Development Of Spontaneous Colitis Research Articles

Alzheimer’s disease-related presenilins are key to intestinal epithelial cell function and gut immune homoeostasis

ObjectiveMutations in presenilin genes are the major cause of Alzheimer’s disease. However, little is known about their expression and function in the gut. In this study, we identify the presenilins...

Claudin-7 is essential for the maintenance of colonic stem cell homoeostasis via the modulation of Wnt/Notch signalling

Intestinal stem cells (ISCs) play a crucial role in the continuous self-renewal and recovery of the intestinal epithelium. In previous studies, we have revealed that the specific absence of Claudin-7 (Cldn-7) in intestinal epithelial cells (IECs) can lead to the development of spontaneous colitis. However, the mechanisms by which Cldn-7 maintains homeostasis in the colonic epithelium remain unclear. Therefore, in the present study, we used IEC- and ISC-specific Cldn-7 knockout mice to investigate the regulatory effects of Cldn-7 on colonic Lgr5+ stem cells in the mediation of colonic epithelial injury and repair under physiological and inflammatory conditions. Notably, our findings reveal that Cldn-7 deletion disrupts the self-renewal and differentiation of colonic stem cells alongside the formation of colonic organoids in vitro. Additionally, these Cldn-7 knockout models exhibited heightened susceptibility to experimental colitis, limited epithelial repair and regeneration, and increased differentiation toward the secretory lineage. Mechanistically, we also established that Cldn-7 facilitates the proliferation, differentiation, and organoid formation of Lgr5+ stem cells through the maintenance of Wnt and Notch signalling pathways in the colonic epithelium. Overall, our study provides new insights into the maintenance of ISC function and colonic epithelial homoeostasis.

Biochanin a ameliorates DSS-induced ulcerative colitis by improving colonic barrier function and protects against the development of spontaneous colitis in the Muc2 deficient mice

There is an increasing appreciation that colonic barrier function is closely related to the development and progression of colitis. The mucus layer is a crucial component of the colonic barrier, responsible for preventing harmful bacteria from invading the intestinal epithelium and causing inflammation. Furthermore, a defective mucus barrier is also a significant characteristic of ulcerative colitis (UC). Biochanin A (BCA), an isoflavonoid, has garnered increasing interest due to its significant biological activities. However, the impact of BCA on UC has not been reported yet. In this study, we used a dextran sodium sulfate (DSS)-induced ulcerative colitis model and the Muc2 deficient (Muc2−/−) mice spontaneous colitis model to explore the mechanisms of BCA in the treatment of UC. Here, we verified that DSS-induced UC was observably attenuated and spontaneous colitis in Muc2−/− mice was relieved by BCA. Treatment with BCA improved colitis-related symptoms and reduced intestinal permeability by upregulating the levels of goblet cells and tight junction (TJ) proteins. In addition, we confirmed that BCA promotes autophagy through the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) pathway, thereby alleviating DSS-induced UC. In addition, the administration of BCA was able to reduce apoptosis and promote proliferation by suppressing Cleaved Caspase-3 (Cleaved Cas-3) expression, and increasing PCNA and Ki67 levels. Further research revealed that BCA treatment ameliorated spontaneous colitis and alleviated epithelial damage in Muc2−/− mice by restoring the intestinal barrier and promoting autophagy. Our results demonstrated that BCA alleviated UC by enhancing intestinal barrier function and promoting autophagy. These findings indicate that BCA may be a novel treatment alternative for UC.

Specific deletion of Mettl3 in IECs triggers the development of spontaneous colitis and dysbiosis of T lymphocytes in mice.

