ELUCIDATING THE POTENTIAL ROLE OF ARYL HYDROCARBON RECEPTOR IN THE PATHOGENESIS OF CAMPYLOBACTER JEJUNI. By Husnain Ahmed A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Comparative Medicine and Integrative Biology—Doctor of Philosophy 2022 ABSTRACT ELUCIDATING THE POTENTIAL ROLE OF ARYL HYDROCARBON RECEPTOR IN THE PATHOGENESIS OF CAMPYLOBACTER JEJUNI. By Husnain Ahmed Campylobacter jejuni is a leading cause of human foodborne gastroenteritis in the US, with an incidence rate of 13.6 diagnosed cases per 100,000 individuals. The most frequent cause of C. jejuni infection in the US is the consumption of chicken contaminated during processing. Macrolide antibiotics such as azithromycin and ciprofloxacin are the drug of choice to treat C. jejuni infection in human populations. However, the over-use of antibiotics has led to the emergence of antimicrobial-resistant C. jejuni strains and reduced treatment efficacy. The development of antimicrobial resistance traits in C. jejuni isolates has augmented the need to develop innovative strategies to treat drug-resistant C. jejuni infections in human and animal populations. Members of the genus Lactobacillus are commonly used as probiotics, however the mechanisms by which they provide protective health effects remain elusive. In the first study, we described a novel mechanism by which L. murinus attenuates pro- inflammatory responses in the human intestinal epithelial cells. The results showed that L. murinus activates aryl hydrocarbon receptor (AHR) to decrease the secretion of IL-8 in response to exogenous stimulation by TNF-alpha in the human intestinal epithelial cells. Furthermore, activating the AHR with its defined ligand also reduced the secretion of IL- 8 upon TNF-alpha stimulation. These results suggest that AHR can a novel target for inflammatory bowel disease (IBD) treatment. Furthermore, these results suggest that L. murinus can be a novel probiotic for treating IBD. In the 2nd study, we determined the effect of prophylactic inoculation of L. muirnus on the pathogenesis of C. jejuni in the BALB/c IL-10-/- mice. A total of 41 BALB/c IL-10-/- mice were used in this study. 11 mice were sham inoculated, 10 mice received only L. murinus, 10 mice received only C. jejuni, and 10 mice in the test group received both L. murinus and C. jejuni such that L. murinus was inoculated 32 days before C. jejuni infection. In addition, 30 days post-C. jejuni challenge mice were sacrificed and assessed for gut pathology. Fecal samples were also collected to access bacterial colonization levels in the gut through routine culture techniques and 16S sequence analysis. Both positive control group for C. jejuni and test groups mice developed severe colitis. 16S analysis of fecal DNA revealed that bacterial diversity in the test and positive control group for C. jejuni was significantly less (P<0.001) than in the Lactobacillus only and negative control group. These results suggest that prophylactic administration of L. murinus does not protect BALB/c IL-10-/- mice from developing disease following C. jejuni infection. Overall, this dissertation contains identification of a novel mechanism of action of L. murinus. The results provide insights for the identification of novel targets to treat C. jejuni disease without using antibiotics. This dissertation provides a basis for the future studies to further dissect the role of the AHR in the pathogenesis of C. jejuni. Copyright by HUSNAIN AHMED 2022 This thesis is dedicated to my grandmother (late) and my daughters, Khadija and Aima. v ACKNOWLEDGMENTS I am grateful to my advisor Dr. Linda Mansfield for her support throughout my academic career. I am thankful to all my graduate committee members, including Dr. James Pestka, Dr. John LaPres and Dr. Norbert Kaminski. Each member provided me guidance throughout this study. I am eternally grateful to Dr. Norbert Kaminski for allowing me to use his laboratory equipment to generate data for this project. I also extend my thanks to the members of the Kaminski Lab, including Robert Crawford, for his technical assistance. I am obliged to Dr. Srinand Sreevatsan and Dr. Colleen Hegg for their consistent support throughout my graduate studies. My sincere thanks also go to Dr. Julia Bell for her assistance in multiple aspects of this study. Furthermore, I am thankful to the graduate and undergraduate students of Mansfield’s Lab for their support. I am indebted to my wife and to my daughters for their love and support. Furthermore, I am eternally grateful to my father and mother for their love, support, and belief in me to make this work possible. vi TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ x LIST OF FIGURES ..........................................................................................................xi CHAPTER 1: LITERATURE REVIEW. ............................................................................ 1 Campylobacter species disease burden in animals ..................................................... 2 Campylobacter jejuni in chickens: a commensal or a pathogen? ................................ 2 Chickens: a leading cause of C. jejuni transmission to humans .................................. 4 C. jejuni: a human pathogen ........................................................................................ 5 C. jejuni pathogenesis in humans ................................................................................ 6 The current treatment regimen for C. jejuni and its challenges .................................... 8 Alternatives to antibiotics to reduce C. jejuni prevalence ............................................. 9 The classical role of AHR in xenobiotic metabolism .................................................. 12 The emerging role of AHR in the gut immune system ............................................... 13 Sources of AHR ligands in the gut ............................................................................. 14 AHR promotes host resistance against bacterial pathogens ..................................... 15 Does AHR plays a role in promoting host resistance against C. jejuni? .................... 19 Concluding remarks ................................................................................................... 19 Specific Aim 1 ............................................................................................................ 20 Hypothesis ............................................................................................................. 20 Specific Aim 2 ............................................................................................................ 20 Hypothesis ............................................................................................................. 20 REFERENCES .......................................................................................................... 21 CHAPTER 2: LACTOBACILLUS MURINUS ACTIVATES THE ARYL HYDROCARBON RECEPTOR TO ATTENUATE TNF-ALPHA-INDUCED PRO-INFLAMMATORY RESPONSES IN A HUMAN INTESTINAL EPITHELIAL CELL MODEL. ...................... 33 Abstract ..................................................................................................................... 34 Background ............................................................................................................ 34 Methods ................................................................................................................. 34 Results ................................................................................................................... 35 Conclusions ........................................................................................................... 35 Introduction ................................................................................................................ 35 Materials and methods .............................................................................................. 38 Chemicals and Reagents ....................................................................................... 38 Bacterial Culture..................................................................................................... 38 Cell culture ............................................................................................................. 39 Cell treatments ....................................................................................................... 40 Cell viability assay .................................................................................................. 40 Measurement of TEER........................................................................................... 40 vii Quantification of IL-8 protein through Enzyme-Linked Immunosorbent Assay (ELISA) .................................................................................................................. 41 Quantitative reverse transcription PCR .................................................................. 41 Statistical Analysis ................................................................................................. 42 Results....................................................................................................................... 42 Caco-2 monolayers form strong tight junctions when grown on Transwell inserts . 42 TNF-  decreased intestinal epithelial barrier integrity in a concentration-dependent manner ................................................................................................................... 43 TNF-  does not affect the mRNA expression of occludin and tight junction protein 1 (TJP-1) in a concentration dependent manner .................................................... 44 TNF- induced secretion of IL-8 from the intestinal epithelial cells in a concentration-dependent manner .......................................................................... 44 TCDD and FICZ activate the AHR in the Caco-2 cells in a concentration-dependent manner ................................................................................................................... 44 Lactobacillus murinus activate the AHR in MOI dependent manner ...................... 45 AHR activation attenuated TNF- induced gut barrier disruption and pro- inflammatory response in Caco-2 monolayers ....................................................... 45 Discussion ................................................................................................................. 46 APPENDIX ................................................................................................................ 55 REFERENCES .......................................................................................................... 65 CHAPTER 3: CO-INOCULATION OF LACTOBACILLUS MURINUS AND CAMPYLOBACTER JEJUNI ENHANCED COLONIZATION LEVELS OF BOTH TAXA AND CHANGED THE GUT MICROBIOTA LANDSCAPE BUT DID NOT PROTECT BALB/C IL-10-/- MICE FROM DEVELOPING COLITIS. ................................................ 73 Abstract ..................................................................................................................... 74 Background ............................................................................................................ 74 Methods ................................................................................................................. 74 Results ................................................................................................................... 75 Conclusions ........................................................................................................... 75 Introduction ................................................................................................................ 76 Materials and methods ........................................................................................... 78 Animal Handling Protocols ..................................................................................... 78 Experimental Design .............................................................................................. 79 Growing bacterial cultures and their inoculation in mice ........................................ 80 Lactobacillus murinus............................................................................................. 80 Campylobacter jejuni.............................................................................................. 81 Necropsy, sample collection, and gross histopathological scoring ......................... 81 Histological staining of ICC junction ....................................................................... 83 Histopathological Scoring ....................................................................................... 84 16S sequence analysis .......................................................................................... 84 Semi-quantitative scoring for L. murinus colonization ............................................ 85 Semi-quantitative scoring for C. jejuni colonization ................................................ 86 Statistical analysis .................................................................................................. 86 Results....................................................................................................................... 86 viii Prophylactic inoculation of L. murinus did not prevent C. jejuni induced gross pathology in mice ................................................................................................... 86 Prophylactic inoculation of L. murinus did not reduce histopathologic lesions in the ileocecocolic junctions............................................................................................ 87 L. murinus pre-enrichment increased C. jejuni colonization in the cecum .............. 88 Increased L. murinus colonization in the test group ............................................... 88 C. jejuni infection changed the gut microbiota composition .................................... 89 Discussion ................................................................................................................. 90 APPENDIX ................................................................................................................ 96 REFERENCES ........................................................................................................ 105 CHAPTER 4: SUMMARY AND FUTURE DIRECTIONS. ............................................ 112 REFERENCES ........................................................................................................ 118 ix LIST OF TABLES Table 1) Groups of mice used in this study ................................................................... 79 x LIST OF FIGURES Figure 2.1) Resistance readings of Caco-2 monolayer .............................................. 56 Figure 2.2) Resistance readings of HT-29-MTX-E12 monolayer ............................... 57 Figure 2.3) TNF- decreases epithelial barrier integrity in a concentration-dependent manner………………………………………………………………………………………….58 Figure 2.4) TNF- does not affect the mRNA expression of Occludin and TJP-1 ...... 59 Figure 2.5) TNF- induces IL-8 secretion from Caco-2 monolayers in a concentration- dependent manner ........................................................................................................ 60 Figure 2.6) AHR ligands increase CYP1A1 transcription in a concentration-dependent manner………………………………………………………………………………………….61 Figure 2.7) L. murinus upregulates CYP1A1 transcription in MOI dependent manner………………………………………………………………………………………….62 Figure 2.8) AHR activation attenuates epithelial barrier disruption ............................ 63 Figure 2.9) AHR activation attenuates pro-inflammatory response ............................ 63 Figure 3.1) Gross pathology scoring .......................................................................... 97 Figure 3.2) Histopathological scoring of the ileocecocolic junctions ........................... 98 Figure 3.3) Hematoxylin and Eosin-stained (H&E) sections of the ileocecocolic junctions……..………………………………………………………………………………….99 Figure 3.4) C. jejuni colonization scores .................................................................. 100 Figure 3.5) L. murinus colonization scores............................................................... 101 Figure 3.6) 16S rRNA gene sequence analysis of fecal DNA .................................. 102 Figure 3.7) Shannon-Diversity Index ........................................................................ 103 Figure 3.8) Principal Component Analysis ............................................................... 104 xi CHAPTER 1: LITERATURE REVIEW. 