of COPD by impacting inflammatory and/or immune processes in the lungs. Lung macrophages play a central role in clearing harmful bacteria such as *Haemophilus influenzae, Moraxella catarrhalis* and *S. pneumoniae,* from the lungs, and this macrophage function deteriorates as the disease progresses

#### **Figure 5.**

*Phagocytic activity of airspace macrophages, harvested from bronchial alveolar lavage, in subjects with COPD. CD40 was used to label M1 and CD163 to label M2 macrophages. The phagocytosis of flourescentlabelled StapholococcusAureas was measured using flow cytometry. Phagocytosis was reduced in COPD subjects compared to control subjects in all the different macrophage populations but was particular lowin the most abundant non-labeled or double negative cells.*

[63] leading to colonization of airspaces and exacerbations of COPD [65]. Therefore, the defective phagocytic function of macrophages in COPD could contribute to the colonization of the airways with various bacteria, specifically those known to cause acute exacerbations and pneumonia during COPD.

One of the key functions of lung macrophages is to remove and clear cellular debris as well as dead or damage cells following an inflammatory insult to the lungs. This process, which is termed efferocytosis, is defective in subjects with COPD [66]. In most subjects with COPD, there are an access of neutrophils in the airspaces (as measured by bronchial alveolar lavage), which further increase during acute COPD exacerbations. Defective clearance of these recruited neutrophils results in the accumulation of necrotic neutrophils that indiscriminately release toxic granule proteins containing neutrophil elastase and proteases that has been associated with tissue damage and COPD progression [67]. Since LMs are the primary "janitors" of the lungs, dysfunctional processing and clearance of apoptotic and necrotic cells and cellular debris could contribute to ongoing lung tissue inflammation in subjects with COPD, even long after they stop smoking [68].

Lung macrophages are primary responsible for processing and removing of inhaled irritants and particulate matter from the lungs. In this process they release proinflammatory mediators that could also inflict damage to lung tissues, promoting a dysregulated inflammatory response, which may lead to dysfunctional tissue repair and a persistent state of chronic low-grade lung inflammation, a hallmark of COPD. Studies that unravel the mechanisms promoting macrophage anti-inflammatory and reparative functions could contribute to the development of more targeted therapeutic interventions to reduce the destructive inflammatory response induced by cigarette smoke and environmental exposures that eventually lead to COPD.

*Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

#### **5. Macrophages and lung cancer**

The majority of lung cancers (~80%) are diagnosed at an advanced stage with >50% in older subjects who are ineligible for surgery [69, 70] leaving chemotherapy as their primary treatment modality. A better understanding of tumor immunology and our body's natural immune response to combat cancer over the last two decades have highlighted the key role macrophages play in containing the progression and metastasis of tumor cells. The tumor microenvironment (TME), characterized by low levels of nutrients, hypoxia and acidity, promotes tumor growth, invasion and metastasis [71]. The most abundant immune cells in or surrounding lung tumors are "tumor-associated macrophages" (TAMs), and the functions of these macrophages are determined by the TME [72].

The tumor microenvironment recruits both innate and adaptive immune cells to the tumor site, with macrophages abundant at all stages of tumorigenesis. Evidence suggests that TAMs originate predominantly from blood monocytes, and are recruited to tumor sites by tumor-derived chemotactic signals, including monocyte chemo-attractant protein-1 (MCP-1), which is also known as CCL 2 [73]. Initially these macrophages have an M1-like phenotype, activated by interferon-γ (IFN-γ), demonstrating pro-inflammatory functions with the capacity to facilitate tumor cell destruction. They are also characterized by a high production of nitric oxide (NO) and reactive oxygen intermediates (ROI), and pro-inflammatory cytokines, including TNF-α, IL- 1, IL- 12 and IL- 23 and MHC molecules [74]. These mediators recruit cytotoxic CD8+ T and NK cells that destroy the tumor cells [72] (**Figure 6**).

With tumor progression the TME changes to a milieu that converts macrophages to a more M2-like phenotype macrophages, which suppress anti-tumor immune responses. This in turn promotes cell proliferation, angiogenesis and ultimately

#### **Figure 6.**

*The effects of tumor associated macrophages (TAMs) on tumor growth, progression and metastasis. The immune response elicited by lung cancer cells included recruitment of monocytes from the blood (via CCL2/ CCR2 interaction) that convert to macrophages and are recruited tothe tumor niche where they become either M1 type TAMs under the influence of mediators such as GM-CSF and INF-λand inhibit tumor growth via secreting mediators such as TNF-α, IL-1β, IL-12 and 23. Mediators such as IL-10, IL-4 and M-CSF will change the macrophages to a more M2 type TAMs that promote tumor growth via immuno-suppressive properties that include blocking NK-cell and other T-cells tumorcidaleffects. The M2-TAMsenvironment will also promote angiogenesis via mediators such as VEGF and PDGF and tumor invasion and metastasis via mediators such as MMPs and TGF-B. other mediators in the tumor environment produced by M2 macropahagessuch as IL-6, TGF-βand MMPs have also been shown to elicit chemotherapy resistance of tumors.*

metastasis. Damage-associated molecular patterns (DAMPs) from dead or dying cells in the tumor microenvironment promote polarization of macrophages to immunosuppressive TAM [75]. These M2-like TAM have a similar phenotype as LPS-tolerant macrophages and is thought to contribute to the immunosuppression in the tumor microenvironments. They express a variety of mediators that inhibit the host antitumor immune responses. These include cell surface receptors, cytokines, chemokines and a variety of enzymes. This anti-tumor immune response is via inhibition of direct cell-to-cell contact between TAM receptors and their ligand counterpart death/ inhibitory receptors expressed by the target immune effector cells. For example, TAMs express the ligand receptors for PD-1 and CTLA-4 that upon activation suppress cytotoxic functions of T- cell and NK cells. They also express the ligand for the death receptors FAS and TRAIL that triggers T-cells and induce caspase dependent apoptosis of tumor cells. The TAMs produce TGF-β that impedes the cytotoxicity of NK cells, and promotes expression of PD-L1 that impedes the anti-tumor activity of T cells [76]. They also secrete cytokines IL-10 that inhibit T cells effector functions and chemokines such as CCL5, CCL20, CCL22 that recruit Treg cells that are immunosuppressive.

The density of macrophages, in particular M2 phenotypes, has been associated with a poor prognosis in almost all human cancer types including lung cancers in clinical trials [77]. The CD68+ CD163+ or CD68+ CD206+ markers on TAM are used to identify M2-like macrophages and these macrophages are associated with more dense peritumoral lymphatic microvessels, a pathological feature that relate to poor patients' prognoses in subjects with lung cancer [78]. Furthermore, an increased density of CD68<sup>+</sup> CD163+ macrophages in tumor nests and stroma was associated with lymph node metastases [78] and Cao *et al* showed expression levels of CD 68+ CD163+ on M2 macrophages were inversely correlated with overall survival, and disease free survival in non-small cell lung cancer (NSCLC) [77].

The overwhelming evidence that TAMs and especially M2 macrophages promote tumorigenesis has made TAMs a target for a novel anti-tumor strategy in lung cancer. Several strategies that have been explored include blockade of the CCL2-CCR 2 interaction and the CSF1-CSF1R recruitment of monocyte pathways that decrease TAM infiltration, thereby reversing their immunosuppressive effects [73]. Mu and co-workers have suggested that reprogramming TAM macrophages can be a promising approach to address immunosuppressive failure in the cancer environment [79]. Re-educating TAMs to a M1 phenotype or switching M2 to M1 macrophages with several drugs has also shown promise including the use of BTH1677 (a yeast β-glucan immunomodulator), hydroxychloroquine, and celecoxib [80, 81]. Another approach is to block the levels of critical TAM-secreted cytokines involved in tumor biology such as CCL18, CCL 22, and MIP-3α, which are mainly produced by M2-type macrophages, and promote malignant behavior of tumors [82, 83]. Furthermore, nanoparticles or nanoparticle-based drug delivery are more reliable and effective in regulating the macrophage phenotype by ensuring that the drug reaches the cancer site without off-target activities [84, 85]. In addition, materials used in nanoparticle production, including TiO2 and Ag, may preferentially polarize TAMs towards an M1 phenotype [86, 87].

#### **6. Macrophages and their role in asthma**

Asthma is characterized by chronically inflamed airways, leading to remodeling and constriction in response to a wide variety of stimuli. One of the prototypical

#### *Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

traits of asthma is airway hyperresponsiveness. Typical triggers of bronchoconstriction are aero-allergens and viral pathogens but non-specific stimuli such as irritating chemicals, cold air, and exercise (increase flow) can also trigger this response. Airway inflammation is characterized by increased mucus secretion, thickening of all the components of the airway wall and luminal narrowing, leading to symptoms of shortness of breath, chest tightness, wheezing, and cough [88]. The inflammatory response in the airways is typically type 2 (Th2) in which allergens are detected by pattern recognition receptors (PRRs) on epithelial cells, which, upon activation, secrete alarmins such as interleukin (IL)-33, IL-25 and thymic stromal lymphopoietin (TSLP) and cytokines such as GM-CSF. These mediators induce type 2 inflammation by activating dendritic cells (DCs) and type 2 innate lymphoid cells (ILC2s) and differentiating naïve T cells into T helper (Th) 2 cells, which produce IL-4, IL-5, and IL-13 [88, 89]. These type 2 cytokines are involved in producing IgE and recruiting eosinophils into the airways. A classic type 2 inflammatory response can be suppressed by treatment with corticosteroids. In adults a substantial subset of patients with non-atopic asthma may be driven by a Th1 response, which is characterized by infiltration of neutrophils. Th1 inflammation is more difficult to treat, as it is often resistant to corticosteroids [90].

Macrophages are the most abundant leukocytes found in alveoli, and in small as well as conducting airways, suggesting that they have an important role in providing protection against foreign inhaled particulate matter including allergens, pathogens and noxious gasses. Links between lung macrophages and airway inflammation, including eosinophilic inflammation and airway remodeling are well documented in asthma [91–95]. However, it is not still unclear whether macrophages have a predominant pro-inflammatory or regulatory role in asthma.

As discussed previously, macrophages have the ability to adapt to their microenvironment (plasticity). In asthmatic lung inflammation this plasticity of macrophage function is most likely responsible for their apparent dual or contrasting roles as pro-inflammatory versus immunosuppressive effector immune cells. Zaslona and co-workers showed that during allergic inflammation, resident alveolar macrophages proliferate locally and exert a protective effect on allergic inflammation, whereas recruited monocytes/macrophages aggravate allergic inflammation [96]. These recruited monocytes are also involved in the characteristic chronic remodeling of airways [97]. When circulating monocytes were depleted by intravenous injection of clodronate, there was significant attenuation of allergic inflammation in the airways and when clodronate was administered via the intratracheal route to deplete resident airspace macrophages, eosinophilic inflammation was enhanced [97]. Collectively, these data suggest that resident macrophages serve to maintain lung homeostasis by suppressing inflammatory responses, while recruited monocytes primarily promote allergic inflammation. The picture that emerges is one of rapid recruitment of monocytes to fight the perceived dangers of the allergen by mounting an inflammatory response and then subsequently expanding the pool of suppressive AMs in an attempt to restore homeostasis (**Figure 7**). The dominant macrophages in allergic asthma are alternatively activated AMs, which respond to IL-4/IL-13. Although the presence of these macrophages correlates well with the severity of airway inflammation, it is not clear whether these macrophages significantly contribute to the allergic inflammatory response or are the downstream consequence of the allergic inflammation [98–100]. This issue has been addressed in several experimental studies using transgenic murine models of asthma [101–103]. Together, these studies have shown distinct differences between resident and recruited macrophages in contributing to asthmatic

#### **Figure 7.**

*After allergen exposure, there is rapid recruitment of monocytes from the blood that become inflammatory macrophages that predominantly promote acute inflammatory responses in the airspace. Resident macrophages (ResMac), which are largely self-replicating, act to suppress the acute inflammation in an attempt torestore homeostasis. It is as yetunclear whether ResMaccan arise directly from recruited monocytes or from IMs as intermediate progenitors. These newly recruited monocyte-derived cells appear to oppose the suppressive actions of resident AMs.* **b** *inflammation becomes chronic after repeated exposures to allergen with the increased recruitment of immune cells and consequent elevated levels of cytokines such as IL-4, IL-13, and IFNγ. In response to these signals, ResMaccan polarize across a continuum of activation phenotypes, losing their suppressive functions and gaining pathogenic functions. IL-4/IL-13-induced AMs promote type 2/eosinophilic inflammation, and IFNγinduced AMs are associated with type I/neutrophilic inflammation. It is as yetunclear whether these activated phenotypes can arise from recruited monocytes directly or from IMs as an intermediate progenitor.*

inflammation. However, as there may be significant differences between mice and humans, these findings should be interpreted cautiously. Recent studies have shown that there is a mixed population of macrophage phenotypes in both human and mouse models of allergic inflammation [101–103] with functional studies showing that IFNγstimulated macrophages (M1) prevent the development of allergic inflammation in mice by suppressing DC maturation [104]. However in human studies of subjects with established asthma, there is a higher number of macrophages expressing the IFNγ-activated transcription factor, interferon regulatory factor 5 (IRF5), in airways which in turn correlates with the severity of airflow obstruction [102]. These studies highlight the potential dual role of classically activated macrophages (M1) in asthma.

The presence of type 1 cytokines, such as IL-6, would be expected during host responses to viruses and to a variety of exogenous and endogenous ligands that trigger asthma exacerbations [88, 105]. Infections with common respiratory viruses such as rhinovirus are a major trigger of asthma exacerbations and in these virus-induced exacerbations, there is direct interaction of the rhinovirus with airspace macrophages [105]. Asthma models in mice, support the role of macrophages in viral induced exacerbations, but interestingly, the pathogenic macrophage phenotype involved varies with the underlying inflammatory milieu. In mice with a predominant type 2 inflammatory response, viral infection induces activation of alternative AMs, which amplifies eosinophil recruitment into the airways. However, mice with predominantly type 1 inflammation demonstrate mostly classical activation of AMs, skewing the phenotype to a more neutrophilic inflammation upon virus infection [106].

