Diarrheal disease causes 1.5 million deaths each year. Among the causative agents, enteropathogenic and enterohemorrhagic Escherichia coli are of particular concern. Citrobacter rodentium is a murine pathobiont used to model enteropathogenic E. coli and enterohemorrhagic E. coli infections because of shared virulence mechanisms and disease features, with infection exhibiting a distinct spatial distribution within the colon, by an unknown mechanism. The hypothesis of this study was that regional innate immune responses mediated by colonic goblet cells and proinflammatory cytokines contribute to the spatial distribution of C. rodentium in the colon. To interrogate this, mucus production, proinflammatory responses, and goblet cell lineage activity were assessed as potential drivers of C. rodentium site selection in the colon. C57BL/6 mice were infected with either DBS100 or fluorescent C. rodentium, and bacterial burden, localization, intestinal permeability, and cytokine expression were analyzed. Whole-body imaging showed high bacterial infection in the mid and distal colon, with corresponding elevated expression of proinflammatory cytokines in these regions. In contrast, analysis of key mucus proteins revealed region-specific differences, with goblet cell lineage markers and Muc2 expression significantly up-regulated only in the proximal colon, associated with enhanced protection against bacterial colonization in this region. These findings demonstrate that goblet cells and proinflammatory cytokines influence the regional distribution of C. rodentium infection and that mucus-associated protein regulation occurred independently of MUC2.

Colonic goblet cells play a crucial role in mucosal defense by secreting Muc2 mucin and other proteins that entrap and expel enteropathogens. However, the role of innate effectors in the gut like cathelicidin peptides in regulating the mucus barrier during infections remains unclear. In this study, we used cathelicidin-deficient (Camp-/-) littermates, colonoids, and human LS174T goblet-like cells to investigate how cathelicidin modulates goblet cell function and mucosal defense against attaching/effacing enteropathogen Citrobacter rodentium. We showed that increased fecal shedding and epithelial colonization by C. rodentium in Camp-/- littermates was accompanied by impaired mucus secretion and higher retention of mucin granules and trefoil factor 3 (Tff3) in bloated colonic goblet cells. Reduction in mucus secretion by goblet cells was accompanied by reduced reactive oxygen species (ROS) production during C. rodentium infection in Camp-/- as compared to Camp+/+ littermate controls. In LS174T goblet-like cells, human cathelicidin LL-37 stimulated the secretion of TFF3 and resistin-like molecule β (RELMβ) in a ROS-dependent manner. These findings reveal that cathelicidin regulates goblet cell mucus and mucus-associated protein secretion through a ROS-mediated mechanism critical for bacterial clearance and maintenance of gut homeostasis.

Goblet cells are the guardians that produce MUC2 mucin that forms the protective mucus barrier as the first line of innate host defense against pathogen invasion while sustaining a healthy gut microbiota. Using an unbiased screen, we cloned a high LS174T colonic adenocarcinoma MUC2 mucin-producing goblet-like cell that revealed by whole genome sequencing, mutations that upregulated the expression of tumor suppressor TP53, anterior gradient 2 (AGR2) and mitogen-activated protein kinase kinases (MEK and MAPK) genes that enhanced MUC2 biosynthesis and secretion. Hypersecretory mutant (Mut) cells exhibited a phenotype characterized by high constitutive MUC2 mRNA, upregulation of glycosyltransferases, and hypersecretion of sialylated and fucosylated MUC2 mucus enumerated by glycomics analysis. Mut secreted mucus was altered quantified by increased permeability to fluorescence microsphere (0.2 μm and 1 μm) beads and to Salmonella enterica adherence, invasion, and cell death as compared to WT controls. Shotgun proteomic analysis revealed that Mut cells were upregulated in metabolic pathways for oxidative stress, HIF pathways and growth factors that enhanced wound healing, and tumorigenesis. These results highlight the importance of regulatory genes for proper mucus production in providing barrier function and homeostasis in the gastrointestinal tract that is markedly affected by metabolic stress and glycosylation prevalent in colonic carcinomas.

The mucus layer produced by highly stressed goblet cells forms a protective shield in the gut to protect the underlying mucosal epithelial cells from external threats. Hypersecretion and depletion of mucin-2 (MUC2) mucin from goblet cells is characteristic of symptomatic Entamoeba histolytica infections. It was hypothesized that MUC2 depleted goblet cells could mount a second line of innate host defense by producing proinflammatory cytokines. To investigate this, whether E. histolytica could stimulate proinflammatory responses in wild-type (WT) high MUC2 mucin-producing goblet-like cells and in clustered regularly interspaced palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) gene-edited MUC2KO cells was investigated. In response to live E. histolytica and soluble E. histolytica proteins, WT, and to a lesser extent, MUC2KO cells produced high levels of CXCL8. Entamoeba histolytica temporally induced greater levels of CXCL8 mRNA expression and protein secretion in WT versus MUC2KO cells, which was abrogated with alleviation of endoplasmic reticulum stress with the NADPH-oxidase inhibitor diphenyleneiodonium chloride. WT cells produced elevated reactive oxygen species that induced longer half-lives of CXCL8 transcripts, which was abrogated with diphenyleneiodonium chloride. Western blot and proteomic analyses revealed that WT cells, but not MUC2KO cells, were basally primed to respond to external stressors and responded to E. histolytica through rapid activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase, mitogen-activated protein kinase/p38, and phosphatidylinositol 3-kinase/Akt pathways, to induce CXCL8. These results suggest that colonic goblet-like cells defend against E. histolytica infections by hypersecreting mucus and produce the chemokine, CXCL8, to recruit neutrophils.