1 Campylobacter species disease burden in animals Campylobacter spp. are ubiquitous and colonize the gut of many animals and birds such as cattle, pigeons, water-fowls, and crows [1]. The Campylobacter spp. constitute a diverse group of bacteria, some of which cause severe diseases in humans and animals; others reside in the gut as commensals or lack clear association with overt disease manifestation [2]. A cross-sectional study described the prevalence of Campylobacter spp. in various animal host species. The prevalence in cattle, sheep, ducks, and pigeons ranged from 21.9% to 27.8% [3]. Campylobacter spp. causes disease in cattle, goats, and sheep. The two main Campylobacter spp. associated with disease in ruminants are C. fetus subsp. fetus and C. fetus subsp. venerealis [4]. In sheep and goats, C. fetus subsp. fetus causes abortion, whereas C. fetus subsp. venerealis causes infertility and embryonic mortality in cattle [4]. Chickens were found to have the highest prevalence (41%) of Campylobacter spp. [3]. In chickens Campylobacter spp. (such as C. jejuni) are thought to reside as a commensal [5]; however, recent literature has challenged this paradigm by reporting that in susceptible breeds of chicken C. jejuni damaged gut mucosa and induced a prolonged inflammatory response in the gut [6]. Moreover, C. jejuni infection caused diarrhea in the susceptible breeds of chicken [6]. Campylobacter jejuni in chickens: a commensal or a pathogen? Early studies reported that C. jejuni infection does not lead to the development of clinical disease in chickens [7, 8]. This observation led scientists to hypothesize that C. jejuni resides as a commensal in the chicken gut [9]. When chickens were experimentally infected with clinical C. jejuni strains isolated from patients who developed enteritis, none 2 of the infected chickens developed enteritis, nor did they manifest any clinical signs of disease [8]. Similarly, in another study, 9-day old chickens were inoculated with C. jejuni by crop gavage. No clinical signs of illness or gross pathological lesions were found in any of the infected groups at necropsy despite successful colonization of C. jejuni in the chicken gut [10]. Since then, various studies have reported the ability of C. jejuni to colonize the chicken gut, but the persistent lack of clinical disease manifestation in chickens, i.e., enteritis and bloody diarrhea, led to development of the paradigm that C. jejuni acts as a commensal in chickens [7, 11]. Recent studies have challenged this long-standing paradigm of C. jejuni commensalism in the chicken. Suzanne et al. [6] infected different commercial breeds of chicken having varying abilities to gain weight. It was found that breeds having a faster weight gain ability developed diarrhea and inflammatory response when challenged with C. jejuni. Whereas the breed having slower weight gain ability had no clinical signs of the disease and developed a less pronounced inflammatory response as compared to the fast-growing breed. Furthermore, they found that the slow-growing breed had a significantly higher induction of IL-10 (50-fold higher) as compared to the fast-growing breed. Xi et al. [12] also reported that the genetics of the chicken plays an important role in determining the resistance and susceptibility of different chicken breeds to C. jejuni infection. Day-old broilers from two different parental lines (Line A and Line B) were infected with C. jejuni. On days 7 and 14 post-infection, the cecal contents from both lines 3 were cultured for C. jejuni. It was found that Line A (resistant breed) had significantly fewer C. jejuni colony forming units in their ceca as compared to Line B (susceptible breed). Also, gene expression profiling of Line A (resistant breed) and Line B (susceptible breed) indicated that genes responsible for host defense responses were upregulated in Line A as compared to Line B. Interestingly, circadian rhythm genes were also significantly upregulated in Line A (resistant breed) birds as compared to Line B (susceptible breed) birds [13]. These results indicate that C. jejuni does not merely reside as a commensal in the chicken gut, rather is fully capable of causing enteritis in a susceptible host. Chickens: a leading cause of C. jejuni transmission to humans Over the past decades, advances in genetics and nutrition have enabled large- scale poultry production. Per capita availability of chicken meat has significantly increased with the advent of large-scale poultry production [14]. Unfortunately, some pathogens, such as C. jejuni, can also spread more easily between birds in large-scale poultry production because more birds are housed in these intensive poultry farming units [15]. Furthermore, during processing, chicken meat can get contaminated with C. jejuni present in intestinal contents [16]. Scalding, evisceration, washing, and chilling are the major cross-contamination points during chicken slaughter processing [17]. Chicken contaminated during processing is the principal source of C. jejuni transmission to humans [18, 19]. However, C. jejuni can also infect humans through contaminated water, milk, other dairy products and by direct contact with infected animals [5]. 4 C. jejuni: a human pathogen C. jejuni is a leading cause of foodborne gastroenteritis in the US, with an incidence rate of 13.6 diagnosed cases per 100,000 individuals [20]. Because many sporadic human infections go undiagnosed, it is predicted that the actual incidence is much higher, with estimates of approximately 1.5 million cases of campylobacteriosis in the US per annum [20]. C. jejuni is a broad host range pathogen residing as a commensal in the gastrointestinal tract of many agricultural animals [5]. Thus, human infections most commonly occur from the consumption of contaminated water or animal products [21, 22]. The most frequent cause of Campylobacter infection in the US is the consumption of chicken contaminated during processing [21]. Campylobacteriosis (a disease in humans) is characterized by mild to severe bloody diarrhea, abdominal pain, fever, and severe intestinal inflammation that lasts from 7-10 days followed by resolution [5]. C. jejuni infection can also lead to the autoimmune disease Guillain- Barré syndrome (GBS), a paralytic illness resulting from an immune system attack on the peripheral nervous system, which results in flaccid paralysis [23]. GBS has been documented to occur due to molecular mimicry between oligosaccharide motifs on the outer surface of C. jejuni and gangliosides on the surface of peripheral nerves [24]. Antibodies especially of the IgG1 subclass are generated against the lipooligosaccharides of certain C. jejuni strains that also bind to GM1 and GD1a gangliosides on peripheral nerves which precipitates complement-mediated damage [25, 26]. Other autoimmune sequelae to C. jejuni infection include reactive arthritis, and Reiter’s syndrome [27, 28]. 5 C. jejuni pathogenesis in humans In the intestinal epithelial tract, the mucus layer is considered the first line of defense against enteric pathogens. The average thickness of mucus in the gut is 700 m and it consists of mucin glycoproteins, lysozymes, defensins, and antibodies that function in the clearance of enteric pathogens [29, 30]. However, certain gut pathogens and C. jejuni, in particular, have adapted sophisticated mechanisms to breach the protective mucus barrier and establish colonization. For instance, C. jejuni has shown chemotactic motility towards mucins [31]. Once it encounters this layer, the fluid nature of the mucus aids in C. jejuni motility towards intestinal epithelial cells powered by the flagella [32, 33]. All of these factors facilitate C. jejuni colonization in the mucus of the intestinal epithelial tract where it can persist for long periods of time. After establishing itself in the mucus, C. jejuni then invades the intestinal epithelial cells (IECs). IECs serve as a barrier between the mucus layer and intestinal immune cell populations residing in the lamina propria [34]. This barrier property of the intestinal epithelial cells is mainly due to the presence of junctional complexes between the epithelial cells. These junctional complexes consist of specialized intercellular structures, formed by tight junctions, adherens junctions, and desmosomes. Tight junctions between cells consist of proteins including claudins, occludin, and junctional adhesion molecules, whereas adherens junctions mainly consist of cadherin molecules [35]. Thus, tight junctions between neighboring intestinal epithelial cells are formed by the assembly of multiple proteins that controls the permeability of ions, nutrients and water [36]. 6 “Leaky gut” syndrome is often considered a hallmark of human campylobacteriosis, facilitating the translocation of C. jejuni from inflamed intestines to deeper body tissues [37]. C. jejuni has multiple molecular mechanisms to breach the intestinal tight junction barrier. Harrer et al. [38] reported that C. jejuni secretes a serine protease (htrA) that cleaves occludin, thereby facilitating pathogen translocation across the intestinal epithelial barrier. The role of htrA in translocation across the intestinal epithelial barrier was further confirmed by using the htrA knockout mutant (ΔhtrA). ΔhtrA showed a significantly reduced ability to cross the tight junction barrier in-vitro. These results indicate that C. jejuni possesses intricate mechanisms to breach tight junctions in order to invade deeper body tissues [38]. Once C. jejuni reaches the basolateral layer of the IEC’s, it expresses the adhesin CadF to bind to the fibronectin complex to invade IEC’s [39]. Thereafter, C. jejuni expresses a type three secretion system (T3SS) virulence protein CiaD that is required for the invasion of host cells [40]. IEC’s respond to C. jejuni invasion by producing proinflammatory cytokines such as IL-8, also known as a “neutrophil chemotactic factor” as it specifically attracts neutrophils [41-43]. Neutrophils kill bacterial pathogens by phagocytosis, secretion of anti-bacterial proteins, or by releasing NETs (Neutrophil Extracellular Traps) [44]. S. Callahan et al. [45] recently showed that C. jejuni was internalized by neutrophils derived from healthy donors. Moreover, neutrophil-derived proteins such as myeloperoxidase, neutrophil elastase, and lipocalin-2 negatively impacted C. jejuni growth in-vitro. Furthermore, ferrets infected with C. jejuni showed increased neutrophil activation levels in the colon that directly correlated with intestinal 7 inflammation and pathology [45]. These results indicate that where neutrophils function to kill C. jejuni, their increased activation also results in intestinal tissue damage [45]. C. jejuni also invades the antigen-presenting cells residing in lamina propria, such as macrophages and dendritic cells [46]. It was shown that C. jejuni can replicate in human mononuclear cells and induce cytokine secretion including IL-1 and tumor necrosis factor-alpha [47, 48]. C. jejuni can also induce apoptosis in these mononuclear cells by secreting cytolethal distending toxin (CDT) [49]. CDT has DNAase activity and is one of the major virulence factors of C. jejuni [50, 51]. The inflammatory reaction initiated in response to C. jejuni infection is generally considered host protective and helps clear the infection. However, excessive inflammation that is not downregulated can be detrimental to the host [52-54]. Therefore, mice deficient in anti-inflammatory cytokines such as IL-10 are a robust C. jejuni disease model and display clinical features of human campylobacteriosis upon infection [55]. These findings indicate that excessive host inflammation contributes to the pathogenesis of C. jejuni-mediated colitis. The current treatment regimen for C. jejuni and its challenges Treatment options for Campylobacter infection primarily include the use of antibiotics such as azithromycin and ciprofloxacin. However, the over-use of antibiotics has led to the emergence of antimicrobial-resistant C. jejuni strains and has reduced treatment efficacy [56, 57]. In 2015, the National Antimicrobial Resistance Monitoring System (NARMS) reported that 47% of C. jejuni isolates were resistant to tetracycline, whereas 25% of the isolates were resistant to ciprofloxacin [58]. More than 35,000 people 8 die each year in the United States from antibiotic-resistant infections [59]. The Centers for Disease Control and Prevention (CDC) has categorized drug-resistant C. jejuni as a serious public health threat [57, 59]. The development of antimicrobial resistance traits in C. jejuni isolates has augmented the need to develop innovative strategies to treat drug- resistant C. jejuni infections in human populations. Alternatives to antibiotics to reduce C. jejuni prevalence Amid increasing concerns about antibiotic-resistant C. jejuni isolates, there is a dire need to develop alternatives to antibiotics to reduce the C. jejuni bacterial burden in all of its hosts. Probiotics are commonly used in poultry husbandry and human medicine to maintain gut health [60]. A large body of work has been performed in the last two decades to validate probiotics as a means to reduce C. jejuni prevalence in chickens. Lactobacillus spp. (LAB) are the most commonly used commensal bacterial genera to be used as probiotics in poultry and humans [61-63]. Several studies have confirmed that LAB can significantly decrease the growth of C. jejuni in-vitro. Chaveerach et al. [64] described that the growth of C. jejuni was negatively affected by the chicken LAB isolate (P93) in-vitro. It was found that the production of volatile fatty acids (VFA) from the P93 LAB strain was a major mechanism in inhibiting C. jejuni growth in-vitro. Several other studies have confirmed the role of VFA in inhibiting the growth of C. jejuni in-vitro [65-67]. Other mechanisms by which LAB spp. can inhibit the growth of C. jejuni in-vitro, includes the production of hydrogen peroxide [65] and bacteriocins [68]. 9 LAB strains have also been shown to attenuate the ability of C. jejuni to adhere to or invade human intestinal epithelial cells cultured in-vitro. In a recent study by Taha- Abdelaziz et al., [69] the LAB strains (L. salivarius, L. johnsonii, L. reuteri, L. crispatus, and L. gasseri) alone or in combination reduced the ability of C. jejuni to adhere to and invade Caco-2 cells cultured as a confluent monolayer. Moreover, co-incubation of LAB strains alone (except L. reuteri) or in combination reduced the expression of C. jejuni virulence genes, including motility associated genes (flaA, flaB, and flhA) and the gene responsible for invasion (CiaB). These findings indicate that LAB strains can attenuate C. jejuni virulence properties. Similarly, Wang et al. [70] screened seventy-eight LAB strains for anti-Campylobacter activity. They found that L. casei ZL4 was able to reduce the adhesion and invasion of C. jejuni to HT-29 cells. The high concentration of lactic and acetic acid produced by L. casei ZL4 in these adherence invasion assays was responsible for inhibiting C. jejuni growth in-vitro. These results demonstrate that LAB strains possess multiple mechanisms to affect C. jejuni growth, adherence, and invasion capabilities in- vitro. In-vivo effects of LAB strains on reducing Campylobacter spp. colonization in the gut has also been reported [71]. Smialek et al., [72] reported that the addition of selected LAB strains (L. lactis, L. casei, L. plantarum) into chicken feed resulted in decreased Campylobacter spp. colonization in the gut. Furthermore, less Campylobacter spp. carcass contamination was observed in the experimental group given probiotic (LAB strains) as compared to the sham-fed control group. Saint-Cyr [61] also reported similar findings when 107 CFU of L. salivarius SMXD51 were given by oral gavage to broiler 10 chickens followed by oral challenge with C. jejuni. At 35 days of age, the group that was given L. salivarius SMXD51 had a 2.81 log reduced load of C. jejuni in the cecal contents as compared to the control group. Furthermore, the 16S sequence analysis of the chicken gut microbiota revealed that certain bacterial genera were more abundant in the L. salivarius treated group as compared to the control group. A higher abundance of Anaerotruncus, Escherichia and Flavonifractor species were found in the L. salivarius treated group as compared to the control group [61]. In the developed world, chicken contaminated during processing is the principal source of C. jejuni infection in humans [5]. Decreasing the bacterial load of Campylobacter spp. in the chicken can significantly reduce human exposure to C. jejuni. Rosenquist et al. [73] developed a mathematical risk model to assess the human exposure to C. jejuni from processed chicken meat. They found that the incidence of human campylobacteriosis could be reduced 30 times if one obtains a 2 -log reduction of C. jejuni contamination in the chicken carcass. The results of this quantitative risk assessment model are important for effective implementation of prevention methods because probiotic supplementation to chickens has been shown to result in a significant reduction