The role of lung macrophages in allergic asthma is still evolving. Current paradigms suggest that macrophages of different origins or phenotypes have the potential to be either protective and/or harmful in different stages of allergic airway disease. In asthma, macrophages may have a dual role: with induction of the allergic

*Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

inflammatory response they may be predominantly regulatory to resolve the inflammation but when persistently activated, they may contribute to chronic inflammation and further damage of the airways (**Figure 7**). The role of resident and recruited macrophages as well as the macrophage phenotypes in the pathogenesis of allergic airways disease requires additional studies.

#### **7. Macrophages in interstitial lung disease**

Chronic and aberrant lung repair responses that lead to irreversible scarring and remodeling of the airways and lung parenchyma are hallmarks of pulmonary fibrotic diseases. These diseases are characterized by excessive deposition of ECM leading to fibrotic remodeling of lungs and irreversible lung dysfunction [107]. Alveolar macrophages have been shown to be involved in ECM processing by secreting matrix metalloproteinases (MMPs) such as MMP9, a type IV collagenase known to degrade extracellular matrix, and numerous non-matrix protein, which have been demonstrated in a murine model of lung fibrosis induced by bleomycin [108, 109] These macrophages also endocytose collagen and produce soluble mediators required for collagen-degradation, and enhance the activity of fibroblast-specific protein-1 (FSP-1) that increases the proliferation and production of ECM by lung fibroblasts [110]. Recent animal studies have shown that the AMs involved in the bleomycin murine lung fibrosis model are monocyte derived and not resident AMs. Depletion of resident AMs by intratracheal instillation of liposomal clodronate before bleomycin administration does not alter the fibrotic response, indicating that resident AMs are dispensable for the development of fibrosis [111]. To support this concept, deletion of the anti-apoptotic protein c-Flip in circulating monocytes (the precursors of monocyte-derived AMs) in mice showed that the number of monocyte-derived AMs decreased, which was accompanied by a reduction in lung fibrosis with bleomycin injury [112] Gene expression studies of resident AMs and monocyte-derived AMs also indicate that only monocyte-derived AMs have a profibrotic gene profile in bleomycin induced model of lung fibrosis [112] Single cell transcriptomic studies support these findings [113]. Transgenic reporter mice that marked the Cx3cr1-expressing transitional macrophages showed that these macrophages localize to fibrotic niches suggesting that these transitional macrophages arise from monocytes and interact with fibroblasts to drive fibrosis, in concordance to studies of Misharin et al. and McCubbrey et al. [111, 112].

In human interstitial lung diseases, AMs show greater heterogeneity compared with healthy lungs. In subjects with idiopathic pulmonary fibrosis, lung tissues have a higher proportions of AMs lacking CD71, a transferrin receptor. These CD71 negative macrophages were also more immature and showed impaired phagocytosis and enhanced expression of profibrotic genes [114]. Single cell RNA sequencing analysis from eight normal compared to eight lungs from advanced lung fibrosis subjects (from patients undergoing lung transplantation) supports the idea of increased heterogeneity of macrophages in fibrotic lungs and subsets of AMs that were enriched with pro-fibrotic genes [115]. Together these studies suggest lung macrophages contribute significantly to the pathogenesis of interstitial fibrotic lung disease. However, it is still unclear whether these macrophages are from the resident pool or newly recruited into the lung, what signals attract these macrophages or whether they are just secondary to the change in microenvironment, and lastly, what their contribution is to progression of the disease [116].

Interstitial macrophages (IMs) are ideally positioned to participate in the lung fibrotic processes. In a radiation-induced lung fibrosis (RIF) mouse models, IMs have acquired a pro-fibrotic phenotype, and express high levels of CD206, a marker of alternatively activated M2 macrophages [117]. Interstitial macrophages isolated from RIF lungs promote fibrosis by inducing the differentiation myocytes to myofibroblasts. Myofibroblasts have been shown to be key players in the initiation and progression of lung fibrosis [118]. Depletion of IMs in the RIF mouse model with CSF1-R specific mAb exerts an anti-fibrotic effect, while the depletion of AMs by intranasal administration of clodronate liposomes has no effects on RIF [117]. Recent studies showed that lung fibrosis was exacerbated after depletion of Lyve1hiMHCIIlo IM1s during the induction of fibrosis, which suggested that these IM1 might have an early antifibrotic role. This is in keeping with expression of high levels of genes associated with wound healing, repair, and fibrosis in this subset of IMs [20].

#### **8. Conclusion**

It is clear that lung macrophages have an essential role in both lung homeostasis and in disease states. Here we have highlighted the specific origins, unique phenotypes and functions of the two main populations of lung macrophages, AMs and IMs, and emphasized the distinct roles in common lung diseases. It is still not clear if lung macrophages derived from circulating monocytes eventually become indistinguishable from embryonically derived resident AMs in chronic lung diseases or if they are long lived in the lungs with a slightly different genomic and functional profile thereby changing the lung macrophage landscape. There is still a lack of knowledge of IMs in terms of how they are maintained and their importance in lung conditions. With the increasing power of phenotyping and genomic techniques, there will be opportunities to better characterize the origin, subtypes and functions of AMs and IMs in both health and disease. There is a pressing need to focus strongly on macrophage function, especially in regards to mechanisms of their role in inflammatory and antiinflammatory pathways, their turnover and survival during the immune response, and their interactions with recruited macrophages in promoting wound healing. Refining our understanding of macrophage plasticity and the role of distinct populations of macrophages in various pulmonary diseases will lead to the identification of novel macrophage-targeted therapies.

#### **Acknowledgements**

This work was grant supported by Canadian Institute of Health Research, the British Columbia Lung Association, and the Providence Airway Centre. Stephan F. van Eeden is the Canadian Institute for Health Research/Glaxo Smith Kline (CIHR/ GSK) Professor in Chronic Obstructive Pulmonary Disease and Don D. Sin holds a Tier 1 Canada Research Chair in COPD and the HLI De Lazzari Family Chair.

*Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

#### **Author details**

Stephan F. van Eeden1,2\* and Don D. Sin1,2

1 Department of Medicine (Respirology), University of British Columbia, Vancouver, BC, Canada

2 Centre for Heart Lung Innovation, St. Paul's Hospital, Vancouver, BC, Canada

\*Address all correspondence to: stephan.vaneeden@hli.ubc.ca

© 2022 The Author(s). Licensee IntechOpen. 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.

## **References**

[1] Wynn TA, Chawla A, Pollard JW. Origins and hallmarks of macrophages: Development, homeostasis, and disease. Nature. 2013;**496**:445-455

[2] Epelman S, Lavine KJ, Randolph GJ. Origin and functions of tissue macrophages. Immunity. 2014;**41**:21-35

[3] Shi T, Denney L, An H, Ho L-P, Zheng Y. Alveolar and lung interstitial macrophages: Definitions, functions, and roles in lung fibrosis. Journal of Leukocyte Biology. 2021;**110**:107-114

[4] Lehnert BE, Valdez YE, Sebring RJ, Lehnert NM, Saunders GC, Steinkamp JA. Airway intraluminal macrophages: Evidence of origin and comparisons to alveolar macrophages. American Journal of Respiratory Cell and Molecular Biology. 1990;**3**:377-391

[5] Warner AE, Molina RM, Brain JD. Uptake of bloodborne bacteria by pulmonary intravascular macrophages and consequent inflammatory responses in sheep. The American Review of Respiratory Disease. 1987;**136**:683-690

[6] Dehring DJ, Wismar BL. Intravascular macrophages in pulmonary capillaries of humans. The American Review of Respiratory Disease. 1989;**139**:1027-1029

[7] van Furth R, Cohn ZA. The origin and kinetics of mononuclear phagocytes. The Journal of Experimental Medicine. 1968;**128**:415-435

[8] Landsman L, Jung S. Lung macrophages serve as obligatory intermediate between blood monocytes and alveolar macrophages. Journal of Immunology. 2007;**179**:3488-3494

[9] Guilliams M, De Kleer I, Henri S, Post S, Vanhoutte L, De Prijck S, et al. Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF. The Journal of Experimental Medicine. 2013;**210**:1977-1992

[10] Hashimoto D, Chow A, Noizat C, Teo P, Beasley MB, Leboeuf M, et al. Tissue resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity. 2013;**38**:792-804

[11] Hoeffel G, Wang Y, Greter M, See P, Teo P, Malleret B, et al. Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages. The Journal of Experimental Medicine. 2012;**209**:1167-1181

[12] Gomez Perdiguero E, Klapproth K, Schulz C, Busch K, Azzoni E, Crozet L, et al. Tissue-resident macrophages originate from yolksac- derived erythro-myeloid progenitors. Nature. 2015;**518**(7540):547-551

[13] Aggarwal NR, King LS, D'Alessio FR. Diverse macrophage populations mediate acute lung inflammation and resolution. American journal of Physiology, Lung Cellular and Molecular Physiology. 2014;**306**(8):L709-L725

[14] Cai Y, Sugimoto C, Arainga M, Alvarez-Hernandez X, Didier ES, Kuroda MJ. In vivo characterization of alveolar and interstitial lung macrophages in rhesus macaques: Implications for understanding lung disease in humans. Journal of Immunology. 2014;**192**:2821-2829

[15] Goto Y, Hogg JC, Whalen B, Shih CH, Ishii H, Van Eeden SF.

*Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

Monocyte recruitment into the lungs in pneumococcal pneumonia. American Journal of Respiratory Cell and Molecular Biology. 2004;**30**(5):620-626. DOI: 10.1165/rcmb.2003-0312OC

[16] Schneider C, Nobs SP, Kurrer M, et al. Induction of the nuclear receptor PPAR- gamma by the cytokine GM-CSFis critical for the differentiation of fetal monocytes into alveolar macrophages. Nature Immunology. 2014;**15**:1026-1037

[17] Gourbal B, Pinaud S, Beckers GJM, et al. Innate immune memory: An evolutionary perspective. Immunological Reviews. 2018;**283**:21-40

[18] Yao Y, Jeyanathan M, Haddadi S, et al. Induction of autonomous memory alveolar macrophages requires T cell help and is critical to trained immunity. Cell. 2018;**175**:1634-1650

[19] Gibbings SL, Thomas SM, Atif SM, et al. Three unique interstitial macrophages in the murine lung at steady state. American Journal of Respiratory Cell and Molecular Biology. 2017;**57**:66-76

[20] Chakarov S, Lim HY, Tan L, et al. Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches. Science. 2019;**363**:eaau0964

[21] Lambrechts D, Wauters E, Boeckx B, et al. Phenotype molding of stromal cells in the lung tumor microenvironment. Nature Medicine. 2018;**24**:1277-1289

[22] Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: Cancer as a paradigm. Nature Immunology. 2010;**11**:889-896

[23] Biswas SK, Gangi L, Paul S, Schioppa T, Saccani A, Sironi M, et al. A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-kappaB and enhanced IR F-3/STAT1 activation). Blood. 2006;**107**:2112-2122

[24] Nelson MP, Christmann BS, Dunaway CW, Morris A, Steele C. Experimental pneumocystis lung infection promotes M2a alveolar macrophage-derived MMP12 production. American Journal of Physiology. Lung Cellular and Molecular Physiology. 2012;**303**:L469-L475

[25] Zhang W, Xu W, Xiong S. Blockade of Notch1 signaling alleviates murine lupus via blunting macrophage activation and M2b polarization. Journal of Immunology. 2010;**184**:6465-6478

[26] Koscsó B, Csóka B, Kókai E, Németh ZH, Pacher P, Virág L, et al. Adenosine augments IL-10-induced STAT3 signaling in M2c macrophages. Journal of Leukocyte Biology. 2013;**94**:1309-1315

[27] Olmes G, Buttner-Herold M, Ferrazzi F, Distel L, Amann K, Daniel C. CD 163+ M2c-like macrophages predominate in renal biopsies from patients with lupus nephritis. Arthritis Research & Therapy. 2016;**18**:90

[28] Wang Q, Ni H, Lan L, Wei X, Xiang R, Wang Y. Fra-1 protooncogene regulates IL-6 expression in macrophages and promotes the generation of M2d macrophages. Cell Research. 2010;**20**:701-712

[29] Helm O, Held-Feindt J, Grage-Griebenow E, Reiling N, Ungefroren H, Vogel I, et al. Tumor-associated macrophages exhibit pro- and antiinflammatory properties by which they impact on pancreatic tumorigenesis. International Journal of Cancer. 2014;**135**:843-861

[30] Akata K, Yamasaki K, Filho FSL, Yang CX, Takiguchi H, Sahin B, et al. Abundance of non-polarized lung macrophages with poor phagocytic function in chronic obstructive pulmonary disease (COPD). Biomedicine. 2020;**8**(10):398. DOI: 10.3390/biomedicines 8100398

[31] Baßler K, Fujii W, Kapellos TS, Horne A, Reiz B, Dudkin E, et al. Alterations of multiple alveolar macrophage states in chronic obstructive pulmonary disease. bioRxiv. **2020**. DOI: 10.1101/2020.05.28.121541

[32] Naessens T et al. Innate imprinting of murine resident alveolar macrophages by allergic bronchial inflammation causes a switch from hypo-inflammatory to hyper-inflammatory reactivity. The American Journal of Pathology. 2012;**181**:174-184

[33] Kumagai Y et al. Alveolar macrophages are the primary interferon-α producer in pulmonary infection with RNA viruses. Immunity. 2007;**27**:240-252

[34] Guarda G et al. Type I interferon inhibits interleukin-1 production and inflammasome activation. Immunity. 2011;**34**:213-223

