Bruce Vallance

The intestinal mucus layer is an essential structure that is well recognized, not only as a key physiochemical barrier that limits direct contact between noxious agents within the intestinal lumen and the underlying epithelium, but also as an important interface between the resident gut microbiota and the host. Mucus is primarily comprised of the mucin (Muc)-2 protein, that is heavily O-glycosylated by five major sugar monomers, including sialic acid. To establish infections, enteric pathogens must evolve strategies to adapt to the intestinal environment, overcome the mucus barrier and microbiota-mediated colonization resistance to successfully infect their hosts. I investigated the interactions between the pathogen and mucus, as well as the mucus-degrading commensals during Citrobacter rodentium infection. C. rodentium is an attaching and effacing pathogen that must cross the colonic mucus layer to infect intestinal epithelial cells (IEC). I demonstrated that upon entering the host, C. rodentium localized to the colonic mucus layer and required mucin-derived sialic acid to fuel its growth and virulence. A C. rodentium strain deficient in sialic acid uptake (ΔnanT) was dramatically impaired in infecting mice. Sensing of sialic acid also enabled the pathogen to migrate towards mucus and promoted C. rodentium’s virulence by inducing the secretion of two key virulence factors, which enhanced the translocation across the mucus layer and increased adhesion to the epithelium. Mucus-degrading commensal bacteria mediated C. rodentium’s access to sialic acid by releasing sialic acid from mucin glycans. Correspondingly, the intestines of germ-free (GF) mice contained very low levels of free sialic acid and in line with this, C. rodentium displayed impaired virulence when infecting them. However, mice mono-colonized with the commensal bacterium Bacteroides thetaotaomicron that can readily degrade mucin glycans displayed increased susceptibility to C. rodentium colonic infection. Overall, my research contributes to a better understanding of how enteric bacterial pathogens interact with mucin glycans, and further emphasizes the crucial role played by mucin-degrading microbiota in enabling these interactions.

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Ultraviolet B (UVB) light phototherapy has been successfully used to treat cutaneous disorders. In contrast, little is known about its systemic immunomodulatory effects, and if UVB light exposure on the skin is able to influence the gastrointestinal barrier and the intestinal microbiome. This thesis research investigates the influence of cutaneous UVB light exposure on the microbiome in humans, as well as vitamin D independent effects on the murine gastrointestinal environment during health and disease. The results from a clinical pilot study in healthy women indicate that three sub-erythemal UVB light exposures within the same week significantly improves serum vitamin D concentrations and is able to modulate the microbiome composition of those who started the study with vitamin D insufficiency. UVB light exposures in individuals who have been taking vitamin D supplementation over the winter months did not show any significant change in microbiome composition. The results suggest that the increase in serum vitamin D is responsible for taxa specific modulation of the microbiome, predominantly in the Lachnospiraceae genera. We also investigated if, and how cutaneous UVB light exposure affects the gastrointestinal environment in mice. C57BL/6 mice were repeatedly exposed to a sub-erythemal dose of UVB light which strongly attenuated concurrent DSS-induced colitis. This treatment was not associated with elevated production of vitamin D, but instead induced increased release of microbiome-derived acetate and aryl hydrocarbon receptor (Ahr) ligands, and subsequent Ahr activation as measured by downstream enzyme CYP1A1 activity. Chronic UVB light exposure also led to increased differentiation of intestinal goblet cells and other secretory epithelial subtypes in the colon and ileum. These responses to UVB light, including protection against colitis were abrogated in Ahr deficient mice. Similarly, stool metabolites from UVB exposed mice promoted goblet cell differentiation in colon derived murine organoids. This study demonstrates that exposure to UVB light induces the release of Ahr ligands that promote the differentiation of secretory epithelial cells, thereby protecting against colitis. The results from this thesis research indicate that cutaneous UVB light exposure affects the intestinal environment. Future studies should investigate the feasibility of clinical application of UVB phototherapy.