in the C. jejuni bacterial load in the chicken gut and carcass contamination [61, 71, 72]. Probiotics possess multiple mechanisms that function to reduce C. jejuni invasion. For example, LAB strains (L. salivarius, L. johnsonii, L. reuteri, L. crispatus, and L. gasseri) upregulated the phagocytic activity of chicken macrophage-like cells (MQ- 11 NCSU) [69]. LAB strains were also able to upregulate the expression of proinflammatory cytokines (IFN-gamma, IL-1beta, and IL-20p40) in chicken macrophages. Moreover, a combination of L. salivarius, L. reuteri, and L. crispatus was able to upregulate the expression of co-stimulatory molecules CD40, CD80, and CD86 in chicken macrophages [69]. However, the immunomodulatory mechanisms by which probiotics manifest anti- Campylobacter mechanisms are not yet completely understood. Recently Lamas et al. [74] reported a novel mechanism of action for Lactobacillus- based probiotics. They reported that three LAB strains (L. murinus CNCM I-5020, L. reuteri CNCM I-5022, and L. taiwanensis CNCM I-5019) were able to metabolize tryptophan into ligands that activated the aryl hydrocarbon receptor (AHR) to protect mice from colitis. These results suggested a novel mechanism of action of such probiotics in maintaining gut homeostasis by activating AHR. AHR was initially defined for its role in xenobiotic metabolism [75], however, recent literature has identified the emerging immuno-modulatory roles of AHR [76]. Identification of AHR in certain immune cell types has posed the possibility of AHR as a drug target for novel therapeutics against bacterial pathogens [77-79]. The classical role of AHR in xenobiotic metabolism The AHR was first identified due to its binding with toxicant 2,3,7,8- Tetrachlorodibenzo-p-dioxin (TCDD) [75]. Dioxins are a group of chemically related compounds that are environmental pollutants. Dioxins are generated as by-products in the industrial manufacturing of various products such as chlorophenols, herbicides, and 12 pesticides [80]. Apart from dioxin release as industrial by-products, occasional industrial accidents also lead to environmental contamination with dioxins which can cause lethal toxicity in humans and animals. One such incident occurred in Seveso, Italy, where due to an industrial accident, large amounts of TCDD was released into the environment, and thousands of people were exposed to the toxic levels [81]. Epidemiological studies confirmed that this cohort of people living in the TCDD contaminated zones were more susceptible to cancer, diabetes, and chronic obstructive pulmonary disease (COPD) as compared to people living in TCDD free zones [82]. In mammalian cells, TCDD specifically binds to the AHR, which is a ligand- activated transcription factor. In the cytoplasm, the AHR is retained in its inactive form as a complex with heat-shock protein 90 (hsp90) and AHR-interacting protein (AIP/XAP) [83]. Upon binding to its ligand, the AHR translocate to the nucleus and dimerizes with the AHR nuclear translocator (ARNT) and regulates the transcription of a battery of genes, including cytochrome P450 superfamily members, i.e., CYP1A1, CYP1A2, and CYP1B1 [84]. Cytochrome P450 (CYPs) are enzymes that can metabolize xenobiotics by oxidizing them into harmless and excretable metabolites. The CYP enzymes are the most abundant of all the xenobiotic-metabolizing enzymes [85]. Therefore, CYP1A1 is considered a biomarker for AHR activation [86]. The emerging role of AHR in the gut immune system With the discovery of AHR expression in the cells of the immune system, this receptor has received significant attention from immunologists regarding its potential as 13 a therapeutic target for immunomodulation [76]. AHR is heavily expressed at mucosal barrier sites such as lungs, skin, and the gastrointestinal (GI) tract. In the GI tract, AHR is expressed by certain innate and adaptive immune cell types. In innate immune cells, AHR is highly expressed by dendritic cells [87]. Among innate lymphoid cells (ILCs), AHR is expressed by ILC2 [88] and ILC3 [89]. AHR is also expressed by cells of the adaptive immune system, such as T and B cells. AHR is highly expressed by T-helper 17 cells (Th17) [90] and marginally expressed by T-regulatory cells [91]. Furthermore, AHR is also expressed by innate TCR T-cells [76]. However, AHR is not expressed by naïve T-cells, Th1 and Th2 cells [92]. It appears that all B cells express AHR, but certain types of B cells, such as marginal B cells and B1 B cell subsets, have higher AHR expression. The presence of AHR in the vast majority of gut resident immune cells suggest that it may function in a regulatory role and makes it an ideal target for immunomodulation against gut pathogens. Sources of AHR ligands in the gut Aside from synthetic ligands such as TCDD and FICZ, many naturally occurring ligands in the gut also activate AHR. The two most important sources for the production of AHR ligands in the gut arise from diet and the members of the gut microbiome [93]. In the diet, cruciferous vegetables contain high amounts of AHR ligands. Broccoli is a rich source of glucobrassicin, which is cleaved into indole-3-carbinol (I3C) by digestive enzymes. It has been found that I3C activates AHR [94]. Flavonoids are another group of compounds that can interact with AHR. Flavonoids are found in fruits and tea. Most of the flavonoids are AHR antagonists, but some of them can activate AHR, such as diosmin, 14 tamarixetin, tangeritin, and quercetin [95-97]. Curcumin which is found in turmeric can also activate AHR [98]. Furthermore, several dietary plant compounds, such as carotenoids and tryptophan, can induce AHR activation [99]. Gut microbiota is another important source for the production of AHR ligands. This is because some micro-organisms can produce metabolites that can act as AHR ligands [93]. Members of the gut microbiota documented to produce AHR ligands include Enterococcus faecalis [100], Bacteroides spp. [101] and Citrobacter spp. [102]. However, certain probiotic Lactobacillus spp. can also produce AHR ligands, such as L. reuteri [74, 103], L. murinus [74], L. taiwanensis [74] and L. bulgaricus OLL1181 [104]. Zelante et al. [103] described that L. reuteri was able to produce indole-3-aldehyde (I3A) when mice were given an unrestricted supply of tryptophan in the diet. I3A was produced as a by- product of the tryptophan-indole pathway. However, L. johnsonii was not able to metabolize tryptophan to I3A under carbohydrate starvation conditions. These results indicate that not all members of the genus Lactobacillus are able to produce AHR ligands. In another study, supplementation of L. murinus, L. reuteri, and L. taiwanensis were able to protect mice from developing colitis by the activation of AHR [74]. These results suggest that activation of AHR by probiotics can be a potential mechanism to protect against intestinal inflammatory insults. AHR promotes host resistance against bacterial pathogens The role of AHR in promoting host resistance against several bacterial pathogens has been reported. Vorderstrasse et al. [77] described the TCDD (potent AHR agonist) 15 induced protection of C57BL/6 mice against lethal Streptococcus pneumoniae challenge. Mice were given TCDD or vehicle one day prior to the S. pneumoniae challenge. Only 35% of the vehicle-treated mice survived, whereas TCDD treatment increased mouse survival rate to 75%. Furthermore, TCDD treatment significantly reduced the S. pneumoniae bacterial count in the lungs. It was found that the protective effect of AHR activation was not associated with increased inflammatory response in the lungs as proinflammatory cytokines such as IFN-gamma, TNF-alpha, and IL-1 were significantly reduced in the TCDD treated group as compared to the control group. However, the mechanistic basis for AHR-induced protection against a lethal S. pneumoniae challenge remained elusive. The mechanistic basis for AHR-induced protection against bacterial infections [89, 105] began to be revealed when cytokine production was compared between immune cells (isolated from lamina propria) of AHR-/- and AHR+/+ mice. In AHR+/+ mice, the ILC3 produced more IL-22 as compared to ILC3 isolated from AHR-/- [89]. These results indicated that AHR plays an important role in intestinal IL-22 expression. Also, the antimicrobial peptide RegIII produced by mammalian cells was significantly reduced in AHR-/- mice compared to AHR sufficient mice. There was a 100% survival rate in AHR+/+ mice (n=6) when they were given a challenge infection with Citrobacter rodentium. Whereas all of the AHR-/- mice (n=4), when challenged with C. rodentium, succumbed to death by ten days post-infection. However, ectopic expression of IL-22 in AHR-/- mice increased mouse survival rate to 60% upon challenge with C. rodentium. A higher C. rodentium bacterial count was present in the feces of AHR-/- mice as compared to AHR+/+ 16 mice. Furthermore, a significantly reduced C. rodentium bacterial count was detected in AHR-/- mice having ectopic expression of IL-22 [89]. These results indicate that AHR is essential for protection against a gastrointestinal C. rodentium infection. Furthermore, AHR modulates ILC3 in the gut to increase the production of IL-22 that protected mice from lethal C. rodentium infection. Activation of AHR also negatively affects the pathogenesis of Listeria monocytogenes. L. monocytogenes is a gram-positive foodborne pathogen that causes gastroenteritis. The most susceptible population to L. monocytogenes infection include newborns, adults over the age of 65, and people with a weakened immune system [106]. Homozygous AHR-/-mice on C57BL/6J background were more susceptible to L. monocytogenes than heterozygous AHR+/- mice. Upon infection with L. monocytogenes, AHR-/- mice had a higher bacterial load of this bacterium in the spleen and liver as compared to AHR+/-. However, proinflammatory cytokine production in response to L. monocytogenes infection was not affected in AHR-/- mice as serum levels of MCP-1, IFN- gamma, and TNF-alpha were comparable between L. monocytogenes infected AHR-/- and AHR+/- mice. These results demonstrate that AHR activation is required for host resistance against L. monocytogenes infection, which cannot be mediated by cytokine production alone [107]. In another study, Kimura et al. [78] also examined effects of AHR activation on the L. monocytogenes pathogenesis in mice. C57BL/6 WT and C57BL/6 AHR-/- were infected with L. monocytogenes intra-peritoneally. AHR-/- mice succumbed to death at five days 17 post-infection, whereas no significant decrease in survival rate was observed in WT mice infected with L. monocytogenes. A significantly higher L. monocytogenes bacterial load was found in the spleen and liver of AHR-/- mice as compared to the WT mice. Furthermore, AHR activation promoted the survival of macrophages when infected with L. monocytogenes. AHR activation induced the expression of an anti-apoptotic factor in macrophages, thereby preventing macrophage cell death. AHR activation also promoted the production of reactive oxygen species (ROS) in these macrophages, which resulted in L. monocytogenes clearance from the cells [78]. These results shed light on possible mechanisms by which AHR enhances host resistance against bacterial pathogens. The effect of AHR activation against Clostridium difficile has also been reported [108]. Two groups of C57BL/6 mice were either given regular chow or custom chow having 1000 ppm of I3C. I3C is a dietary AHR agonist which is naturally found in cruciferous vegetables [93]. 66% of the mice survived C. difficile infection, which were given custom chow having I3C. In contrast, only 20% of the mice survived C. difficile infection when given regular chow. Mice given I3C in the diet had an increased number of FoxP3+ CD4+ T-regulatory cells, ILC3, and neutrophils in the cecal lamina propria as compared to the mice on regular chow. Furthermore, mice fed I3C in the diet also had a significantly reduced translocation of C. difficile to the spleen and lungs as compared to the controls. These results indicate that activation of AHR through dietary components can be a novel therapy for the amelioration of C. difficile disease that is often resistant to all available antibiotics. 18 Does AHR plays a role in promoting host resistance against C. jejuni? There is a critical knowledge gap in understanding of the direct effects of AHR activation during C. jejuni mediated disease. Elucidating the role of AHR activation in C. jejuni pathogenesis may lay the foundations for enhancing the host resistance mechanisms to ameliorate C. jejuni disease without the use of antibiotics. It also holds potential for decreasing the significant tissue damage that can accompany infection with this foodborne pathogen. If AHR activation plays an important role in limiting C. jejuni colonization it may also be important in blocking the initiation of the autoimmune consequences of this pathogen. Concluding remarks With the advent of antimicrobial-resistant traits in bacterial pathogens such as C. jejuni [59], there is a dire need to develop alternatives to antibiotics. Five decades after the identification of AHR, its important immunological roles have started to emerge. Given the established role of AHR against certain human bacterial pathogens such as S. pneumoniae [77], L. monocytogenes [107] and C. difficile [108], it is plausible to determine the effect of AHR activation on other human bacterial pathogens such as C. jejuni. AHR can be a novel drug target to treat or ameliorate human bacterial diseases (such as campylobacteriosis) without the use of antibiotics. The probability of AHR as a therapeutic target against C. jejuni prompts new research questions, such as 1) What will be the effect of AHR activation on C. jejuni pathogenesis? 2) How does probiotic supplement capable of AHR activation affect C. 19 jejuni disease? Answers to these questions may guide future research efforts to discover novel AHR targeted therapeutics against C. jejuni and disease it causes. To know the answers to the afore-mentioned questions, this study was conducted with the following aims. Specific Aim 1 To determine the effect of AHR activation on inflammation and intestinal epithelial barrier integrity in an in-vitro model of colitis. Hypothesis Activation of AHR by probiotic or defined AHR ligands will attenuate pro- inflammatory responses and epithelial barrier damage in a cell model of colitis. Specific Aim 2 To determine the effect of probiotic supplementation on C. jejuni disease in a mouse model. Hypothesis Inoculation of L. murinus having AHR activation potential will attenuate C. jejuni induced colitis in IL-10-/- mice. 20 REFERENCES 21 REFERENCES 1. Authority, E.F.S., E.C.f.D. Prevention, and Control, The European Union one health 2018 zoonoses report. EFSA Journal, 2019. 17(12): p. e05926. 2. Scallan, E., et al., Foodborne illness acquired in the United States—major pathogens. Emerging infectious diseases, 2011. 17(1): p. 7. 3. 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Shi, L.Z., et al., The aryl hydrocarbon receptor is required for optimal resistance to Listeria monocytogenes infection in mice. Journal of immunology (Baltimore, Md. : 1950), 2007. 179(10): p. 6952-6962. 108. Julliard, W., et al., Amelioration of Clostridium difficile Infection in Mice by Dietary Supplementation With Indole-3-carbinol. Ann Surg, 2017. 265(6): p. 1183-1191. 32 CHAPTER 2: LACTOBACILLUS MURINUS ACTIVATES THE ARYL HYDROCARBON RECEPTOR TO ATTENUATE TNF-ALPHA-INDUCED PRO-INFLAMMATORY RESPONSES IN A HUMAN INTESTINAL EPITHELIAL CELL MODEL. 33 Abstract Background Anti-tumor necrosis factor-alpha (anti-TNF-) therapy is an established treatment modality for inflammatory bowel disease (IBD). Yet up to 30% of patients do not respond to anti-TNF- therapy (primary non-responders), and almost 50% of responders lose clinical efficacy over time (secondary non-responders). A potential alternative to anti-TNF-  therapy is the use of novel probiotics capable of attenuating the damaging effects of TNF- on the intestinal epithelium. We hypothesized that Lactobacillus murinus attenuates TNF- induced pro-inflammatory responses in human intestinal epithelial cell model of colitis in vitro by activating a novel transcription factor, aryl hydrocarbon receptor (AHR). Methods We used Caco-2 cells grown on Transwell inserts to model the human intestinal epithelial barrier. To develop an in vitro model of colitis, Caco-2 monolayers were treated with increasing concentrations of TNF-. Caco-2 monolayers were also treated with L. murinus or defined ligands (exogenous or endogenous) to determine the activation of AHR in Caco-2 cells. Next, we pre-treated the Caco-2 cells with either L. murinus or defined AHR ligands for 12 hours. After pre-treatment, Caco-2 monolayers were then stimulated with TNF- for 24 hours. Finally, IL-8 protein and transepithelial electrical resistance (TEER) were quantified as endpoints to determine the pre-treatment efficacy. 