Abstract Background MUC2 mucin produced by colonic goblet cells, form a mucus bilayer that provides a physical barrier between potential pathogens in the lumen and the underlying epithelial cells. Mucus is thus the first line of innate host defense in the gastrointestinal (GI) tract. Many GI diseases including inflammatory bowel disease and colon cancer affect the glycosylation of mucus. Goblet cells produce a variety of proteins that are associated with the mucus layer. Of these proteins, FCGBP is of significant interest due to its structural similarities to MUC2 mucin with unknown functions. In this study, we investigated if a missense mutation in FCGBP altered the glycosylation of goblet cell MUC2 and affected its barrier functions. Aims Aims: 1) To determine mechanistically how FCGBP impeded the structural integrity of the mucus layer 2) To quantify MUC2 glycoprotein modifications in the altered mucus layer Methods To investigate whether FCGBP impaired mucus barrier functions, two cell types were investigated: wildtype LS174T (WT) MUC2 mucus-producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP-Mut). To determine if FCGBP-Mut led to loss in barrier function in the mucus layer, the penetration of 0.2, 1, and 2 μm fluorescent beads (to mimic bacteria) through the mucus layer were quantified. To determine if the differences in penetrability were caused by differences in MUC2 glycosylation in the goblet cell lines, sensitive glycomic analyses were performed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Both cells and purified MUC2 mucin granules were analyzed and confirmed by lectin binding assays. To enumerate differences in the glycomics profiles, RT-PCR was performed on over 30 human glycosyltransferases. Results FCGBP-Mut cells exhibited an altered glycomics profile with a significant increase in sialylated/fucosylated glycans as quantified by HPLC-MS and an increase in sialyl- and fructosyltransferase mRNA expression. In contrast to WT goblet cells, FCGBP-Mut cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetration through the mucus layer temporally. The altered glycosylation in FCGBP-Mut cells triggered increased metabolic stress and ROS production that enhanced susceptibility to Salmonella enterica binding, invasion and cell death. Conclusions This study demonstrates that a single missense mutation in FCGBP altered the penetrability of the mucus layer associated with an increase in sialylated/fucosylated glycans, a signature hallmark of numerous colonic diseases. These findings underscore the importance of FCGBP in providing structural integrity of the mucus layer and homeostatic maintenance of goblet cell glycosylation profiles. Funding Agencies CIHR

Co-existing chronic hepatitis B virus (CHB) infection and metabolic dysfunction associated steatotic liver disease (MASLD) can exert complex effects on hepatic metabolism, requiring mechanistic study. CHB participants were assessed for MASLD and the impact of hepatic steatosis/metabolic syndrome (MetS) on novel viral and immunological markers. In this prospective, cohort study, untreated CHB subjects were assessed for liver disease by non-invasive tests (i.e. FibroScan, controlled attenuation parameter, CAP). Subjects were tested for cytokines and IFN-gamma ELISPOT assay to HBV Surface (S) and Core (C) proteins. Standard HBV serological, exploratory biomarkers and deep sequencing of HBV S and C genes were performed. In 53 subjects (median age 45 years [SD = 10.6], 35% F, 56% Asian, 20% Black, 3% White), 94% (50) HBeAg negative, 63% genotype B/C, mean HBV DNA 3.2 log10 IU/mL (SD = 1.8), quantitative HBsAg 2.9 log(10) IU/mL (SD = 1.2) and HBV pgRNA 2.1 log(10) copies/mL (SD = 1.3). In enrolled subjects, the mean ALT was 41.9 U/L (SD = 24.0), FibroScan was 5.7 kPa (SD = 1.9) and CAP was 306.4 dB/m (SD = 49.0). The mean BMI was 28.2 kg/m(2) (SD = 4.2), 20% (11/53) had diabetes, 35% (19/53) dyslipidaemia and 24% (13/53) hypertension. Subjects with MetS and steatosis showed lower HBV markers (p < .01), higher HBV S diversity (p = .02) and greater frequency of HBV variants associated with host-anti-viral immune escape. Pro-inflammatory cytokine levels and HBV-specific cellular responses were higher in participants with hepatic steatosis. In CHB, MASLD/hepatic steatosis was associated with HBV variants and systemic immune responses potentially impacting liver disease progression despite low-level viraemia.