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We investigated the role of the inflammatory caspases during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Streptomycin-pretreated wildtype C57BL/6, Casp1/11 deficient (−/−), Casp1−/− and Casp11−/− mice were orally infected and S. Typhimurium burdens determined at 18h-7d post infection (p.i.). Increased cecal and luminal pathogen burdens were observed for all caspase-deficient mice as compared to wildtype, which correlated with increased intracellular S. Typhimurium loads in the crypt IECs. Interestingly, cecal pathology scores for all inflammatory caspase mice were decreased compared to wildtype mice, especially with regard to ‘epithelial integrity’ and ‘goblet cell loss’. To determine if the increased intracellular pathogen burdens were due to the loss of IEC-intrinsic inflammasomes, cell lines and enteroid monolayers derived from each genotype and infected. These studies revealed significantly increased intracellular burdens in caspase-deficient monolayers in concert with a marked decrease in IEC sloughing and cell death. In human epithelial monolayers, siRNA-depletion of caspase-4, a human ortholog of caspase-11, led to increased bacterial colonization as well as increased secretion of the proinflammatory cytokine, interleukin (IL)-18. Inflammatory caspase activity was measured in enteroid monolayers and peak activity in wildtype cells correlated with shedding, suggesting IEC-intrinsic inflammasome-based restriction of S. Typhimurium occurs through infected IEC expulsion. To examine the effect of inflammasome signaling on overall mucosal defense, mucus layer thickness was evaluated by immunofluorescence staining. At 18h p.i., wildtype tissues demonstrated a dramatic increase in mucus thickness while only a marginal increase was observed in caspase deficient mice. Also, expression of the antimicrobial lectins Reg3γ and β were attenuated in all caspase-deficient mice. Mucin release and Reg3γ and β induction has been previously linked to the cytokine IL-22. We detected higher IL-22 levels in infected wildtype mice and when IL-22 was neutralized, wildtype mice carried increased S. Typhimurium burdens and decreased infection-induced mucin secretion and Reg3γ and β induction. No differences were observed in Casp1/11−/− mice treated with neutralizing antibody or isotype control. These results thus indicate that the intestinal mucosa utilizes inflammasome signaling to coordinate multiple layers of innate defense at the gut surface to ultimately restrict enteric pathogen infections and systemic spread.

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The intestinal tract harbours the largest population of microbes in the human body where they play an important role in promoting the health of their host. If the composition of these microbes is altered, this may lead to dysbiosis that triggers or exacerbates intestinal and extra-intestinal diseases. Probiotics have been investigated as a complementary therapy in dysbiosis-related diseases. However, their effectiveness in treating severe conditions such as Inflammatory Bowel Disease (IBD) is quite variable and have shown controversial results. To address the importance of a personalized probiotic approach to treat intestinal inflammation, we first examined the effect of personalized bacteria using a model of chemical induced colitis. The animals that received commensals isolated from their own feces were more protected against inflammation as they showed reduced signs of colitis, less histological damage and lower levels of inflammatory markers as compared to mice given a commercial probiotic strain. Next, the role of the intestinal mucin Muc2 and the Core-1 enzyme that glycosylates it were explored using the Citrobacter rodentium model of infectious colitis. The intestinal mucus layer is the first line of defense in the intestine and is largely composed of the secreted mucin Muc2. Since almost all enteric bacteria must cross the overlying mucus layer to infect the host, the mucus-enteric bacterial interactions provide fundamental knowledge about infectious diseases as well as inflammatory conditions linked to dysbiosis (e.g. IBD). Specifically, we compared C. rodentium susceptibility by infecting WT, Muc2 -/-, core 3 (C3GnT) -/-, core -1 (C1galt1) -/-, and C1galt1 f/f mice. While C3GnT -/- mice showed a very similar phenotype to WT mice with only mild inflammation, complete absence of Muc2 or just core 1 derived O-glycans resulted in significantly higher histological damage, barrier disruption, and increased pathogen burdens. Interestingly, the supplementation of tributyrin protected mice against infection resulting in less histological damage and lower C. rodentium colonization as compared to control groups. These studies highlight a novel personalized therapy that may be considered relevant to diseases affected by dysbiosis as well as the key role of Muc2 and its core 1 glycosylation in host defense against enteric infections.