34 Results TNF- stimulation decreased TEER and induced the secretion of IL-8 in a concentration-dependent manner. AHR was significantly activated in monolayers treated with L. murinus or defined AHR ligands. Furthermore, pre-treating the monolayers with L. murinus or with endogenous AHR ligand attenuated the TNF- induced pro-inflammatory response and decreased epithelial barrier disruption. Conclusions These results indicate that TNF- induced damage to the gut wall can be attenuated using novel probiotics such as L. murinus. These data suggest that AHR could be a novel drug target for treating IBD. Keywords: IBD, probiotics, inflammation. Introduction Inflammatory bowel disease (IBD) is an umbrella term used to describe the chronic inflammatory disorders of the digestive tract [1]. The two major forms of IBD are Crohn’s Disease (CD) and Ulcerative Colitis (UC) [2]. It is estimated that 1 in 209 adults in the US currently suffers from some form of IBD. There is a high incidence of IBD as 70,000 new patients are diagnosed with IBD each year in the US [3]. CD causes inflammation in the whole or any part of the gastrointestinal (GI) tract whereas, UC causes inflammation in the colon. Furthermore, CD and UC differ in their signs and symptoms; as symptoms such as pain in the lower right abdomen and thickening of the bowel wall are mainly associated 35 with CD whereas, symptoms such as pain in the lower left abdomen, diarrhea, weight loss, and rectal bleeding are primarily associated with UC [4]. Furthermore, certain gut bacterial pathogens such as Campylobacter jejuni have been associated with initiation of the pathogenesis of IBD [5, 6]. Current treatment options for IBD include the use of aminosalicylates, immunosuppressants, and monoclonal antibodies [7]. The aminosalicylates maintain remission in IBD by inhibiting the upregulation of leukocyte adhesion molecule; thereby, preventing the recruitment of leukocytes in the inflamed bowel [8]. The use of monoclonal antibodies has revolutionized IBD treatment. Infliximab was the first FDA-approved monoclonal antibody against TNF- for the treatment of IBD [9]. Infliximab targets TNF-  which is one of the key cytokines that is upregulated in IBD [10]. At present, anti-TNF therapy is an established treatment modality for IBD. Yet up to 30% of patients do not respond to anti-TNF- therapy (primary non-responders), and almost 50% of responders lose clinical efficacy over time (secondary non-responders). Moreover, numerous safety concerns, including increased risk for opportunistic infections, are associated with long-term use of anti-TNF- agents [11]. Additionally, many of these IBD medications have side effects such as headache, nausea, vomiting and diarrhea [4]. Increased incidence of these medicinal side-effects reduces patient compliance and thus treatment efficacy. Side effects and the safety concerns of current IBD therapy have driven the need to develop innovative strategies to treat IBD. 36 Many factors play a role in the onset of IBD such as genetic predisposition, environmental factors and perturbations in the intestinal microflora [12]. For instance, mutations in the NOD2 gene are associated with increased susceptibility to CD [13]. Previous studies have reported an association between dysbiosis of the gut microbiome and IBD. Manichanh et al. [14] reported a decreased diversity of the bacterial phylum Firmicutes in the stool samples of patients suffering from CD. Ott et al. [15] analyzed the mucosa-associated colonic microflora of patients suffering from active IBD. A reduced bacterial diversity was found in the IBD patients as compared to healthy controls. All of these results suggest a role for the gut microbiota in the development of IBD. Gut microbiota dysbiosis and disruption in mucosal immunity have been described as triggers for IBD [16]. Many scientists have suggested employing probiotics to restore the gut microbial balance and attenuate the excessive inflammatory response at the gut epithelial surface as a novel approach to treating IBD. The clinical efficacy of specific probiotic preparations has been reported in patients with mild to moderately active UC [17]. However, the mechanisms by which probiotics induce protection against some forms of IBD are not yet completely understood. In this study we report that Lactobacillus murinus (a putative probiotic strain) attenuates TNF- induced inflammatory insults in human intestinal epithelial cells by activating a novel transcription factor. We hypothesized that L. murinus attenuates TNF-  induced pro-inflammatory responses in the human intestinal epithelial cells by activating the AHR. AHR is a ligand-activated transcription factor that belongs to the basic-helix- 37 loop-helix family of transcription factors [18]. Caco-2 cells were grown on Transwell inserts to model the human intestinal epithelial barrier. The monolayers were pre-treated with either L. murinus or with defined AHR ligands (exogenous or endogenous) or with sham treatments. After pre-treatment, the cells were then stimulated with TNF-. The results indicate that TNF- induced pro-inflammatory responses and damage to the gut wall can be attenuated using L. murinus or an endogenous AHR ligand. The results from this study identify a novel mechanism of action for probiotics to attenuate TNF- induced damage to the gut wall. Materials and methods Chemicals and Reagents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in dimethyl sulfoxide (DMSO) was a kind gift from Dr. Norbert Kaminski, Michigan State University. 6-formylindolo[3,2-b] carbazole (FICZ) was purchased from Sigma-Aldrich (St. Louis, MO), Catalog no. SML1489. DMSO (Catalog no. D8418) was also purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human TNF- protein (Catalog # 210-TA-005) was purchased from R&D systems (Minneapolis, MN). Bacterial Culture L. murinus bacterial strain was grown on De Man, Rogosa and Sharpe (MRS) agar (Neogen, Lansing, MI). The plates were incubated at 37C under microaerophilic conditions (5% CO2) for 48 hours. Bacterial colonies were then suspended in sterile PBS 38 (Millipore-Sigma, Catalog no. 806552) and OD600 was measured using spectrophotometry. Co-relation between OD600 reading and colony-forming units (CFU)/ml was made by plating serial dilutions on MRS agar and enumerating the colonies. Next, the L. murinus colonies were suspended in sterile PBS, adjusted to OD600 of  1 (5  106 CFU/ml) and then added to cell culture medium containing the Caco-2 cells. Cell culture Caco-2 cells were purchased from ATCC (Gaithersburg, MD, USA) and were used between passages 6 to 10. The Caco-2 cells were cultured with Minimum Essential Medium Eagle (Millipore-Sigma, Catalog no. M2279), supplemented with 20% Fetal Bovine Serum (Rocky Mountain Biologics, Catalog no. FBS-CBT), 1% L-glutamine (Thermofisher, Catalog no. 25030-081), and 1% MEM Non-essential amino acids solution (Thermofisher, Catalog no. 11140050). For the undifferentiated use of Caco-2 cells, the cells were cultured on 24 well tissue culture plates (Alkali Scientific, Catalog no. TPN 1024) at the density of 5  104 cells per well. The cells were given treatments upon reaching 90% confluence. HT-29-MTX-E12 cells were a kind gift from Dr. Leslie Bourquin at Michigan State University. E12 cells were originally purchased from Sigma-Aldrich (St. Louis, MO) and were used between 51 – 60 passages. The HT-29-MTX-E12 cells were cultured using Dulbecco’s Modified Eagle Medium (DMEM, GibcoTM, Catalog # 11995073) with high glucose (4500 mg/L) and sodium pyruvate (110 mg/L). The cells were supplemented with 39 10% Fetal Bovine Serum (Rocky Mountain Biologics, Catalog no. FBS-CBT), 1% L- glutamine (Thermofisher, Catalog no. 25030-081), and 1% MEM Non-essential amino acids solution (Thermofisher, Catalog no. 11140050). Cell treatments All treatments were given for 12 hours except for TNF- treatment which was given for 24 hours. Caco-2 monolayers were treated with increasing concentrations of TCDD (0.1 nM, 1 nM and 10 nM) and FICZ (1 nM, 10 nM and 100 nM) as a positive control to activate AHR. Vehicle control cells were treated with DMSO having a final concentration of 0.02% in cell culture medium. Cell viability assay Caco-2 cells were treated with increasing doses of TNF- (0.1, 1 and 10 ng/ml), TCDD (0.1 nM, 1 nM and 10 nM) and FICZ (1 nM, 10 nM and 100 nM) for 6 hours. Lactate dehydrogenase (LDH) was measured using CytoTox 96® non-radioactive cytotoxicity assay kit (Catalog # G1780) by Promega (Madison, WI). Percent cytotoxicity was calculated according to kit protocol. Measurement of TEER Caco-2 cells were cultured on Transwell inserts at the density of 5  104 cells per Transwell. 6.5 mm Transwell inserts were purchased from Corning Life Sciences (Catalog no. 3470), having 0.4 um pore size on the polyester membrane. TEER was measured using an EVOM2 epithelial volt/ohm meter (World precision instruments, Sarasota, FL). 40 Once the TEER reached 500 ohms/cm2 the monolayers were considered to have formed tight junctions. Based on preliminary trials on average it took between 16 – 21 days for the monolayers to form tight junctions in Caco-2 monolayers. Quantification of IL-8 protein through Enzyme-Linked Immunosorbent Assay (ELISA) Cell culture medium was collected from the basal chamber of the Transwell inserts after treating monolayers with TNF- for 24 hours. IL-8 protein was quantified using Invitrogen IL-8 human ELISA kit (Catalog # BMS 204-3) according to the manufacturer’s instructions. Optical density (OD) was measured at 450 nm on a Bio-Tek microplate reader (Winooski, VT). The concentration of the samples was calculated using the standard curve generated from the known controls. Quantitative reverse transcription PCR Total RNA was isolated using an Qiagen RNeasy mini kit. The samples were processed according to the manufacturer’s instructions. The RNA purity was determined by measuring absorbance ratios using a NanoDrop ND-1000 spectrophotometer (Wilmington, DE). The samples having A260/280 absorbance ratio of ~ 2.0 and A260/230 absorbance ratio ~ 2.2 were considered pure for RNA. 300 ng of total RNA was used for cDNA synthesis using a high-capacity cDNA reverse transcription kit (Catalog # 4368814) by Applied BiosystemsTM (Waltham, MA). TaqManTM Universal PCR Master Mix (Catalog # 4304437) was used for qRT-PCR. Furthermore, TaqManTM gene expression assays for human CYP1A1 (Hs00153120-m1, Catalog # 4453320), 18S rRNA (Hs03003631-g1, 41 Catalog # 4448489), tight junction protein 1 (Hs01551871-m1, Catalog # 4448892) and occludin (Hs00170162-m1, Catalog # 4453320) were used for specific gene amplifications. qRT-PCR measurements were made on QuantStudioTM 3 Real-Time PCR machine. Thermal cycling conditions consisted of initial denaturation at 95 C for 10 minutes followed by 40 cycles of denaturation at 95 C for 15 seconds and annealing at 60 C for 1 minute. The fold-change in gene expression was calculated using ∆∆ Ct method [19]. Statistical Analysis GraphPad Prism version 9.2.0 was used to perform statistical analysis and to prepare graphs. The data is displayed as Mean ± S.E.M in all graphs. The Kolmogorov– Smirnov test was used to test for the normality of data. For the comparison between two groups, student’s t-test was used. For the comparison between more than two groups one-way analysis of variance (ANOVA) was performed. Bartlett’s test was used to determine the homogeneity of variance across samples before using ANOVA. The P values between different groups were summarized as ns: P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Results Caco-2 monolayers form strong tight junctions when grown on Transwell inserts We initially selected Caco-2 [20] and HT-29-MTX-E12 [21] cell lines to model human gut barrier in vitro. The monolayers for both cell-lines were grown separately on 42 Transwell inserts for 21 days. To access the formation of tight junctions TEER was measured between the apical and basolateral compartments of the Transwell insert. 500 ohms per cm2 was considered a threshold for the formation of tight junctions [22]. Caco- 2 cells formed strong tight junctions as their TEER was significantly above 500 ohms per cm2 by day 21 (Fig 2.1). However, HT-29-MTX-E12 cells failed to form strong, tight junctions as their average TEER was  40 ohms per cm2 by day 21 (Fig 2.2). Therefore, we chose the Caco-2 cell line to model the human intestinal epithelial barrier in vitro based on their ability to form strong tight junctions when grown on Transwell inserts. TNF-  decreased intestinal epithelial barrier integrity in a concentration- dependent manner We then determined the concentration-dependent effect of TNF- on the resistance readings of Caco-2 monolayers. Increasing concentrations of TNF- caused a concentration-dependent decrease in relative TEER at 24 hours (Fig 2.3). Groups that were given 0.1 and 1 ng/ml of TNF- had a relative change in TEER of -9.81% and - 36.77%, respectively. The highest decrease in relative TEER was observed in the group given 10 ng/ml of TNF- (-55.76%). However, there was an increase in relative TEER in the control group. These results indicate that TNF- decreases intestinal epithelial barrier integrity concentration-dependent. 43 TNF-  does not affect the mRNA expression of occludin and tight junction protein 1 (TJP-1) in a concentration dependent manner The expression of mRNA for occludin and TJP-1 was determined by qRT-PCR with 18S rRNA as an internal control. Results showed that increasing concentrations of TNF- does not affect the mRNA expression of occludin (Fig 2.4A) and TJP-1 (Fig 2.4B). TNF- induced secretion of IL-8 from the intestinal epithelial cells in a concentration-dependent manner Next, we sought to determine the concentration-dependent effect of TNF- on the secretion of IL-8 from Caco-2 monolayers. Caco-2 monolayers were treated with 10-fold increasing concentrations of TNF- (0.1, 1 and 10 ng/ml) for 24 hours. No IL-8 protein was detected in the negative control group (0 ng/ml of TNF-) and cells treated with a 0.1 ng/ml concentration of TNF-. However, there was a significant induction of IL-8 protein in groups treated with 1 and 10 ng/ml of TNF- (Fig 2.5). These results indicate that TNF-  induces the secretion of IL-8 protein from Caco-2 cells in a concentration-dependent manner. TCDD and FICZ activate the AHR in the Caco-2 cells in a concentration- dependent manner After developing an in vitro model of colitis by stimulating Caco-2 cells with TNF- , we next asked if AHR can be activated by its defined exogenous and endogenous ligands in the Caco-2 cell model in vitro. We selected TCDD as it is a well-characterized exogenous synthetic agonist of the AHR [23]. Furthermore, we selected FICZ produced 44 by tryptophan under ultra-violet radiation because it is the most potent endogenous ligand of AHR [24]. After treating the Caco-2 cells for 12 hours with increasing concentrations of TCDD (0.1 nM, 1 nM and 10 nM) and FICZ (1 nM, 10 nM and 100 nM), we then quantified the mRNA of the CYP1A1 gene. CYP1A1 has been shown to be a reliable biomarker for AHR activation [25]. Our results showed that TCDD and FICZ activated AHR in a concentration-dependent manner (Fig 2.6). Lactobacillus murinus activate the AHR in MOI dependent manner After determining that AHR can be stimulated in the Caco-2 cells by its defined agonists; we then sought to determine if L. murinus can activate the AHR in Caco-2 cells. L. murinus was suspended in PBS and the OD600 was measured to estimate the colony forming units per ml. The inoculum was then plated on MRS agar after limiting dilution assay to determine the absolute CFU. Caco-2 monolayers were stimulated with L. murinus in increasing multiplicity of infection (MOI). After 24 hours of stimulation, we then quantified the mRNA of the CYP1A1 gene. The results showed that L. murinus increased AHR activation in an MOI dependent manner (Fig 2.7). AHR activation attenuated TNF- induced gut barrier disruption and pro- inflammatory response in Caco-2 monolayers Next, we asked if AHR activation can attenuate TNF- induced decrease in TEER and IL-8 secretion in Caco-2 monolayers. To determine this, we first activated the AHR in the Caco-2 monolayers by pre-treating with TCDD (10 nm), FICZ (100 nm) or L. murinus (MOI 1:100). After 12 hours of pre-treatment the monolayers were then 45 stimulated with TNF- (10 ng/ml) for a period of 24 hours. Relative TEER percentage and IL-8 protein were quantified as a measure of efficacy of the pre-treatment. L. murinus pre-treatment significantly attenuated the TNF- induced TEER disruption. However, pre-treating the monolayers with TCDD or FICZ was not effective in attenuating the TNF- induced epithelial barrier damage (Fig 2.8). These results showed that L. murinus pre-treatment was significantly effective in attenuating the TNF- induced damage to the intestinal