The O-glycoprotein Mucin-2 (MUC2) forms the protective colon mucus layer. While animal models have demonstrated the importance of Muc2, few studies have explored human MUC2 in similar depth. Recent studies have revealed that secreted MUC2 is bound to human feces. We hypothesized human fecal MUC2 (HF-MUC2) was accessible for purification and downstream structural and functional characterization. We tested this via histologic and quantitative imaging on human fecal sections; extraction from feces for proteomic and O-glycomic characterization; and functional studies via growth and metabolic assays in vitro. Quantitative imaging of solid fecal sections showed a continuous mucus layer of varying thickness along human fecal sections with barrier functions intact. Lectin profiling showed HF-MUC2 bound several lectins but was weak to absent for Ulex europaeus 1 (α1,2 fucose-binding) and Sambucus nigra agglutinin (α2,6 sialic acid-binding), and did not have obvious b1/b2 barrier layers. HF-MUC2 separated by electrophoresis showed high molecular weight glycoprotein bands (∼1-2 MDa). Proteomics and Western analysis confirmed the enrichment of MUC2 and potential MUC2-associated proteins in HF-MUC2 extracts. MUC2 O-glycomics revealed diverse fucosylation, moderate sialylation, and little sulfation vs. porcine colonic MUC2 and murine fecal Muc2. O-glycans were functional and supported the growth of Bacteroides thetaiotaomicron (B. theta) and short-chain fatty acid (SCFA) production in vitro. MUC2 could be similarly analyzed from inflammatory bowel disease stools, which displayed an altered glycomic profile and differential growth and SCFA production by B. theta vs. healthy samples. These studies describe a new non-invasive platform for human MUC2 characterization in health and disease.

Abstract Background Colonic goblet cells by secreting Muc2 mucin and specific proteins is critical for physically entrapping and expelling invading enteropathogens. Thus, is not surprising that Muc2-/- littermates exhibit increased susceptibility to attaching/effacing Citrobacter rodentium colonization. The colonic epithelium also secretes small cathelicidin peptide, which potentially interacts intimately with goblet cells and was presumed to accumulate within the sterile inner mucus layer as a simple antimicrobial peptide defense. Aims To determine the effects of cathelicidin on mucin secretion in goblet cells during C. rodentium-induced colitis and the impact on the mucus barrier defense. Methods We used cathelicidin-deficient (Camp-/-) mice, mouse colonoids and human colonic LS174T like-goblet epithelial cells to elucidate the mechanisms by which cathelicidin regulates goblet cell secretions. Results Camp -/- littermates infected with C. rodentium displayed increased fecal shedding and epithelial colonization. Camp-/- littermates at the peak of C. rodentium infection (7 dpi) showed a deficient mucin layer with fewer Alcian blue/PAS filled goblet cells and a reduction in fucose (UEA-1+) and N-acetylglucosamine (WGA+) glycoproteins. By transmission electron microscopy (TEM), goblet cells in Camp-/- colons were swollen and retained a large number of mucus granules during C. rodentium infection. C. rodentium infected Camp-/- littermates showed impaired reactive oxygen species (ROS) production and a transcriptomic profiling associated with decreased ROS biosynthesis and an increase in ROS negative regulators. In mucin producing LS174T colonic epithelial cells, human cathelicidin LL-37 promptly induced the secretion of goblet cell-associated TFF3 and RELMβ, via a ROS-dependent mechanism. Conclusions These findings revealed that mice lacking cathelicidin (Camp-/-) were more susceptible to C. rodentium colonization caused by defective goblet cell mucus and mucin-associated protein secretion via a ROS-dependent mechanism. Importantly, cathelicidin regulated mucus secretion revealing a non microbicidal action of this peptide with homeostatic properties on the colonic mucus barrier, critical in excluding luminal microbiota away from the epithelia to clear bacterial infections and restore gut homeostasis. Funding Agencies NSERC

The colonic mucus bilayer is the first line of innate host defense that at the same time houses and nourishes the commensal microbiota. The major components of mucus secreted by goblet cells are MUC2 mucin and the mucus-associated protein, FCGBP (IgGFc-binding protein). In this study, we determine if FCGBP and MUC2 mucin were biosynthesized and interacted together to spatially enhance the structural integrity of secreted mucus and its role in epithelial barrier function. MUC2 and FCGBP were coordinately regulated temporally in goblet-like cells and in response to a mucus secretagogue but not in CRISPR-Cas9 gene-edited MUC2 KO cells. Whereas ~85% of MUC2 was colocalized with FCGBP in mucin granules, ~50% of FCGBP was diffusely distributed in the cytoplasm of goblet-like cells. STRING-db v11 analysis of the mucin granule proteome revealed no protein-protein interaction between MUC2 and FCGBP. However, FCGBP interacted with other mucus-associated proteins. FCGBP and MUC2 interacted via N-linked glycans and were non-covalently bound in secreted mucus with cleaved low molecular weight FCGBP fragments. In MUC2 KO, cytoplasmic FCGBP was significantly increased and diffusely distributed in wounded cells that healed by enhanced proliferation and migration within 2 days, whereas, in WT cells, MUC2 and FCGBP were highly polarized at the wound margin which impeded wound closure by 6 days. In DSS colitis, restitution and healed lesions in Muc2+/+ but not Muc2-/- littermates, were accompanied by a rapid increase in Fcgbp mRNA and delayed protein expression at 12- and 15-days post DSS, implicating a potential novel endogenous protective role for FCGBP in wound healing to maintain epithelial barrier function.