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Enteric infections and neonatal intestinal development both represent times of significant change in the intestine. Mode of delivery as well as food source, i.e. breast milk or formula, may impact later susceptibility to the development of allergies, asthma and other inflammatory diseases. Similarly, the development of pathological conditions such as irritable bowel syndrome and inflammatory bowel disease have been reported as long-term sequelae of infection by various enteric pathogens. The single cell layer of epithelial cells lining the intestine are in closest proximity to the luminal changes that occur, be it interaction with a bacterial pathogen or the introduction of breast milk. This positions intestinal epithelial cells (IEC)s to serve as key players in shaping responses during these times. To address the importance of IEC responses during enteric infections, we first examined the intricacies of IEC innate immune-mediated responses (MyD88-dependent) using in vivo models. Following infection with the common food poisoning pathogen Salmonella Typhimurium, IEC-specific MyD88 signalling was required to limit pathogen penetration of intestinal crypts and mediate goblet cell/antimicrobial responses. IECs lacking MyD88 also displayed decreased bactericidal capacity against Salmonella and Citrobacter rodentium, a close relative of enteropathogenic Escherichia coli. Thus, IEC MyD88 signalling promotes early, protective responses during enteric infection. Next, IEC changes during intestinal development, as well as the impact of food source during this process (i.e. mother’s milk versus formula) were explored using a neonate pup-in-a-cup model. Specifically, the role of milk fats found in mammalian milk (and currently not widely used in formulas), referred to as milk fat globule membrane (MFGM), were assessed for their ability to normalize intestinal development to that seen with mother’s milk. Interestingly, MFGM addition to formula resulted in similar villus and crypt development, as well as goblet cell, Paneth cell and enterocyte numbers and/or expression to that of mother’s milk fed pups. Further, addition of MFGM protected the formula fed neonate from intestinal damage by bacterial toxins. These studies highlight IECs as key players in shaping beneficial responses during enteric infection and intestinal development and have important implications for newborn health as well as for populations vulnerable to enteric infections.

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The intestinal mucus layer, which is largely composed of the secreted mucin Muc2 provides a first line of defense in the intestine. Muc2 is a heavily O-glycosylated protein with core 1 and core 3 derived O-glycans as primary constituents. It plays an important role in host defense against the attaching/effacing (A/E) pathogen Citrobacter rodentium. However whether it provides protection against the invasive human pathogen Salmonella is still unclear. Furthermore, the role of O-glycosylation in mediating the protective role played by the Muc2 mucin against enteric pathogens has not been investigated. Likewise, although almost all enteric bacterial pathogens must cross the overlying mucus layer to infect the intestinal epithelium, there is very little known about mucus-enteric bacterial interactions and virulence strategies used to accomplish this feat. We began our investigations by comparing Salmonella-induced colitis and mucus dynamics in Muc2-deficient (Muc2 -/-), C3GnT -/-, and C57BL/6 (WT) mice. While absence of core 3 derived O-glycosylation only impacted epithelial barrier integrity, absence of Muc2 resulted in significantly higher barrier disruption, host mortality rates, and increased colonic and systemic Salmonella burdens. Likewise, absence of core 1 derived O-glycans (C1galt1 -/- mice) resulted in heightened susceptibility to C. rodentium, characterized by impaired mucus levels in the lumen, and bacterial aggregation in close proximity to the intestinal epithelial surface, phenotypes not seen in WT or C3GnT -/- counterparts. To understand if the non-motile pathogen C. rodentium used bacterial proteases/mucinases as a mucus degrading strategy to gain access to the underlying epithelium, we investigated the role of a putative mucinase and a class 2 SPATE PicC. While PicC did not affect C. rodentium’s ability to colonize the colon, it appeared to have an unprecedented role in regulating C. rodentium’s activation of the innate receptor TLR2, suggesting that despite its mucinase activity, PicC's major roles in vivo may be to limit C. rodentium aggregation and its recognition by the host's innate immune system. Overall these studies highlight a novel protective role of Muc2 and its O-linked glycosylation in host defense against enteric infections and the importance of Muc2-mediated regulation of pathogen burdens at the intestinal epithelial surface.

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Vitamin D deficiency increases the risk of developing inflammatory bowel disease (IBD), a disease characterized by exaggerated immune responses to luminal bacteria. While it is unclear how vitamin D impacts IBD development, it is recognized that vitamin D plays an important role in host defense against pathogenic microbes. However, the mechanisms underlying vitamin D’s ability to affect a host’s susceptibility to infection is poorly understood. Escherichia coli is a pathobiont associated with IBD. Intestinal mucosa associated E. coli have been observed in greater numbers in patients with IBD compared to healthy controls, and these bacteria have been shown to play a role in driving intestinal inflammation. Since clinically important strains of E. coli generally do not colonize mice, researchers often rely on the related but mouse-specific attaching and effacing bacterial pathogen Citrobacter rodentium. This thesis explores the impact of vitamin D in modulating host defenses and intestinal homeostasis during infection with C. rodentium. In chapter 2, I describe how treatment with active vitamin D, calcitriol worsens colitis during C. rodentium infection. Surprisingly, calcitriol treatment of infected mice led to increased pathogen burdens, exaggerated tissue pathology and mucosal erosions. In association with their increased susceptibility, calcitriol-treated mice had substantially reduced numbers of Th17 T-cells within their infected colons and defects in their production of the antimicrobial peptide RegIIIγ. In chapter 3, I describe how dietary induced vitamin D3 deficiency also increases susceptibility to C. rodentium infection. Vitamin D3 deficient mice carried higher C. rodentium burdens, developed worsened histological damage and had higher inflammatory tone. Notably, these exaggerated inflammatory responses accelerated the loss of commensal microbes and were associated with an impaired ability to detoxify bacterial lipopolysaccharide. Together, these studies show that vitamin D plays an important role in regulating host response during enteric infection. Vitamin D deficiency impairs host defense, yet treatment with the active vitamin D also suppresses Th17 T-cell responses in vivo, and may impair mucosal host defense against bacteria. These findings have important implications for patients with IBD who suffer from overactive immune responses, yet are also have a high risk of enteric infection.

