*Acidithiobacillus* Its Application in Biomining Using a Quorum Sensing Modulation Approach

*Juan Carlos Caicedo and Sonia Villamizar*

## **Abstract**

A group of particular acidophiles microorganisms (bacteria and archaea) known as chemolithoautotrophs are capable of using minerals as fuel. Its oxidation generates electrons to obtain energy and carbon that is obtained by fixing CO2 from the air. During this aerobic mineral oxidation, metals are solubilized or biodegraded. Metal bioleaching usually is used in biomining and urban biomining approaches to recovery metals such as copper, gold and zinc. Several species of bacterial genus *Acidithiobacillus* display a great bioleaching activity. Bacterial attachment and biofilm formation are the initial requirements to begin a successful bioleaching process. Biofilm formation in *Acidithiobacillus* bacteria is strongly regulated by cell to cell communication system called Quorum Sensing. The goal of this chapter is to review the Quorum Sensing system mediated by the autoinducer N-acyl- homoserine-lactones in the Bacterium *Acidiothiobacillus ferroxidans*, in order to enhance and to boost the bioleaching technologies based in the use of this bacterium. The main applications of the cell-to-cell communication system concepts in *A. ferrooxidans* are reviewed in this chapter. It is that the addition of synthetic autoinducers molecules, which act as agonist of quorum sensing system, especially those with long acyl chains, both as single molecules (C12-AHL, 3-hydroxy-C12-AHL, C14-AHL, and 3-hydroxy-C14-AHL) or as a mixture (C14-AHL/3- hydroxy-C14-AHL/3-oxo-C14- AHL) increased the adhesion to sulfur and pyrite and enhance the metal bioleaching in urban biomining approaches.

**Keywords:** AfeI/R, Biofilm, Bioleaching, EPS, Autoinducer, Synthetic Agonist

## **1. Introduction**

As the world population grows, the reduction of several natural sources becomes more evident. One of the major concerns is the fastest decrease of metals ores. Currently, the hyper-technological society demands several metals in order to attend the rising request for electrical and electronical devices. The fact of the production of these devices is gradually cheapest and its useful life is increasingly shorter. Equally, the advertising campaigns increase the people desires to renew the older devices by a brand-new much faster and modern. It has been triggering the generation of waste of electrical and electronical devices (E-waste). It has been rising 5 folds in the last 20 years [1]. The organic and inorganic fractions of E-waste could be a serious threat to environment and public health if its final disposition

is not done accurately. The incineration and landfill are the global strategies frequently applied in a whole world for final disposal of E-waste [2]. Although of its highly toxic nature, E-waste particularly, printed circuit boards (PCBs) are a promising secondary source of metals. The concentrations of copper (Cu) and precious metals, such as gold (Au), platinum (Pt) and palladium (Pd), are high compared to natural mines [3]. Urban E-waste mining knowing as Biomining is expected to be an important secondary source of metals in the near future. PCBs are electronic components whose most abundant metal is Cu (roughly 20–25% by weight). PCBs are also composed of a substantial amount of precious metals. Precious metals constitute the largest fraction of the value of discarded PCBs and are the main economic driver of metal recovery [4]. In the last 20 years, great efforts have been focused in the recovery of these metals from the E-waste, mainly based in traditional approaches such as pyrometallurgy and hydrometallurgy. However, these two approaches have been inconvenient, due to the high consumption of energy and the high emission of polluting gases in the first one approach and in the second one the high generation of acid leachates, these leachates could reach easily the subterranean and surface water bodies [5].

Recent biotechnological developments have made possible the adaptation of various microorganisms to hostile bioleaching environments. Thus, emerging a new approach for the metal recovery from E-waste called biohydrometallurgy. This process involve microorganism in order to make use of metal elements for their structural and metabolic functions. This process based on the use of biomass has the advantages over pyrometallurgy and hydrometallurgy processes such as: it does not require the construction of a huge infrastructure, it is not necessary to deal with a large amount of harmful residual pollutants generated in the process. However, the main bottleneck that prevents this process from being attractive at an industrial level despite its high efficiency, it is the drop in yield of metal recovery when using large amounts of E-waste. However, the main bottleneck that prevents this process from being attractive at an industrial level despite its high efficiency, it is the drop in yield of metal recovery when using large amounts of E-waste. The cell-to-cell communication system known as Quorum sensing, it allows bacteria to colonize new ecological niches, to resist environmental changes and toxic substances, enhances its competitiveness and to resist host defenses [6]. As this quorum sensing system regulates near to 25% of non-essential genes, its knowledge, understanding and modulation could help to enhance the bioleaching reactions at industrial level. In this chapter, we focused to review the quorum sensing system in the bacterium *Acidithiobacillus ferrooxidans*, which bacterium is widely recognized by its tremendous bioleaching ability, and discussed the application potential of QS of this bacterium in bioleaching.

## **2.** *Acidiothibacillus ferroxidans*

The gram-negative bacterium *A. ferrooxidans* belongs to a gamma-proteobacterium group. *A. ferrooxidans* is a facultative anaerobe bacterium, which is able to grow from oxic to anoxic environments. *A. ferrooxidans* is acidophile, mesophilic and chemolithoatotrophic, that obtain energy mainly by the oxidation of ferrous iron (Fe2+), elemental and inorganic sulfur and other sulfur compounds [7]. *A. ferrooxidans* grows optimally at temperature of around 30°C and pH below 2. This bacterium is a natural inhabitant of ecological niches associated to pyritic ores, coal deposits and acid drainages [8]. Another exceptional aspect of A. *ferrooxidans* is its outstanding ability to thrive in environments with higher concentration of dissolved Fe2+ around of 1016 folds superior that neutral environments. Opposing

Acidithiobacillus *Its Application in Biomining Using a Quorum Sensing Modulation Approach DOI: http://dx.doi.org/10.5772/intechopen.98774*

to what would expect that this massive soluble Fe concentration leads to bacterial DNA and protein damage, *A. ferrooxidans* uses Fe as micronutrient and energy source, which makes it an exceptional model microorganism for the homeostasis and assimilation of Fe [9].

The genome size of *A. ferrooxidans* is 2.98 MB with 58.7% of G + C, a total of 3217 protein encoding genes of which 2070 have a putative function [10]. Like others bioleaching bacteria, *A. ferrooxidans* must face with huge heavy metal concentration in their natural niches. Diverse gene cluster have been related with the tolerance to mercury, arsenic and copper. These genes including: copper extrusion system, resistance-nodulation cell division family transporters and encoding copper translocating ATPases [10]. Two Quorum Sensing systems mediated by the autoinducer acyl homoserine lactones molecules (AHLs) have been described in *A. ferrooxidans,* the first one AfeI/R resembling to classical *LuxI/R* system presents in numerous gram-negative bacteria [11] and the second one called *act* for de acyl transfer function also produces AHL autoinducer molecules [12]. The review of these quorum sensing systems in *A. ferrooxidans*, its conformation likewise, the biological traits regulated by these communication systems such as: energy metabolism, attachment, biofilm production and toxic tolerance are the main purpose of this chapter.

## **3. Bioleaching reaction by** *A. ferrooxidanss*

In the bacterium *Acidithiobacillus ferroxidans*, bioleaching is carried out by three types of mechanisms: (i) contact, (ii) independent contact and (iii) cooperative. These three mechanisms are not mutually exclusive and generally work synergistically. The contact mechanism is characterized by the imperative need for the bacterial production of Extracellular Polymeric Substances (EPS). This EPS is essential for the subsequent formation of biofilms that will facilitate the contact between the bacterial wall and the surface of the E-waste and thus achieve the oxidation of the metal.

Previous studies have shown that *A. ferroxidans* bacteria do not adhere randomly to solid surfaces, but there is still a fine chemotaxis mechanism involved in preferential adherence to metals [13]. In the independent contact mechanism, the bacteria are not attached to the surface and the oxidation of Fe2+ in solution to Fe3+ occurs. Fe will subsequently act as an oxidizing agent, solubilizing the metals contained in the E-waste particles [14]. The cooperative mechanism is a combination of the two previous mechanisms in which the attached bacteria and the oxidizing agent Fe3+ in solution cooperate to oxidize the metals present in the E-waste particles. In the E-waste, specifically in PCI, metals are not present in the form of metallic sulphides. These are present as zero valence metals such as Cu0 , Zn0 , Ni0 , etc. Thus, the ferric ions and/or protons produced biologically from ferrous ions or the reduced sulfur compounds are responsible for the conversion of insoluble neutral metals to water soluble metallic ions.