epithelial barrier function in vitro. There was a significant increase in IL-8 production upon the treatment of monolayers with TNF- (10 ng/ml). However, L. murinus pre-treatment, significantly reduced the secretion of IL-8 upon TNF- stimulation (P > 0.0001). Furthermore, activating the AHR by FICZ also significantly reduced IL-8 production upon TNF- stimulation (P = 0.0176). However, pre-treating the monolayers with TCDD did not reduce the IL-8 secretion when stimulated with TNF- (Fig 2.9). These results suggest that activation of the AHR by some but not all AHR ligands attenuate the TNF- induced pro- inflammatory response in the human intestinal epithelial cells. Thus, these results also suggest that these protective effects of AHR activation against TNF- induced pro- inflammatory response are ligand dependent. Discussion The incidence of IBD in the US is high with estimates of 1 in 209 people suffering from some form of IBD [3]. Immunosuppressants and anti-TNF- drugs are the current 46 treatment modalities for IBD; however, their associated safety concerns along with increasing risks for opportunistic infections has augmented the need to develop innovative treatments for IBD. Yet, this is a difficult task because the pathogenesis of IBD is complex where genetics (mutations in NOD2), epigenetics, host immune system factors such as detrimental mucosal immune responses or imbalances between pro- inflammatory and anti-inflammatory cytokines, and alterations in gut microbiota leading to dysbiosis have all been considered as primary factors for IBD [26]. One novel approach for treating IBD that has been recently sought is identification and understanding of the role of commensal members of the gut microbiota [27]. Several studies have reported alterations in the human gut microbiota (such as lower abundance of firmicutes) and dysbiosis in patients suffering from IBD [14, 27, 28]. Frank et al., [28] performed rRNA sequence analysis of intestinal tissue samples obtained from patients suffering from IBD. They found a decreased abundance of bacteria belonging to phyla Firmicutes and Bacteroidetes in patients suffering from IBD as compared to healthy controls. They also showed that bacteria belonging to phyla Actinobacteria and Alphaproteobacteria were increased in IBD patients as compared to healthy controls. These results indicate that alterations in gut microbiota mediating dysbiosis play a role in the pathogenesis of IBD, but more work is needed to study bacterial taxa at a phylogenetic level where functional attributes can be identified. Given the role of dysbiosis in the pathogenesis of IBD, therapeutics employing the commensal gut microbiota such as probiotics, are a logical approach for treating IBD. 47 Many studies have reported little or no beneficial effect of probiotics in treating CD [29, 30]. However, as opposed to CD, studies have shown efficacy of certain probiotics in patients suffering from UC [31]. VSL # 3 is one of the most studied probiotic preparation in treating UC. VSL # 3 consists of four strains of Lactobacilli (L. paracasei, L. plantarum, L. acidophilus, and L. delbrueckii subsp. bulgaricus), three strains of Bifidobacteria (B. longum, B. breve, and B. infantis), and one strain of Streptococcus salivarius subsp. thermophilus (S. thermophilus). Miele et al., [32] conducted a one-year-long, placebo-controlled, double-blind study to test the efficacy of VSL # 3 on induction and maintenance of remission in children with active UC. It was found that 92.8% of patients achieved remission upon treatment with VSL # 3 and IBD therapy whereas, only 36.4% of patients achieved remission upon treatment with placebo and IBD therapy. In another study, a randomized clinical trial was conducted to test the effectiveness of Lactobacillus reuteri ATCC 55730 in patients suffering from active distal UC [33]. Patients suffering from active distal UC received a rectal enema of L. reuteri ATCC 55730 or placebo for 8 weeks. Investigators found that clinical and endoscopic score for UC (Mayo score) decreased significantly in the group that was given L. reuteri as compared to the placebo- controlled group. The mucosal expression level of anti-inflammatory cytokine (IL-10) was increased significantly in the L. reuteri-treated group as compared to the placebo-treated group. Furthermore, the mRNA expression of pro-inflammatory cytokines such as TNF- , IL-1 and IL-8 were significantly reduced in the L. reuteri group compared to the placebo-controlled group [33]. These results indicate that intestinal microbiota manipulation through probiotics can be effective in treating some forms of IBD. 48 While probiotics are becoming an attractive therapy to treat UC, there is also a great need to understand the molecular mechanisms that govern probiotic induced beneficial effects against chronic intestinal inflammation. Previous studies have elucidated some mechanisms for probiotic-induced protection, including the production of short-chain fatty acids (SCFA) [34], increased production of mucin [35], enhancement of the intestinal epithelial barrier function [36], modulation of the immune system including production of immunoglobulins [37] and modulation of negative TLR regulators [38]. However, the mechanisms by which probiotics attenuate chronic gut inflammation are not yet fully understood. Our study has identified a novel mechanism of action of a putative probiotic strain, L. murinus. We showed that L. murinus activates AHR in the human intestinal epithelial cells in vitro. To determine the activation of AHR in the Caco-2 monolayers, we quantified the CYP1A1 mRNA fold-change, since CYP1A1 is considered a biomarker for AHR activation [25]. Using 18S rRNA as a house-keeping gene [39], we used 10-fold increasing MOIs of L. murinus on Caco-2 monolayers to determine the activation level of AHR with increasing L. murinus MOIs. Our results indicate that AHR activation is directly proportional to the MOI of L. murinus in the Caco-2 cells (Fig 2.7). We then treated the monolayers with defined AHR agonists in a concentration dependent manner. We chose to treat cells with TCDD or FICZ in 10-fold increasing concentrations. We specifically chose TCDD because it is the most comprehensively studied exogenous AHR agonist [23]. We also chose a second known AHR agonist, FICZ, 49 because it is a tryptophan derivative and an endogenous ligand having the highest affinity for binding to AHR among all other endogenous ligands [40]. Our results indicate that AHR can be activated in the Caco-2 human intestinal epithelial cell monolayer model by its defined exogenous and endogenous ligand in a concentration-dependent manner (Fig 2.6). Next, to optimize the in vitro model of IBD, we treated the Caco-2 cells with increasing concentrations of TNF- because this is one of the main cytokines that is upregulated in the gut epithelium of IBD patients [41]. Our strategy was to use TNF- as a surrogate for C. jejuni infection because it is the principal cytokine that signals early inflammatory responses after infection with this bacterium [42]. Other investigators have employed TNF- in this manner to model C. jejuni infection outcomes in in vitro models [43]. This is effective because exogenous TNF- induces a leaky gut barrier and mimics the mucosal cytokine storm which is a prominent feature in C. jejuni pathogenesis [44, 45]. Campylobacteriosis is very common in the US and worldwide and previous studies have associated a role for C. jejuni induced enteritis in the pathogenesis of IBD [5, 6]. Therefore, stimulating Caco-2 cells with TNF- mimics C. jejuni infections conditions as well as models IBD conditions in vitro. Our results indicate that a decrease in barrier integrity is directly proportional to the increasing concentrations of TNF- (Fig 2.3). These findings are consistent with findings reported by Cui et al.,[46] in which Caco-2 monolayer permeability was increased when treated with TNF-. However, the increasing concentrations of TNF- had no effect on 50 the mRNA expression of the occludin (Fig 2.4A) and TJP-1 (Fig 2.4B). These results suggest that TNF- directly acts on tight junction proteins to induce barrier dysfunction and does not affect the mRNA expression of these barrier proteins. Our results also indicate that IL-8 secretion from Caco-2 cells is directly proportional to the TNF- concentration (Fig 2.5). Sonnier et al., [47] also reported similar findings where basolateral stimulation of Caco-2 cells with TNF- resulted in apical and basolateral secretion of IL-8; however, no concentration-dependent effect of TNF- on IL-8 secretion was determined in their studies. Next, we determined the effect of AHR activation—either by defined AHR agonists or by L. murinus—on IL-8 secretion and gut barrier function in vitro. Our results indicate that pre-treating the monolayers with L. murinus attenuated the TNF- induced decrease in barrier integrity when compared to the positive control i.e., monolayers given TNF- only (Fig 2.8). However, pre-treating the monolayers with defined AHR agonists (TCDD and FICZ) did not protect against the TNF- induced barrier disruption. Interestingly, we also observed a significant disruption in epithelial barrier integrity in groups given either FICZ or TCDD. One explanation for the disruption of intestinal epithelial barrier integrity with TCDD and FICZ is that AHR ligands can also activate several mitogen-activated protein kinase (MAPK) pathways [48]. MAPK signaling pathways play a dual role in regulating intestinal barrier permeability [49]. For example, it has been reported that activation of MAPK through the ERK1/2 pathway leads to intestinal barrier disruption [50]. In another study, inhibition of MAPK pathway resulted in a reduced loss of barrier function 51 in Caco-2 cells [51]. Furthermore, we did not observe any cytotoxic effects of TCDD and TNF- at concentrations of 0.1 nM, 1 nM and 10 nM and 0.1, 1 and 10 ng/ml respectively (data not shown). Whereas no cytotoxic effects of FICZ were observed at 1nm concentration. However, a cytotoxicity of 4.88% and 7.47% was observed in the Caco-2 cells treated with FICZ concentrations of 10 nM and 100 nM respectively (data not shown). The cytotoxic effects of FICZ on epithelial cells at higher concentrations has also been reported by Walczak et al., [52]. Pre-treating the cells with L. murinus or FICZ also significantly reduced the secretion of IL-8 from Caco-2 monolayers upon stimulation with TNF- (Fig 2.9). This is of significance since IL-8 is also known as a “neutrophilic chemotactic factor” as it attracts neutrophils, basophils and T-cells to the site of inflammation [53]. Neutrophils are also the primary cell type recruited to the inflamed bowel [54]. Furthermore, neutrophils contribute to the gut epithelial barrier damage by producing reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) [55]. Assessing levels of neutrophil infiltration in gut mucosa is widely considered a useful means to determine disease severity in IBD [56, 57] and is employed routinely to score for UC disease severity [58, 59]. Therefore, attenuating the IL-8 secretion by L. murinus or endogenous AHR agonist (FICZ) may decrease the neutrophil-induced damage to the gut wall. On the other hand, activation of AHR by TCDD did not result in decreased IL-8 response. The reason for the differential FICZ and TCDD response to IL-8 secretion is not clear. However, recent evidence suggests that endogenous AHR ligands may induce differential cell type responses compared to synthetic ligands such as TCDD [60, 61]. The reason for these divergent 52 effects between the endogenous and exogenous AHR ligands is not completely understood; however, the differences between the half-lives of TCDD and FICZ may explain this dichotomy. TCDD is resistant to AHR-induced metabolism and has a long half-life therefore it leads to sustained AHR activation. On the other hand, FICZ is rapidly metabolized, has a short half-life and leads to a transient AHR activation [62]. In future, conducting proof-of-concept studies will further dissect the role of AHR in the pathogenesis of IBD. To the best of our knowledge, at present, AHR-/- Caco-2 cell- line is not commercially available. Another way to conduct proof-of-concept studies is to use AHR ligand-selective antagonists. CH223191 is a ligand-selective antagonist that inhibits the TCDD mediated nuclear translocation and DNA binding of AHR [63]. In our preliminary experiments CH223191 blocked TCDD mediated CYP1A1 upregulation in the HT-29 cells (data not shown). However, we did not use CH223191 in designing subsequent proof-of-concept studies as we did not observe any protective effect of TCDD pretreatment in attenuating TNF- induced pro-inflammatory responses in in vitro model of colitis. In future, gene editing techniques such as CRISPR-Cas9 [64] can be used to develop AHR-/- Caco-2 cell-line to perform in vitro proof-of-concept studies. In short, our results indicate a novel mechanism of action of probiotics to alleviate damaging effects of TNF- on the gut wall. The results from this study may lead to the development of microbiome-based interventions to potentially treat IBD without the use of immunosuppressants or anti-TNF-. Furthermore, the results from this study may lead 53 to the development of novel probiotics capable of treating bacterial disease which manifests similar disease pathogenesis as IBD such as human campylobacteriosis. 54 APPENDIX 55 Figure 2.1) Resistance readings of Caco-2 monolayer Transepithelial electrical resistance (TEER) of the Caco-2 monolayer was measured when grown on the TranswellTM inserts. The monolayer was grown on the TranswellTM insert for the period of 21 days. The resistance was measured every couple of days. Resistance readings are shown as mean ± SEM. 56 Figure 2.2) Resistance readings of HT-29-MTX-E12 monolayer The TEER values of the E12 monolayer were measured when grown on the Transwell inserts. The monolayer was grown on the Transwell insert for a period of 21 days. The resistance was measured every couple of days. Resistance readings are shown as mean ± SEM. 57 Figure 2.3) TNF- decreases epithelial barrier integrity in a concentration-dependent manner Caco-2 cells monolayers were treated with 10-fold increasing concentrations of TNF-. TEER was measured at 24 hours post TNF- stimulation. Relative TEER % was calculated as the percent change of TEER at 0 hours. The results are expressed as mean ± SEM. There were at least 3 replicates per group. The statistical analysis was performed using one-way ANOVA with post-hoc Tukey’s HSD test for computing statistically significant differences relative to the control group (0 ng/ml). 58 Figure 2.4) TNF- does not affect the mRNA expression of Occludin and TJP-1 Caco-2 cells monolayers were treated with 10-fold increasing concentrations of TNF-. The mRNA of occludin and TJP-1 was quantified by qRT-PCR using 18S rRNA as an internal control. The results are expressed as mean ± SEM. There were at least 3 replicates per group. The statistical analysis between different groups was performed using one-way ANOVA. There was no statistically significant difference between any of the groups. 59 Figure 2.5) TNF- induces IL-8 secretion from Caco-2 monolayers in a concentration- dependent manner The Caco-2 monolayers were treated with increasing concentrations of TNF- for 24 hours. IL-8 protein was measured from the cell culture medium collected from the basal chamber of the Transwell inserts. There were at least 3 replicates per group. The statistical analysis was performed using unpaired students t-test. 60 Figure 2.6) AHR ligands increase CYP1A1 transcription in a concentration-dependent manner The expression of CYP1A1 was measured in the Caco-2 cells treated with different concentrations of the two defined AHR agonists, i.e., FICZ and TCDD for 12 hours. The expression of the CYP1A1 was determined by qRT-PCR and the results were normalized using 18S rRNA as a house keeping gene. The results are expressed as mean ± SEM. There were at least 3 replicates per group. The statistical analysis between different groups was performed using one-way ANOVA with post-hoc Tukey’s HSD test for comparing multiple treatments. 61 Figure 2.7) L. murinus upregulates CYP1A1 transcription in MOI dependent manner Caco-2 cells were treated with L. murinus in 10-fold increasing MOI. CYP1A1 activity was measured by qRT-PCR with 18S rRNA used as an internal control. Cells were treated with 100 nm FICZ as positive control and 0.02% DMSO as vehicle control. All treatments were given for 24 hours. There were at least 3 replicates per group. The results are expressed as mean ± SEM. The statistical analysis between different groups was performed using one-way ANOVA with post-hoc Tukey’s HSD test for comparing multiple treatments. 