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The attaching and effacing (A/E) bacterial pathogen enteropathogenic Escherichia coli (EPEC), targets the intestinal epithelial cells (IEC) lining the gastrointestinal tract, causing severe diarrhea and potentially death. Although IEC express Toll like receptors (TLRs), they are hypo-responsive to most bacterial products, thereby preventing overt inflammatory responses against commensal bacteria. Single Ig IL-1-Related Receptor (SIGIRR) is a negative regulator of interleukin (IL)-1 and TLR expressed by IECs. Its expression by IEC may limit their ability to respond to invading pathogens, potentially increasing host susceptibility to infection. To address whether SIGIRR expression influences host defense against enteric pathogens, Sigirr deficient (-/-) mice were infected with the mouse adapted A/E pathogen Citrobacter rodentium. Sigirr -/- mice responded with accelerated IEC proliferation, and strong pro-inflammatory and antimicrobial responses in this study. Yet, they were highly susceptible to infection. Exaggerated IEC response of Sigirr -/- mice were primarily dependent on IL-1R signaling leading to the rapid and dramatic loss of competing commensal microbes from the infected intestine. Thus, SIGIRR promotes commensal-based resistance to pathogen colonization despite limiting IEC responses to infection. Besides the host, A/E pathogens also actively suppress IEC inflammatory and anti-microbial responses using a type 3 secretion system to deliver bacterial effector proteins into infected IEC. To identify these effector(s), I tested an array of EPEC mutants and identified that non-LEE encoded effector (Nle)C suppressed the release of the chemokine IL-8 from infected IEC in vitro. NleC localized to EPEC-induced pedestals and inhibited both NF-ΚB and p38MAP kinase activation. Comparison between mice infected by ΔnleC to wildtype C. rodentium demonstrated that loss of NleC did not impact pathogen burdens but did result in more severe colitis. Furthermore, ΔnleC compared to wildtype C. rodentium induced significantly greater chemokine responses. Thus, innate IEC responses are actively suppressed by the host in an attempt to prevent enteric infections. However, once A/E pathogens succeed in infecting their host, they try to suppress IEC responses to prolong the infection. These studies highlight the importance of IEC as the key player in controlling host susceptibility to pathogens and in maintaining a mutualistic relationship with the intestinal commensal microbiota.

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The non-invasive attaching and effacing (A/E) pathogens Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are a prominent subgroup of diarrheagenic E. coli, and remain an important cause of morbidity and mortality worldwide. A/E pathogens intimately attach to the surface of intestinal epithelial cells, and efface or destroy their microvilli. The intestinal epithelium is the first line of defense against A/E and all pathogens, and evidence is implicating epithelial secretory cells as playing a key role in this regard. Goblet cells are specialized secretory epithelial cells that are the sole producers of the mucus barrier lining the intestinal tract through the release of the polymeric Muc2 mucin. Goblet cells also secrete the small peptide Resistin-like Molecule-{beta} (RELMβ), which plays a direct role in host defense against parasitic helminths. Despite the abundance of which these molecules are released, little is known of their role in host-protection against A/E pathogens. I hypothesized that goblet cells play a critical role in host defense against A/E bacteria by secretion of Muc2/mucus and RELMβ into the intestinal tract. Using Citrobacter rodentium, a murine A/E pathogen that is an established model of EPEC and EHEC, my results demonstrate that goblet cells are a critical component of innate host-defense against an A/E pathogen. Studies with Muc2⁻/⁻ mice show that Muc2/mucus production was critical for limiting luminal burdens, and for flushing away pathogenic bacteria as well as commensal bacteria from the mucosa. Moreover, RELMβ was highly induced and secreted into the lumen during the first week of infection. Studies with Retnlb⁻/⁻ mice demonstrated that RELMβ production limited cecal burdens, deep penetration of colonic crypts, and severe inflammatory damage following infection. Lastly, adaptive immunity plays a role in modulating goblet cell function, by promoting a down-regulation of goblet cell-specific gene expression and protein production, including Muc2. This effect appears to reflect a generalized adaptive immunity-mediated epithelial proliferative response and is associated with clearance of surface-associated pathogens. Thus, goblet cells are critical for managing infection by an A/E bacterial pathogen. These studies highlight a novel and previously unappreciated function of goblet cells in host defense against enteric bacterial infection.