## **4. Quorum sensing systems in extremophiles and its biotechnological applications**

Cooperative bacterial behavior is one of the most intensively studied topics of microbial ecology in recent decades. The understanding of this lifestyle allows us to revel the fitness strategies of bacteria to thrive in very different environmental niches. Quorum sensing (QS) is a system of bacterial cell–cell communication

that enables the microorganism to sense a minimum number of cells (quorum) in order to respond to external stimuli in a concerted fashion. QS system is based on the production, releasing to the extracellular environment and perception of small diffusible molecules known as auto-inducers (AI) [15]. Thus, this communication allows the bacteria to detect changes in the density of the population, which leading to generate variations in the concentration of nutrients, oxygen, inorganic molecules, pH and osmolarity in the extracellular environment. In this way, an increase in the bacterial population causes the accumulation of the AI molecule in the medium, which, when detected by the bacteria, generates changes in the expression of several target genes in order to regulate phenotypes traits in pathogenic or environmental bacteria. The main traits regulated are: attachment, virulence, resistance and bioluminescence among others [16].

In Gram-negative bacteria including the extremophiles (i.e. thermophiles, halophiles, psychrophiles and acidophiles) the most studied system involves AI molecules of the N-acyl homoserine lactones (AHLs) or autoinducer-1 (AI-1) type. This QS system is involved in intra-species and inter-species communications [17]. It was first described in the bioluminescent marine bacterium *Vibrio fischeri*. This system is considered as the QS paradigm in Gram-negative bacteria, which includes at least four elements: (i) AHLs as signal molecule or AI-1, (ii) an AHL synthase protein responsible for the synthesis of AI-1, (iii) a transcriptional regulator (belonging to the R protein family) and (iv) a cis-active palindromic DNA sequence that is the target of the R-AHL binary complex [18]. The general mechanism of action of AHLs molecules is to diffuse into the target cell and bind to an R-type transcriptional regulator to generate its dimerization (**Figure 1**). This occurs when the concentration of AHLs in the extracellular medium is adequate to generate a gradient towards the intracellular space [19]. The concentration of AHL molecules in the extracellular environment not only depends on their synthesis, but also on their diffusion, transport and degradation. The diffusion of AHL molecules depends on their size.

#### **Figure 1.**

*Schematic representation of Quorum Sensing system type I AI-1. AHLs are synthesized inside the bacteria by protein I. The AHLs molecules diffuse towards the extracellular environment, increasing their concentration both in the internal and external environment of the cell in proportion to the increase in the cell population. Upon reaching the threshold concentration, the AHLs molecules bind with the R protein, which dimerizes and promotes or represses the transcription of target genes. Some of these target genes have a binding site for the R/AHL complex in their promoter region.*

#### Acidithiobacillus *Its Application in Biomining Using a Quorum Sensing Modulation Approach DOI: http://dx.doi.org/10.5772/intechopen.98774*

Short acyl chain AHLs diffuse freely across the cell membrane [20], while long chain AHLs are excreted by transporters [21].

The biological meaning of cell to cell communication in extreme environments is not completely comprehended, however, this communication systems play a pivotal role in the survival to fitness to these restrictive habitats. However, a better understanding about the adaptation strategies regulated by quorum sensing in these extreme ecological niches, could possibly contribute to the design and development of innovative approaches for the biotechnological solutions with industrial, medical and research applications.

Most of the Quorum sensing systems present in mesophyll bacteria are shared by thermophilic bacteria. Bioinformatic analysis revealed the existence of complete AI-2 systems in 17 thermophilic bacteria from phyla Deinococcus-Thermus.

For other hand, 18 show incomplete QS systems having only LuxS [22]. Particularly interesting is the AI-2 production in the bacteria *Thermotoga maritima* and *Pyrococcus furiosus.* These two hyperthermophiles bacteria produce AI-2 signals despite lacking of synthase enzyme LuxS [23]. *T. maritima* and *P. furiosus* have genes that encode for the SAH (S-adenosylhomocysteine) hydrolase, which catalyzes the cleavage of SAH producing adenosine and homocysteine. Then, the adenosine produced is transformed to AI-2. This production of AI-2 in independent fashion of luxS could be explained by the action of the rearrangement of phosphorylated ribose, which rearrangement seems to be modulated by temperature depending mechanism [24].

Psychrophile bacteria produce DiKetoPiperazine (DKPs) molecules, which can activate the LuxR circuit. *Pseudoalteromonas haloplanktis* bacterium produce eight different types of DKPs [25]. DKPs shows a great potential for disruption of the AHLs quorum sensing system in the pathogenic bacteria *Burkholderia cenocepacia* and *Pseudomonas aeuroginosa.* It results from the direct inhibition of AHL synthase. This fact represents a great potential for the alternative treatment of disease such as cystic fibrosis [26]. Another industrial application mediated by QS in psychrophile bacteria is the alginate overproduction at low temperature by the *Pseudomonas mandelii.* Pseudomonads bacteria produce alginate by the expression of genes *algU* and *mucA*. These genes expression are QS regulated [27]. In the biofilm formation process at low temperature (4–15°C) the alginate operon transcription is keeping active because its repressor is downregulated. Biofilm formation shows to be an adaptation strategy for survival at low temperatures [28].

Acidophiles bacteria have endowed with Quorum Sensing systems such as: AI-1, AI-2 and CAI-1. The genome of acidophilic iron oxidizing bacterium *Ferrovum* sp. harbors a thermophilic ene-reductase (ERs). The ERs Family enzymes has shown that it could play a role in the Quorum Sensing mediation of oxidative stress response [29]. *Ferrovum* sp. lacks of encoding gene for the AHL synthase LuxI. The gene *foye-1* in the genome of *Ferrovum* is located directly downstream of the LuxR. A previous study suggests that ERs FOYE-1 in this bacterium could be responsible for the production of HLA and triggers the LuxR perception [30]. The ene-reductase enzymes have increasing relevance in the field of bio-catalysis because of its superior stability. This fact rises the biotechnological potential of these ERs.

Enteric pathogenic bacterium *Vibrium cholerae* produces and senses three AIs such as: AI-1, DPO and CAI-1. The first two are recognized for the interspecies communication role, while the last one is employed as intra-genus communication AI. These all three QS systems work together for the transition from an aerobic to an anaerobic environment. This QS represses the virulence and biofilm formation in the *Vibrium cholerae* bacterium at high cell density [31].

The bacterium *Acidithiobacilus ferrooxidans* widely recognized by its bioleaching ability displays two quorum sensing systems, (i) a completely functional

LuxI/R like, encoding the gene cluster *afeI*-*orf3*-*afeR* [32]. *At. ferrooxidans* is able to produce nine types of AHLs with acyl chains, whose length ranges between 8 and 16 carbons. A recent study shows that depending on energy substrate, the protein AfeI synthetizes different types of acyl-HSLs, similarly, the regulation of different metabolic systems also depends on substrate energy [33]. (ii) The second Quorum Sensing system in *A. ferrooxidans* has been identified using a reporter strain strategy. This QS system was termed *act* QS system. This reporter strain evidenced the production of HLA molecule e.g. C14 acyl-HSL by *E. coli* cloned with the *act* gene [34]. The quorum sensing system *act* is mainly expressed when *A. ferrooxidans* cells are grown in culture medium enriched with Fe+2 than when the cells are cultivated in microbiological medium enriched with sulfur. The role of *act* Quorum Sensing system of *A. ferrooxidans* remains uncertain.

Synthetic biology is based on the use of engineering principles to design and apply new biological components, besides to integrate functions and traits into the present ones in order to standardize or modulate their behavior. The completely understanding of a particular Quorum sensing circuit enable to design tools to precisely and predictably manipulate cell responses under quorum sensing regulation. That is, QS and synthetic biology research have been highly complementary, with QS research expanding the synthetic biology toolkit and synthetic biology providing new tools for investigating QS [35]. The HLA QS system is a relatively simple system to synthetic biology approaches, due to few components to comprise the circuit. Several studies using synthetic biology to engineer cell were performed to modulate behavior when the phenotype is depending on cell density. One of the most usual approaches in this case is the manipulation of the regulator protein LuxR i.e. modifying its affinity to is cognate or not cognate AHL signals, regulating the transcription to control the number of copies of LuxR and by modifying the AHL binding site [36, 37]. Some of the above mentioned approaches were used with success in the biomining sewage bioremediation [38].