BACKGROUND & AIMS: Inflammatory bowel diseases (IBD) are affected by dietary factors, including nondigestible carbohydrates (fibers), which are fermented by colonic microbes. Fibers are overall beneficial, but not all fibers are alike, and some patients with IBD report intolerance to fiber consumption. Given repro-ducible evidence of reduced fiber-fermenting microbes in patients with IBD, we hypothesized that fibers remain intact in select pa-tients with reduced fiber-fermenting microbes and can then bind host cell receptors, subsequently promoting gut inflammation. METHODS: Colonic biopsies cultured ex vivo and cell lines in vitro were incubated with oligofructose (5 g/L), or fermentation su-pernatants (24-hour anaerobic fermentation) and immune re-sponses (cytokine secretion [enzyme-linked immunosorbent assay/meso scale discovery] and expression [quantitative poly-merase chain reaction]) were assessed. Influence of microbiota in mediating host response was examined and taxonomic classifi- cation of microbiota was conducted with Kraken2 and metabolic profiling by HUMAnN2, using R software. RESULTS: Unfermented dietary b-fructan fibers induced proinflammatory cytokines in a subset of IBD intestinal biopsies cultured ex vivo, and immune cells (including peripheral blood mononuclear cells). Results were validated in an adult IBD randomized controlled trial examining b- fructan supplementation. The proinflammatory response to intact b-fructan required activation of the NLRP3 and TLR2 pathways. Fermentation of b-fructans by human gut whole microbiota cul-tures reduced the proinflammatory response, but only when mi-crobes were collected from patients without IBD or patients with inactive IBD. Fiber-induced immune responses correlated with microbe functions, luminal metabolites, and dietary fiber avoidance. CONCLUSION: Although fibers are typically beneficial in individuals with normal microbial fermentative potential, some dietary fibers have detrimental effects in select patients with active IBD who lack fermentative microbe activities. The study is publicly accessible at the U.S. National Institutes of Health data-base (clinicaltrials.gov identification number NCT02865707).

Abstract Background Dietary fibers are not digested in the bowel; they are fermented by microbes, typically promoting gut health. However, IBD patients experience sensitivity to consumption of fibers. Our previous findings offered the first mechanistic evidence demonstrating that unfermented dietary β-fructans (inulin and oligofructose) can induce pro-inflammatory cytokines and altered epithelial barrier integrity in a subset of pediatric IBD colonic biopsies cultured ex vivo, and in the SYNERGY-1 (β-fructan) clinical study of adult remission UC patients. Fermentation of β-fructan by whole-microbiota intestinal washes from non-IBD or remission IBD patients (but not non-IBD microbes) reduced pro-inflammatory responses. Purpose Here we aimed to expand our findings to uncover the physiologically relevant gut immune and epithelial responses to over 50 unfermented and partially fermented dietary fibers (arabinoxylans, β-glucans, β-mannans, galatooligosaccharides, inulins, oligofructoses, pectins, raffinooligosaccharides, xyloglucans) sourced from commonly consumed fruits, grains, and vegetables to better understand which foods are safe for IBD patients, and in which disease state settings. Method Colonic biopsies cultured ex vivo, peripheral blood mononuclear cells (PBMCs), colonic organoids, and cell lines were incubated with individual dietary fibers or mixture of fibers extracted from commonly consumed fruits, grains, and vegetables. Epithelial barrier integrity (TEER, microscopy, FITC-dextran) and immune responses (cytokine secretion [ELISA/MSD] and expression [qPCR]) were assessed. Structural features of the different fibers (e.g., degree of polymerization, phenolic/phytic content, branching, sugar content) were measured by HPLC and gas chromatography and correlated to host cell responses. Result(s) Most significantly unfermented inulin, oligofructose, and arabinoxylan induced pro-inflammatory responses, particularly in myeloid cells. Pectin and galatooligosaccharides were either non-inflammatory or anti-inflammatory depending on the food source. The epithelial barrier response to select dietary fibers correlated more significantly with the chemical properties of the fibers; longer fibers (greater degree of polymerization; e.g., inulin) displayed improved barrier integrity while shorter dietary fibers with higher phenolic content displayed reduced barrier integrity. Fiber structural properties varied significantly between different fiber subtypes along with the same fiber subtype sourced from different foods. Conclusion(s) Our findings suggest that intolerance and avoidance of fibers in select IBD patients occurs in patients whose gut microbiota do not support fermentation of fibers resulting in increased presence of unfermented dietary fibers in the gut. Here we show which specific dietary fibers from specific food items can elicit gut barrier damage and inflammation in the gut dependent on fiber structural features, suggesting mechanisms underlying IBD patient avoidance of specific high-fiber foods. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; Weston Family Foundation, MMSF, NSERC, CRC Disclosure of Interest None Declared