62 Figure 2.8) AHR activation attenuates epithelial barrier disruption Figure 2.9) AHR activation attenuates pro-inflammatory response 63 AHR activation attenuates epithelial barrier disruption and pro-inflammatory response in Caco-2 monolayers Caco-2 monolayers were given respective pre-treatments for 12 hours with either L. murinus (MOI 1:100) or FICZ (100 nM) or TCDD (10 nM); followed by 24 hours of treatment with TNF- (10 ng/ml). At the end of 24 hours of treatments, TEER and IL-8 were measured to determine the effect of AHR activation on barrier dysfunction (Fig 2.8) and IL-8 secretion (Fig 2.9) from Caco-2 cells. The results are expressed as mean ± SEM. There were at least 3 replicates per group. 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Nature Protocols, 2013. 8(11): p. 2281-2308. 72 CHAPTER 3: CO-INOCULATION OF LACTOBACILLUS MURINUS AND CAMPYLOBACTER JEJUNI ENHANCED COLONIZATION LEVELS OF BOTH TAXA AND CHANGED THE GUT MICROBIOTA LANDSCAPE BUT DID NOT PROTECT BALB/C IL-10-/- MICE FROM DEVELOPING COLITIS. 73 Abstract Background Interleukin-10 deficient mice are a robust model for investigating inflammatory bowel disease (IBD). When infected with the foodborne pathogen Campylobacter jejuni, IL-10 deficiency leads to enhanced inflammation in the gut resulting in colitis [1]. In a previous experiment, C57BL/6 IL-10-/- mice unexpectedly failed to develop colitis upon infection with C. jejuni 11168. This result contrasted with the previously reported findings where C57BL/6 IL-10-/- mice developed severe colitis upon inoculation with C. jejuni 11168 [1, 2]. Coincident with this finding, Lactobacillus murinus was isolated from the mice protected from C. jejuni induced colitis, suggesting a role for L. murinus in suppressing C. jejuni induced colitis [3]. This outcome was consistent with the ability of this L. murinus strain isolate to attenuate inflammatory responses in an in vitro intestinal cell model of colitis [4]. In this study, we hypothesized that prophylactic administration of L. murinus would attenuate C. jejuni induced colitis in an IL-10 deficient mouse model. Methods A total of 41 BALB/c IL-10-/- mice were used to test this hypothesis. 11 mice were sham inoculated (negative control), 10 mice received only L. murinus, 10 mice received only C. jejuni (positive control), and 10 mice in the test group received both L. murinus and C. jejuni. L. murinus was inoculated 32 days before C. jejuni infection. Thirty days post-C. jejuni challenge mice were sacrificed and assessed for gut pathology. During necropsy intestinal tissues and fecal samples were collected from mice to determine 74 histopathological changes and bacterial colonization levels in the gut through 16S sequence analysis and bacterial culture. Results Both the C. jejuni positive control and the co-inoculated (L. murinus and C. jejuni) test group mice developed severe colitis. Furthermore, both C. jejuni and L. murinus colonized more densely in the co-inoculated test group than in the C. jejuni only control or L. murinus only groups. 16S sequencing analysis of fecal DNA of all groups revealed that the Shannon-alpha diversity in the C. jejuni infected control and the co-inoculated (L. murinus and C. jejuni) test groups were significantly less (P<0.001) than in the Lactobacillus only and negative control groups. Presence of C. jejuni caused dysbiosis in the gut bacterial community with increased abundance of Enterococcus and Lactobacillus and decreased abundance of Lachnospiraceae. Conclusions These results indicate that gut microbial diversity was significantly decreased in the presence of C. jejuni. The microbiota landscape was also significantly altered in the presence of C. jejuni, particularly in co-infected mice. The increase of colony forming units of both C. jejuni and L. murinus in the co-infected group suggests an ecological interaction between these two taxa. These results showed that prophylactic administration of L. murinus did not protect BALB/c IL-10-/- mice from developing inflammatory bowel disease following C. jejuni infection despite its ability to attenuate inflammatory responses in an in vitro intestinal cell model of colitis. 75 Introduction Campylobacter jejuni is a leading cause of human foodborne gastroenteritis in the US, with an incidence rate of 13.6 diagnosed cases per 100,000 individuals. Because many sporadic infections go undiagnosed, the Centers for Disease Control predict that the actual incidence is much higher, with estimates of approximately 1.5 million cases of campylobacteriosis in the US per annum [5]. C. jejuni resides as a commensal in the gastrointestinal tract of many agricultural animals [6]. The most frequent cause of C. jejuni infection in the US is the consumption of chicken contaminated during processing because C. jejuni resides as a commensal in chicken gut [7]. Campylobacteriosis in humans is characterized by mild to severe bloody diarrhea, abdominal pain, and severe intestinal inflammation that lasts from 7 – 10 days [6]. Various post-infectious auto- immune diseases such as Miller-Fisher syndrome, Guillain-Barre syndrome, and reactive arthritis have been associated with Campylobacter infections increasing its impact as an intestinal pathogen [2, 8, 9]. Macrolide antibiotics such as azithromycin and ciprofloxacin are the drugs of choice to treat C. jejuni infection in human populations [10]. However, over-use of antibiotics has led to the emergence of antimicrobial-resistant C. jejuni strains and reduced treatment efficacy with long-term shedding in patients [10, 11]. In 2015, the National Antimicrobial Resistance Monitoring System (NARMS) reported that 47% of C. jejuni isolates were resistant to tetracycline, whereas 25% of the isolates were resistant to ciprofloxacin [12]. More than 35,000 people die each year in the US from antibiotic- resistant infections [13]. Thus, the Centers for Disease Control (CDC) declared C. jejuni 76 a serious health threat [13]. The development of antimicrobial resistance traits in C. jejuni isolates has augmented the need to develop innovative strategies to prevent and treat drug-resistant C. jejuni infections in human and animal populations. Probiotics can be an attractive therapy to reduce or eliminate the use of antibiotics to limit gut bacterial pathogens such as C. jejuni [14]. Certain members of the Lactobacillus spp. are commonly used as probiotics [15]. Previous studies have elucidated several mechanisms by which particular Lactobacillus spp. can attenuate C. jejuni virulence properties in vitro [16, 17]. Different Lactobacillus strains (L. salivarius, L. johnsonii, L. reuteri, L. crispatus, and L. gasseri) alone or in combination reduced the ability of C. jejuni to adhere and invade human intestinal epithelial cells in vitro. Furthermore, incubating these Lactobacillus strains (except L. reuteri) with C. jejuni reduced the expression of C. jejuni virulence genes, including genes responsible for motility such as flaA, flaB, and flhA and gene responsible for invasion (CiaB) [16]. Similarly, Wang et al. [17] reported that L. casei ZL4 reduced the adhesion and invasion of C. jejuni in cultured HT-29 cells, a mucus-producing human intestinal epithelial cell line. These results suggest that certain members of the Lactobacillus spp. possess mechanisms that can attenuate C. jejuni virulence. Previously, L. murinus was isolated from C57BL/6 IL-10-/- mice that failed to develop colitis when infected with a colitogenic strain of C. jejuni, thus, suggesting a possible role for the L. murinus isolate to suppress C. jejuni induced colitis [3]. We also reported the ability of this isolate of L. murinus to attenuate TNF-alpha-induced pro- 77 inflammatory responses in cultured human intestinal epithelial cells by activating the aryl hydrocarbon receptor (AHR) [1]. TNF-alpha was used as a surrogate to C. jejuni infection to provide an in vitro model of colitis [1]. Therefore, in this study we hypothesized that prophylactic inoculation of L. murinus will protect BALB/c IL-10-/- mice against C. jejuni induced colitis. To test this hypothesis, we challenged BALB/c IL-10-/- mice with a colitogenic strain of C. jejuni known to induce TNF-alpha during infection [2-4]. We used BALB/c IL-10-/- mice to model C. jejuni disease because IL-10-/- mice are a robust model to study C. jejuni pathogenesis [3, 5]. Upon infection with C. jejuni, IL-10-/- mice develop clinical symptoms and histopathologic lesions consistent with those in humans with campylobacteriosis [3]. Yet, in this study, results indicated that prophylactic administration of L. murinus was not effective in attenuating C. jejuni induced colitis in BALB/c IL-10-/- mice. Interestingly, C. jejuni infected groups had reduced bacterial diversity of the gut microbiota when compared to the non-C. jejuni infected groups. Further, co-infected mice had the highest levels of C. jejuni and L. murinus and the landscape of the microbial community was significantly altered. These results demonstrate that C. jejuni mediates dysbiosis of the gut microbial community with reduced microbial diversity during its pathogenesis and that L. murinus was not capable of preventing this outcome in an IL-10 deficient environment. Materials and methods Animal Handling Protocols Animal protocols were approved by Michigan State University (MSU). C.129P2(B6)-Il10tm1Cgn/J (referred to as BALB/c IL-10-/-) mice were originally obtained 78 from the Jackson Laboratories (Bar Harbor, ME). Mice were housed in a Campylobacter/Helicobacter-free facility at MSU. Mice were kept in the Flex-Air ventilated mouse rack (Alternate Design Manufacturing & Supply Inc., Siloam Spring, AR) with an ad-libitum supply of feed and water. The mice were routinely monitored for the presence of a variety of bacterial, viral, and protozoan agents by routine monitoring of the dedicated sentinel mice. Furthermore, mice developing signs of spontaneous colitis were screened for the presence of colitogenic bacteria, including Campylobacter spp., Enterococcus faecalis, Citrobacter rodentium, and Helicobacter spp., by PCR [6-9]. The detailed protocols for maintaining mice in specific pathogen-free facility at MSU has been described previously [3]. Experimental Design A total of 41 BALB/c IL-10-/- mice were used in this experiment. The mice were divided into four groups. The table below describes the groups of mice for this experiment. Table 1) Groups of mice used in this study Group Group Description Inoculation Strain / Vehicle (Day of (# of mice) inoculation) Group A (10) Control group for L. murinus L. murinus (1) Group B (10) Control group for C. jejuni C. jejuni 11168 (32) Group C (10) Test group L. murinus (1) and C. jejuni 11168 (32) Group D (11) Vehicle control group Sham inoculation, Tryptic Soy Broth (1,32) 79 Mice in all groups were sacrificed 30 days post-C. jejuni infection. Fecal and intestinal tissue samples were taken from mice during necropsy. Growing bacterial cultures and their inoculation in mice Lactobacillus murinus L. murinus was initially isolated from C57BL/6 mice. The detailed description regarding the isolation and characterization of L. murinus isolated from C57BL/6 mice has been described by J. Brudvig [1]. In brief, L. murinus was recovered from glycerol stocks stored at -80 C and then streaked onto De Man, Rogosa, and Sharpe (MRS) agar plates. The MRS agar plates were incubated at 37 C for 48 hours. After 48 hours, individual colonies were picked and Gram-stained to check for L. murinus morphology and culture purity. L. murinus were identified to species using MALDI-TOF mass spectrometry at the Veterinary Diagnostic Laboratory, Michigan State University [1]. L. murinus growth was harvested from the MRS agar plates and diluted in tryptic soy broth (TSB) to an optical density co-related with 1  109 CFU of L. murinus per 100 ul of TSB. Each mouse in groups A and C received 100 ul of TSB containing 1  109 CFU of L. murinus in the stomach through a sterile 3.5-French feeding tube (Kendall Sovereign; Tyco Healthcare Group, Mansfield, MA) attached to a 1-ml Luer-Lok syringe (Becton Dickinson and Company, Franklin Lakes, NJ). The mice in groups B and D were sham inoculated with 100 ul of TSB without L. murinus. 80 Campylobacter jejuni C. jejuni 11168 was initially obtained from American Type Culture Collection (Manassas, VA) and stored in glycerol stock cultures at -80C. C. jejuni ATCC 700819 (referred to as C. jejuni 11168) was streaked onto tryptone soy agar (TSA) (Oxoid, Basingstoke, United Kingdom) that was supplemented with 5% defibrinated sheep's blood (Cleveland Scientific, Bath, OH) [1]. The plates were then incubated for 48 hours at 37C in sealed containers containing CampyGen sachet (Oxoid, Basingstoke, United Kingdom). After 48 hours, individual colonies were picked and Gram-stained to check for C. jejuni spiral morphology and culture purity. Furthermore, wet mount preparations were made to verify C. jejuni darting motility under the microscope. C. jejuni colonies were picked from TSA agar plates using a sterile cotton swab (Puritan Medical Products, Guilford, ME) and were resuspended in TSB to an optical density that correlated with 1  1010 CFU of C. jejuni per 100 ul of TSB. Each mouse in groups B and C received 100 ul of TSB (containing 1  1010 CFU of C. jejuni) in the stomach by a sterile 3.5-Fr feeding tube (Kendall Sovereign; Tyco Healthcare Group, Mansfield, MA) attached to a 1-ml Luer- Lok syringe (Becton Dickinson and Company, Franklin Lakes, NJ). Mice in groups A and D were sham inoculated with 100 ul of TSB not containing C. jejuni. Necropsy, sample collection, and gross histopathological scoring Fecal samples from each mouse were collected prior to euthanasia in 1.5 ml Eppendorf  tubes (Hamburg, Germany). Mice were euthanized using a gradually administered overdose of CO2 gas in a sealed chamber according to AVMA guidelines [2]. Immediately after euthanasia, blood was obtained by cardiac puncture and 81 immediately mixed with 0.68% sodium citrate. Plasma was collected from blood centrifugation and quickly stored at -80 C until further analysis could be performed. Mice were sprayed with alcohol, placed in dorsal recumbency and the abdomen opened to remove the gut in its entirety from esophagus to rectum. The gut was placed on a sterile field, and gross histopathological changes were observed, including the thickened gut wall, enlarged ileocecocolic lymph nodes, and blood in the lumen. Gross pathological changes were graded as follows: Grade 0 = no gross pathological findings were observed, Grade 1 = One of the following findings were observed, enlarged ileocecocolic (ICC) lymph node or thickened gut wall (TGW) or enlarged (ENL) colon or cecum, Grade 2 = Two of the following gross pathological findings were observed, enlarged ICC lymph node or TGW or ENL colon or cecum, Grade 3 = Three of the following findings were observed, enlarged ICC lymph node or TW or ENL colon and cecum including bloody feces or luminal contents. The cecum and colon were divided into 3 sections; one section of each organ was stored in formalin; the second section was flash-frozen, and the third section was streaked onto TSA plates. The TSA-CVA plates contained 10 g vancomycin per ml 20 g cefoperazone per ml, and 2 g amphotericin B per ml (all antibiotics were purchased from Sigma-Aldrich, St-Louis, MO). The plates were then incubated in a sealed container containing CampyGen sachet for 48 hours. 82 Histological staining of ICC junction Cecum, along with 1 cm of the terminal ileum and 1 cm of the proximal colon, was excised from each mouse, and its contents were removed by placing it in phosphate- buffered saline (PBS). It was then preserved for histopathology by placing it inside a histopathological cassette (Histocette II; Simport Plastics, Beloeil, Quebec, Canada) and immersing into 10% phosphate-buffered formalin. After 24 hours the formalin was then replaced with 60% ethanol. The intestinal tissues were then sent to the Investigative Histopathology Laboratory at MSU. The samples were then vacuum infiltrated with paraffin using Sakura VIP 2000 tissue processor, followed by embedding with ThermoFisher HistoCentre III embedding station. The blocks were finely sectioned at 4-5 microns using Reichert Jung 2030 rotary microtome. Sections were then dried at 56°C using a slide incubator. Once dried, the slides were then removed from the incubator and stained on a Leica Autostainer XL using Hematoxylin and Eosin (H&E) staining method which is as follows: Two changes of Xylene – 5 minutes each, two changes of absolute ethanol – 2 minutes each, two changes of 95% ethanol – 2 minutes each, running tap water rinse for 2 minutes, endure Hematoxylin (Cancer Diagnostics – Durham, NC) for 1 ½ minute followed directly by a 10 – 15 second differentiation in 1% aqueous glacial acetic acid and place slides under running tap water for 2 minutes to enhance nuclear detail. Slides were then given the following treatments for a defined length of time; placed in 95% ethanol for 2 minutes, 1% Alcoholic Eosin-Phloxine B for 2 minutes to stain cytoplasm, placed in 95% ethanol for 2 minutes, four changes of 100% ethanol for 2 minutes each, four changes of Xylene for 2 minutes each followed by coverslip with synthetic mounting media for permanent retention and visualization with light microscopy. 