## **5. Improving of bioleaching ability through EPS regulation synthesis in**  *A. ferrooxidans*

Secretion of EPS (Extracellular Polymeric Substances) by *A. ferrooxidans* is a determinant factor for biofilm formation and an essential feature for mineral dissolution in bioleaching process [39]. Bioleaching denotes direct and indirect actions exerted by microorganisms that lead to the dissolution of metals in ores or urban biomining strategies. It has been widely demonstrated that bacterial attachment to various mineral surfaces and the formation of a well-established biofilm, contribute significantly to enhancing bioleaching activity, since, the biofilm allows the formation of reaction space identified as "surface conditioning layer" between the ores or E-waste and bacteria [40]. A previous urban bioleaching study was done using a partition system based on a semipermeable layer, in order to avoid the contact between bacteria and E-waste. This study showed that the lack of bacterial attachment reduced 25% the recovery of copper [41]. These physiological steps are mediated by extracellular polymeric substances (EPS), which are composed of polysaccharides, proteins, and lipids. Furthermore, by increasing EPS secretion and biofilms formation on mineral surfaces, attached cells extend their reactive space and obtain significantly higher amounts of substrate than planktonic cells [42]. Thus, the increase of EPS secretion could increase bioerosion capacity of the overexpression strain on the substrates and enhance the efficiency of the bioleaching process.

Acidithiobacillus *Its Application in Biomining Using a Quorum Sensing Modulation Approach DOI: http://dx.doi.org/10.5772/intechopen.98774*

Early studies showed that it is feasible to modulate *A. ferrooxidans* adhesion to pyrite particles by addition of synthetic AHL and HLSs analogues. Nevertheless, the questionable point of this study was that the results were obtained using an indirect approach, i.e. the number of the residual planktonic cells was employed in order to calculate the number of cells attached to the surface of pyrite [43]. Subsequent studies based on the use of different microscopy techniques provided conclusive evidence on the effect of bacterial adhesion on bioleaching efficiency [44]. In the bioleaching process, a bacterial attachment to copper sulfide minerals occurs selectively [45]. Thus, in the cell population that interacts electrostatically or hydrophobically with pyrite or sulfur surfaces, an increase in cell densities is observed on these surfaces. It triggers the activation of Quorum sensing regulon in those cells that remaine attached to solid surfaces much faster than in planktonic cells. This activation could contribute to favor the biofilm producer phenotype at the cell population. As it was mentioned above, the AfeI/R Quorum Sensing system promotes the EPS production and consequently contributes to the biofilm formation. Based on these observations, several studies have been implemented the addition of synthetic auto-inducers in order to make the bioleaching reaction more efficient and get a higher yield of metals. The QS agonists employed were principally those with Carbon long length e.g. C12-C14 [46]. In a previous study using direct microscopy technics, the researchers reported that AHLs with long acyl chains used as single molecules (C12-AHL, 3-hydroxy-C12-AHL, C14-AHL, and 3-hydroxy-C14- AHL) or as a mixture (C14-AHL/3- hydroxy-C14-AHL/3-oxo-C14-AHL) increased the adhesion to sulfur and pyrite [47]. Though, studies focused on elucidating and clearly understating all molecular steps produced by the addition of different AHLs autoinducers, and its effects on biofilm formation are required.

## **6. Conclusions**

The cell communication system Quorum sensing is a ubiquitous phenomenon in prokaryotes. This communication system modulates near to 25% of no essential genes. Induction or quenching of quorum sensing system could be a high valuable tool to select the desire trait in bacteria. The bacterium *A. ferrooxidans* is equipped with a fully functional Quorum Sensing system AfeI/R, which promotes the EPS secretion and biofilm formation. These traits are extremely desirable for the bioleaching reaction both for natural ores and urban biomining processes. However, special care must be taken in order to choose the energy source for the media culture where the bacteria will be grown and the bioleaching reaction will take place. As mentioned before, long chain synthetic agonists of AHL Quorum sensing system could favor the biofilm formation when the Sulfur is provided as energy source, otherwise, the addition of AHL synthetic agonist of long chain in microbiological media culture using iron as energy source, reduce drastically the bacterial growth and repress the genes responsible for the EPS and biofilm formation. Act, the second Quorum system in *A. ferrooxidans* has shown an over expression cultured in the media enriched with iron. Nevertheless, the mechanistic details about its encoding genes and their biological roles remain unclear. In addition to modulating the biofilm formation, AfeI/R Quorum Sensing system in *A. ferrooxidans* controls the grown rate, the metabolic systems and membrane permeability. Finally, the synergism between synthetic biology and Quorum sensing research enables a wide specter of approaches, in order to modulate the behavior of acidophile microorganisms with potential applications in the biomining industry.

## **Acknowledgements**

The author thanks to all researchers of department of microbiology and biotechnology at Gujarat University for their unavailable help and disposition. This work was supported by Administrative Department of Science and Technology MINCIENCIAS and Energy Mining Planning Unit UPME, Grant No 80740-008-2020.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Juan Carlos Caicedo1 \* and Sonia Villamizar2

1 Universidad de Santander, Faculty of Exact, Natural and Agricultural Science, Bucaramanga, Colombia

2 School of Agricultural and Veterinarian Sciences, São Paulo State University (UNESP), Jaboticabal, Brazil

\*Address all correspondence to: jua.caicedo@mail.udes.edu.co

© 2021 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.

Acidithiobacillus *Its Application in Biomining Using a Quorum Sensing Modulation Approach DOI: http://dx.doi.org/10.5772/intechopen.98774*

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[34] Gao, X.-Y., Fu, C.-A., Hao, L., Gu, X.-F., Wang, R., Lin, J.-Q., Liu, X.-M., Pang, X., Zhang, C.-J., Lin, J.-Q. and Chen, L.-X. (2021), The substratedependent regulatory effects of the AfeI/R system in Acidithiobacillus ferrooxidans reveals the novel regulation strategy of quorum sensing in acidophiles. Environ Microbiol, 23: 757-773. https://doi.org/10.1111/ 1462-2920.15163

[35] Stephens K, Bentley WE. Synthetic Biology for Manipulating Quorum Sensing in Microbial Consortia. Trends Microbiol. 2020 Aug;28(8):633-643. doi: 10.1016/j.tim.2020.03.009. Epub 2020 Apr 24. PMID: 32340782.

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[38] Hong, S. H., Hegde, M., Kim, J., Wang, X., Jayaraman, A., and Wood, T. K. (2012). Synthetic quorum- sensing circuit to control consortial biofilm formation and dispersal in a microfluidic device. Nat. Commun 3, 613.

[39] Rivas, M., Seeger, M., Jedlicki, E., and Holmes, D.S. (2007) Second acyl homoserine lactone production system in the extreme acidophile *Acidithiobacillus ferrooxidans*. Appl Environ Microbiol 73: 3225 doi. org/10.1128/aem.02948-06.

[40] Gao, X.-Y.; Liu, X.-J.; Fu, C.-A.; Gu, X.-F.; Lin, J.-Q.; Liu, X.-M.; Pang, X.; Lin, J.-Q.; Chen, L.-X. Novel Strategy for Improvement of the Bioleaching Efficiency of Acidithiobacillus ferrooxidans Based on the AfeI/R Quorum Sensing System. Minerals 2020, 10, 222. https://doi.org/10.3390/ min10030222

[41] Liu, H.L.; Chen, B.Y.; Lan, Y.W.; Cheng, Y.C. (2003). SEM and AFM images of pyrite surfaces after bioleaching by the indigenous *Thiobacillus thiooxidans*. Appl. Microbiol. Biotechnol. 62, 414-420

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[44] Ruiz LM, Valenzuela S, Castro M, Gonzalez A, Frezza M, Soulere L, Rohwerder T, Sand W, Queneau Y, Jerez CA, Doutheau A, Guiliani N (2008) AHL communication is a widespread phenom- enon in biomining bacteria and seems to be involved in mineral- adhesion efficiency. Hydrometallurgy 94:133-137

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## **Chapter 6**

## Immunomodulatory Potential of *Lactobacillus acidophilus*: Implications in Bone Health

*Asha Bhardwaj, Leena Sapra, Bhupendra Verma and Rupesh K. Srivastava*

## **Abstract**

*Lactobacillus acidophilus* is homofermentative anaerobic rod-shaped gram-positive bacteria. *L. acidophilous* is one of the most common probiotics and is used for the treatment of various gastrointestinal, metabolic and inflammatory disorders. *L. acidophilous* produces antimicrobial compounds, maintains gut permeability and prevents dysbiosis. *L. acidophilus* also shows various other properties such as: it is anticarcinogenic, lowers serum cholesterol level and improves lactase metabolism of host. One of the most significant property of *L. acidophilous* is that it modulates the immune system and can prevent various inflammatory disorders. *L. acidophilous* influences several immune cells such as Th17 cells and Tregs. Various studies reported that inflammation induces bone loss and leads to several bone pathologies such as osteoporosis, rheumatoid arthritis and periodontitis. Recent studies have shown the potential of probiotics in preventing inflammation mediated bone loss. *L. acidophilous* is one of these probiotics and is found capable in inhibition of various bone disorders. *L. acidophilous* restores the dysregulated immune homeostasis and prevents inflammatory bone loss. Thus, *L. acidophilous* can be a potential therapeutic for the management of various bone pathologies. In this book chapter we reviewed various immunomodulatory properties of *L. acidophilous* along with its efficacy in preventing dysbiosis and maintaining gut permeability. We also discussed the potential role of *L. acidophilous* as a therapeutic for the management of inflammation induced bone disorders.