Abstract Background MUC2 mucin, produced by colonic goblet cells, forms a mucus bilayer that provides a physical barrier between potential pathogens in the lumen and the underlying epithelial cells. Mucus is thus the first line of innate host defense in the gastrointestinal (GI) tract. Many GI diseases including inflammatory bowel disease and colon cancer affect the glycosylation of mucus. Goblet cells produce a variety of proteins that are associated with the mucus layer. Of these proteins, FCGBP is of significant interest due to its structural similarities to MUC2 mucin with unknown functions. In this study, we investigated how a missense mutation in FCGBP altered the glycosylation of goblet cell MUC2 and affected its barrier functions. Purpose Hypothesis: A missense mutation in FCGBP results an impaired mucus layer by the altering glycomic profiles of goblet cell mucins. Specific aims: 1) To determine mechanistically how FCGBP impeded the structural integrity of the mucus layer 2) To quantify MUC2 glycoprotein modifications in the altered mucus layer Method To investigate whether FCGBP impaired mucus barrier functions, two cell types were investigated: wildtype LS174T (WT) MUC2 mucus-producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS). To determine if FCGBP MS led to loss in barrier function in the mucus layer, the penetration of 0.2, 1, and 2 μm fluorescent beads (to mimic bacteria) through the mucus layer were quantified. To determine if the differences in penetrability were caused by differences in MUC2 glycosylation in the goblet cell lines, sensitive glycomic analyses were performed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Both intact cells and isolated MUC2 mucin granules were analyzed. To determine if differences in the glycomic profiles was caused by differences in glycotransferases, RT-PCR was performed on over 30 human glycosyltransferases. Result(s) FCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetration through the mucus layer in a temporal manner. FCGBP MS cells exhibited an altered glycomic profile with a notable increase in sialylated glycans as quantified by HPLC-MS. The increase in sialylated glycans was associated with a significant increase in sialyl-transferase expression in FCGBP MS cells. Conclusion(s) These data demonstrate that a single missense mutation in FCGBP altered the penetrability of the mucus layer associated with an increase in sialylated proteins, a signature hallmark of numerous colonic diseases. FCGBP was critical in providing structural integrity of the mucus layer and maintenance of goblet cell glycosylation profiles. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

Abstract Background The colonic mucus bilayer is an integral innate host defense mechanism that provides a physical barrier separating the lumen and its contents from the underlying epithelium. This essential barrier is produced by specialized secretory goblet cells of which Muc2 mucin is its primary product. IgG-Fc-binding protein (Fcgbp) is the second most abundant protein produced by goblet cells, which has a suggested function of crosslinking with Muc2 to stabilize the structural integrity of mucus. FCGBP is observed to decrease preceding the onset of inflammation in ulcerative colitis patients, leading to spatially distinct structural mucus weakening to contribute to the pathogenesis of the disease. Purpose Fcgbp is altered regionally in the gut and plays a role in the pathogenesis of dextran sulfate sodium (DSS)-induced colitis. The specific aims are: To characterize the spatial expression of Muc2 and Fcgbp basally in goblet cells To quantify alterations in Muc2 and Fcgbp in response to DSS-induced colitis and at restitution of disease Method mRNA and protein expression in Muc2+/+ and Muc2-/- C57BL/6 littermates were analyzed by RT-qPCR and Western blotting, respectively. Mucin granules were isolated from colonic goblet cells and the proteome quantified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Colitis was induced in Muc2+/+ mice with 3.5% (w/v) DSS in tap water for five days, whereas Muc2-/- littermates were given 1.5% (w/v) DSS in tap water for three days, ad libitum. Mice were given regular tap water for the remainder of the experiment to allow restitution of inflammation. Disease activity index (DAI) was scored based on weight loss. Mice were sacrificed at various time points up to 10 days, and colons excised and sectioned for histopathology analysis. Result(s) LC-MS/MS of mucin granules run under reducing and non-reducing conditions confirmed that Muc2 and Fcgbp were the most abundant proteins in mucin granules and were non-covalently bound to each other. mRNA and protein expression of Muc2 and Fcgbp were highly expressed in the mid colon, and regulation of Fcgbp was unaffected in Muc2-/- littermates. In response to DSS-induced colitis in Muc2+/+ mice, Muc2 transcription rapidly increased in all regions of the colon with highest expression in the mid colon. In contrast, Fcgbp transcription increased in the mid and distal colons and peaked during highest disease activity. Interestingly, Fcgbp protein expression was abrogated in the mid colon even at restitution. In Muc2-/- mice, Fcgbp transcription decreased during disease onset but returned to normal levels following removal of DSS. Image Conclusion(s) This study demonstrates that in response to DSS, Fcgbp expression was spatially degraded at the onset of disease and remained low at restitution. The disappearance of Fcgbp in the mid colon of Muc2+/+ littermates, despite Muc2 restoration, suggests that the mucus barrier remains structurally altered and functionally impaired at restitution. Supported by CIHR Disclosure of Interest None Declared