83 Histopathological Scoring Histopathological scoring for the H&E stained ileocecocolic junction of each mouse was performed by a single veterinary investigator who was blinded to the sample identity. The criteria for histopathological scoring have been described in detail elsewhere [1]. In brief, the ileocecocolic junction of each mouse was scored on a scale from 0 to 44. Features used for scoring the ileocecocolic junction include the presence of mucus and inflammatory exudate in the lumen. The intestinal epithelium was evaluated for its integrity, hypertrophy of goblet cells, depletion of goblet cells, presence of intraepithelial lymphocytes, crypt hyperplasia and crypt atrophy, and crypt inflammation. The lamina propria was evaluated on the presence and distribution of immune cells and the sub- mucosa was evaluated for the development of inflammation and/or fibrosis. 16S sequence analysis Genomic DNA was isolated from the fecal samples collected from each mouse using a FastDNA SPIN Kit for stool (MP Biomedicals, Solon, OH) according to the manufacturer’s protocol. Careful attention was given to preventing laboratory contamination by the operator or within the laminar flow hood. All reagents were validated free of 16S DNA before use. For all samples, standard controls (Mock Community, ATCC/BEI) were run on reagents and kits to control for random effects of the reagents and procedures used. Once DNA was extracted from the samples, Qubit assay (Thermofisher, Waltham, MA) was performed to ascertain the concentration and 16S rRNA gene PCR was performed [3] to validate the quality of the DNA samples prior to submission to the Michigan State University Research Technology Support Facility 84 (RTSF) for Illumina MiSeq sequencing. At RTSF, the V4/V6 regions of the 16S rRNA gene were amplified by PCR in triplicate using two sets of barcoded primers, and the PCR products purified, combined and sequenced using Illumina MiSeq and then demultiplexed. Then the raw sequences were imported into QIIME2 for quality control. The reads were denoised with Deblur, then clustered into OTU’s with vsearch, both using QIIME 2. The reads were then filtered for chimeras which were removed along with borderline chimeras. Then taxonomy was classified at 97% similarity to Silva database release 128. The quality-controlled sequences were imported into R Studio. Raw read counts were converted into relative abundances using base R studio functions, then filtered for any reads with relative abundance higher than 1% in at least one mouse. The filtered reads were subjected to principal component analysis (PCA) and visualized using package factoextra version 1.0.5. Relative abundance bar plots were created using Microsoft Excel. Package vegan version 2.5-3 was used to run similarity percentage analysis (SIMPER) and calculate the Shannon diversity index and Bray-Curtis dissimilarity index. Package ggplot2 version 3.0.0 was used for visualization. Semi-quantitative scoring for L. murinus colonization Fecal samples were suspended in PBS and 20 ul were suspended onto MRS agar containing 10 g vancomycin per ml, 20 g cefoperazone per ml, and 2 g amphotericin B per ml (all antibiotics were purchased from Sigma-Aldrich, St-Louis, MO). The plates were then incubated at 37 C for 48 hours. The plates were then ranked from 0 – 4 85 according to number of L. murinus colonies per plate as 0 (no CFU), 1 (1 – 20 CFU), 2 (20 – 200 CFU), 3 (>200 CFU) and 4 (confluent growth) [4]. Semi-quantitative scoring for C. jejuni colonization At necropsy small snips of colon tissue were streaked onto TSA-CVA agar plates. The plates were then incubated in a sealed container containing CampyGen sachet at 37 C for 48 hours. The plates were then ranked from 0 – 4 according to the number of C. jejuni colonies per plate as 0 (no CFU), 1 (1 – 20 CFU), 2 (20 – 200 CFU), 3 (>200 CFU), and 4 (confluent growth) [3]. Statistical analysis GraphPad Prism version 9.2.0 was used for the preparation of graphs and statistical analysis. Statistical analysis was performed using Kruskal-Wallis one-way- analysis of variance. The P values between different groups were summarized as ns: P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Results Prophylactic inoculation of L. murinus did not prevent C. jejuni induced gross pathology in mice Prophylactic treatment of L. murinus did not prevent presence of gross pathology in C. jejuni infected BALB/c IL-10-/- mice (Fig 3.1). 2/10 mice in the positive control group for C. jejuni (group B) had enlarged ICC lymph nodes, whereas 7/10 mice in group B had 86 enlarged ICC lymph nodes and thickened gut wall (TGW). In the test group (group C), 3/10 mice had enlarged ICC lymph node and 3/10 mice had enlarged ICC lymph nodes and TGW. However, 1/10 mice in group C presented all 3 gross histological findings i.e., enlarged ICC lymph node, TGW and bloody intestinal contents. Interestingly, 3/10 mice in the test group did not present gross pathological lesions, whereas only 1/10 mice in the positive control group for C. jejuni remained protected from developing gross pathological lesions. Furthermore, no gross pathological lesions were observed in the L. murinus control group (group A). However, one mouse developed mild symptoms of spontaneous colitis in the vehicle control group (group D). These results indicate that prophylactic inoculation of L. murinus was not effective in preventing C. jejuni induced gross pathological lesions in the BALB/c IL-10-/- mice. Prophylactic inoculation of L. murinus did not reduce histopathologic lesions in the ileocecocolic junctions L. murinus pre-enrichment in the test group mice had no significant effect on reducing the histopathologic lesions in ileocecocolic (ICC) junctions of mice in group C as compared to the mice in the C. jejuni positive control group B (Fig 3.2). The mean histopathology score in the test group was 22.8 ± 7.45, whereas the mean histopathology score in the positive control group was 21.9 ± 7.35. There was no significant difference (P > 0.99) between the histopathology scores of the test group (group C) and the positive control group for C. jejuni (group B). Histopathological scores below 10 are considered normal in IL-10-/- mice [1]. 1/10 mice in group C received a score below 10. However, no mouse in group B received a score below 10. The mean histopathology scores in the L. 87 murinus control group (group A) and the vehicle control group (group D) were 7.1 and 9.2 respectively. These results indicate that prophylactic administration of L. murinus was not effective in attenuating C. jejuni induced gut pathology in BALB/c IL-10-/- mice. Furthermore, these results also indicate that L. murinus is a commensal and does not induce gastrointestinal pathology in the BALB/c IL-10-/- mice. L. murinus pre-enrichment increased C. jejuni colonization in the cecum Mice in the test group (group C) had a higher C. jejuni count in the cecum as compared to the mice in the positive control group for C. jejuni (group B). 8/10 mice in the C. jejuni infected group received a score of 1. In comparison, 8/10 mice in the L. murinus treated C. jejuni infected group (group C) received a score of 2. Interestingly, no C. jejuni was detected from the cecum of 2/10 mice in the C. jejuni infected group. As expected, no C. jejuni was detected in the L. murinus control group or vehicle control group. These results indicate that prophylactic administration of L. murinus increases cecal colonization of C. jejuni in the BALB/c IL-10-/- mice. Increased L. murinus colonization in the test group Increased L. murinus colonization was found in the test group as compared to the L. murinus control group (Fig 3.5). In group A, 1/10 mice received a score of 2, 2/10 mice received a score of 3, and 4/10 mice received a score of 4. Whereas in group C, 1/10 mice received a score of 2, 4/10 mice received a score of 3, and 5/10 mice received a score of 4. Furthermore, no L. murinus was detected from group B and group D mice. 88 These results indicate that L. murinus colonization was enhanced in the test group as compared to the L. murinus control group. C. jejuni infection changed the gut microbiota composition 16S sequence analysis revealed that the gut microbiota composition was significantly changed in the C. jejuni infected groups (group B and C) as compared to the non-C. jejuni infected groups (group A and D). The relative abundance of the genus Enterococcus was also higher in the C. jejuni infected groups as compared to non-C. jejuni infected groups (Fig 3.6). The relative abundance of genus Enterococcus was 21.68% in group B and 17.45% in group C. The relative abundance of the genus Enterococcus in group A was 0.022%, and in group D was 0.015%. However, the relative abundance of the Lachnospiraceae NK4A136 was higher in the non-C. jejuni infected groups as compared to the C. jejuni infected groups. The relative abundance of the bacterium Lachnospiraceae NK4A136 was 19.62% in group A, and 18.89% in group D. Whereas, the relative abundance of the bacterium Lachnospiraceae NK4A136 was 4.2% and 2.4% in group B and C respectively. Interestingly, members of the genus Lactobacillus were also identified in the 16S sequence analysis of the C. jejuni only group (group B). However, no bacterial growth was observed when the fecal samples of group B mice were cultured on MRS agar plates. A principal component analysis (PCA) plot based on the genus level relative abundance profile showed that group A and group D overlapped with each other (Fig 3.8). However, groups B and C were relatively dispersed in the PCA plot. Dimension 1 of the 89 PCA plot (Dim 1) explained 58.8% of the variability and dimension 2 of the PCA plot (Dim 2) explained 17.4% of the variability. Furthermore, the Shannon-diversity index (Fig 3.7) revealed that C. jejuni infected groups (group B and C) had a significantly decreased bacterial diversity as compared to the non-C. jejuni infected groups (Group A and D). There was a statistically significant difference (P < 0.05) in the Shannon-diversity scores between group A and group C. These results suggest that C. jejuni infection significantly changes the gut microbiota composition in the BALB/c IL-10-/- mice. Discussion C. jejuni is the leading cause of bacterial diarrheal illness in the United States [5]. C. jejuni infection can lead to Guillain-Barré syndrome, an auto-immune disease in which molecular mimicry between C. jejuni outer surface and nerve gangliosides causes patients to produce autoantibodies against the peripheral nerves, which can lead to paralysis [6]. At present, macrolide antibiotics are the drug of choice to treat C. jejuni infections, yet, the high rates of antibiotic resistance in C. jejuni have led to reduced treatment efficacy [7]. Therefore, the CDC have categorized drug-resistant Campylobacter as a serious public health threat [8]. It is important to identify alternatives to antibiotics to attenuate C. jejuni pathogenesis in humans and animal populations. Some members of the Lactobacilli are commonly used as probiotics [9]. Previous studies have also reported the antimicrobial activity of Lactobacillus spp. against C. jejuni in vitro [10, 11]. In this study, we determined the effect of prophylactic administration of a putative probiotic strain (L. murinus) on the pathogenesis of C. jejuni in a mouse model. 90 These results indicate that L. murinus pre-treatment was not able to reduce the intestinal burden of C. jejuni in BALB/c IL-10-/- mice. In fact, there were higher C. jejuni counts in the co-infected test group C as compared to the C. jejuni alone control group B. Similar findings were reported by Bereswill et al., [12] where they tested the efficacy of another species L. johnsonii on C. jejuni virulence in a secondary abiotic mouse model (generated by broad-spectrum antibiotic treatment). L. johnsonii was administered orally to abiotic mice in a prophylactic or a therapeutic manner. Neither the prophylactic nor the therapeutic administration of L. johnsonii was able to decrease the intestinal burden of C. jejuni [12]. However, a previous study reported a beneficial effect of a combination of probiotics against C. jejuni in BALB/c mice. BALB/c mice were transplanted with the defined human gut microbiota to generate a human-microbiota-associated (HMA) mouse model. HMA mice were given a combination of probiotic strains containing L. acidophilus, L. rhamnosus, L. casei, L. gasseri, L. reuteri, Bifidobacterium thermophilus, B. adolescentis, and B. longum before C. jejuni challenge. The probiotic-fed mice were able to completely eradicate C. jejuni in one week after challenge [13]. However, the use of a single probiotic strain such as L. johnsonii was not effective in eradicating C. jejuni from secondary abiotic mice [12], whereas a combination of probiotics successfully eradicated C. jejuni in HMA mice [13]. Our results from these studies provide an example where the use of a single probiotic strain was not able to eradicate C. jejuni from BALB/c IL-10-/- mice. Although using a combination of probiotic strains may produce colonization resistance against C. jejuni in the BALB/c IL-10-/- mice, it may be more fruitful to focus on determining why some strains of Lactobacillus spp. enhance the growth of C. jejuni under in vivo conditions. Importantly, the differential response of probiotics in eradicating C. 91 jejuni may be due to the use of mouse models having different immunological features. BALB/c IL-10-/- mice are immune compromised mice and have a reduced population of intestinal Treg (CD4+ CD25+ Foxp3+ T-cells) cells [14] and are therefore prone to develop spontaneous colitis [15]. Furthermore, IL-10 deficiency in BALB/c mice renders them more susceptible to developing spontaneous colitis as compared to C57BL/6 IL-10-/- mice [15]. In contrast, HMA mice are immune-competent mice having a normal population of intestinal Treg cells. Although a portion of patients that develop inflammatory bowel disease are deficient in IL-10 [16]. These patients and BALB/c IL-10-/- mice may not be an appropriate model for dissecting the role of probiotics in attenuating the C. jejuni induced inflammatory responses in the gut mucosa. This is particularly true if the mechanism of action involves an active role for IL-10 downregulation in the colon where C. jejuni resides. The results from the 16S sequence analysis suggest that members of the genus Lactobacillus were present in all four groups. However, we were only able to detect L. muirnus in the fecal samples of L. murinus only group (group A) and the test group (group C) mice. The disparity between the 16S and bacterial culture results can be explained by the fact that 16S sequencing relies on amplifying the hypervariable regions (V1 – V9) of the bacterial 16S rRNA gene [17]. One of the limitations of the 16S sequence analysis is that the resolution is not high enough to differentiate the closely related species in a particular genus [18]. Therefore, it is likely that the Lactobacillus genus reads in the 16S analysis (Fig 3.6) consist of species other than L. murinus. However, those putative Lactobacillus species did not grow on the MRS agar supplemented with antibiotics such 92 as vancomycin and cefoperazone and thus were not successfully isolated from these fecal samples. The antibiotic resistant characteristics of the inoculated L. murinus strain has been described by Jean Brudvig [4]. Further work is needed to determine if they were actually Lactobacilli or another closely related bacterial species. C. jejuni colonization was increased in the test group (group C) as compared to the C. jejuni positive control group (Fig 3.4). The reason for the increased C. jejuni load in the test group is not clear. However, a recent finding by Sinha et al., [19] help explain this phenomenon. C. jejuni contain a phosphate-transporter PstSCAB that utilize lactate as a substrate. Sinha et al., reported that the PstSCAB transporter was crucial for the growth of C. jejuni in the lactate containing medium [19]. Furthermore, deletion of PstSCAB transporter resulted in reduced fitness of C. jejuni colonization in chickens. The bacterial load of the PstSCAB mutants were significantly less in the cecum of chickens as compared to wild-type C. jejuni [19]. It is known that Lactobacillus species produce lactate as a major end product of fermentation [20]. Therefore, it is likely that the lactate produced by L. murinus was being utilized as a substrate by C. jejuni in the test group, thereby enhancing the colonization levels of C. jejuni in the test group. Results from 16S sequence analysis also show that C. jejuni 11168 colonization of the BALB/c IL-10-/- mice was associated