**Keywords:** probiotics, *Lactobacillus acidophilus*, immune cells, dysbiosis, gut permeability, bone

## **1. Introduction**

The word "Probiotics" is derived from Latin language meaning life [1] and came into attention in 1953 by the German scientist Werner Kollath who defined them as "active substances that are essential for healthy development of life". Later on, in 1992 Fuller defined them as "a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance" [2]. Currently probiotics are defined as "live organisms that when administered in adequate amounts confer health benefits on the host" and are specified by the Food and Agriculture Organization of the United Nations and the World Health Organization (FAO/WHO, 2001). Probiotics are present mainly in fermented foods like cheese, bread, wine, kefir and kumis and are commercially available in the market as powders, tablets and packets [1]. Probiotics are used from centuries for the treatment of various diseases but it was not known until the 20th century that probiotics are healthy bacteria that replace harmful microbes in the gut and regulate gut flora [3]. The most extensively used probiotics are *Lactobacillus* and *Bifidobacterium.* Other common probiotics are *Bacillus*, *Streptococcus*, *Enterococcus* and the fungus *Saccharomyces* [4]. Probiotics are used for the treatment of various gastrointestinal disorders like irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), infectious diarrhea, *Clostridium difficile* colitis and antibiotic associated diarrhea and many other metabolic disorders such as obesity, diabetes and non-alcoholic fatty liver disease [5, 6]. Several mechanisms are involved in preventive activities of probiotics such as they modulate the immune system, regulate gut barrier and protect from pathogens [7]. For a probiotic to be successful it should have various qualities like it should be resistant to the low pH present in gastrointestinal tract, able to colonize in the gut, adhere to the epithelium and be able to activate the immune system. It should also have several other qualities such as it should be of human origin, non-pathogenic, noncariogenic and influence the local metabolic activity [4]. *Lactobacillus acidophilus* (LA) is one of the most common probiotics and is present in several commercially available food products and dietary supplements [8]. LA exhibits antimicrobial, anticarcinogenic and anti-inflammatory properties [8, 9]. LA has various properties that make it a good probiotic such as it is acid tolerant, bile tolerant, has lactase activity, can adhere to the human epithelial cells, lowers serum cholesterol level, prevents infection, modulates immune response, improves lactose metabolism of host, etc. [10]. Several commercially accessible strains of LA have probiotics ability like LA-1 to LA-5 (Chr. Hansen, Demark), NCFM (Dansico, Madison), SBT-2026 (Snow brand milk products, Japan), DDS-1 (Nebraska cultures, Nebraska), etc. LA NCFM is the most common LA strain and is regarded safe by the US Food and Drug Administration (FDA) [10]. LA has immunomodulatory properties and is considered for the treatment of various inflammatory diseases such as IBD, cancer, etc. [11, 12]. Use of probiotics for the prevention of bone loss has recently gain much attention. Probiotics prevent osteoporosis and other bone diseases like arthritis and periodontitis by influencing the immune system or via other mechanisms. Various studies have shown the potential of LA in preventing bone diseases [9]. Bone disorders like osteoporosis, rheumatoid arthritis (RA) and periodontitis are immune disorders and it is observed that LA has potential of preventing these disorders by modulating the immune system. Thus, LA can act as a therapeutic for the treatment of various bone fragilities.

In this chapter we summarized some of the mechanisms which are responsible for the health promoting effects of LA focussing primarily on immunomodulatory properties of LA. We also discussed the role of LA in preventing inflammatory bone loss and how modulation of gut microbiota and maintenance of gut integrity by LA can play a role in regulating bone health.

### **2.** *Lactobacillus acidophilus*

LA is a type of lactic acid bacteria (LAB). LAB constitute a group of grampositive, acid tolerant, catalase negative, non-sporulating and generally rod-shaped bacteria [13] that are frequently associated with dairy, meat and plants [14]. LAB produce lactic acid from carbohydrate fermentation which make them important in fermentation and agriculture-based industries. They are used for imparting unique textures and flavors and for preservation and acidification of different food

*Immunomodulatory Potential of* Lactobacillus acidophilus*: Implications in Bone Health DOI: http://dx.doi.org/10.5772/intechopen.97063*

items [10]. LAB comprised a number of genera such as *Lactobacillus*, *Enterococcus*, *Streptococcus*, *Cornobacterium*, *Leuconostoc*, *Lactococcus*, *Bifidobacterium* and *Sporolactobacillus* which are further subdivided into species, subspecies, variants and strains [15, 16]. *Lactobacillus* is the largest genus of lactic acid bacteria having more than 145 species [17]. *Lactobacilli* is part of human microbial flora which colonizes in the human gastrointestinal and urinary tract [18]. *Lactobacillus* species are the first ones to colonize the gut after birth where they provide various health promoting effects. *Lactobacillus* species have various qualities that make them suitable as probiotics. They are resistant to stomach pH and bile juices, can adhere to the mucosa, inhibit growth of other harmful bacterial species and have immunomodulatory properties [19]. *Lactobacilli* encompass a wide range of species that have role in various biochemical and physiological functions [10]. LA is one of the most known species belonging to *Lactobacillus* genus. LA was earlier named as *Bacillus acidophilus* and first isolated in 1900 from the human infant feces by Moro [19]. Almost 80% of the yogurts in America have LA [19]. LA is rod shaped homofermentative anaerobic having size of approximatively 2–10 μM. LA is a thermophile and grows optimally at a temperature of 37 to 450 C and at pH range of 4–5 [16]. Highest growth is observed at pH between 5.5 and 6.0 whereas growth ceases at pH 4. Diet is one of the major source of LA in gut. Various commercially available food products such as yogurt and milk are supplemented with LA due to its probiotic value [19]. LA is part of human microbiota and is isolated from digestive, oral and vaginal areas but Claesson's characterization revealed that gastrointestinal tract is its main environment [19]. It is observed that LA supplementation to humans in heat killed form is completely safe. It is observed that heat killed LA provides protection to immunodeficient mice infected with *Candida albicans* [20]. Simakachorn et al. also reported that LA supplementation induces no adverse effect in children having diarrhea [21]. LA is found effective in the treatment of various inflammatory disorders like IBD, diabetes, cancer, etc. [19]. LA prevents these inflammatory disorders by regulating the immune homeostasis. Therefore, the immunomodulatory potential of LA can be used for the management of various disorders. From recent studies it is observed that LA also inhibits inflammatory bone loss and can prevent various bone fragilities such as osteoporosis, RA and periodontitis. Below we reviewed the various immune modifying properties of LA. Later in the chapter, we discussed the role of immune modification by LA in prevention of inflammation induced bone loss. Apart from immunomodulation LA also prevents dysbiosis and increase in gut permeability which are discussed later in the chapter.

### **2.1 LA role in modulating the immune system**

LA has great immunomodulatory properties. Because of the immune modifying properties of LA it is considered for the treatment of various inflammatory diseases. LA can be an inexpensive therapeutic for treatment of numerous clinical manifestations involving malfunctioning of the immune system. Here we discuss various studies highlighting the importance of LA as a potential therapeutic for the prevention of several immune related disorders. It is observed that LA and *L. plantarum* supplementation for 60 days enhanced the expression of genes related to innate immune response in crayfish [22]. Feeding of probiotic "dahi" consisting of LA and *Bifidobacterium bifidum* reversed decrease in immune response in aging mice [23]. LA and *Bifidobacterium animalis* subspecies lactis decreased inflammation of intestinal epithelial cells by modifying the toll like receptor 2 (TLR2) mediated Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways [24]. Feeding of milk fermented with LA and *L. casei* increased both the phagocytic and lymphocytic

activity in swiss mice [25]. LA strain NCFM increased gram-positive immune response in *C. elegans* by modulating key immune signaling pathways such as p38 MAPK and β-catenin signaling pathways [26]. LA can be used for the prevention of obesity related effects. LA-KCTC3925 supplementation significantly attenuated the levels of splenic and hepatic cyclooxygenase-2 (COX-2) mRNA expression and intracellular adhesion molecule-1 (ICAM-1) expression in high fat diet induced obese mice [27]. LA supplementation generated non-specific immune response in germ free mice [28].