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Enteropathogenic Escherichia coli (EPEC) belongs to the attaching and effacing (A/E) family of bacterial pathogens, which infect intestinal enterocytes with type three secretion system (T3SS), leading to diarrheal disease. While A/E bacteria also express flagellin (FliC) which inducessecretion of pro-inflammatory interleukin (IL)-8 from intestinal epithelial cells (IECs), it is unclear if flagellin is the sole trigger of epithelial responses. IECs express innate toll-like receptors (TLRs)to recognize flagellin and trigger inflammatory responses. Activation of TLRs is strictly controlled in IECs producing a state of hyporesponsiveness which prevents unwanted inflammation. The Single IgG IL-1 related receptor (Sigirr) is described as a negative regulator of IL-1β and TLR4 responses. While IECs express Sigirr, it is unknown if Sigirr inhibits innate responses to bacterial flagellin. The aims of this study were to characterize the role of flagellin in innate responses to EPEC infection and determine how Sigirr regulates these responses. Following infection of Caco-2 IECs by wild type and △fliC EPEC, activation of several proinflammatorygenes including IL-8, MCP-1 and MIP3α occurred in a FliC-dependent manner. Atlater time points, a subset of these pro-inflammatory genes (IL-8, MlP3α) was also induced in cells infected with △fliC EPEC. The mouse adapted A/E pathogen Citrobacter Rodentium, triggered asimilar innate response through a TLR5-independent but partially NF-kB-dependent mechanism. Moreover, the EPEC F1iC-independent responses increased in the absence of T3SS, suggesting that translocated bacterial effectors attenuated these response. While exploring regulatory mechanisms, we found that exposure of non-transformed TECs to bacterial flagellin transiently downregulated Sigirr expression correlating with IL-8 response. Transient silencing of the Sigirr gene augmented IL-S responses to flagellin, whereas stable over-expression of Sigirr diminished theNF-kB mediated IL-8 response to TLR ligands and inflammatory cytokines. The expression of Sigirr increased as IECs differentiated in tissue culture. Similarly, Sigirr expression was prominent in differentiated cells on the apex while diminished at the base of intestinal crypts in human colonic tissues. Thus, we demonstrate that A/E pathogens trigger pro-inflammatoryresponses through both FliC-dependent and -independent pathways that are regulated by Sigirr in differentiated human IEC, with clinical implications for infectious and inflammatory bowel diseases.

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Salmonellosis poses a global threat to human health. Host resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) in the murine model is mediated by Natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical for host defense, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S. Typhimurium. Despite this critical role, the mechanisms underlying Nramp1’s protective effects are unclear. This thesis presents a detailed analysis of Nramp1 expression in the murine gastrointestinal tract and its impact on S. Typhimurium infection following oral infection. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium and play an important role in Salmonella pathogenesis. Intestinal, splenic and bone marrow derived DCs (BMDCs) all expressed Nramp1 protein. In intestinal DCs, Nramp1 expression is restricted to a discrete subset of DCs (CD11c⁺ CD103-) that express elevated levels of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in DCs, infected Nramp1⁺/⁺ DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-α) than Nramp1-/- DCs. This suggests that Nramp1 expression promotes accelerated inflammatory responses to S. Typhimurium. This hypothesis was tested using the Salmonella-induced colitis model, where pre-treatment of mice with antibiotics enhances colonization of the cecum/colon and induces massive inflammation. We found that Nramp1⁺/⁺ mice mounted a faster and more robust inflammatory response characterized by elevated pro-inflammatory cyto/chemokines (IFN-γ, TNF-α and MIP1-α) and recruitment of neutrophils and macrophages, thereby limiting spread of S. Typhimurium to systemic sites and ultimately protecting the host.Nramp1⁺/⁺ mice also developed a chronic Salmonella infection of the gastrointestinal tract that led to severe tissue fibrosis. Intestinal fibrosis is a serious complication of Crohn’s disease, often requiring surgical intervention but the mechanisms underlying its development are poorly understood due to the lack of relevant animal models. A novel model of severe and persistent intestinal fibrosis caused by chronic bacterial induced colitis was developed. Since the pathology closely resembles human fibrosis, we present a valuable tool for investigating host and bacterial contributions to inflammatory bowel diseases, as well as infectious colitis.

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