with gut microbiota dysbiosis. Shannon- diversity index scores were significantly lower in the C. jejuni infected groups (group B and C) as compared to non-C. jejuni infected groups. The gut microbiome composition was significantly changed in the C. jejuni infected groups as compared to the non-C. jejuni 93 infected groups. C. jejuni infected mice groups were having a higher relative abundance of the genus Enterococcus. Members of the genus Enterococcus can be opportunistic pathogens [21], particularly E. faecalis and E. faecium are important nosocomial infections [22]. The role of Enterococcus in the pathogenesis of C. jejuni warrants further investigation. C. jejuni associated gut microbiota dysbiosis has also been reported by lone et al [23]. Interestingly, certain members of the gut microbiota were severely depleted in the C. jejuni infected groups as compared to the non-C. jejuni infected groups. The relative abundance of the genus Lachnospiraceae NK4A136 group was 19.62% and 18.89% in groups A and D respectively, whereas groups B and C were only having a relative abundance of 4.2% and 2.4% respectively. L. NK4A136 group is associated with health and anti-inflammatory properties [24]. Members of the genus Lachnospiraceae are important butyrate producers residing in the colonic gut mucosa [25]. Butyrate is one of the most abundant short-chain fatty acids (SCFA’s) found in the gut, along with acetate and propionate [26]. The functional role of SCFA’s in protection against intestinal inflammation is well-known [27]. However, the mechanisms by which SCFA’s attenuate inflammatory responses at the gut mucosa has remained elusive. A novel role of butyrate as a ligand of AHR has been described recently [28]. AHR is a ligand-dependent transcription factor, and its role in maintaining intestinal homeostasis has recently begun to be unraveled [29]. AHR appears as a promising target to develop novel therapeutics to attenuate pro-inflammatory responses in the gut [30]. In 94 the previous study, we reported that isolated L. murinus strain activated AHR in the intestinal epithelial cells to protect them from TNF-alpha-induced inflammatory insults [31]. In this study, we do not know if L. murinus was able to activate AHR in BALB/c IL- 10-/- mice. However, the ability of Lactobacillus strains, including L. murinus CNCM 1-509 to activate AHR has been reported by Lamas et al [32]. Inoculating mice with L. murinus CNCM I-5020, L. reuteri CNCM I-5022, and L. taiwanensis CNCM I-5019 or treatment with AHR agonist attenuated chemically induced colitis in Card9-deficient mice [32]. These results suggest that L. murinus can activate AHR and attenuate some forms of colitis in mice. Taken together, our results indicate that prophylactic inoculation of L. murinus alone was not effective in providing colonization resistance against C. jejuni in the BALB/c IL-10-/- mice. Furthermore, the results demonstrate that C. jejuni infection induces gut microbiota dysbiosis and inflammation in the BALB/c IL-10-/- mice and show the dominant bacterial taxa associated with that outcome. These results are also important because they show that C. jejuni colonization of the gut of an immunocompromised mouse model produces dysbiosis and reduce butyrate producing commensals in the gut. Furthermore, these results shed light on the role of probiotics in exacerbating C. jejuni disease under IL-10 deficient conditions and demonstrate the need to discern the underlying L. murinus gene expression and ecological conditions that fostered this outcome. Future research to study the effect of a combination of probiotics on the pathogenesis of C. jejuni is warranted. 95 APPENDIX 96 Figure 3.1) Gross pathology scoring Mice were scored for gross pathological lesions at necropsy on a scale of 0 – 3. Statistical analysis was performed using Kruskal-Wallis one-way analysis of variance (P = 0.00026). Pairwise comparisons were made by Mann-Whitney U-test with Bonferroni correction. There was no statistically significant difference between the C. jejuni positive control group (group B) and the test group (group C). There was a statistically significant difference (P < 0.001) between the L. murinus control group (group A) and the C. jejuni control group (group B). Furthermore, there was a statistically significant difference (P < 0.01) between the test group (group C) and the vehicle control group (group D). 97 Figure 3.2) Histopathological scoring of the ileocecocolic junctions Mice were scored for histopathological lesions on a scale from 0 to 44. Statistical analysis was performed using Kruskal-Wallis one-way analysis of variance (P < 0.0001). The pairwise comparisons between different groups were performed using Dunn’s multiple comparison test. There was no statistically significant difference between the group B and group C mice. However, there was a statistically significant difference (P < 0.001) between the L. murinus control group and the C. jejuni control group. Furthermore, there was a statistically significant difference (P < 0.05) between the test group (group C) and the vehicle control group (group D). 98 Figure 3.3) Hematoxylin and Eosin-stained (H&E) sections of the ileocecocolic junctions Images of the H&E-stained sections of the ileocecocolic junctions. Figure 3.3A, represents the H&E-stained ICC junction of a mouse in group A. No significant histopathological lesions were seen in group A mice. In figure 3.3B, erosion of the intestinal epithelium and hyperplasia can be seen in a group B mouse. Severe histopathological lesions were observed in group B mice. In figure 3.3C, erosion of the intestinal epithelium and infiltration of monocytes can be seen in a group C mouse. Similarly, severe histopathological lesions were observed in group C mice. In figure 3.3D, no significant histopathological lesions were seen in vehicle control group mouse. 99 Figure 3.4) C. jejuni colonization scores Cecal tissue was collected from each mouse during necropsy and was cultured for C. jejuni under microaerophilic conditions. The plates were ranked from 0 – 4 according to number of C. jejuni colonies per plate as 0 (no CFU), 1 (1 – 20 CFU), 2 (20 – 200 CFU), 3 (>200 CFU) and 4 (confluent growth). Statistical analysis between groups was performed using Kruskal-Wallis one-way analysis of variance (P < 0.0001). Pairwise comparisons were made by Mann-Whitney U-test with Bonferroni correction. There was a statistically significant difference (P < 0.001) between the C. jejuni control group (group B) and the test group (group C). 100 Figure 3.5) L. murinus colonization scores Fecal samples were taken from each mouse during necropsy and were cultured for L. murinus. The plates were then ranked from 0 – 4 according to the number of L. murinus colonies per plate as 0 (no CFU), 1 (1 – 20 CFU), 2 (20 – 200 CFU), 3 (>200 CFU), and 4 (confluent growth). Statistical analysis between groups was performed using Kruskal- Wallis one-way analysis of variance (P < 0.0001). Pairwise comparisons were made by Mann-Whitney U-test with Bonferroni correction. There was no statistically significant difference between the L. murinus control group (group A) and the test group (group C). 101 Figure 3.6) 16S rRNA gene sequence analysis of fecal DNA 16S rRNA gene sequence analysis was performed on fecal samples. The bar plot describes the percent relative abundance of OTU’s on the y-axis. The different groups are plotted against the x-axis. The landscape of the gut microbiome composition was significantly changed in the C. jejuni infected groups (B and C). 102 Figure 3.7) Shannon-Diversity Index The Shannon-diversity index was calculated for all the experimental groups. Statistical analysis was performed using Kruskal-Wallis one-way-analysis of variance (P = 0.0004). The pairwise comparisons between different groups were performed using Dunn’s multiple comparison test. There was a statistically significant difference (P < 0.05) between the L. murinus control group and the test group. Furthermore, there was a statistically significant difference (P < 0.01) between the vehicle control group and the test group. 103 Groups L. murinus only (A) C. jejuni only (B) L. murinus + C. jejuni (C) Vehicle (D) Figure 3.8) Principal Component Analysis Principal Component Analysis (PCA) of the 16S rRNA gene sequencing of the fecal samples. Dimension 1 explains 58.8% of variation whereas dimension 2 explains 17.4% of variation between the samples. 104 REFERENCES 105 REFERENCES 1. Mansfield, L., et al., C57BL/6 and congenic interleukin-10-deficient mice can serve as models of Campylobacter jejuni colonization and enteritis. 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Marinelli, L., et al., Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells. Scientific reports, 2019. 9(1): p. 643. 46. Rothhammer, V. and F.J. Quintana, The aryl hydrocarbon receptor: an environmental sensor integrating immune responses in health and disease. Nature Reviews Immunology, 2019. 19(3): p. 184-197. 47. Cannon, A.S., P.S. Nagarkatti, and M. Nagarkatti, Targeting AhR as a Novel Therapeutic Modality against Inflammatory Diseases. International Journal of Molecular Sciences, 2022. 23(1): p. 288. 110 48. Lamas, B., et al., CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands. Nature medicine, 2016. 22(6): p. 598. 111 CHAPTER 4: SUMMARY AND FUTURE DIRECTIONS. 112 IBD is an umbrella term encompassing ulcerative colitis and Crohn's disease [1]. Chronic, relapsing inflammation of the gut is a typical manifestation of IBD. In addition, the mechanisms of disease manifestation in human campylobacteriosis, such as damage to the gut epithelium [2] and dysbiosis of the gut microflora [3] are similar to the pathogenesis of IBD. Furthermore, C. jejuni infection may contribute to the pathogenesis of IBD [4]. Therefore, the pathogenesis of C. jejuni infection and disease manifestation of IBD greatly overlap one another. In the 2nd chapter, we described a novel mechanism for the beneficial effect of probiotics against inflammatory responses operating at the gut epithelial surface. We reported that probiotics such as L. murinus activate AHR to maintain intestinal homeostasis against inflammatory insults [5]. We also described the role of AHR activation (by defined AHR ligands or L. murinus) in the attenuation of pro-inflammatory responses in the in-vitro model of colitis [5]. This finding is significant as it identifies a novel therapeutic target to attenuate inflammation in IBD. However, we still don't know what specific secreted product by L. murinus was able to activate the AHR. In the future, identifying the secreted product(s) from L. murinus that act(s) as AHR ligand(s) would be significant. To identify the secreted products of L. murinus, we can use fractionation-based assays such as liquid-chromatography mass- spectrometry (LCMS) [6]. LCMS technique is best suited for bacterial secreted proteins since identifying membrane proteins by LCMS is challenging [6]. Since we will be interested in identifying the secreted products of L. murinus, the LCMS technique can be 113 a powerful approach. After identifying secreted proteins through LCMS, we can employ in-vitro assays to validate if a secreted protein(s) activates AHR. Once we identify the secreted protein(s) that acts as an AHR ligand, we can generate L. murinus mutants lacking the AHR ligand protein to conduct proof-of-concept assays. Previously, we have reported that L. muirnus activates AHR in an MOI (multiplicity of infection) dependent manner, and L. murinus pre-treatment of the human intestinal epithelial cells attenuated the TNF-alpha induced inflammatory responses [5]. Therefore, generating L. murinus mutants lacking the AHR binding protein(s) and evaluating their pre-treatment efficacy in the attenuation of colitis can be valuable in providing a proof-of-concept for the functional role of these secreted protein(s). Furthermore, identifying specific secreted products from L. murinus having AHR activation potential may result in the development of synthetic dietary compounds with beneficial health effects. The results in the 2nd chapter indicate an association of AHR activation with the attenuation of pro-inflammatory responses in the epithelial cell model of colitis [5]. However, a proof-of-concept for the role of AHR in the attenuation of colitis is lacking. Future experiments can be designed to provide a proof-of-concept for the role of AHR in gut homeostasis. An AHR deficient intestinal epithelial cell-line can be used to conduct in-vitro proof-of-concept studies. To the best of our knowledge AHR-/- Caco-2 cell line is not commercially available. However, gene-editing techniques such as CRISPR-Cas9 can be used to knock out (KO) AHR gene in the Caco-2 cells. The development of AHR- /- Caco-2 cell line will be a valuable resource for elucidating the role of AHR in maintaining gut homeostasis. 114 In the 3rd chapter, we tested the ability of L. murinus to protect against C. jejuni induced colitis in BALB/c IL-10-/- mice. The results indicate that prophylactic inoculation of L. murinus did not protect BALB/c IL-10-/- mice from developing C. jejuni induced colitis. Furthermore, the gut microbiome composition was significantly changed in the C. jejuni infected groups compared to the non-C. jejuni infected groups. These findings are of significance as they indicate that probiotics, although "generally recognized as safe" (GRAS) [7] may complement the growth and pathogenesis of pathogenic bacteria such as C. jejuni in IL-10-/- mice. We used BALB/c IL-10-/- mice to determine the prophylactic effect of L. murinus on the C. jejuni pathogenesis in a mouse model. We chose IL-10-/- mice based upon their ability to mimic clinical features of human Campylobacteriosis (such as enteritis) upon infection with a colitogenic strain of C. jejuni [8]. Since wild-type (WT) mice serves as C. jejuni colonization models but do not develop enteritis upon infection with C. jejuni [9]. Although IL-10 deficient mice are a robust model for C. jejuni induced colitis [8] they also have certain limitations. One limitation of the IL-10-/- mouse model is that they have a reduced population of CD4+ CD25+ Foxp3+ T-reg cells in the gut compared to WT mice [10]. This limitation of the IL-10-/- deficient mice hinders their use to study the beneficial effect of the class of probiotics which specifically function by targeting CD4 + CD25+ Foxp3+ T-reg cells in the gut [11]. Most probiotic strains including those of genus Lactobacillus target T-reg cells in the gut to provide beneficial health effects [11, 12]. A previous study has reported that oral inoculation of L. rhamnosus significantly increased the percentage of CD4+ CD25+ Foxp3+ T-reg cells in the intestinal tissues of the wild-type 115 (WT) BALB/c mice [13]. These results indicate that IL-10-/- mice are not a suitable model to study the beneficial effect of probiotics which function by stimulating CD4 + CD25+ Foxp3+ T-reg cells in the gut. To address this problem, future studies can be designed to determine the direct effect of AHR activation on C. jejuni colonization in the WT mice. WT C57BL/6 mice are a model for C. jejuni colonization, however, they do not develop enteritis upon infection with a colitogenic strain of C. jejuni [14]. AHR can be activated in WT C57BL/6 mice by oral gavage of TCDD for multiple days [15]. After multiple gavages of TCDD for consecutive days, mice will be challenged with C. jejuni. Mice will be sacrificed 5-weeks post-C. jejuni challenge and intestinal samples will be collected to access C. jejuni colonization in the gut. Furthermore, intestinal tissue sample will also be subjected to flow cytometry to determine the effect of TCDD on CD4+ CD25+ Foxp3+ T-reg cells in the lamina propria. Fecal samples will also be collected from mice during the necropsy to assess the level of IgA in mice in response to the TCDD treatment. The results from this experiment will determine if AHR activation by TCDD reduces C. jejuni colonization in a mouse model. Due to rising antimicrobial resistance in C. jejuni, it is imperative to identify novel therapeutic targets to ameliorate C. jejuni pathogenesis. Recently, AHR appeared as a promising target to enhance host resistance against lethal bacterial pathogens such as Streptococcus pneumoniae [16, 17], Listeria monocytogenes [18] and Clostridium difficile [19]. However, there is a knowledge gap in elucidating the role of the AHR in enhancing 116 host resistance against C. jejuni infection. The results of this thesis provide an initial understanding of the role of AHR in C. jejuni pathogenesis. However, future studies are warranted to further dissect the role of this receptor in the C. jejuni disease manifestation. 117 REFERENCES 118 REFERENCES 1. Ananthakrishnan, A.N., Epidemiology and risk factors for IBD. Nature Reviews Gastroenterology & Hepatology, 2015. 12(4): p. 205-217. 2. 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