Abstract Background The gel-forming O-glycoprotein Mucin-2 (MUC2) is a key mediator of host-microbe homeostasis in part by forming a barrier to segregate inflammatory microbes from distal colon tissues. While animal models have provided insights into how Muc2 functions, relatively few studies have explored human MUC2. This is due to difficulty in accessing primary MUC2 which has been traditionally seen as firmly adherent to tissues and thus attainable mainly through invasive approaches (e.g. surgery, biopsies, etc), or via transformed cell lines (e.g. LS174T). This highlights a need to find alternative sources of MUC2. Purpose The purpose of this study is to develop a non-invasive method to analyze human MUC2 from healthy persons and determine if this can be applied to disease states. Recent studies have shown a significant portion of MUC2 is bound to feces (Bergstrom and Shan et al, 2020). We therefore reasoned this fecal MUC2 was accessible for both purification and structural and functional characterization. Method We purified MUC2 from feces via established extraction methods used for tissues. The mucins were resolved by composite urea agarose polyacrylamide gel electrophoresis (UreaAgPAGE) and analyzed by in-gel staining with Alcian Blue (AB), or Western blot for lectins and MUC2. Mucins were subjected to both proteomics to confirm enrichment of MUC2; and O-glycomics via non-reductive ammonia-catalyzed β-elimination followed by capillary electrophoresis (CE) and mass spectrometry in parallel with Type III porcine gastric mucin (PGM) O-glycans for comparison. Purified O-glycans were tested functionally via microbial growth assays with Bacteroides spp, and the ability to be metabolized into short-chain fatty acids (SCFA). Mucus barrier function was also visualized directly on Carnoy's-fixed paraffin-embedded (CFPE) fecal sections followed by dual staining for bacteria by FISH, and MUC2 and/or lectins. Result(s) Confocal imaging of CFPE fecal sections revealed a microbiota-encapsulating barrier layer of varying thickness among various healthy human subjects. UreaAgPAGE showed high molecular weight bands (~1 – 2 MDa) by AB staining. Proteomics and western analysis confirmed MUC2 enrichment in fecal mucin preparations. Western analysis via a lectin panel showed human MUC2 bound several lectins but was notably lacking in signal for Sambucus nigra lectin (SNA; α2,6-linked sialic acid) or Ulex europaeus agglutinin I (UEA1; α1,2-linked fucose). O-glycomics revealed extensive sialylation, moderate sulfation, and very little fucosylation vs. PGM. Functionally, the glycans supported growth of Bacteroides thetaiotaomicron as well as B.theta-dependent SCFA production in vitro. Pilot studies with human Ulcerative Colitis (UC) showed intact MUC2 with a differential O-glycosylation profile by glycan "fingerprinting" via CE. Conclusion(s) These studies highlight a new way to access primary human MUC2 for downstream functional analyses and pave the way for characterizing MUC2 dysfunction in diseases including IBD. Disclosure of Interest None Declared A71 SIGNIFICANT RACIAL/ETHNIC DIFFERENCES EXIST IN THE RECEIPT OF IBD-RELATED SURGERY A SYSTEMATIC REVIEW AND META-ANALYSIS T. Chhibba*, P. Tandon, N. Natt, G. Brar, G. Malhi, G. Nguyen Background Patients with inflammatory bowel disease (IBD) may require surgical intervention for management of their disease. There is a rising incidence of IBD in racial and ethnic minorities but studies regarding healthcare utilization patterns in these populations have yielded variable results. Purpose We aimed to examine the differences in surgical rates of ethnic and racial groups compared to White patients with IBD. Method Electronic databases were searched through December 20, 2021. Studies that compared ulcerative colitis (UC) or Crohn’s disease (CD) surgery rates between different racial/ethnic groups were included. Both pediatric and adult studies were included. Pooled event rates were generated and p-value < 0.05 was considered statistically significant in generating odds ratios (OR) with 95% confidence interval (CI). We also compared differences in disease location, phenotype, and IBD-medication exposure amongst different groups included. Result(s) Forty-one studies stratified rates of IBD-related surgeries by race or ethnicity (n=1,094,693 patients). Black patients were less likely to undergo IBD-related surgeries compared to White patients (pooled OR 0.70, 95% CI, 0.55-0.89, I2=87.0%). Black patients were also less likely compared to White patients to undergo an emergent colectomy with an incidence rate ratio of 0.43 (95% CI, 0.32-0.58). Furthermore, Hispanic patients were less likely to undergo a CD-related surgery (pooled OR 0.57, 95% CI, 0.48-0.68, I2=0%) compared to White patients. Finally, Asian patients had no significant difference in likelihood of CD-related and UC-related surgeries compared to White patients. Black patients were more likely to have perianal disease (pooled OR 1.40, 95% CI, 1.06-1.86), I2=58.2%) but otherwise disease characteristics and phenotypes were similar across all populations compared to Caucasians. Conclusion(s) Black and Hispanic patients with IBD are less likely to have surgery, including emergent surgery, for IBD compared to White patients with IBD, despite similar disease phenotype characteristics. Disparities in access to care may be contributory toward these findings and efforts should be made to provide equitable care to all persons living with IBD, regardless of race and ethnicity. 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Abstract Background The colonic mucus bilayer is the first line of innate defense against pathogen invasion in the gut by forming a physical barrier between the lumen and the underlying single layer of mucosal epithelial cells. While MUC2 mucin is the primary component of the mucus layer, it also contains several mucus associated proteins of which FCGBP is of significant interest due to its structural similarities to MUC2 with unknown functions. Here we elucidated the functions of FCGBP in MUC2 mucin in response to S. typhimurium ( St) adherence and invasion in human colonic goblet cells. Aims Hypothesis: FCGBP is coordinately produced with MUC2 mucin and plays an important role in innate host defense. The specific aims are: 1. To determine if FCGBP alters the structural integrity of the mucus layer 2. To determine the role of FCGBP in St adherence and invasion of goblet cells Methods To investigate whether FCGBP impaired mucus barrier functions, wildtype LS174T (WT) MUC2 mucus-producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS) were used. To determine if FCGBP MS led to loss in barrier function, St adherence and invasion, and 0.2, 1, and 2 μM fluorescent bead penetration through the mucus monolayers were quantified. MUC2 and FCGBP mRNA and protein expression induced by St were analysed by RT-PCR and Western blotting. St-induced pro-inflammatory cytokines responses were analyzed by RT-PCR and human focus 15-plex cytokine and chemokine array. Cell killing was enumerated by LDH assay. Results FCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetration closer towards the epithelial cell surface temporally as compared to WT cells independent of bead size. Adherence of St was significantly increased in FCGBP MS cells and induced robust MUC2 mRNA and protein expressions in a time-dependent manner. Similarly, St elicited robust expression of pro-inflammatory cytokine mRNA and protein release in FCGBP MS as compared to WT cells. FCGBP MS were readily invaded by St that resulted in increased cell death as compared to WT cells. Conclusions Loss of function by FCGBP MS, but not in WT cells, showed increased penetration of fluorescent beads through the mucus layer that resulted in increased St adherence, invasion, and cell death. In WT cells, FCGBP and MUC2 were coordinately upregulated in response to St. The concurrent increase in pro-inflammatory cytokine expression in FCGBP MS cells in response to St suggests that bacteria directly interacted with the cell surface indicative of an impaired mucus layer. The overall trend for increased bacterial invasion, pro-inflammatory response and cell death in FCGBP MS demonstrates that FCGBP was critical in providing structural integrity and protective functions of the mucus layer. Funding Agencies CIHR