[35] Aegerter H et al. Influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection. Nature Immunology. 2020;**21**(2):145-157. DOI: 10.1038/ s41590-019-0568-x

[36] Bost P et al. Host–viral infection maps reveal signatures of severe COVID-19 patients. Cell. 2020;**181**:1475-1488.e12

[37] Gao T et al. Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation. medRxiv. 2020. DOI: 10.1101/2020.03.29.20041962

[38] Carvelli J et al. Association of COVID-19 inflammation with activation of the C5a–C5aR1 axis. Nature. 2020;**588**:146-150

[39] Knapp S et al. Alveolar macrophages have a protective anti-inflammatory role during murine pneumococcal pneumonia. American Journal of Respiratory and Critical Care Medicine. 2003;**167**:171-179

[40] Cohen SB et al. Alveolar macrophages provide an early *Mycobacterium tuberculosis* niche and initiate dissemination. Cell Host & Microbe. 2018;**24**:439-446. e4

[41] Misharin AV et al. Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. The Journal of Experimental Medicine. 2017;**214**:2387-2404

[42] Machiels B et al. A gammaherpesvirus provides protection against allergic asthma by inducing the replacement of resident alveolar macrophages with regulatory monocytes. Nature Immunology. 2017;**18**:1310-1320

[43] Ghoneim HE, Thomas PG, McCullers JA. Depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections. Journal of Immunology. 2013;**191**:1250-1259

[44] Yao Y et al. Induction of autonomous memory alveolar macrophages requires T cell help and is critical to trained immunity. Cell. 2018;**175**:1634-1650. e17

[45] Roquilly A et al. Alveolar macrophages are epigenetically altered *Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

after inflammation, leading to longterm lung immune-paralysis. Nature Immunology. 2020;**21**:636-648

[46] Goulding J et al. Respiratory infections: Do we ever recover? Proceedings of the American Thoracic Society. 2007;**4**:618-625

[47] Blériot C, Chakarov S, Ginhoux F. Determinants of resident tissue macrophage identity and function. Immunity. 2020;**52**:957-970

[48] Kulikauskaite J, Wack A. Teaching old dogs new tricks? The plasticity of lung alveolar macrophage subsets. Trends in Immunology. 2020;**41**:864-877

[49] Eisner MD, Anthonisen N, Coultas D, Kuenzli N, Perez-Padilla R, Postma D, et al. An official American Thoracic Society public policy statement: Novel risk factors and the global burden of chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine. 2010;**182**(5):693-718

[50] Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. The New England Journal of Medicine. **2004**;**350**:2645-2653

[51] Hogg JC, Paré PD, Hackett TL. The contribution of small airway obstruction to the pathogenesis of chronic obstructive pulmonary disease. Physiological Reviews. 2017;**97**(2):529-552

[52] Ando M, Sugimoto M, Nishi R, Suga M, Horio S, Kohrogi H, et al. Surface morphology and function of human pulmonary alveolar macrophages from smokers and non-smokers. Thorax. 1984;**39**:850-856

[53] Finkelstein R, Fraser RS, Ghezzo H, Cosio MG. Alveolar inflammation and its relation to emphysema in smokers. American Journal of Respiratory and Critical Care Medicine. 1995;**152**:1666-1672

[54] Barnes PJ. Alveolar macrophages as orchestrators of COPD. COPD: Journal of Chronic Obstructive Pulmonary Disease. 2004;**1**:59-70

[55] Russell RE, Culpitt SV, DeMatos C, Donnelly L, Smith M, Wiggins J, et al. Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease. American Journal of Respiratory Cell and Molecular Biology. 2002;**26**(5):602-609

[56] Nakajima T, Nakamura H, Owen CA, Yoshida S, Tsuduki K, Chubachi S, et al. Plasma cathepsin S and cathepsin S/cystatin C ratios are potential biomarkers for COPD. Disease Markers. 2016;**2016**:4093870

[57] Donnelly LE, Barnes PJ. Defective phagocytosis in airways disease. Chest. 2012;**141**(4):1055-1062

[58] Zakharkina T, Koczulla AR, Mardanova O, Hattesohl A, Bals R. Detection of microorganisms in exhaled breath condensate during acute exacerbations of COPD. Respirology. 2011;**16**(6):932-938

[59] Eurlings IM, Dentener MA, Mercken EM, et al. A comparative study of matrix remodeling in chronic models for COPD; mechanistic insights into the role of TNF-α. American Journal of Physiology. Lung Cellular and Molecular Physiology. 2014;**307**(7):557-565

[60] Stout RD, Suttles J. Functional plasticity of macrophages:

Reversible adaptation to changing microenvironments. Journal of Leukocyte Biology. 2004;**76**(3):509-513

[61] Stewart JI, Criner GJ. The small airways in chronic obstructive pulmonary disease: Pathology and effects on disease progression and survival. Current Opinion in Pulmonary Medicine. 2013;**19**(2):109-115

[62] Dewhurst JA, Lea S, Hardaker E, et al. Characterization of lung macrophage subpopulations in COPD patients and controls. Scientific Reports. 2017;**7**(1):7143

[63] Berenson CS, Kruzel RL, Eberhardt E, et al. Phagocytic dysfunction of human alveolar macrophages and severity of chronic obstructive pulmonary disease. The Journal of Infectious Diseases. 2013;**208**(12):2036-2045

[64] Mammen MJ, Sethi S. COPD and the microbiome. Respirology. 2016;**21**:590-599

[65] Naito K, Yamasaki K, Yatera K, Akata K, Noguchi S, Kawanami T, et al. Bacteriological incidence in pneumonia patients with pulmonary emphysema: A bacterial floral analysis using the 16S ribosomal RNA gene in bronchoalveolar lavage fluid. International Journal of Chronic Obstructive Pulmonary Disease. 2017;**12**:2111-2120

[66] Kirkham PA, Spooner G, Rahman I, Rossi AG. Macrophage phagocytosis of apoptotic neutrophils is compromised by matrix proteins modified by cigarette smoke and lipid peroxidation products. Biochemical and Biophysical Research Communications. 2004;**318**:32-37

[67] Pandey KC, De S, Mishra PK. Role of proteases in chronic obstructive pulmonary disease. Frontiers in Pharmacology. 2017;**8**:512

[68] Hodge S, Hodge G, Holmes M, Reynolds PN. Increased airway epithelial and T-cell apoptosis in COPD remains despite smoking cessation. The European Respiratory Journal. 2005;**25**:447-454

[69] American Cancer Society. Cancer facts and figures. 2015. Available from: http://www.cancer. org/research/cancerfactsstatistics/ cancerfactsfigures2015/ [Accessed: July 13, 2015]

[70] Gridelli C, Perrone F, Monfardini S. Lung cancer in the elderly. European Journal of Cancer. 1997;**33**:2313-2314

[71] Goswami KK, Ghosh T, Ghosh S, Sarkar M, Bose A, Baral R. Tumor promoting role of anti-tumor macrophages in tumor microenvironment. Cellular Immunology. 2017;**316**:1-10

[72] Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nature Reviews. Immunology. 2008;**8**:958-969

[73] Li X, Yao W, Yuan Y, Chen P, Li B, Li J, et al. Targeting of tumourinfiltrating macrophages via CCL 2/CCR 2 signalling as a therapeutic strategy against hepatocellular carcinoma. Gut. 2017;**66**:157-167

[74] Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: Cancer as a paradigm. Nature Immunology. 2010;**11**:889-896

[75] Ostuni R, Kratochvill F, Murray PJ, Natoli G. Macrophages and cancer: From mechanisms to therapeutic implications. Trends in Immunology. 2015;**36**:229-239. DOI: 10.1016/j.it.2015.02.004

[76] Sumitomo R, Hirai T, Fujita M, Murakami H, Otake Y, Huang CL.

*Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

PD-L1 expression on tumor-infiltrating immune cells is highly associated with M2 TAM and aggressive malignant potential in patients with resected non-small cell lung cancer. Lung Cancer. 2019;**136**:136-144

[77] Cao L, Che X, Qiu X, Li Z, Yang B, Wang S, et al. M2 macrophage infiltration into tumor islets leads to poor prognosis in non-small-cell lung cancer. Cancer Management and Research. 2019;**11**:6125-6138

[78] Zhang B, Yao G, Zhang Y, Gao J, Yang B, Rao Z, et al. M2-polarized tumor-associated macrophages are associated with poor prognoses resulting from accelerated lymphangiogenesis in lung adenocarcinoma. Clinics (São Paulo, Brazil). 2011;**66**:1879-1886

[79] Mu Q, Najafi M. Modulation of the tumor microenvironment (TME) by melatonin. European Journal of Pharmacology. 2021;**907**:174365

[80] Li Y, Cao F, Li M, Li P, Yu Y, Xiang L, et al. Hydroxychloroquine induced lung cancer suppression by enhancing chemo-sensitization and promoting the transition of M2-TAMs to M1-like macrophages. Journal of Experimental & Clinical Cancer Research. 2018;**37**:259

[81] Brandão RD, Veeck J, Van de Vijver KK, Lindsey P, de Vries B, van Elssen CH, et al. A randomised controlled phase II trial of pre-operative celecoxib treatment reveals anti-tumour transcriptional response in primary breast cancer. Breast Cancer Research. 2013;**15**:R29

[82] Zhu B, Zou L, Cheng X, Lin Z, Duan Y, Wu Y, et al. Administration of MIP-3alpha gene to the tumor following radiation therapy boosts anti-tumor immunity in a murine model of lung

carcinoma. Immunology Letters. 2006;**103**:101-107

[83] Zhou Z, Peng Y, Wu X, Meng S, Yu W, Zhao J, et al. CCL 18 secreted from M2 macrophages promotes migration and invasion via the PI3K/Akt pathway in gallbladder cancer. Cellular Oncology (Dordrecht). 2019;**42**:81-92

[84] Han S, Wang W, Wang S, Wang S, Ju R, Pan Z, et al. Multifunctional biomimetic nanoparticles loading baicalin for polarizing tumorassociated macrophages. Nanoscale. 2019;**11**:20206-20220

[85] Cao M, Yan H, Han X, Weng L, Wei Q, Sun X, et al. Ginseng-derived nanoparticles alter macrophage polarization to inhibit melanoma growth. Journal for Immunotherapy of Cancer. 2019;**7**:326

[86] Zhang J, Song W, Guo J, Zhang J, Sun Z, Li L, et al. Cytotoxicity of different sized TiO2 nanoparticles in mouse macrophages. Toxicology and Industrial Health. 2013;**29**:523-533

[87] Park J, Lim DH, Lim HJ, Kwon T, Choi JS, Jeong S, et al. Size dependent macrophage responses and toxicological effects of Ag nanoparticles. Chemical Communications. 2011;**47**:4382-4384

[88] Holgate ST. Pathogenesis of asthma. Clinical and Experimental Allergy. 2008;**38**:872-897

[89] Licona-Limon P, Kim LK, Palm NW, Flavell RA. TH2, allergy and group 2 innate lymphoid cells. Nature Immunology. 2013;**14**:536-542

[90] Peters SP. Asthma phenotypes: Nonallergic (intrinsic) asthma. The Journal of Allergy and Clinical Immunology. In Practice. 2014;**2**:650-652 [91] Arjomandi M et al. Repeated exposure to ozone increases alveolar macrophage recruitment into asthmatic airways. American Journal of Respiratory and Critical Care Medicine. 2005;**172**:427-432

[92] Gordon S. Alternative activation of macrophages. Nature Reviews. Immunology. 2003;**3**:23-35

[93] Leung TF, Wong GW, Ko FW, Lam CW, Fok TF. Increased macrophagederived chemokine in exhaled breath condensate and plasma from children with asthma. Clinical and Experimental Allergy. 2004;**34**:786-791

[94] Mautino G et al. Increased expression of tissue inhibitor of metalloproteinase-1 and loss of correlation with matrix metalloproteinase-9 by macrophages in asthma. Laboratory Investigation. 1999;**79**:39-47

[95] Moon KA et al. Allergen-induced CD11b+ CD11c(int) CCR3+ macrophages in the lung promote eosinophilic airway inflammation in a mouse asthma model. International Immunology. 2007;**19**:1371-1381

[96] Zasłona Z, Przybranowski S, Wilke C, van Rooijen N, Teitz-Tennenbaum S, Osterholzer JJ, et al. Resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in murine models of asthma. Journal of Immunology. 2014;**193**:4245-4253

[97] Lee YG, Jeong JJ, Nyenhuis S, Berdyshev E, Chung S, Ranjan R, et al. Recruited alveolar macrophages, in response to airway epithelial-derived monocyte chemoattractant protein 1/ CCl2, regulate airway inflammation and remodeling in allergic asthma. American Journal of Respiratory Cell and Molecular Biology. 2015;**52**:772-784

[98] Chung S, Lee TJ, Reader BF, Kim JY, Lee YG, Park GY, et al. FoxO1 regulates allergic asthmatic inflammation through regulating polarization of the macrophage inflammatory phenotype. Oncotarget. 2016;**7**:17532-17546

[99] Nieuwenhuizen NE, Kirstein F, Jayakumar J, Emedi B, Hurdayal R, Horsnell WGC, et al. Allergic airway disease is unaffected by the absence of IL-4Rα-dependent alternatively activated macrophages. The Journal of Allergy and Clinical Immunology. 2012;**130**:743-750

[100] Gundra UM, Girgis NM, Ruckerl D, Jenkins S, Ward LN, Kurtz ZD, et al. Alternatively activated macrophages derived from monocytes and tissue macrophages are phenotypically and functionally distinct. Blood. 2014;**123**:e110-e122

[101] Draijer C, Robbe P, Boorsma CE, Hylkema MN, Melgert BN. Characterization of macrophage phenotypes in three murine models of house-dust-mite-induced asthma. Mediators of Inflammation. 2013;**2013**:632049