The enzymatic core component of m6A writer complex, Mettl3, plays a crucial role in facilitating the development and progress in gastric and colorectal cancer (CRC). However, its underlying mechanism in regulating intestinal inflammation remains unclear and poorly investigated. Firstly, the characteristics of Mettl3 expression in IBD patients were examined. Afterwards we generated the mice line with IECs-specific deletion of Mettl3 verified by various experiments. We continuously recorded and compared the physiological status including survival rate etc. between the two groups. Subsequently, we took advantage of staining assays to analyze mucosal damage and immune infiltration of Mettl3WT and Mettl3KO primary IECs. Bulk RNA sequencing was used to pursuit the differential expression of genes (DEGs) and associated signaling pathways after losing Mettl3. Pyroptosis-related proteins were to determine whether cell death was caused by pyroptosis. Eventually, CyTOF was performed to probe the difference of CD45+ cells, especially CD3e+ T cells clusters after losing Mettl3. In IBD patients, Mettl3 was highly expressed in the inner-nucleus of IECs while significantly decreased upon acute intestinal inflammation. IECs-specific deletion of Mettl3 KO mice triggered a wasting phenotype and developed spontaneous colitis. The survival rate, body weight and intestinal length observed from 2 to 8-week of Mettl3KO mice was significantly lower than Mettl3WT mice. The degree of mucosal damage and immune infiltration in Mettl3KO were even more serious than their WT littermate. Bulk RNA sequencing demonstrated that DEGs were dramatically enriched in NOD-signaling pathways due to the loss of Mettl3. The colonic epithelium were more prone to pyroptosis after losing Mettl3. Subsequently, CyTOF revealed that T cells have altered significantly in Mettl3KO. Furthermore, there were abnormal proliferation of CD4+ T and markedly exhaustion of CD8+ T in Mettl3KO mice. In severe IBD patients, Mettl3 located in the inner-nucleus of IECs and declined when intestinal inflammation occurred. Subsequently, Mettl3 prevented mice from developing colitis.

A211 CAN AN EXPERIMENTAL MOUSE MODEL BE USED TO CHARACTERIZE BEHAVIOURAL COMORBIDITIES ASSOCIATED WITH INFLAMMATORY BOWEL DISEASES?

Abstract Background Inflammatory Bowel Diseases (IBD) are increasing globally with westernized countries experiencing a dramatic increase in cases of ulcerative colitis and Crohn’s disease. The corresponding rise in the comorbidities of anxiety and depression are linked to inflammation and microbiome dysbiosis; impacting the gut-brain axis (GBA). Another facet of the GBA are the tryptophan metabolites serotonin (5HT) and kynurenine (Kyn). 5HT and Kyn are metabolites implicated in anxious behaviours and memory deficiencies with dysbiosis influencing metabolite production. In inflammatory conditions, upregulation of Kyn impacts brain homeostasis as Kyn has both positive and negative brain metabolites. As disease outcomes are tied to the severity of depression/anxiety, characterization of the behavioural and tryptophan metabolite profile of an IBD model can allow us to develop better IBD therapies. Purpose To characterize the anxiety and memory phenotype with corresponding assessment of tryptophan metabolites 5HT and Kyn across tissues of interest in an spontaneous colitis model. Method Mucin 2 knockout mice (Muc2-/-) lack the protective mucin 2 layer in the distal colon, allowing for direct contact between the gut contents and the gut epithelial layer. Over time this contact results in the development of spontaneous colitis. Muc2-/- n=12 per sex were compared to C57Bl/6 mice for behaviour and tryptophan metabolites across brain, colon and serum. Open Field Test and Light/Dark tests were used to assess anxiety levels, Novel Object and Radial Arm maze were performed to assess memory deficits. Mice underwent one anxiety and one memory test and were euthanized following their last maze. Enzyme-linked immunosorbent assay (ELISA) assessment of 5HT and Kyn were performed on brain, colon and serum samples. T-tests were performed and graphs created using PRISM (v9.4.1). Result(s) Muc2-/- mice had decreased anxiety (p=0.0113) compared to C57Bl/6 wild type mice in the light/dark maze. Muc2-/- displayed no significant differences in novel object preferences or the number of memory errors in novel object recognition test and radial arm maze respectively. Serum kynurenine was found to be significantly elevated (p=0.0444) in Muc2-/- mice. The sex factor has to be mentioned here in the results not in the conclusions. Conclusion(s) IBD is increasing globally and problems with mental health is often comorbid. Within our model we were able to show that behavioural and biochemical differences do exist between our Muc2-/-and wild-type mice with depressed anxiety responses and heightened serum kynurenine, however the implications of these findings require further investigation. Understanding how inflammation is driving behavioural alterations can provide pathways to ameliorate the behavioural comorbidities in IBD patients suffering from anxiety of memory deficits. Disclosure of Interest None Declared