LA regulates the secretion of cytokines from various immune cells and maintains the balance between inflammatory and anti-inflammatory cytokines. It is observed that LA treatment significantly altered the production of interleukin (IL)-4 and interferon (IFN)-γ from splenocytes in the presence of purified tumor antigen. LA and *L. reuteri* modulated the cytokine response in neonatal gnotobiotic pigs infected with human rotavirus (HRV). LA and *L. reuteri* treatment in HRV infected pigs significantly enhanced the production of Th1 and Th2 cytokine responses as indicated by the higher concentration of IL-12, IL-10, IL-4 and INF-γ in these pigs. Treated pigs also had higher concentration of transforming growth factor (TGF)-β as compared to the controls. Thus, LA and *L. reuteri* supplementation can maintain immune homeostasis by regulating TGF-β level after HRV infection [29]. LA induced the production of cytokines such as IL-1β, TNF-α, IL-10 and IFN-γ from human peripheral blood mononuclear cells (PBMCs) [30]. LA significantly downregulated the production of anti-inflammatory cytokines and reactive oxygen species (ROS) whereas increased the production of anti-inflammatory cytokines from PBMCs isolated from Parkinson's disease patients [31]. Chen et al. showed that LA suppressed IL-17 production in experimental colitis model by suppressing expression of IL-23 and TGF-β1 and downstream phosphorylation of phospho-signal transducer and activator of transcription 3 (p-STAT3) [11]. It is observed that LA downregulated the expression of IL-1α, IL-1β, IL-8, monocyte chemoattract protein (MCP)-1 and C-X-C motif ligand 3 (CXCL3) in bovine mammary epithelial (BME) cells after Lipopolysaccharide (LPS) challenge. LA also increased the expression level of negative regulators of TLRs viz. toll interacting protein, ubiquitin-editing enzyme A20 and single immunoglobulin IL-1 single receptor in BME cells after LPS challenge [32]. Thus, LA can be a treatment option for bovine mastitis which is characterized by inflammation of the mammary glands. LA treatment significantly enhanced the expression of IL-1β, IFN-α, IFN-γ, interferon regulatory factor (IRF)-7, interferon-inducible transmembrane protein M3 and 2′,5′-oligoadenylate synthetase in chicken macrophages in response to avian influenza virus [33]. LA induced the production of TGF-β and inflammatory cytokines such as IL-6 and tumour necrosis factor (TNF-α) in dendritic cells (DCs) cocultured with intestinal epithelial cells [34]. It is observed that administration of LA strain L36 to germ-free mice induced higher expression of cytokines associated with Th2 cells such as IL-6, IL-5 and TGF-β and Th17 cells like IL-17A, IL-6 and TNF-α [35]. In dextran sodium sulphate (DSS) induced colitis LA administration suppressed the production of pro-inflammatory cytokines such as IL-6, TNF-α and IL-17 in colon tissues. *In vitro* LA treatment stimulated Tregs and the production of IL-10 [36]. LA treatment downregulated the expression of inflammatory cytokines, chemokines and myeloperoxidase in mice model of DSS induced colitis [37]. LA treatment also restored the number of colon goblet cells by inducing IL-10 expression and suppressing proinflammatory cytokines expression in DSS induced colitis [38]. LA treatment induced various antiviral cytokines and chemokines such as IL-1β, regulated upon activation, normal T cell expressed and presumably secreted (RANTES), macrophage colony stimulating factor (MCSF), eotaxin and IFN-α in lung and IL-17 in peyer's patches of influenza virus infected mice [39].

*Immunomodulatory Potential of* Lactobacillus acidophilus*: Implications in Bone Health DOI: http://dx.doi.org/10.5772/intechopen.97063*

One of the mechanisms by which LA inhibits the progression of inflammatory diseases is by modulating T cells (**Figure 1**). It is observed that LA-CGMCC 7282 along with *C. butyricum* CGMCC 7281 exerts strong anti-inflammatory effects and can prevent Th1 and Th2-type ulcerative colitis [40]. LA protected the β-lactoglobulin sensitized mice by reducing the allergic inflammation [41]. LA treatment was found to be positively associated with decreased mRNA expression of IL-17 and RORγt and reduced proliferation of Th17 cells under both *in vitro* and *in vivo* models of β-lactoglobulin allergy [41]. In case of HRV infection it is observed that varied doses of LA induced different effects. Wen et al. showed that low dose of LA enhanced IFN-γ producing T cell response but decreased Tregs response the production of TGF-β and IL-10 from Tregs. On the other hand, higher dose of LA upregulated Tregs response in gnotobiotic pigs infected with HRV [42].

#### **Figure 1.**

*Schematic diagram depicting immumodulatory properties and effect of LA on gut permeability and dysbiosis.* **(A)** *LA influence the activity of various immune cells such as Tregs and Th17 cells, dendritic cells, macrophages, natural killer cells,* γδ *T cells and B cells.* **(B)** *LA prevents the increase in gut permeability which leads to various diseases such as IBD, IBS, etc.* **(C)** *LA restores gut microbiota composition in dysbiotic conditions.*

LA strain L-92 attenuated the progression of 2, 4-dinitroflurobenzene induced contact dermatitis by regulating Tregs in spleen and cervical lymph nodes. LA-L-92 administration also enhanced FoxP3, IL-10 and TGF-β levels as compared to the controls [43]. LA and *B. longum* administration to the colitis mice model upregulated the number of Tregs and γδ T cells in intraepithelial lymphocytes [44]. Li et al. showed that LA prevents β-immunoglobulin allergy by regulating the balance the Tregs/Th17 cells and activation of TLR2/NF-Κb signaling pathways [45]. It is observed that LA lysates administration in DSS induced colorectal cancer mice model suppressed macrophage (type 2 i.e. M2) polarization, increased the number of CD8<sup>+</sup> T cells and effector memory T cells and decreased the number of Tregs in tumor microenvironment [12]. It is reported that when LA is administered after saline challenge in pigs, it increased the number of leucocytes and CD4+ T cells whereas when challenged with LPS decreased the number of both CD4<sup>+</sup> and CD8<sup>+</sup> T lymphocytes, leukocytes, expression of IL-6 and TNFα as compared to the control diet. LA modulates the activity of other immune cells also. LA enhanced the production of IL-10 and IFN-γ from splenocytes induced with concanavalin A (Con A) and significantly increased the phagocytic activity of peritoneal macrophages [46]. Surface layer protein (Slp) isolated from LA-NCFM reduced the production of IL-1β, TNF-α and ROS in LPS induced RAW 264.7 cells via suppression of MAPK and NF-κB signaling pathways. Slp also attenuated the production of nitic oxide (NO) and prostaglandin E2 (PGE2) by inhibiting the expression of inducible nitric oxide synthase (iNOS) and COX-2 [40]. SLP derived from the LA-CICC6074 also like LA-NCFM decreased the secretion of TNF-α and enhanced the secretion of NO in RAW 264.7 cells [47]. LA stimulated M2 macrophages in peritoneal cavity and Tregs and Th2 cells in spleen of DSS treated mice [38]. Administration of LA, *L. rhamnosus* and *Bifidobacterium lactis* to the mice significantly enhanced the phagocytic activity of leukocytes and peritoneal macrophages as compared to the controls [48]. It is observed that non-LPS component of LA strain DSS-1 induced the IL-1α and TNF-α production by macrophages [49]. Moreover, LA treated macrophages showed higher expression of IFN-γ and costimulatory molecule CD40 [33]. LA induces activation and maturation of DCs [50]. LA stimulated the IFN-β response in DCs in a myeloid differentiation primary response 88 (Myd88) dependent manner [51]. Konstantinov et al., showed that major SlpA of LA-NCFM interacted with the DCs via their receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and modulated the function of DCs and T cells [52]. LA-NCFM upregulated the expression of defense genes in DCs such as IL-12 and IL-10 in a TLR-2 dependent manner [51]. LA administration also decreased the degranulation of mast cells and eosinophils [53]. It is observed that pre-treatment with LA-L-92 enhanced the natural killer (NK) cells activity in lung [39]. L-92 also reduced the number of neutrophils in lung of influenza treated mice [39]. It is reported that heat killed LA 205 increased the cytolytic activity of NK cells in a time and dose dependent manner. LA enhanced the cytotoxicity of NK cells by elevating the expression of granulysin which is cytolytic granule component in NK cells [54]. In elderly population administration of LA and *Bifidobacterium bifidum* enhanced the frequency of B cells in peripheral blood [55]. LA prevents various diseases by modulating the level of antibody production. LA can be beneficial in preventing food allergy. It is observed that LA-AD031 and *Bifidobacterium lactis* ADO11 administration significantly decreased the ovalbumin (OVA) specific IgE, IgA and IgG1 in OVA and cholera toxin sensitized mice [53]. Oral administration of heat killed LA attenuated hypersensitivity responses in bovine β-lactoglobulin sensitized mice model. LA administration decreased inflammatory cells and IgE production. Along with IgE production LA treatment enhanced mRNA expression levels of