[102] Draijer C, Boorsma CE, Robbe P, Timens W, Hylkema MN, Ten Hacken NHT, et al. Human asthma is characterized by more IRF5+ M1 and CD206+ M2 macrophages and less IL10+ M2-like macrophages around airways compared to healthy airways. The Journal of Allergy and Clinical Immunology. 2016;**140**(1):280-283

[103] Kim Y-K, Oh S-Y, Jeon SG, Park H-W, Lee S-Y, Chun E-Y, et al. Airway exposure levels of lipopolysaccharide determine type 1 versus type 2 experimental asthma. Journal of Immunology. 2007;**178**:5375-5382

[104] Bedoret D, Wallemacq H, Marichal T, Desmet C, Quesada *Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases DOI: http://dx.doi.org/10.5772/intechopen.102420*

Calvo F, Henry E, et al. Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice. The Journal of Clinical Investigation. 2009;**119**:3723-3738

[105] Karta MR, Wickert LE, Curran CS, Gavala ML, Denlinger LC, Gern JE, et al. Allergen challenge in vivo alters rhinovirus-induced chemokine secretion from human airway macrophages. The Journal of Allergy and Clinical Immunology. 2014;**133**:1227-1230.e4

[106] Hong JY, Chung Y, Steenrod J, Chen Q, Lei J, Comstock AT, et al. Macrophage activation state determines the response to rhinovirus infection in a mouse model of allergic asthma. Respiratory Research. 2014;**15**:63

[107] Chanda D, Otoupalova E, Smith SR, et al. Developmental pathways in the pathogenesis of lung fibrosis. Molecular Aspects of Medicine. 2019;**65**:56-69

[108] Dancer RC, Wood AM, Thickett DR. Metalloproteinases in idiopathic pulmonary fibrosis. The European Respiratory Journal. 2011;**38**:1461-1467

[109] Craig VJ, Zhang L, Hagood JS, et al. Matrix metalloproteinases as therapeutic targets for idiopathic pulmonary fibrosis. American Journal of Respiratory Cell and Molecular Biology. 2015;**53**:585-600

[110] Zhang W, Ohno S, Steer B, et al. S100a4 is secreted by alternatively activated alveolar macrophages and promotes activation of lung fibroblasts in pulmonary fibrosis. Frontiers in Immunology. 2018;**9**:1216

[111] Misharin AV, Morales-Nebreda L, Reyfman PA, et al. Monocyte derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. The Journal of Experimental Medicine. 2017;**214**:2387-2404

[112] McCubbrey AL, Barthel L, Mohning MP, et al. Deletion of c-FLIP from CD11b(hi) macrophages prevents development of bleomycin-induced lung fibrosis. American Journal of Respiratory Cell and Molecular Biology. 2018;**58**:66-78

[113] Aran D, Looney AP, Liu L, et al. Reference-based analysis of lung singlecell sequencing reveals a transitional profibrotic macrophage. Nature Immunology. 2019;**20**:163-172

[114] Allden SJ, Ogger PP, Ghai P, et al. The transferrin receptor CD71 delineates functionally distinct airway macrophage subsets during idiopathic pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine. 2019;**200**:209-219

[115] Reyfman PA, Walter JM, Joshi N, et al. Single-cell transcriptomic analysis of human lung provides insights into the pathobiology of pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine. 2019;**199**:1517-1536

[116] Shi T, Denney L, An H, Ho L-P, Zheng Y. Alveolar and lung interstitial macrophages: Definitions, functions, and roles in lung fibrosis. Journal of Leukocyte Biology. 2021;**110**:107-114

[117] Meziani L, Mondini M, Petit B, et al. CSF1R inhibition prevents radiation pulmonary fibrosis by depletion of interstitial macrophages. The European Respiratory Journal. 2018;**51**:1702120

[118] Wynn TA, Barron L. Macrophages: Master regulators of inflammation and fibrosis. Seminars in Liver Disease. 2010;**30**:245-257

#### **Chapter 5**

## Macrophages in the Smooth Muscle Layers of the Gastrointestinal Tract

*Gianluca Cipriani and Suraj Pullapantula*

#### **Abstract**

Muscularis macrophages are a newly discovered population of immune cells populating the smooth muscle layers of the gastrointestinal tract. Beyond their well-established role in modulating innate immunity, these cells are emerging for their ability to communicate with cells required for gastrointestinal motility. This chapter will describe the factors contributing to muscularis macrophages' phenotype and the functional connections these cells established with different cell types.

**Keywords:** macrophages, gut, enteric neurons, enteric glia, gastrointestinal motility

#### **1. Introduction**

The gut is home to the body's largest population of immune cells [1, 2]. Beyond the frontline defenses against unparalleled exposure to foreign antigens, gut macrophages (MΦ) also constantly communicate with an intricate network of cell types orchestrating gastrointestinal (GI) functions [3, 4]. By now, the plasticity of MΦ in different organs of the body (inter-diversity) and within tissue layers (intra-diversity) has been established. While the M1/M2 dogma has served to advance the field of Immunology and understand MΦ phenotype and function [5, 6], immunologists now generally agree that there is a spectrum of phenotypes between the two classifications [7–9]. This chapter will outline the phenotype and function of a population of MΦ in the GI tract, called muscularis macrophages (MMΦ). As the name states, MMΦ resides in the muscularis layers of the GI tract, called muscularis propria. MMΦ fulfills multiple functions across development, adulthood, and under disease conditions. This chapter will report the factors contributing to MMΦ' phenotype heterogeneity and describe the functional interaction MMΦ establish with cells populating the same environment.

#### **2. Identification of MMΦ: a new population of macrophages**

Mikkelsen and colleagues first identified "macrophage-like" cells in the muscularis propria of the small intestine using combination of electron microscopy and immunohistochemistry [10]. Subsequent studies by the same group confirmed these cells to be MMΦ after endocytosis of FITC-dextran and F4/80 labeling co-labeled. In addition to the identification of MMΦ by immunohistochemistry, electron microscopy analysis revealed distinct morphological features based on location within the muscularis

**Figure 1.** *Different regions of the gastrointestinal tract along with the varying morphologies of MΦ.*

propria. These cells were noted to contain a nucleus, Golgi bodies, smooth and rough endoplasmic reticulum, and enveloped by the processes of interstitial cells of Cajal (ICC). The GI muscularis propria (**Figure 1**) is separated from the external environment by the mucosa, which is in constant contact with ingested food along with the gut microbes and other pathogens.

The primary function of the mucosa is to absorb the dietary nutrients and protect against the different external stimuli [11–13]. On the other hand, the muscularis propria is mainly responsible for coordinating contractions for proper food movement through the GI tract. The cellular anatomy of the muscularis propria is complex and characterized by different regions. The two muscular layers are called longitudinal and circular, respectively, based on their orientation. The myenteric plexus, also known as the Auerbach plexus, contains a significant number of enteric neurons (ENs) and pace-making ICC, which regulate peristalsis between the two muscle regions [14–17] (**Figure 1**). Advanced technologies further demonstrated MMΦ's heterogeneity within the muscularis propria, as recent studies described differences in MMΦ distribution and phenotype within different regions of the GI muscularis propria. Current understanding revolves around MMΦ acquiring distinct phenotypes upon exposure to intrinsic GI cues. However, emerging evidence suggests that MMΦ display differences in their functions and phenotypes that are not exclusively driven by the GI milieu but also by their ontogeny.

#### **3. MMΦ heterogeneity: ontogeny vs. environmental cues**

For the longest time, the origin of MΦ was attributed entirely to circulating blood monocytes, which after engrafting the tissue, acquired tissue-resident MΦ resemblance [18–20]. However, studies in the late 2010s challenged this paradigm as scientists theorized that populations of resident MΦ in homeostatic tissues also derived

#### *Macrophages in the Smooth Muscle Layers of the Gastrointestinal Tract DOI: http://dx.doi.org/10.5772/intechopen.102530*

from embryonic progenitors of the yolk sac and fetal liver [21–23]. After this finding, there is now a consensus on the double origin of tissue-resident MΦ in multiple organs, where monocyte-derived- and embryonic MΦ coexist [24]. Embryonic MΦ are established before birth and their number is maintained by cell division, independently of circulating monocytes' recruitment. On the other hand, a population of MΦ is continuously replenished by a monocyte waterfall wherein circulating adult monocytes ingress into tissues and differentiate to tissue-resident MΦ [24].

While MMΦ ontogeny in homeostatic condition has been recently studied [25], most of the research in the last couple of decades has primarily been focused on disease models. Like microglia in the central nervous system (CNS), MMΦ contains populations of different origins, as cells of both embryonic and circulating monocyte origin constitute the entire pool of tissue-resident MMΦ. MMΦ as microglia in the brain express the high level of CX3CR1 as a canonical marker of tissue-resident cells.

Using a lineage tracing mouse model, CX3CR1 MMΦ were followed from the embryonic stage to adulthood. This population represents the totality of tissue-resident MMΦ at the embryonic stage and declines with time. This decline starts between 4 and 20 weeks after birth and stops after this timepoint. Consequently, the population that remains seeded has come to be known as long-lived MMΦ. Importantly, this population of embryonic MMΦ has a different transcriptional profile compared with monocyte-derived MMΦ. In fact, a population of tissue-resident MMΦ expresses high levels of CX3CR1, also known as CX3CR1hi. CX3CR1low MMΦ, on the contrary—as the name suggests—express low levels of CX3CR1 and highly express C-C chemokine receptor 2 (CCR2). CCR2 is involved in monocyte homing in response to inflammation in the local tissue environment [26, 27]. Resident MMΦ exhibits a general anti-inflammatory phenotype at steady-state conditions compared to the more inflammatory phenotype of mucosal MΦ. This is underscored by the expression of their wound healing and tissue-protective genes.

Long-lived MMΦ express genes responsible for cell-to-cell adhesion, cytoskeletal anchoring, and neuron development, suggesting their anatomical association with ENs. In addition, 26% of the genes enriched in a subpopulation of long-lived MMΦ is unique compared to the data set from tissue-resident MΦ populating other tissues. However, most of this population express gene previously associated with microglia in the brain, such as Fc receptor-like scavenger (*FCRLS*), cystatin C (*CST3*), platelet factor 4 (*PF4*), apolipoprotein E (*ApoE*), and disabled-2 (*Dab2*). Although a subtype of MMΦ is maintained by cell division, most tissue-resident MMΦ are continuously replenished by circulating monocytes. In fact, bone marrow transplanted CX3CR1 EGFP cells engrafted into the muscle layers, and within 4 weeks of transplantation, they re-established the number of tissue-resident MMΦ [28].

A study aimed at differentiating between long-lived MΦ and monocyte-derived MΦ used Tim4 and CD4 as surface markers to separate the two populations [29]. Long-lived MΦ were Tim-4<sup>+</sup> /CD4+ as evidenced by the slower rate of turnover compared to Tim-4− /CD4− cells as expressed by chimerism of cells post-irradiation. Although the total pool of small bowel MΦ was considered, this segregation is largely driven by mucosal tissue-resident MΦ in commensal-rich areas as noted in the study due to the higher rate of monocyte-macrophage turnover. Interestingly, Tim-4 is as an important apoptotic cell uptake receptor indicating that these long-lived MΦ might be playing a crucial role in efferocytosis, a process which has been shown to resolve inflammation in the brain by microglia [30], and other tissue types [31].

A new population of embryonic MMΦ called perivascular (PVMs) was recently identified in anatomical association with blood vessels within the muscularis propria of the ileum and small intestine [32]. The authors identified a gene—musculoaponeurotic fibrosarcoma (*Maf*)—which is required for the development of this subpopulation in white adipose tissue (WAT). This population, named vasculature-associated macrophages (VAMs), existed in all organs in proximity to blood vessels. This commonality led them to understand the effect of *Maf* regulation in the muscularis propria of the ileum and small intestine since they harbor MMΦ expressing similar markers to VAMs, specifically, VAM2. Furthermore, when the *Maf* gene was deleted, there was a total loss of CD206+ MMΦ in the small and large intestines, implicating its critical role in their phenotype and function.

All these data suggest the bivalent origin of tissue-resident MMΦ with the coexistence of monocyte- and embryonic-derived MMΦ. This level of heterogeneity has been recently described in the single-cell transcriptomic analysis of colonic MMΦ. Colonic MMΦ can be divided into three different populations, including "transient" monocyte-like MMΦ expressing high levels of calprotectin (heterodimer of S100A8 and S100A9) and long-lived calprotectin-negative MΦ expressing a TRM phenotype [33, 34].

Further investigation is needed to outline the key similarities and differences between murine and human MMΦ distribution, morphology, and composition. Although the murine monocyte waterfall in the intestine depicts a detailed transition of Ly6Chi CX3CR1int monocytes to CX3CR1hi MHC-IIhi CD64+ MMΦ, information on human MMΦ is sparse, generally relied upon immunohistochemistry and morphological studies.

Another study by Bernardo and colleagues tracked the transition phenotype of human monocytes to tissue-resident MΦ [35]. It was found that CD14+ monocytes differentiated into inflammatory monocyte-like cells upon entering healthy and inflamed colonic mucosa. These cells, identified by CD11chi CCR2hi CX3CR1+ expression, then transitioned through an intermediate phenotype of CD11cdim CCR2low CX3CR1low before finally becoming tissue-resident—or tolerogenic—MΦ with a CD11c− CCR2− CX3CR1− signature. As is true in the mouse model, CCR2 remains critical in recruiting monocytes in humans. Furthermore, the authors found that homing was abrogated when it was blocked on monocytes before migration. Whether this is true in MMΦ is yet to be elucidated. Changes to monocyte-derived or long-lived embryonic MMΦ can alter the total MMΦ number in diseases where homeostasis is challenged. For example, in diabetic and diabetic gastroparetic mice [36], there is an increase in MMΦ which is linked to the recruitment of inflammatory circulating monocytes.