Abstract Background A hallmark of Entamoeba histolytica ( Eh) invasion in the gut is acute intestinal inflammation dominated by the secretion of pro-inflammatory cytokines. Live Eh in contact with macrophages activates caspase-1 by the recruitment of the NLRP3 inflammasome in a Gal-lectin and Eh cysteine proteases 5 ( EhCP-A5)-dependent manner, resulting in the maturation and secretion of IL-1β. Eh in contact with macrophages also activates caspase-4 by outside-in signaling but it is unclear how Eh-induced caspase-1/4 regulates gasdermin D (GSDMD) cleavage to drive both pore formation and IL-1β secretion without causing cell death. In this study, we interrogated the requirements and mechanism of Eh-induced caspase-4 activation in cleaving GSDMD to mediate bioactive IL-1β release. Aims Hypothesis: Eh-induced activation of caspase-4 regulates GSDMD mediated pro-inflammatory responses. Specific aim: To quantify caspase-1/4 cleavage of GSDMD in Eh-induced pro-inflammatory responses. Methods Human PMA-differentiated THP-1 macrophages were used for Eh-macrophage studies. Caspase-1/4 activation and GSDMD cleavage were detected by immunoblot analysis. Bioactive IL-1β secretion was quantified by HEK-BlueTM IL-1β reporter cells via the measurement of secreted embryonic alkaline phosphatase (SEAP) and cell pyroptosis (inflammatory cell death) was determined by LDH assay. Immunoprecipitation was performed in HEK 293T cells transfected with human GSDMD plasmid followed by in vitro caspase cleavage assay. Results Unlike caspase-1, Eh-induced caspase-4 activation and IL-1β secretion was independent of the NLRP3 inflammasome as revealed with the use of CRISPR-Cas9 gene edited caspase-1, 4, ASC and NLRP3 macrophages. In the absence of caspase-1, caspase-4 activation was significantly upregulated that promoted the cleavage of GSDMD to induce robust IL-1β secretion. Eh-induced caspase-4 played a major role in triggering IL-1β release and GSDMD pore formation as quantified by SEAP assay and immunoprecipitation of overexpressed GSDMD in HEK 293T cells followed by in vitro caspase cleavage assay. Pharmacological inhibition of GSDMD pore formation and in CRISPR-Cas9 gene edited GSDMD macrophages, Eh-induced IL-1β secretion was highly dependent on GSDMD pore formation and independent of pyroptosis. This was in marked contrast to the positive control, LPS + Nigericin that induced high expression of caspase-1 but not caspase-4 that enhanced GSDMD cleavage and IL-1β secretion and induced massive pyroptosis. Conclusions These results suggest that Eh induced a state of “hyperactivated macrophages” that led to caspase-4 dependent GSDMD cleavage and IL-1β secretion in the absence of pyroptosis important in disease pathogenesis. Funding Agencies NSERC