*Immunomodulatory Potential of* Lactobacillus acidophilus*: Implications in Bone Health DOI: http://dx.doi.org/10.5772/intechopen.97063*

CD25, FoxP3 and TGF-β whereas decreased the expression of IL-17A, RORγt and IL-10 in allergic group [56]. Intermediate dose of LA increased rotavirus specific IgA and IgG antibody secreting cells and memory B cells in response to rotavirus vaccine. Thus, LA administration can be used to improve the efficacy of rotavirus vaccine and thus can be effective against rotavirus diarrhea [57]. LA increase the IgA, IL-10 and IFNγ producing cells in small intestine [58]. LA improved the immunogenicity of Newcastle Disease vaccines (NDV). Chicks treated with both LA and vaccine have increased IgG and NDV antibody titres than the only vaccinated group [59]. Feeding of probiotic "dahi" (curd) containing LA and *L. casei* ameliorated the secretory IgA and lymphocyte proliferation in *Salmonella enteritidis* infected mice [60]. These probiotics also increased the proliferative response of splenocytes to LPS and con A [48]. Su et al. showed that LA SW1 could function as a promising immune adjuvant in DNA vaccine against foot and mouth disease (FMD). Oral LA-SW1 enhanced the levels of anti-FMDV antibody titres and FMDV neutralizing antibodies [61]. LA lysates also increased the antitumor activity of CTLA-4 monoclonal antibody [58]. LA not only promotes immune response but also inhibits unnecessary lethal immune responses. It is observed that LA along with *B. bifidus* or *B. infantis* suppressed the mitogen activated cell proliferation of splenocytes and PBMCs and arrested the cell cycle at G0/G1 phase. At higher concentrations these probiotics inhibited the mitogen activated overactive immune response and at lower concentration skewed the balance of Th1/Th2 balance towards Th1 [62].

On the basis of these above discussed studies, we can consider that LA has immune regulatory properties. LA has capabilities of regulating various innate and adaptive immune cells and therefore can maintain immune homeostasis. Altogether, these studies suggest that immunomodulatory properties of LA can be employed for regulating the disrupted immune homeostasis in various inflammatory diseases.

### **2.2 LA role in preventing dysbiosis**

Trillions of microbes reside in human gastrointestinal tract. These microbes contribute in number of vital functions related to health. These are source of essential vitamins and nutrients. Microbes extract energy from food, modulate immune system and maintain gut permeability. Gut microbiota usually promotes human health but alteration in the gut lead to various clinical manifestations. Alteration in gut microbiota is termed as dysbiosis. Gut microbiota can be altered by various factors like diet, toxins, pathogens, drugs, antibiotics, etc. [63]. Dysbiosis is reported in various diseases like IBS, IBD, diabetes, obesity, cardiovascular diseases, asthma, allergy, etc. [63–67]. Dysbiosis is also observed in leukemia where selective modulation of *Lactobacillus* species is reported [68]. Dysbiosis is the reason for various vaginal diseases like aerobic vaginitis, bacterial vaginosis and vulvovaginal candidiasis. In vagina of reproductive aged women microbial homeostasis is maintained by the mutualistic relationship between microbes and the host which provide protection against vaginal infections by preventing the colonization of opportunistic pathogens [69]. LA, *L. iners* and *L. crispatus* are the most abundant bacterial species in vaginal tract [70]. Role of LA in preventing dysbiosis is reported by various studies. LA-DSS-1 administration improved the abundance of beneficial bacteria like *Lactobacillus* spp. and *Akkermansia* spp. in caecum [71]. In ulcerative colitis patients, supplementation of LA, *Lactobacillus salivarius* and *Bifidobacterium bifidus* along with anti-inflammatory drug mesalazine prevented intestinal dysbiosis [72]. LA also decreased dysbiosis and inflammation induced by *Salmonella typhimurium* infection in Th1 and Th2 biased mice [73]. LA reversed the alterations in the gut

microbiota composition caused due to administration of high fat diet in animals [74]. Oral administration of LA along with cobiotic ginger extract encapsulated in calcium-alginate beads modulated gut microbiota and prevented 1,2 dimethylhydrazine (DMH)/DSS induced colitis and precancerous lesions in rats [75]. Probiotic combination consisting of LA, *L. helveticus*, *L. gassari*, *L. crispatus* and *L. salivarius* prevented vaginal dysbiosis by restoring the altered vaginal microbiota to normal level. Probiotics combination enhanced the abundance of *Lactobacillus* while decreased the abundance of *Enterobacter* and *Enterococcus* [76]. Antibiotics use provide protection against wide number of pathogens but also disturb the intestinal microflora balance. On the contrary LA is found to be capable of restoring intestinal microbial homeostasis. It is observed that LA prevent the dysbiosis induced by antibiotic Azithromycin [77]. Synbiotic consisting of inulin, LA, *L. plantarum* W21, *L. lactis* and *Bifidobacterium lactis* W51 prevented stress induced dysbiosis and thus can be useful in preventing dysbiosis induced in stress related diseases like IBS and IBD [78]. LA administration is found effective in treatment of dyspepsia caused by dysbiosis [79]. It is observed that the oral intake of LA-GLA-14 and *L. rhamnosus* HN001 mixture along with bovine lactoferrin prevent vaginal dysbiosis and improve vaginal health. After oral ingestion both the LA-GLA-14 and *L. rhamnosus* HN001 colonize and restore the vaginal microbiota [69]. LA supplementation in mice increases short chain fatty acid (SCFA) producing bacteria and thus decreases the gram-negative bacteria [80].

#### **2.3 LA role in maintaining gut permeability**

Gut barrier is very important for the regulation of the immune homeostasis and for preventing the access of pathogens into the gut lumen. Through the leaky gut, pathogens invade into the lumen and lead to uncontrolled inflammation. Gut barrier is regulated by tight Junctions (TJs) which are present between the intestinal epithelial cells. TJs are transmembrane proteins and are divided into four groups: claudins, occludin, tricullin and junctional adhesion molecules (JAMs). Transmembrane TJs are linked with the actin cytoskeleton through the cytosolic scaffold proteins like zona occludens (ZOs) which are of three types ZO-1, ZO-2 and ZO-3 [81]. Alteration in expression of TJs leads to increase in gut permeability and intestinal inflammation which is responsible for various inflammatory diseases like IBD [82–86], colon cancer [87] and RA [88].

Several studies have shown that LA administration maintain gut permeability. It is observed that the mixture of LA-KLDS1.0901 and *L. plantarum* KLDS1.0344 prevents chronic alcohol liver injury in mice by improving the gut permeability. *Lactobacillus* mixture inhibits the increase in gut permeability and reduces the abundance of gram-negative bacteria resulting in decrease of LPS entering the portal vein thereby suppressing alcohol promoted liver inflammation [80]. LA along with *L. rhamnosus* and *B. bifidumi* prevented high fat diet induced increase in gut permeability and LPS translocation [74]. LA in combination with ginger extract restored colonic permeability in DMH-DSS induced colon cancer in Wistar rats [75]. Conditioned media of LA significantly prevented the increase in IL-1β induced increase in gut permeability. Conditioned media of LA inhibits IL-1β stimulated decrease in occludin and increase in claudin-1 expression and thus preserve intestinal permeability by normalizing the expression of occludin and claudin-1 [89]. Probiotic combination of LA, *L. reuteri*, *L. casei*, *Streptococcus thermophiles* and *Bifidobacterium bifidum* significantly reduced diabetes incidence and gut permeability [90]. Administration of probiotics LA and *Bifidobacterium infantis* to the pregnant women daily from embryonic day 15- to 2-week-old postnatally maintained the intestinal integrity of preweaned offspring. Thus, LA supplementation

to pregnant women can promote barrier function of developing offsprings [91]. LA and *Streptococcus thermophilus* enhanced the barrier function of epithelial cells and protected the epithelial cells from infection induced by enteroinvasive *E. coli* by limiting its adhesion and invasion [92].