MMΦ phenotype depends on regional distribution across the muscularis propria and the interaction MMΦ established with other cell types populating the same environment. MMΦ have a different morphology [37] in the different regions of the muscularis propria. MMΦ located in the myenteric plexus and serosal regions is multipolar, with many branches originating from the main body (**Figure 1**).

On the other hand, MMΦ distributed within the muscular layers have a bipolar shape that follows muscle cells' orientation. Further data are needed to understand if morphological differences between these diverse MMΦ populations translate into functional changes. Such differences have been easier to study in the brain since the various CNS regions are accessible and can be separated. Whereas this has enabled the study of microglial phenotype residing in each area, it is more complicated to get the same type of information from the muscularis layers of the GI tract due to the technical difficulties in separating the different regions.

Long-lived MMΦ occupy a specific anatomical niche within the GI muscularis propria. De Schepper and colleagues [25] identified this MMΦ population as essential

#### *Macrophages in the Smooth Muscle Layers of the Gastrointestinal Tract DOI: http://dx.doi.org/10.5772/intechopen.102530*

for maintaining ENs, located within the myenteric plexus region where they interact with ENs. The critical role of the environmental cue in shaping MMΦ phenotype is evident as embryonic MΦ are also present in the mucosa, but they have an overall distinct phenotype compared to long-lived MMΦ. Interestingly, another population of embryonic MMΦ does not express CX3CR1, but in this case, it appeared to be anatomically coupled with blood vessels [32].

Another essential feature contributing to MMΦ heterogeneity is the location of MMΦ in different regions of the muscularis propria. Whereas in the small intestine MMΦ are mostly concentrated in the myenteric plexus, gastric MMΦ are evenly distributed between the myenteric plexus and smooth muscle layers [38]. Further studies are needed to understand if this difference in MMΦ distribution is also responsible for functional changes. Phenotypically, at resting, gastric MMΦ do not express high levels of CD206 as MMΦ from the small intestine does. MMΦ density also differs between the small intestine and colon in young mice, with a reduction of MMΦ density observed in the colon [39]. However, this type of difference was no longer observed in adult mice. It also appeared that MMΦ in the different gut regions responds to external stimuli differently, suggesting a possible intrinsic phenotypic difference. For example, in diabetes, gastric MMΦ change their phenotype leading to gastric dysfunction, whereas, in the context of the same disease, MMΦ in the small intestine are unchanged [36].

Although we have a clearer picture of MMΦ distribution in different smooth muscle layers at steady-state conditions, we have only partial information about their distribution in states of altered homeostasis and disease. In aging, clusters of CD163-IR immune cells are visualized in proximity to sympathetic hyperinnervation in the jejunum of rats [40]. In a mouse model of diabetic gastroparesis, an increased number of MMΦ has been described with the onset of diabetes, but no changes in the MMΦ distribution have been reported [41].

More studies are needed to understand the differences between the populations of MMΦ residing in the different gut regions looking at (1) phenotypic changes, (2) changes in response to inflammation/stimuli, and (3) origin.

#### **4. MMΦ: new players of gastrointestinal motility**

As anticipated in the previous section of the chapter, the muscularis propria contains numerous ENs that work in concert with the CNS to control digestive function. The enteric nervous system (ENS) has 200–600 million ENs distributed in thousands of small ganglia [15, 16]. Importantly, the ENS can function independently from the CNS to control digestive function. The GI tract is innervated by intrinsic ENs and the CNS axons of extrinsic sympathetic and parasympathetic neurons.

Since some MMΦ are closely associated with ENs, this raises the following questions: Do these MMΦ functionally interact with enteric nerves, and what does such communication entail?

#### **4.1 Functional interaction between MMΦ and intrinsic innervation**

MMΦ-ENs functional interaction has been studied extensively in the homeostatic and diseased gut. Muller and colleagues showed for the first time an active interaction between MMΦ and ENs in 2014 [42]. In this study, the investigators showed that MMΦ expresses a high level of bone morphogenetic protein 2 (BMP2) compared to mucosal MΦ. Notably, certain ENs express the receptor (BMP2r) that, upon interaction with

BMP2, respond by a pSMAD1/5/8 related mechanism. This type of functional interaction is regulated by microbiota, as microbiota-free mice have reduced BMP2 expression. Depletion of MMΦ results in poorly coordinated colonic contractions in an ex vivo model and abnormal colonic transit time in vivo. Adding exogenous BMP2 to the colonic rings from MMΦ -deficient mice decreases stretch-induced contractions. Enteric neuron number results from a dynamic balance between the ENs dying by apoptosis and the continuous production of new ENs by neurogenesis. As microglia in the CNS, MMΦ played an essential role in clearing cellular debris resulting from neuronal death. In vitro models have shown the bidirectionality of this interaction. Oxytocin (OT) is traditionally considered a nonapeptide hormone synthesized in the hypothalamus that is released from the posterior pituitary into circulation and is involved in milk let-down and uterine contraction. Polarized pro-inflammatory MMΦ regulate the expression of OT and its receptor, OTR in cultured enteric neurons via STAT3 or NF-κB pathway [43].

On the other hand, TGF-β released by anti-inflammatory MMΦ induces the upregulation of OT/OTR [44]. Interestingly higher levels of pro-inflammatory cytokines correlated with a lower level of OT/OTR in DSS-colitis. In the colon, ENS is reduced by pro-inflammatory MMΦ via the GK1-FOXO3 pathway.

Most studies described a close association between MMΦ and nerve fibers. However, recently a paper for the first time also describes a rare population of MMΦ distributed within the ganglia [45, 46], which house the bodies of the ENs. In their study, the authors demonstrated that this population of MMΦ, called intra ganglionic macrophages (IGMs), has processes in this region of mouse colon, suggesting phagocytic capability. Colitis-induced mouse models are characterized by an increased level of pro-inflammatory MMΦ and associated with a reduction of IGMs. Notably, the loss of IGMs in colitis is associated with enteric neuroinflammation, characterized by neuronal hypertrophy.

A series of studies questioned the role of MMΦ in regulating the total number and the genetic coding of ENs. CX3CR1 MMΦ [25] of embryonic origin persisted with aging and remained primarily associated with ENs in the myenteric plexus region. The conditional removal of this population of MMΦ during development results in the overall reduction of ENs, leading to GI dysfunction. Csf1op/op mice, which do not have MMΦ from birth, have an abnormal myenteric plexus and more ENs than control mice [41, 42]. Although the number of nitrergic ENs is increased in Csf1op/op mice [47], the number of cholinergic ENs is not altered, suggesting that MMΦ may regulate different subtypes of ENs (**Figure 2**).

In the same animal model, Cipriani and colleagues also showed that the absence of MMΦ from birth is associated with more ENs sharing cholinergic and nitrergic phenotypes, indicative of a more undifferentiated population of ENs. A reduced number of anti-inflammatory MMΦ in aged mice is linked to ENs loss [48, 49].


**Figure 2.**

*Bidirectional interactions between EN and MMΦ comparing homeostasis and disease.*

#### *Macrophages in the Smooth Muscle Layers of the Gastrointestinal Tract DOI: http://dx.doi.org/10.5772/intechopen.102530*

To further understand this intimate relationship between MMΦ and the ENS, papers understanding their dynamics during development have shed some light on this. MMΦ colonize the gut independently of ENs at E9.5. Although they engraft into the muscularis propria, tissue-resident MMΦ are not close to ENs but are closer to other cells populating the same environment during this period [50]. The investigators identified a plausible explanation for this observation as the absence of CSF1 release by ENs during this period, contrary to adulthood where ENs represent the primary source of CSF1. An increasing number of MMΦ engraft into the muscularis propria during development, where they occupy a distinct niche compared to mucosal MΦ and establish an intimate connection with neuronal processes [51]. Conditional depletion of *irf8*, a gene enriched in this population of MMΦ, leads to impaired intestinal GI motility. More studies are needed to dissect further the possible role of MMΦ in orchestrating ENs differentiation and distribution.

#### **4.2 Functional interaction between MMΦ and extrinsic innervation**

The amount of data describing the functional interaction between tissue MMΦ, and peripheral nerves is limited, mainly because the number of MMΦ is minimal compared to the total number of tissue MΦ. For example, MMΦ expressing CX3CR1 are closely associated with sympathetic nerve fibers of adipose tissue [52]. Precise extrinsic afferent (visceral sensory) and efferent (sympathetic and parasympathetic) innervation of the gut is fundamental for gut-brain cross talk. While the extrinsic nerves do not directly modulate gut motility, they can affect it by regulating other cell types within the ENS [53]. Interactions between MMΦ and extrinsic innervation and the effect of sympathetic and catecholaminergic signaling in the immune cells' modulation of multiple organs have been extensively studied in the past [54]. However, their possible involvement in modulating MMΦ polarization phenotype in the gut has been described only recently. In their study, Gabanyi and colleagues [37] suggest the regulation of MMΦ activation by the β-adrenergic receptor β-2AR receptor (β2AR). Β2AR+ MMΦ resides near neuronal cell bodies or processes of the myenteric ganglia. Because of this interaction, MMΦ expresses higher levels of β2AR, a neuropeptide receptor, than lamina propria MΦ. Notably, adrenergic signaling through this receptor reduces ENs loss following infection [55].

Acetylcholine (Ach) represents the primary parasympathetic neurotransmitter released by preganglionic nerve fibers and the vagus nerve. Ach has been studied for its anti-inflammatory effects in the periphery, as its stimulation is sufficient to suppress systemic inflammation in response to endotoxin. Cholinergic neuronal release during vagal nerve stimulation (VNS) induces an anti-inflammatory MMΦ phenotype activation via the alpha7 nAChR (α7nAChR), ameliorating muscular inflammation [56]. In addition, vagal manipulation leads to an increased number of gastric MHCII+ MMΦ, resulting in delayed gastric emptying [57].

The vagal nerve represents the longest nerve in the body and the main component of parasympathetic innervation. It is well established that the vagal nerve innervating the GI tract originates from 2 central regions of the CNS: the ambiguous nucleus and the dorsal motor nucleus of the vagus. This indeed represents one of the most studied roots to access the ENS from the CNS [58]. VNS mediates MMΦ anti-inflammatory phenotype activation in a model of inflammation induced by mechanical stimulation of the mucosa. This pathway is independent of vagal stimulation from the spleen as vagus denervation from the spleen did not prevent MMΦ activation. In this mouse model, the activation of anti-inflammatory MMΦ through VNS reduced the overall level of inflammation in the tissue. It appeared that this type of pathway

is effective through the α7nAChR since MMΦ from α7nAChR knockout mice did not respond to VNS [56]. Extrinsic vagal innervation is involved in regulating contractions generated by the stomach. Preclinical studies underlined this pathway's involvement and its possible therapeutic role in preventing muscularis propria inflammation in inflammatory bowel disease (IBD). Mice in which the vagus is resected develop severe colitis associated with increased pro-inflammatory cytokine levels such as IL-1β, IL-6, and TNF-α [59].

Interestingly, patients with depression and psychological stress are typically associated with adverse and worst forms of ulcerative colitis. This important association also translates into animal models of depression, which are more prone to develop forms of colitis [60]. Notably, the beneficial effect of antidepressant drug application is abolished after vagotomy. Although this mechanism is not entirely understood, the transfer of MΦ from animal models with depression made the recipient mice more susceptible to developing forms of colitis [61].

Gastroparesis is a disease that affects the stomach and is associated with impaired motility and increased pro-inflammatory MMΦ. VNS stimulation in patients with gastroparesis induced anti-inflammatory MMΦ activation and an incremental improvement in symptom scores [62]. In addition, VNS prevents gastroparesis by inducing antiinflammatory MMΦ through STAT3-JAK2 mediated mechanism. Abdominal surgery is often associated with pro-inflammatory MMΦ activation leading to an overall inflammation of the muscularis propria and affected gastric motility, which VNS3 abolishes [63].

It appears that this interaction is bidirectional, as ENs can also affect MMΦ phenotype, differentiation, and maintenance. In an animal model in which pharmaceutical and genetic sympathetic innervation is deprived, there is an increase of circulating monocytes that ingress into the muscularis propria compared to controls [64]. In addition, isolated MMΦ from sympathetic-deprived mice have anti-inflammatory phenotype and concomitant increment of pro-inflammatory phenotype that led to accelerated GI transit time. Intestinal manipulation in postoperative ileus (POI) promoted an increased level of anti-inflammatory ED1 MMΦ [65] in the colon blocked by an anti-α7nAChR antibody. The authors proposed a mechanism in which ENs released acetylcholine upon intestinal manipulation that binds its receptor (α7nAChR) on the surface [56]. This represents a mechanism that could be targeted to prevent POI. A mouse model of post-infectious irritable bowel syndrome (IBS) is associated with subtype-specific neuronal loss via NRLP6 and caspase 11 mechanism and dysmotility. Notably, in this model, β2-AR signaling depletion in MMΦ resulted in increased loss of ENs in the post-infectious IBS model. These results indicate that, while short-term depletion of MMΦ does not impact intrinsic enteric-associated neurons (iEANs) survival in the unperturbed state, MMΦ may play an iEAN-protective role during enteric infection [56].

#### **5. MMΦ interactions with non-neuronal cells**

#### **5.1 MMΦ-ICC**

The GI tract represents a highly heterogeneous system where multiple cell types coexist and contribute to GI contractility. Similar to the pacemaker cells of the heart, the gut contains cells called ICC that set up the GI contractility pattern. ICC was described more than 100 years ago by Ramon y Cajal. For many years these cells were characterized only by non-specific histological stains and later, more reliably, by electron microscopy. The ultrastructural features and the ICC's anatomical distributions

*Macrophages in the Smooth Muscle Layers of the Gastrointestinal Tract DOI: http://dx.doi.org/10.5772/intechopen.102530*


#### **Figure 3.**

*Bidirectional interactions between ICC and MMΦ in homeostasis and disease.*

suggested their critical physiological roles: (1) they are pacemaker cells and propagate slow waves [66, 67], (2) they mediate both inhibitory and excitatory neurotransmission [68–70], (3) they work as mechanosensory cells.