Gasdermins (GSDMs) are a group of proteins that are cleaved by inflammatory caspases to induce pore formation in the plasma membrane to cause membrane permeabilization and lytic cell death or pyroptosis. All GSDMs share a conserved structure, containing a cytotoxic N-terminal (NT) pore-forming domain and a C-terminal (CT) repressor domain. Entamoeba histolytica ( Eh ) in contact with macrophages, triggers outside-in signaling to activate inflammatory caspase-4/1 via the noncanonical and canonical pathway to promote cleavage of gasdermin D (GSDMD). Cleavage of GSDMD removes the auto-inhibition that masks the active pore-forming NT domain in the full-length protein by interactions with GSDM-CT. The cleaved NT-GSDMD monomers then oligomerize to form pores in the plasma membrane to facilitate the release of IL-1β and IL-18 with a measured amount of pyroptosis. Pyroptosis is an effective way to counteract intracellular parasites, which exploit replicative niche to avoid killing. To date, most GSDMs have been verified to perform pore-forming activity and GSDMD-induced pyroptosis is rapidly emerging as a mechanism of anti-microbial host defence. Here, we review our comprehensive and current knowledge on the expression, activation, biological functions, and regulation of GSDMD cleavage with emphases on physiological scenario and related dysfunctions of each GSDM member as executioner of cell death, cytokine secretion and inflammation against Eh and other protozoan parasitic infections.

Giardia duodenalis cysteine proteases have been identified as key virulence factors and have been implicated in alterations to intestinal goblet cell activity and mucus production during Giardia infection. The present findings demonstrate a novel mechanism by which Giardia cysteine proteases modulate goblet cell activity via cleavage and activation of protease-activated receptor 2. Giardia duodenalis (assemblage A) increased MUC2 mucin gene expression in human colonic epithelial cells in a manner dependent upon both protease-activated receptor 2 activation and Giardia cysteine protease activity. Protease-activated receptor 2 cleavage within the N-terminal activation domain by Giardia proteases was confirmed using a nano-luciferase tagged recombinant protease-activated receptor 2. In keeping with these observations, the synthetic protease-activated receptor 2-activating peptide 2fLIGRLO-amide increased Muc2 gene expression in a time-dependent manner. Calcium chelation and inhibition of the ERK1/2 mitogen activated protein kinase pathway inhibited Muc2 upregulation during Giardia infection, consistent with canonical protease-activated receptor 2 signaling pathways. Giardia cysteine proteases cleaved both recombinant protease-activated receptor 1 and protease-activated receptor 2 within their extracellular activation domains with isolate-dependent efficiency that correlated with the production of cysteine protease activity. Protease-activated receptors represent a novel target for Giardia cysteine proteases, and these findings demonstrate that protease-activated receptor 2 can regulate mucin gene expression in intestinal goblet cells.

A hallmark of Entamoeba histolytica (Eh) invasion in the gut is acute inflammation dominated by the secretion of pro-inflammatory cytokines TNF-α and IL-1β. This is initiated when Eh in contact with macrophages in the lamina propria activates caspase-1 by recruiting the NLRP3 inflammasome complex in a Gal-lectin and EhCP-A5-dependent manner resulting in the maturation and secretion of IL-1β and IL-18. Here, we interrogated the requirements and mechanisms for Eh-induced caspase-4/1 activation in the cleavage of gasdermin D (GSDMD) to regulate bioactive IL-1β release in the absence of cell death in human macrophages. Unlike caspase-1, caspase-4 activation occurred as early as 10 min that was dependent on Eh Gal-lectin and EhCP-A5 binding to macrophages. By utilizing CRISPR-Cas9 gene edited CASP4/1, NLRP3 KO and ASC-def cells, caspase-4 activation was found to be independent of the canonical NLRP3 inflammasomes. In CRISPR-Cas9 gene edited CASP1 macrophages, caspase-4 activation was significantly up regulated that enhanced the enzymatic cleavage of GSDMD at the same cleavage site as caspase-1 to induce GSDMD pore formation and sustained bioactive IL-1β secretion. Eh-induced IL-1β secretion was independent of pyroptosis as revealed by pharmacological blockade of GSDMD pore formation and in CRISPR-Cas9 gene edited GSDMD KO macrophages. This was in marked contrast to the potent positive control, lipopolysaccharide + Nigericin that induced high expression of predominantly caspase-1 that efficiently cleaved GSDMD with high IL-1β secretion/release associated with massive cell pyroptosis. These results reveal that Eh triggered "hyperactivated macrophages" allowed caspase-4 dependent cleavage of GSDMD and IL-1β secretion to occur in the absence of pyroptosis that may play an important role in disease pathogenesis.