## **3. Bone remodeling**

Bone is a dynamic and metabolically active organ that is being remodeled throughout the life of the organisms. Bone remodeling is regulated by three different types of bone cells viz. osteoclasts (bone eating cells), osteoblasts (bone forming cells) and osteocytes. Osteoblasts are derived from multipotent stem cells that also give rise to fibroblasts, adipocytes, chondrocytes and myoblasts [93]. Osteoblasts are responsible for formation of osteoid matrix by depositing collagen which later on get calcified. The major constituent of osteoid matrix is type 1 collagen which provide resistance against fractures. Osteoid matrix also consists of various other non-collagenous proteins which are responsible for various critical functions of bone [94]. Osteoblast differentiation depends on a number of paracrine and transcription factors such as Runx2 and osterix and members of bone morphogenetic protein (BMP) family [94]. Osteoclasts are giant polynuclear cells that have special ability of resorbing bone [95]. Osteoclasts differentiate from monocytic progenitors that also give rise to cells of other monocytic lineages such as macrophages, dendritic cells, granulocytes and microglia. Osteoclast differentiation depends on two important cytokines: MCSF and receptor activator of nuclear factor кb ligand (RANKL). MCSF stimulate proliferation and differentiation of osteoclast progenitors. RANKL acts with the help of its receptor RANK and coupling molecule TNF receptor associated factor 6 (TRAF 6) to promote the differentiation and commitment of precursor cells [95]. Bone resorption starts when osteoclasts attach to the surface of bone and form a unique structure called sealing zone. Sealing zone permit the osteoclasts to form resorption space. Osteoclasts acidifies the resorption space to degrade the mineral and organic compartment of the bone. For this osteoclast secrete various lysosomal enzymes such as cathepsin K into the resorption space. To mediate bone resorption osteoclasts, form a specialized structure called as ruffled border that increase the surface area for active transport of H+ through proton pump. Osteoclasts comparatively resorb a large area of bone and then die by apoptosis [94]. Osteocytes are osteoblasts that have been entrapped in the osteoid matrix during matrix calcification under the influence of bone specific alkaline phosphatase produced by osteoblasts [93, 94]. Osteocytes sense mechanical force and tissue strain and send signal to the other osteocytes and osteoblasts by forming cellular network termed as canaliculi permeating the entire bone matrix [93, 94]. Dynamic equilibrium between the osteoblasts and osteoclasts maintains bone integrity. Multiple interactions take place between the bone forming osteoblasts and bone resorbing osteoclasts to regulate the process of bone remodeling [96]. Osteoblasts positively regulate osteoclast differentiation by secreting RANKL and MCSF at pre-osteoblastic stage and negatively by secreting the RANKL decoy receptor osteoprotegerin (OPG). Bone remodeling restore microdamages and ensure the release of calcium and phosphorus in normal host physiology [97]. Bone remodeling consist of four phases viz. activation phase, resorption phase, reversal phase and formation phase [97]. In activation phase, MCSF and RANKL induce the differentiation of osteoclast progenitors into osteoclasts. During resorption phase pre-osteoclasts migrate at the surface of bone and get differentiated into mature osteoclasts and start resorbing bone. Resorption phase is followed by reversal phase where mononuclear cells remove the collagen remnants and prepare

#### **Figure 2.**

*Schematic representation of bone remodeling. Bone remodeling occurs in four phases viz. 1) Activation phase: MCSF and RANKL induce the differentiation of osteoclast progenitors into osteoclasts. 2) Resorption phase: Mature osteoclast with unique ruffled border starts resorption of bone by secreting cathepsin K, and H+ in sealing zone. After resorption osteoclasts detach from the surface of bone and undergo apoptosis. 3) Reversal phase: During reversal phase osteoblasts precursor get differentiated into mature osteoblasts and are recruited to the resorption site. 4) Formation phase: Osteoblasts get occupied in the resorbed lacuna and start depositing the bone matrix. After formation phase osteoid gets mineralized and bone surface returns to resting phase with bone lining cells.*

the surface for osteoblasts where they can next start the process of bone formation. Mononuclear cell also provides various signals for the differentiation and migration of osteoblasts [93, 97]. During formation phase osteoblasts replace the resorbed bone with new bone [97] (**Figure 2**). Bone remodeling is regulated by various factors such as hormones like estrogen and parathyroid hormone and immune cells like T cells and B cells. Below we next discusse the role of immune system in regulation of bone health and the potential of LA in preventing bone resorption via immunomodulation.

## **4. Bone and immune system**

Bone is an immunomodulatory organ and various immune cells affect the development of bone. Bone cells and immune cells interact with each other in the bone marrow which is the common niche for the development of both bone and immune system. In bone marrow, bone cells and immune cells interact with each other and affects each other development. The interaction between the bone and immune system is now studied under a new field of immunology termed as Osteoimmunology, a term coined by Choi et al. in 2006. Impact of various immune cells and cytokines secreted by immune cells on bone development is now known [97]. It is observed

### *Immunomodulatory Potential of* Lactobacillus acidophilus*: Implications in Bone Health DOI: http://dx.doi.org/10.5772/intechopen.97063*

that cytokines such as IL-1, TNF-α, IL-6, IL-11, IL-15 and IL-17 induce bone resorption whereas cytokines such as IL-4, IL-10, IL13, IL-18, IFN-γ and granulocyte macrophage colony stimulating factor (GM-CSF) prevent bone loss. In various bone disorders the role of immune system has been discovered such as osteoporosis, RA and periodontitis. Osteoporosis is an inflammatory disease and several immune cells affect the development of osteoporosis. To study the immunology of osteoporosis we integrative biology started a novel field termed by us as "Immunoporosis" which deals specifically with the role of immune cells in osteoporosis [96]. Th17 cells and Tregs have most vital role in the development of bones and their balance is required for proper regulation of bone mass. CD4+ FOXP3+ Tregs enhance bone mass by inhibiting osteoclastogenesis by directly suppressing the production of RANKL and MCSF [98]. Another mechanism by which CD4+ FOXP3+ Tregs inhibit osteoclastogenesis or bone loss is by interacting with the CD80 and CD86 present on osteoclast precursors via CTLA-4, thereby inhibiting osteoclast differentiation [96]. Not only CD4+ FOXP3+ Tregs, now the effect of CD8<sup>+</sup> FOXP3<sup>+</sup> Tregs on bone is also discovered. It is observed that the CD8<sup>+</sup> Tregs prevent bone loss by inhibiting the formation of actin ring resulting in suppression of osteoclastogenesis [99]. Unlike Tregs, Th17 cells promote bone loss by inducing osteoclastogenesis via secretion of RANKL. Th17 also secrete IL-17 which induce bone loss by promoting RANKL expression on osteoclastogenesis supporting cells and by stimulating expression of inflammatory cytokines such as TNFα, IL-1 and IL-6 which further upregulate RANKL expression [9, 100]. Imbalance of Tregs and Th17 cells leads to bone loss which occurs during post-menopausal osteoporosis (PMO). Lack of estrogen promotes PMO. Estrogen prevents osteoporosis by inhibiting osteoclastogenesis but estrogen deficiency causes increased osteoclastogenesis by stimulating differentiation of Th17 cells. We also found in our studies that level of Th17 cells and inflammatory cytokines such as TNFα, IL-6, IL-17 and RANKL increased during post-menopausal osteoporosis [9, 101, 102]. Several studies have shown the role of Tregs and Th17 cells imbalanc in pathogenesis of RA [103]. The frequency of Th17 cells are enhanced in the joints and synovial fluid of RA patients [104] whereas the percentage of Tregs get significantly decreased in RA patients [105]. Similarly, the role of Tregs and Th17 cells imbalance is also found to be associated with periodontitis inflammation [106] and osteoarthritis [107]. Apart from these various other immune cells such as Th1, Th2, Th9 cells and γδ T cells are also involved in regulating bone health [97].

## **4.1 Role of LA in regulation of osteoimmune system**

As immune system has such an important role in regulation of bone health, proper maintenance of immune homeostasis is very much required. Immune homeostasis for bone regulation is maintained by various factors such as estrogen hormone. Various strategies are used to prevent bone loss due to immune disruptions such as Denosumab, rituximab and TNF blockers [108, 109]. These strategies are proven effective but they also exert various adverse effects in the long run. Recently the use of probiotics is found to be effective in treatment of various inflammatory disorders such as IBD, obesity, diabetes, etc. [110–112]. Probiotics are also considered for the treatment of various bone disorders. LA is one of these probiotics. It is observed that LA has great potential of treating various bone pathologies. From comparison of different *Lactobacillus* species, it is observed that the effect of *Lactobacillus* on bone health is species dependent and LA has showed the most significant effect on bone parameters such as bone mineral density (BMD) and bone mineral content (BMC) among other *Lactobacillus* species [113]. In rat model of apical periodontitis, it is observed that level of alkaline phosphatase is significantly higher whereas the