Limited data describe MMΦ-ICC interaction in homeostatic conditions in the ENS. Electron microscopy and immunofluorescence analysis showed that MMΦ populations are closely associated with ICC [10, 14], suggesting a potential functional role for this type of interaction. Ji and colleagues [38] recently demonstrated that despite their close association, MMΦ rarely touches ICC, but they are separated by a space of 300 microns. In Csf1op/op mice, that do not have MΦ from birth, ICC appears to have a normal distribution, and the level of expression of kit, a specific ICC marker, is not different from controls (**Figure 3**).

The information relative to MMΦ-ICC functional interaction in GI disease is more extensive. Blockade of IL-17A signal reduced ICC loss in sepsis by affecting the overall number of pro-inflammatory MMΦ [71]. Numerous MMΦ are observed closely associated with ICC in the antiinflammation model compared to controls at ultrastructural level [72]. In the same model with the progression of inflammation, MMΦ has large phagosomes and lysosomes in the proximity of injured ICC, suggesting a possible ongoing phagocytic activity. During development, ICC releases CSF1 in the small intestine, thus contributing to MMΦ migration and survival during this period. IL-6 released by MMΦ during GI surgery promotes upregulation of miR-19a responsible for ICC depletion [73]. An increase of pro-inflammatory cytokines produced by MMΦ is associated with decreased c-kit positive ICC in the dilated colon of Hirschsprung disease (HSCR) associated with enterocolitis [74].

Most of the information acquired in recent years describing a functional interaction between MMΦ and ICC has been produced in the context of diabetic gastroparesis, a functional disease affecting the stomach. One of the main cellular changes observed in both mice and patients with diabetic gastroparesis is ICC depletion and changes to MMΦ composition. Conditioned media from pro-inflammatory activated MMΦ reduces kit positive ICC *in-vitro* [75]. The ineffectiveness of the same conditional media in the presence of a TNF-alpha neutralizing antibody suggests this cytokine's implication in regulating kit expression. Patients with diabetic gastroparesis have fewer CD206+ , anti-inflammatory MΦ. This reduction correlates with ICC loss, suggesting a protective role of anti-inflammatory MMΦ on ICC, that is impaired in diabetic gastroparesis [76]. Anti-inflammatory MMΦ also secrete interleukin 10 (IL-10) to induce heme oxygenase (HO1) expression [77], an enzyme that has a protective effect on ICC. Mice with delayed gastric emptying treated with exogenous IL-10 return to regular gastric emptying with higher levels of HO1 and better connected, more organized, and evenly distributed ICC networks [78]. Thus, treatment to promote MMΦ polarization to an anti-inflammatory phenotype may be a viable treatment for diabetic gastroparesis. Progression of diabetes

and development of delayed gastric emptying is associated with increased levels of proinflammatory MMΦ and reduced anti-inflammatory MMΦ.

A small number of data suggest the contribution of MMΦ-ICC functional interaction in the development of other GI diseases. For example, Crohn's disease is associated with ICC injury and changes to MMΦ morphology [79]. In Achalasia, ICC closely related to immune cells are preserved [80]. Cytokines released by MMΦ have been considered responsible for ICC network disruption in endothelin-B receptor null rat, a model of Hirschsprung's disease [81]. Other functional diseases, such as slow transit constipation, are associated with ICC loss. In this disease, ICC loss depends on the release of exosome miR-34c-5p from MMΦ [82].

#### **5.2 MMΦ/smooth muscle cells and fibroblast-like cells**

Although MMΦ exists in the muscle layers of the gut, the limited research surrounding their spatiotemporal dynamics with smooth muscle cells (SMCs) has hampered their full understanding. Only recently has their spatial interactions with SMCs been characterized in a paper. In it, MMΦ establishes membrane-to-membrane contacts with SMCs forming structures akin to peg-and-socket joints observed using Transmission Electron Microscopy [38] (TEM) in both humans and mice. Due to this tight interaction, it is speculated that chemokines/cytokines that are released by SMCs and vice versa maybe be pertinent for the maintenance of homeostasis in the local environment and ensure proper gut motility (**Figure 4**).

In fact, a study revealed a transient receptor potential vanilloid 4 receptor (TRPV4) mediated interaction between MMΦ and SMCs in the colon of mice [83]. TRPV4 is a biosensor that can detect mechanical, thermal, and chemical cues and has been implicated in various GI disorders. However, because its effect on gut motility was not established, the authors sought to determine its role, specifically in colonic motility. Using TRPV4−/− and TRPV4+/+ as comparisons, they found various indications of impaired colonic motility. For example, they found that TRPV4−/− mice had a significantly increased number of pellets retained in the colons as opposed to their WT controls. This led to the identification of a subtype of MMΦ expressing the TRPV4 channel responsible for colonic contractions independent of ENS input. This independence was confirmed by optogenetic stimulation of the same MMΦ while applying tetrodotoxin (TTX) which resulted in prostaglandin E2 release leading to spontaneous contractions.

Postoperative ileus (POI) is a common abdominal complication almost after every intra-abdominal surgery characterized by a prolonged absence of bowel movement. This leads to symptoms such as a distended abdomen, nausea, vomiting, and other


**Figure 4.** *Bidirectional interactions between SMC and MMΦ comparing homeostasis and disease.*

#### *Macrophages in the Smooth Muscle Layers of the Gastrointestinal Tract DOI: http://dx.doi.org/10.5772/intechopen.102530*

complications that prolong patients' stay in the hospital, furthering the epidemiological burden for all parties involved. Shortly after intestinal manipulation in mice, the MMΦ network in the muscularis propria evokes a localized inflammatory response that recruits neutrophils and mast cells. The extravasation of leukocytes in tandem with MMΦ [84] results in the synthesis of prostaglandins and nitric oxide which directly impair SMCs contractility leading to POI. In the inflammatory cascade that ensues initially, there is an upregulation of MIP-1a, IL-1B, IL-6, ICAM-1, and MCP-1 compared to WT controls, indicating that MMΦ recruits monocytes, which then become resident-MΦ, and subsequently attract more monocytes. The cytokines and chemokine released are injurious to the SMCs resulting in impaired contractility depletion of this MMΦ network has been shown to decrease overall inflammation and prevent POI completely [85].

Crohn's disease is an inflammatory bowel disease (IBD) characterized by persistent inflammation that typically affects both terminal ileum and colon. Due to simplicity, reproducibility, and commonalities associated with humans, TNBScolitis in the murine model is used to study Crohn's disease [86]. In this model, MCP-1 RNA and protein levels are upregulated in the muscularis layer of the gut [46]. Similarly, in humans, inflammation resulting from Crohn's is associated with an increased number of pro-inflammatory MMΦ, neutrophils, and other immune cells recruited in the smooth muscle layers via a CCL2 and MCP-1 dependent mechanism [87]. Consequently, prolonged inflammation and hypertrophy of the surrounding smooth muscle layers have been observed [88]. Further studies are required to properly understand how MMΦ contribute to the pathogenesis of Crohn's disease.

In the process of characterizing ICC at the ultrastructural level, cells with morphological similarities to ICC were discovered [89]. These fibroblast-like cells were shown to contain gap junctions with SMCs in mice, rats, and guinea pigs indicating their potential involvement in GI motility. Years later, they came to be known as PDGFRα positive cells/telocytes (TC) since they were positive for the platelet-derived growth factor receptor alpha (PDGFRα) and negative for c-kit thus differentiating them from ICC. These cells are located all throughout the GI tract but specifically in the muscularis propria, as they can be found encircled around muscle bundles and ganglia. Their long processes form an intricate mesh-like network interacting with ICC networks, once again highlighting their importance in motility and function—specifically, purinergic motor transmission (**Figure 5**) [90].

Recently, it has been shown that MMΦ establishes cell-to-cell contacts with PDGFRα-positive cells using TEM. Due to the close association of PDGFRα-positive cells with SMCs and the connections made with MMΦ, it has further bolstered the notion that MMΦ contributes to homeostasis and GI motility in some fashion that has yet to be elucidated in greater detail.


**Figure 5.**

*Bidirectional interactions between PDGFRα-positive/TC and MMΦ comparing homeostasis and disease.*

#### **5.3 MMΦ: enteric glial cells**

Enteric glial cells (EGCs) are found in the muscularis propria surrounding the ENs, sharing similar features with the brain macroglia, represented by astrocytes and oligodendrocytes. As oligodendrocytes in the brain, EGCs contribute significantly to ENs maintenance, survival, and function [91–94]. As previously described, a subpopulation of MMΦ is specifically situated in this space, making the two cell types sharing the same anatomical space and a functional interaction possible.

EGCs as astrocytes in the brain can shift the phenotype in disrupted homeostasis conditions, such as inflammation. They actively mediate acute and chronic inflammation in the gut by regulating circulating monocyte recruitment during inflammation and the expression of pro-inflammatory cytokines. In vivo and in vitro studies demonstrate that EGCs secrete several cytokines and chemokines, including interferon-γ (IFN-γ), chemokine ligand 20, TNF-α, and prostaglandin D2 [95, 96].

The increased number of CD68 MMΦ during inflammation, partially due to circulating monocyte extravasation, is reduced after conditional depletion of connexin 43 (*Cx43*)—a gene which encodes for gap junctions—from EGCs. IL-1Β level, which is increased in inflammation, binds to its receptor on EGCs and regulates CSF1 expression by a Cx43 dependent mechanism [97].

A similar mechanism involving EGCs-MMΦ crosstalk was also observed in POI, which is associated with an increased level of IL-1β. Intraperitoneal injection of IL-1β promoted the expression of pro-inflammatory genes, and deficient mice for IL-1R1, a receptor for IL-1β, are protected from POI. Interestingly, immunohistochemistry study showed that IL-1R1 in POI co-labeled with EGC labeled with GFAP. Culture enteric glial stimulated with 10 ng/mL of IL1-β for 24 h expressed a high level of MCP-1, suggesting the possible involvement of these cells on circulating monocyte recruitment [98].

Another study showed that EGCs, after inflammation, express CCL2, which promotes monocyte recruitment upon binding with its receptor CCR2 [99]. Another study on POI described the activation of ATP, which through the P2x receptor triggered IL6 production, which is selectively blocked by the P2x2 antagonist ambroxol [100].

In colitis induced by *Heligmosomoides polygyrus* infection, transcriptome analysis of isolated MMΦ revealed the enrichment of IFNγ in the colon. This data is consistent with an enrichment of IFNγ from EGC from patients with undergoing inflammation. Interestingly IFNγ drives a feedback effect on EGC by eliciting chemokine interferon-γ inducible protein 10 kDa (CXCL10) and guanylate-binding protein 10 (GBP10) expression through STAT1, leading to reduced proliferation of EGC. CXCL10 and GBP10 are involved in host defense and mediate immune responses with regards to anti-bacterial immunity and cancer, respectively [101, 102]. In addition to directly impacting ECs, the IFNγ-EGC-Cxcl10 signaling axis regulates tissue repair after helminthes infection through MMΦ via CCL8, CCL7, CXCL2, and CCL2 activation [102].

α-Synuclein (α-Syn) aggregates are found in the brain of patients with Alzheimer's disease. Most patients with this disease suffered from GI functional disorders, however, the mechanism is not understood. Application of α-synuclein (α-Syn) aggregates into the muscularis propria promotes the expansion of EGCs and overall tissue inflammation. Although it is not directly tested, it is possible that in this context, EGCs orchestrate the overall α-Syn mediated inflammation by talking with MMΦ, as multiple genes expressed following EGCs expansion are associated with MMΦ (**Figure 6**) [103].

*Macrophages in the Smooth Muscle Layers of the Gastrointestinal Tract DOI: http://dx.doi.org/10.5772/intechopen.102530*


**Figure 6.**

*Bidirectional interactions between EGC and MMΦ comparing homeostasis and disease.*

Pro-inflammatory MMΦ products, such as IL-1, IL-4, and TNF-α, promote EGCs activation like reactive gliosis in the CNS. It is also evident that MMΦ products can affect EGCs phenotype during inflammation. Esposito and colleagues showed that upon LPS treatment, EGCs acquire an activated phenotype that coordinates the inflammatory response in the ENS. Inhibiting the NF-κB pathway on EGCs ameliorated the overall inflammatory response in colitis and in-vitro models. On the other hand, treatment of EGCs in vitro with LPS promoted the expression of genes associated with an anti-inflammatory response [104].

As in the CNS, EGCs play a role in maintaining gut homeostasis by interacting with ENs. By interacting with EGC, MMΦ can potentially contribute to ENs maintenance. In fact, the application of TNF-α and IL-1β, 2 cytokines produced by proinflammatory MMΦ, can induce the expression of nerve growth factor (NGF), which is implicated in neuronal outgrowth.

#### **6. Conclusion and future directions for MMΦ in the GI tract**

MMΦ are specialized phagocytic cells that fulfill an important role in regulating GI homeostasis and disease. They contain different subpopulations whose phenotype depends on their GI tract location and origin. These unique MMΦ, compared to mucosal MΦ, share an anti-inflammatory phenotype. It is evident now that MMΦ has a dual origin. The MMΦ pool is maintained by both monocytes derived- and embryonic-derived MMΦ. Although there is evidence suggesting the involvement of embryonic-derived MMΦ in regulating ENs, no information supports the possible contribution of circulating monocytes to tissue homeostasis. Depending on their location, MMΦ can interact functionally with cells that are important for GI physiology. Further studies are needed to elucidate the underlying mechanisms regulating this type of interactions.