#### **Figure 3.**

*Role of Tregs/Th17 cells axis in regulation of bone health:* **(A)** *Tregs inhibit the differentiation of osteoclasts by secreting IL-10. Tregs also suppress osteoclastogenesis or bone loss by interacting with the CD80 and CD86 present on osteoclast precursors through cytotoxic T lymphocyte associated antigen 4 (CTLA-4). Th17 promote osteoclastogenesis via secretion of RANKL and IL-17. IL-17 induce expression of RANKL on osteoclastogenesis promoting cells.* **(B)** *In normal healthy conditions there is balance between Tregs and Th17 cells but during osteoporosis or other bone diseases like RA and periodontitis number of Th17 cells is increased which further leads to bone loss. LA treatment in these diseases restores the balance of Tregs and Th17 cells and thus prevent bone resorption.*

level of TRAP and RANKL is significantly lower in LA consumed groups [114]. It is reported that LA has antiarthritic properties and prevented Fruend's complete adjuvant mediated arthritis in female wistar rats [115]. It is observed that LA supernatant increased the proliferation of bone marrow stromal cells derived from rats [116]. It is observed in study from our group that LA can prevent bone loss by modulating the host immune system. We reported that LA improved both cortical and trabecular bone microarchitecture as well as enhanced the BMD and heterogeneity of bone in ovariectomized mice by skewing the Treg-Th17 cell balance (**Figure 3**). LA administration promoted the development of anti-osteoclastogenic Tregs and inhibited the osteoclastogenic Th17 cells in ovariectomized mice. LA supplementation also


*Immunomodulatory Potential of* Lactobacillus acidophilus*: Implications in Bone Health DOI: http://dx.doi.org/10.5772/intechopen.97063*

#### **Table 1.**

*Different strains of LA and their effects on bone.*

attenuated the expression of osteoclastogenic cytokines such as IL-6, IL-17, RANKL, TNF-α and increased the expression of anti-osteoclastogenic cytokines like IL-10 and IFN-γ. Thus, LA has therapeutic effects and it can be used as an osteoprotective agent [9]. LA prevented monosodium iodoacetate induced osteoarthritis and reduced cartilage destruction via inhibition of proinflammatory cytokines production [117]. LA supplementation along with *L. rhamnosus* significantly decreased the inflammatory cytokines IL-1β and IL-6 and enhanced the expression of IL-10 as compared to the controls in experimental apical periodontitis [114]. LA supplementation upregulated anti-inflammatory cytokines and downregulated inflammatory cytokines in serum in experimental arthritis model [118]. Thus, LA has great ability of preventing inflammatory bone loss and of regulating osteoimmune system (**Table 1**).

## **5. Bone and dysbiosis**

A number of microbes are localized in the gut. Some of them are beneficial for health whereas others are pathogenic and a balance of these microbes is required for normal physiological functioning of body. But due to several reasons like surgery, medications, irradiation and antibiotics this balance is dysregulated which leads to modifications in gut microbiota composition [3]. Dysbiosis is observed in various bone pathologies. Normally gut is dominated by four types of microbial phyla: *Firmicutes*, *Bacteroidetes*, *Proteobacteria* and *Actinobacteria*. *Firmicutes* and *Bacteroidetes* constitutes over 90% of the total gut microbiota and dysregulation of *Firmicutes/Bacteroidetes* ratio affect various biological processes like bone remodeling. During osteoporosis *Firmicutes* counts significantly increases whereas counts of *Bacteroidetes* significantly decreases [120–122]. In osteoporosis increase in the number of *Faecalibacterium* and *Dialister* genera is also reported [123]. Dysbiosis is also observed in RA and periodontitis [124, 125]. Although, LA mainly prevents bone loss by regulating immune homeostasis it also restores the gut microbiota composition in various diseases as discussed above. Thus, it can be possible that LA can also inhibit bone resorption by preventing dysbiosis.

## **6. Bone health and gut permeability**

Gut permeability has very important role in regulation of bone health. Various studies have shown that increase in gut permeability is associated with bone loss. Collins et al. group measured the intestinal permeability after 1, 4 and 8 weeks of ovx surgery and they found increased intestinal permeability one week after ovariectomy along with the increase in inflammatory cytokines like IL-1β and TNFα which are responsible for bone loss [126]. Estrogen deficiency during postmenopausal osteoporosis is responsible for increase in gut permeability. Estrogen has very significant role in regulating the gut barrier. It maintains gut barrier through its receptors which are present on the intestinal epithelial cells. There are two types of estrogen receptors: ERα and ERβ. ERβ has very important role in the regulation of TJs as ERβ−/− mice has disrupted expression of tight junction proteins [39]. Various other studies proved the role of estrogen in regulation of gut barrier. Langen et al. reported decreased expression of ERβ and increased gut permeability in IBD patients [127]. Gut permeability decreases during oestrous phase whereas it is increased during dioestrus phase of rats and this increase in intestinal permeability during dioestrus phase can be prevented by treatment with oestradiol which upregulate the expression of occludin [128]. Estrogen and progesterone treatment

*Immunomodulatory Potential of* Lactobacillus acidophilus*: Implications in Bone Health DOI: http://dx.doi.org/10.5772/intechopen.97063*

#### **Figure 4.**

*Proposed mechanism of LA in bone health. In normal healthy condition there is no dysbiosis and no alteration in gut permeability which prevents gut inflammation and thus bone loss. During osteoporosis gut permeability increases which leads to dysbiosis and bone loss. LA regulate Tregs/Th17 cell axis, prevents dysbiosis and maintains gut permeability. Altogether, LA treatment prevents leaky gut and dysbiosis thereby restoring gut immune homeostasis in osteoporosis which inhibit bone loss.*

decreases gut permeability and thus prevent secretion of inflammatory cytokines in IBD models [129]. LA prevents leaky gut in various diseases as discussed above and thus it can be possible that LA is effective in preventing leaky gut induced bone loss also. In summary we can say that by maintaining immune homeostasis and regulating both gut permeability and dysbiosis LA prevents bone resorption (**Figure 4**).

## **7. Conclusion**

In the last few years, several studies have delineated the role of LA in preventing a number of inflammatory and metabolic disorders. LA prevents these disorders through various mechanism such as by modulating the host immune system, by maintaining the gut permeability along with preventing dysbiosis. The role of LA in suppressing bone loss also highlights the importance of LA in regulating bone health. LA enhances bone mass and prevents several bone diseases like osteoporosis, arthritis and periodontitis via regulating the immune homeostasis. Thus, immunomodulatory property of LA is of utmost importance in management of various bone pathologies. LA can also prevent bone resorption by regulating the leaky gut and dysbiosis. Thus, LA has immense potential as a probiotic and can be used as a medical therapy for treatment of bone loss in humans but before that a lot of research is further needed to be done on the efficacy along with the associated pros and cons of LA on human health.

### **Acknowledgements**

This work was financially supported by projects: DST-SERB (EMR/2016/007158), Govt. of India, Intramural project from All India Institute of Medical Sciences (A-798), New Delhi-India and AIIMS-IITD collaborative project

(AI-15) sanctioned to RKS. AB, LS, BV and RKS acknowledge the Department of Biotechnology, AIIMS, New Delhi-India for providing infrastructural facilities. AB thank DST SERB project for research fellowship, LS thank UGC for research fellowship.

## **Author contributions**

RKS contributed in conceptualization and writing of the manuscript. AB, LS and BV participated in writing and editing of the review. RKS suggested and AB and LS created the illustrations. Figures are created with the help of https://smart. servier.com.

## **Conflicts of interest**

The authors declare no conflicts of interest.

## **Author details**

Asha Bhardwaj, Leena Sapra, Bhupendra Verma and Rupesh K. Srivastava\* Department of Biotechnology, All India Institute of Medical Sciences (AIIMS), New Delhi, India

\*Address all correspondence to: rupesh\_srivastava13@yahoo.co.in; rupeshk@aiims.edu

© 2021 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.

*Immunomodulatory Potential of* Lactobacillus acidophilus*: Implications in Bone Health DOI: http://dx.doi.org/10.5772/intechopen.97063*

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*Immunomodulatory Potential of* Lactobacillus acidophilus*: Implications in Bone Health DOI: http://dx.doi.org/10.5772/intechopen.97063*

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## *Edited by Jianqiang Lin, Linxu Chen and Jianqun Lin*

Acidophiles are an important category of microorganisms defined by their ability to withstand and even grow in acidic environments. They are present in terrestrial and marine environments as well as the human body. The diversity, adaptation, and functions of these microorganisms can contribute to the development and application of new biotechnologies for resolving problems of resource exploitation, pollution, and human disease. This book presents breakthroughs and insights into the research on acidophiles. Chapters cover such topics as the two-component system (TCS) in the regulation of the sulfur metabolic process, adaptation mechanisms of acidophiles to low pH, regulation mechanisms and application strategy of quorum sensing in bioleaching bacteria, and *Lactobacillus acidophilus* and its potential role as a therapeutic for human bone disorders.

Published in London, UK © 2021 IntechOpen © Dr\_Microbe / iStock

Acidophiles - Fundamentals and Applications

Acidophiles

Fundamentals and Applications

*Edited by Jianqiang Lin, Linxu Chen and Jianqun Lin*