*Macrophages - Celebrating 140 Years of Discovery*

#### **Author details**

Gianluca Cipriani\* and Suraj Pullapantula Enteric Neuroscience Program, Mayo Clinic, Rochester, Minnesota, United States of America

\*Address all correspondence to: gianluca.cipriani@mayo.edu

© 2022 The Author(s). Licensee IntechOpen. 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.

#### **References**

[1] Wu HJ, Wu E. The role of gut microbiota in immune homeostasis and autoimmunity. Gut Microbes. 2012;**3**(1):4-14. DOI: 10.4161/gmic.19320

[2] Vighi G, Marcucci F, Sensi L, Di Cara G, Frati F. Allergy and the gastrointestinal system. Clinical and Experimental Immunology. 2008;**153**(Suppl. 1):3-6. DOI: 10.1111/ j.1365-2249.2008.03713.x

[3] Shi N, Li N, Duan X, et al. Interaction between the gut microbiome and mucosal immune system. Military Medical Research. 2017;**4**:14. DOI: 10.1186/s40779-017-0122-9

[4] Maloy K, Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature. 2011;**474**:298-306. DOI: 10.1038/ nature10208

[5] Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. Journal of Immunology. 2000;**164**:6166-6173

[6] Mills CD, Shearer J, Evans R, Caldwell MD. Macrophage arginine metabolism and the inhibition or stimulation of cancer. Journal of Immunology. 1992;**149**:2709-2714

[7] Xue J, Schmidt SV, Sander J, Draffehn A, Krebs W, Quester I, et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity. 2014;**40**(2):274-288. DOI: 10.1016/j. immuni.2014.01.006

[8] Gosselin D, Link VM, Romanoski CE, Fonseca GJ, Eichenfield DZ, Spann NJ, et al. Environment drives selection and

function of enhancers controlling tissuespecific macrophage identities. Cell. 2014;**159**(6):1327-1340. DOI: 10.1016/j. cell.2014.11.023

[9] Gautier E, Shay T, Miller J, et al. Geneexpression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nature Immunology. 2012;**13**:1118-1128. DOI: 10.1038/ni.2419

[10] Rumessen JJ, Thuneberg L, Mikkelsen HB. Plexus muscularis profundus and associated interstitial cells. II. Ultrastructural studies of mouse small intestine. The Anatomical Record. 1982;**203**(1):129-146

[11] Caspary WF. Physiology and pathophysiology of intestinal absorption. The American Journal of Clinical Nutrition. 1992;**55**(1):299S-308S. DOI: 10.1093/ajcn/55.1.299s

[12] Blikslager AT, Moeser AJ, Gookin JL, Jones SL, Odle J. Restoration of barrier function in injured intestinal mucosa. Physiological Reviews. 2007;**87**(2):545- 564. DOI: 10.1152/physrev.00012.2006

[13] Macpherson AJ, Geuking MB, McCoy KD. Immune responses that adapt the intestinal mucosa to commensal intestinal bacteria. Immunology. 2005;**115**(2):153-162. DOI: 10.1111/ j.1365-2567.2005.02159.x

[14] Costa M, Brookes SJ, Hennig GW. Anatomy and physiology of the enteric nervous system. Gut. 2000;**47**(Suppl. 4):iv15-iv19; discussion iv26. DOI: 10.1136/gut.47.suppl\_4.iv15

[15] Furness JB. The enteric nervous system and neurogastroenterology. Nature Reviews. Gastroenterology

& Hepatology. 2012;**9**(5):286-294. DOI: 10.1038/nrgastro.2012.32

[16] Furness JB, Callaghan BP, Rivera LR, Cho HJ. The enteric nervous system and gastrointestinal innervation: Integrated local and central control. Advances in Experimental Medicine and Biology. 2014;**817**:39-71. DOI: 10.1007/978-1-4939-0897-4\_3

[17] Faussone-Pellegrini MS, Pantalone D, Cortesini C. Smooth muscle cells, interstitial cells of Cajal and myenteric plexus interrelationships in the human colon. Acta Anatomica. 1990;**139**(1):31-44

[18] Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nature Reviews. Immunology. 2005;**5**(12):953- 964. DOI: 10.1038/nri1733

[19] van oud Alblas AB, van Furth R. Origin, Kinetics, and characteristics of pulmonary macrophages in the normal steady state. The Journal of Experimental Medicine. 1979;**149**(6):1504-1518. DOI: 10.1084/jem.149.6.1504

[20] Bain C, Bravo-Blas A, Scott C, et al. Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nature Immunology. 2014;**15**:929-937

[21] Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;**330**(6005):841-845. DOI: 10.1126/ science.1194637. Epub 2010 Oct 21

[22] Gomez Perdiguero E, Klapproth K, Schulz C, et al. Tissue-resident macrophages originate from yolk-sacderived erythro-myeloid progenitors. Nature. 2015;**518**:547-551

[23] Schulz C, Gomez Perdiguero E, Chorro L, Szabo-Rogers H, Cagnard N, Kierdorf K, et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science. 2012;**336**:86-90

[24] Guilliams M, Thierry GR, Bonnardel J, Bajenoff M. Establishment and maintenance of the macrophage niche. Immunity. 2020;**52**(3):434-451

[25] De Schepper S, Verheijden S, Aguilera-Lizarraga J, Viola MF, Boesmans W, Stakenborg N, et al. Self-maintaining gut macrophages are essential for intestinal homeostasis. Cell. 2019;**176**(3):676

[26] Zigmond E, Varol C, Farache J, Elmaliah E, Satpathy AT, Friedlander G, et al. Ly6C hi monocytes in the inflamed colon give rise to proinflammatory effector cells and migratory antigen-presenting cells. Immunity. 2012;**37**(6):1076-1090

[27] Honda M, Surewaard BGJ, Watanabe M, et al. Perivascular localization of macrophages in the intestinal mucosa is regulated by Nr4a1 and the microbiome. Nature Communications. 2020;**11**:1329. DOI: 10.1038/s41467-020-15068-4

[28] Batra A, Bui TM, Rehring JF, Yalom LK, Muller WA, Sullivan DP, et al. Experimental colitis enhances temporal variations in CX3CR1 cell colonization of the gut and brain following irradiation. The American Journal of Pathology. Feb 2022;**192**(2):295-307. DOI: 10.1016/j. ajpath.2021.10.013. Epub 2021 Nov 10. PMID: 34767810

[29] Shaw TN, Houston SA, Wemyss K, Bridgeman HM, Barbera TA, Zangerle-Murray T, et al. Tissue-resident macrophages in the intestine are long lived and defined by Tim-4 and CD4

*Macrophages in the Smooth Muscle Layers of the Gastrointestinal Tract DOI: http://dx.doi.org/10.5772/intechopen.102530*

expression. The Journal of Experimental Medicine. 2018;**215**(6):1507-1518

[30] Mazaheri F, Breus O, Durdu S, et al. Distinct roles for BAI1 and TIM-4 in the engulfment of dying neurons by microglia. Nature Communications. 2014;**5**:4046

[31] Yanagihashi Y, Segawa K, Maeda R, Nabeshima YI, Nagata S. Mouse macrophages show different requirements for phosphatidylserine receptor Tim4 in efferocytosis. Proceedings of the National Academy of Sciences of the United States of America. 2017;**114**(33):8800-8805

[32] Moura Silva H, Kitoko JZ, Queiroz CP, Kroehling L, Matheis F, Yang KL, et al. c-MAF-dependent perivascular macrophages regulate diet-induced metabolic syndrome. Science Immunology. 2021;**6**(64):eabg7506. DOI: 10.1126/ sciimmunol.abg7506. Epub 2021 Oct 1

[33] Domanska D, Majid U, Karlsen VT, Merok MA, Beitnes AR, Yaqub S, et al. Single-cell transcriptomic analysis of human colonic macrophages reveals niche-specific subsets. Journal of Experimental Medicine. 9 Feb 2022;**219**(3):e20211846. DOI: 10.1084/ jem.20211846. Epub 2022 Feb 9. PMID: 35139155

[34] Bujko A, Atlasy N, Landsverk OJB, Richter L, Yaqub S, Horneland R, et al. Transcriptional and functional profiling defines human small intestinal macrophage subsets. The Journal of Experimental Medicine. 2018;**215**(2):441-458

[35] Bernardo D, Marin AC, Fernández-Tomé S, et al. Human intestinal pro-inflammatory CD11c(high) CCR2(+)CX3CR1(+) macrophages, but not their tolerogenic CD11c(−)CCR2(−) CX3CR1(−) counterparts, are expanded in inflammatory bowel disease. Mucosal Immunology. 2018;**11**:1114-1126

[36] Choi KM, Kashyap PC, Dutta N, Stoltz GJ, Ordog T, Shea Donohue T, et al. CD206-positive M2 macrophages that express heme oxygenase-1 protect against diabetic gastroparesis in mice. Gastroenterology. 2010;**138**(7):2399-409, 2409.e1

[37] Gabanyi I, Muller PA, Feighery L, Oliveira TY, Costa-Pinto FA, Mucida D. Neuro-immune interactions drive tissue programming in intestinal macrophages. Cell. 2016;**164**(3):378-391

[38] Ji S, Traini C, Mischopoulou M, Gibbons SJ, Ligresti G, Faussone-Pellegrini MS, et al. Muscularis macrophages establish cell-to-cell contacts with telocytes/PDGFRα-positive cells and smooth muscle cells in the human and mouse gastrointestinal tract. Neurogastroenterology and Motility. 2021;**33**(3):e13993

[39] Mikkelsen HB, Garbarsch C, Tranum-Jensen J, Thuneberg L. Macrophages in the small intestinal muscularis externa of embryos, newborn and adult germ-free mice. Journal of Molecular Histology. 2004;**35**(4):377-387. DOI: 10.1023/b:hijo.0000039840.86420.b7

[40] Phillips RJ, Hudson CN, Powley TL. Sympathetic axonopathies and hyperinnervation in the small intestine smooth muscle of aged Fischer 344 rats. Autonomic Neuroscience. 2013;**179**(1-2):108-121

[41] Cipriani G, Gibbons SJ, Miller KE, Yang DS, Terhaar ML, Eisenman ST, et al. Change in populations of macrophages promotes development of delayed gastric emptying in mice. Gastroenterology. 2018;**154**(8):2122-2136.e12

[42] Muller PA, Koscsó B, Rajani GM, Stevanovic K, Berres ML, Hashimoto D, et al. Crosstalk between muscularis macrophages and enteric neurons

regulates gastrointestinal motility. Cell. 2014;**158**(2):300-313

[43] Shi Y, Li S, Zhang H, Zhu J, Che T, Yan B, et al. The effect of macrophage polarization on the expression of the oxytocin signalling system in enteric neurons. Journal of Neuroinflammation. 2021;**18**(1):261. DOI: 10.1186/ s12974-021-02313-w

[44] Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA. Antiinflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Current Opinion in Pharmacology. 2009;**9**(4):447-453

[45] Dora D, Arciero E, Hotta R, Barad C, Bhave S, Kovacs T, et al. Intraganglionic macrophages: A new population of cells in the enteric ganglia. Journal of Anatomy. 2018;**233**(4):401-410. DOI: 10.1111/joa.12863. Epub 2018 Jul 18

[46] Dora D, Ferenczi S, Stavely R, Toth VE, Varga ZV, Kovacs T, et al. Evidence of a myenteric plexus barrier and its macrophage-dependent degradation during murine colitis: Implications in enteric neuroinflammation. Cellular and Molecular Gastroenterology and Hepatology. 2021;**12**(5):1617-1641. DOI: 10.1016/j.jcmgh.2021.07.003. Epub 2021 Jul 8

[47] Cipriani G, Terhaar ML, Eisenman ST, Ji S, Linden DR, Wright AM, et al. Muscularis propria macrophages alter the proportion of nitrergic but not cholinergic gastric myenteric neurons. Cellular and Molecular Gastroenterology and Hepatology. 2019;**7**(3):689-691.e4

[48] Becker L, Spear ET, Sinha SR, Haileselassie Y, Habtezion A. Agerelated changes in gut microbiota alter phenotype of muscularis macrophages and disrupt gastrointestinal motility.

Cellular and Molecular Gastroenterology and Hepatology. 2019;**7**(1):243-245.e2

[49] Becker L, Nguyen L, Gill J, Kulkarni S, Pasricha PJ, Habtezion A. Age-dependent shift in macrophage polarisation causes inflammationmediated degeneration of enteric nervous system. Gut. 2018;**67**(5):827-836

[50] Avetisyan M, Rood JE, Huerta Lopez S, Sengupta R, Wright-Jin E, Dougherty JD, et al. Muscularis macrophage development in the absence of an enteric nervous system. Proceedings of the National Academy of Sciences of the United States of America. 2018;**115**(18):4696-4701

[51] Earley AM, Graves CL, Shiau CE. Critical role for a subset of intestinal macrophages in shaping gut microbiota in adult zebrafish. Cell Reports. 2018;**25**(2):424-436

[52] Wolf YS, Boura-Halfon N, Cortese Z, Haimon H, Sar Shalom Y, Kuperman V, et al. Brown-adipose-tissue macrophages control tissue innervation and homeostatic energy expenditure. Nature Immunology. 2017;**18**:665-674

[53] Jacobson A, Yang D, Vella M, Chiu IM. The intestinal neuro-immune axis: Crosstalk between neurons, immune cells, and microbes. Mucosal Immunology. 2021;**14**(3):555-565. DOI: 10.1038/s41385-020-00368-1. Epub 2021 Feb 4

[54] Phillips RJ, Powley TL. Macrophages associated with the intrinsic and extrinsic autonomic innervation of the rat gastrointestinal tract. Autonomic Neuroscience. 2012;**169**(1):12-27. DOI: 10.1016/j.autneu.2012.02.004. Epub 2012 Mar 20

[55] Matheis F, Muller PA, Graves CL, Gabanyi I, Kerner ZJ, Costa-Borges D, et al. Adrenergic signaling in muscularis *Macrophages in the Smooth Muscle Layers of the Gastrointestinal Tract DOI: http://dx.doi.org/10.5772/intechopen.102530*

macrophages limits infection-induced neuronal loss. Cell. 2020;**180**(1):64-78.e16

[56] Matteoli G, Gomez-Pinilla PJ, Nemethova A, Di Giovangiulio M, Cailotto C, van Bree SH, et al. A distinct vagal anti-inflammatory pathway modulates intestinal muscularis resident macrophages independent of the spleen. Gut. 2014;**63**(6):938-948

[57] Lu KH, Cao J, Oleson S, et al. Vagus nerve stimulation promotes gastric emptying by increasing pyloric opening measured with magnetic resonance imaging. Neurogastroenterology and Motility. 2018;**30**(10):e13380

[58] Travagli RA, Anselmi L. Vagal neurocircuitry and its influence on gastric motility. Nature Reviews. Gastroenterology & Hepatology. 2016;**13**(7):389-401

[59] Payne SC, Furness JB, Burns O, Sedo A, Hyakumura T, Shepherd RK, et al. Anti-inflammatory effects of abdominal vagus nerve stimulation on experimental intestinal inflammation. Frontiers in Neuroscience. 2019;**13**:418. DOI: 10.3389/fnins.2019.00418

[60] Filipovic BR, Filipovic BF. Psychiatric comorbidity in the treatment of patients with inflammatory bowel disease. World Journal of Gastroenterology. 2014;**20**(13):3552-3563

[61] Ghia JE, Park AJ, Blennerhassett P, Khan WI, Collins SM. Adoptive transfer of macrophage from mice with depression-like behavior enhances susceptibility to colitis. Inflammatory Bowel Diseases. 2011 Jul;**17**(7):1474-1489. DOI: 10.1002/ibd.21531. Epub 2011 Jan 18

[62] Gottfried-Blackmore A, Adler EP, Fernandez-Becker N, Clarke J, Habtezion A, Nguyen L. Open-label pilot study: Non-invasive vagal nerve stimulation improves symptoms and gastric emptying in patients with idiopathic gastroparesis. Neurogastroenterology & Motility. 2020;**32**(4):e13769. DOI: 10.1111/ nmo.13769. Epub 2019 Dec 5

[63] de Jonge WJ, van der Zanden EP, The FO, Bijlsma MF, van Westerloo DJ, Bennink RJ, et al. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. Nature Immunology. 2005;**6**(8):844-851. DOI: 10.1038/ni1229. Epub 2005 Jul 17. Erratum in: Nature Immunology. 2005;**6**(9):954

[64] Willemze RA, Welting O, van Hamersveld P, Verseijden C, Nijhuis LE, Hilbers FW, et al. Loss of intestinal sympathetic innervation elicits an innate immune driven colitis. Molecular Medicine. 2019;**25**(1):1

[65] Kalff JC, Schraut WH, Simmons RL, Bauer AJ. Surgical manipulation of the gut elicits an intestinal muscularis inflammatory response resulting in postsurgical ileus. Annals of Surgery. 1998;**228**(5):652-663

[66] Sanders KM, Stevens R, Burke E, Ward SW. Slow waves actively propagate at submucosal surface of circular layer in canine colon. The American Journal of Physiology. 1990;**259**(2 Pt 1): G258-G263

[67] Dickens EJ, Hirst GD, Tomita T. Identification of rhythmically active cells in guinea-pig stomach. Journal of Physiology. 1999;**514**(Pt 2):515-531

[68] Lies B, Gil V, Groneberg D, Seidler B, Saur D, Wischmeyer E, et al. Interstitial cells of Cajal mediate nitrergic inhibitory neurotransmission in the murine gastrointestinal tract. American Journal of Physiology. Gastrointestinal and Liver Physiology. 2014;**307**(1):G98-G106

[69] Ward SM, Sanders KM. Involvement of intramuscular interstitial cells of Cajal in neuroeffector transmission in the gastrointestinal tract. The Journal of Physiology. 2006;**576**(Pt 3):675-682

[70] Burns AJ, Lomax AE, Torihashi S, Sanders KM, Ward SM. Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proceedings of the National Academy of Sciences of the United States of America. 1996;**93**(21):12008-12013

[71] Li J, Kong P, Chen C, Tang J, Jin X, Yan J, et al. Targeting IL-17A improves the dysmotility of the small intestine and alleviates the injury of the interstitial cells of Cajal during sepsis. Oxidative Medicine and Cellular Longevity. 2019;**2019**:1475729

[72] Wang XY, Berezin I, Mikkelsen HB, Der T, Bercik P, Collins SM, et al. Pathology of interstitial cells of Cajal in relation to inflammation revealed by ultrastructure but not immunohistochemistry. The American Journal of Pathology. 2002;**160**(4):1529-1540

[73] Deng J, Yang S, Yuan Q, Chen Y, Li D, Sun H, et al. Acupuncture ameliorates postoperative ileus via IL-6-miR-19a-KIT axis to protect interstitial cells of Cajal. The American Journal of Chinese Medicine. 2017;**45**:737-755

[74] Chen X, Meng X, Zhang H, Feng C, Wang B, Li N, et al. Intestinal proinflammatory macrophages induce a phenotypic switch in interstitial cells of Cajal. The Journal of Clinical Investigation. 2020;**130**(12):6443-6456

[75] Eisenman ST, Gibbons SJ, Verhulst PJ, Cipriani G, Saur D, Farrugia G. Tumor necrosis factor alpha derived from classically activated "M1" macrophages reduces interstitial cell of Cajal numbers.

Neurogastroenterology & Motility. Apr 2017;**29**(4):12984. DOI: 10.1111/ nmo.12984. Epub 2016 Oct 25. PMID: 27781339; PMCID: PMC5367986

[76] Grover M, Bernard CE, Pasricha PJ, Parkman HP, Gibbons SJ, Tonascia J, et al. NIDDK Gastroparesis Clinical Research Consortium (GpCRC). Diabetic and idiopathic gastroparesis is associated with loss of CD206-positive macrophages in the gastric antrum. Neurogastroenterology & Motility. Jun 2017;**29**(6):13018. DOI: 10.1111/ nmo.13018. Epub 2017 Jan 9. PMID: 28066953; PMCID: PMC5423829

[77] Kashyap PC, Choi KM, Dutta N, Linden DR, Szurszewski JH, Gibbons SJ, et al. Carbon monoxide reverses diabetic gastroparesis in NOD mice. American Journal of Physiology. Gastrointestinal and Liver Physiology. 2010;**298**(6):G1013-G1019

[78] Choi KM, Gibbons SJ, Sha L, Beyder A, Verhulst PJ, Cipriani G, et al. Interleukin 10 restores gastric emptying, electrical activity, and interstitial cells of Cajal networks in diabetic mice. Cellular and Molecular Gastroenterology and Hepatology. 2016;**2**(4):454-467

[79] Wang XY, Zarate N, Soderholm JD, Bourgeois JM, Liu LW, Huizinga JD. Ultrastructural injury to interstitial cells of Cajal and communication with mast cells in Crohn's disease. Neurogastroenterology and Motility. 2007;**19**:349-364

[80] Zarate N, Wang XY, Tougas G, Anvari M, Birch D, Mearin F, et al. Intramuscular interstitial cells of Cajal associated with mast cells survive nitrergic nerves in achalasia. Neurogastroenterology and Motility. 2006;**18**:556-568

[81] Suzuki T, Won KJ, Horiguchi K, Kinoshita K, Hori M, Torihashi S, et al. *Macrophages in the Smooth Muscle Layers of the Gastrointestinal Tract DOI: http://dx.doi.org/10.5772/intechopen.102530*

Muscularis inflammation and the loss of interstitial cells of Cajal in the endothelin ETB receptor null rat. American Journal of Physiology. Gastrointestinal and Liver Physiology. 2004;**287**:G638-G646

[82] Xu S, Zhai J, Xu K, et al. M1 macrophages-derived exosomes miR-34c-5p regulates interstitial cells of Cajal through targeting SCF. Journal of Biosciences. 2021;**46**:90

[83] Luo J, Qian A, Oetjen LK, Yu W, Yang P, Feng J, et al. TRPV4 channel signaling in macrophages promotes gastrointestinal motility via direct effects on smooth muscle cells. Immunity. 2018;**49**(1):107-119.e4

[84] Iizuka Y, Kuwahara A, Karaki S. Role of PGE2 in the colonic motility: PGE2 generates and enhances spontaneous contractions of longitudinal smooth muscle in the rat colon. The Journal of Physiological Sciences: JPS. 2014;**64**(2):85-96

[85] Wehner S, Behrendt FF, Lyutenski BN, Lysson M, Bauer AJ, Hirner A, et al. Inhibition of macrophage function prevents intestinal inflammation and postoperative ileus in rodents. Gut. 2007;**56**(2):176-185

[86] Antoniou E, Margonis GA, Angelou A, Pikouli A, Argiri P, Karavokyros I, et al. The TNBS-induced colitis animal model: An overview. Annals of Medicine and Surgery. 2016, 2012;**11**:9-15

[87] Grimm MC, Elsbury SK, Pavli P, Doe WF. Enhanced expression and production of monocyte chemoattractant protein-1 in inflammatory bowel disease mucosa. Journal of Leukocyte Biology. 1996;**59**(6):804-812

[88] Chen W, Lu C, Hirota C, Iacucci M, Ghosh S, Gui X. Smooth muscle hyperplasia/hypertrophy

is the most prominent histological change in Crohn's fibrostenosing bowel strictures: A semiquantitative analysis by using a novel histological grading scheme. Journal of Crohn's & Colitis. 2017;**11**(1):92-104

[89] Komuro T, Seki K, Horiguchi K. Ultrastructural characterization of the interstitial cells of Cajal. Archives of Histology and Cytology. 1999;**62**(4):295-316

[90] Kurahashi M, Zheng H, Dwyer L, Ward SM, Koh SD, Sanders KM. A functional role for the 'fibroblast-like cells' in gastrointestinal smooth muscles. The Journal of Physiology. 2011;**589** (Pt 3):697-710

[91] Bassotti G, Villanacci V, Antonelli E, Morelli A, Salerni B. Enteric glial cells: New players in gastrointestinal motility? Laboratory Investigation. 2007;**87**(7):628-632

[92] Seguella L, Gulbransen BD. Enteric glial biology, intercellular signalling and roles in gastrointestinal disease. Nature Reviews Gastroenterology & Hepatology. Aug 2021;**18**(8):571-587. DOI: 10.1038/ s41575-021-00423-7. Epub 2021 Mar 17. PMID: 33731961; PMCID: PMC8324524

[93] von Boyen GB, Steinkamp M, Reinshagen M, Schäfer KH, Adler G, Kirsch J. Proinflammatory cytokines increase glial fibrillary acidic protein expression in enteric glia. Gut. 2004;**53**(2):222-228

[94] Rosenbaum C, Schick MA, Wollborn J, Heider A, Scholz CJ, Cecil A, et al. Activation of myenteric glia during acute inflammation in vitro and in vivo. PLoS One. 2016;**11**(3):-e0151335

[95] Grubišić V, McClain JL, Fried DE, Grants I, Rajasekhar P, Csizmadia E, et al. Enteric glia modulate macrophage phenotype and visceral sensitivity following inflammation. Cell Reports. 2020;**32**(10):108100

[96] Stoffels B, Hupa KJ, Snoek SA, van Bree S, Stein K, Schwandt T, et al. Postoperative ileus involves interleukin-1 receptor signaling in enteric glia. Gastroenterology. 2014;**146**(1):176-87.e1

[97] Brown IA, McClain JL, Watson RE, Patel BA, Gulbransen BD. Enteric glia mediate neuron death in colitis through purinergic pathways that require connexin-43 and nitric oxide. Cellular and Molecular Gastroenterology and Hepatology. 2016;**2**(1):77-91

[98] McClain J, Grubišić V, Fried D, Gomez-Suarez RA, Leinninger GM, Sévigny J, et al. Ca2+ responses in enteric glia are mediated by connexin-43 hemichannels and modulate colonic transit in mice. Gastroenterology. 2014;**146**(2):497-507.e1

[99] Stakenborg M, Abdurahiman S, De Simone V, Goverse G, Stakenborg N, van Baarle L, et al. Enteric glial cells favour accumulation of anti-inflammatory macrophages during the resolution of muscularis inflammation. bioRxiv. DOI: 10.1101/ 2021.06.10.447700

[100] Progatzky F, Shapiro M, Chng SH, et al. Regulation of intestinal immunity and tissue repair by enteric glia. Nature. 2021;**599**:125-130

[101] Liu M, Guo S, Stiles JK. The emerging role of CXCL10 in cancer (review). Oncology Letters. 2011;**2**(4):583-589. DOI: 10.3892/ ol.2011.300

[102] Krapp C, Hotter D, Gawanbacht A, McLaren PJ, Kluge SF, Stürzel CM, et al. Guanylate binding protein (GBP) 5 is an interferon-inducible inhibitor of HIV-1 infectivity. Cell Host & Microbe.

2016;**19**:504-514. DOI: 10.1016/j. chom.2016.02.019

[103] Challis C, Hori A, Sampson TR, et al. Gut-seeded α-synuclein fibrils promote gut dysfunction and brain pathology specifically in aged mice. Nature Neuroscience. 2020;**23**:327-336

[104] Esposito G, Capoccia E, Turco F, Palumbo I, Lu J, Steardo A, et al. Palmitoylethanolamide improves colon inflammation through an enteric glia/ toll like receptor 4-dependent PPAR-α activation. Gut. 2014;**63**(8):1300-1312

## **Chapter 6**