A48 LYOPHILIZED FECAL MICROBIOTA TRANSPLANT (FMT) DELAYS THE ONSET OF SPONTANEOUS COLITIS IN MUC2-/- MICE

Abstract Background Fecal microbiota transplant (FMT) is the transfer of fecal microbes from a healthy donor to a recipient to normalize gut microbiota. FMT therapy has been effectively used to prevent recurrent Clostridium difficile infections. While there are emerging studies applying FMT to other gastrointestinal (GI) disorders including inflammatory bowel disease (IBD), its efficacy in treating IBD, and the mechanisms involved remain unclear. In the GI tract, the mucus barrier plays a critical role in protecting the underlying epithelium against harmful luminal stimuli. The secreted mucin 2 (MUC2) is a glycosylated protein and the major component of the GI mucus layer. Muc2 deficient (-/-) mice are used to model IBD because they develop a leaky gut as well as spontaneous colitis. Purpose We investigated the efficacy of FMT in attenuating the development of spontaneous colitis in Muc2-/- mice. This study also evaluated whether FMT using lyophilized stool can successfully alter the gut microbiome of recipient mice. Method To achieve successful colonization of transplanted microbiota, Muc2-/- mice were pretreated prior to FMT with the antifungal amphotericin B, followed by an antibiotic mixture of metronidazole, ampicillin, neomycin and vancomycin in their drinking water for ten days. For two consecutive days, FMT was administered to Muc2-/- mice via consumption of lyophilized stool cakes created from donor C57BL/6 mice, while sucrose cakes were given as control. The fecal microbiome was analyzed at baseline, post-antibiotics and biweekly post-FMT using 16S rRNA sequencing. We monitored body weight and signs of spontaneous colitis, including rectal prolapse, until the mice reached their humane endpoint or turned 30 weeks old. Result(s) Following antibiotic pretreatment, Muc2-/- mice experienced significant weight loss, with 15-20% requiring euthanization within ten days of receiving antibiotics. Unless FMT was administered post-antibiotics, 95% of the mice developed rectal prolapse or otherwise reached their humane endpoint. In comparison, FMT-treated mice rarely developed rectal prolapse, with 55-80% of the FMT-treated mice surviving until the end of the experiment; some FMT-treated mice had no signs of rectal prolapse when euthanized at 30 weeks. Therefore, FMT significantly improved the survival rate of antibiotic-treated Muc2-/- mice and delayed the onset of rectal prolapse / spontaneous colitis. Microbiome analysis revealed that the lyophilized FMT altered the fecal microbiome as compared to mice receiving the sucrose control. Conclusion(s) Notably, FMT increased the survival rates of antibiotic-pretreated Muc2-/- mice while delaying the onset of rectal prolapse. This indicates that lyophilized FMT counteracts the typical spontaneous colitis and weight loss seen in Muc2-/- mice. Our findings can offer clinically relevant insight into how FMT induces a shift in the gut microbiome and its potential usage in treating IBD, as well as other gastrointestinal conditions. Disclosure of Interest None Declared

Loss of Autotaxin inhibits Toll‐like receptor4‐mediated immune mechanisms and promote spontaneous colitis in Il10‐ko mice

Autotaxin (Atx) is a secreted enzyme converting lysophosphatidylcholine (LPC) and sphingosyl‐phosphorylcholine into lysophosphatidic acid and sphingosine 1‐phosphate, respectively. Given the high affinity of LPC to cholesterol and the enrichment of cholesterol and sphingolipids in lipid rafts wherein LPS sensor Toll‐like receptor 4 (TLR4) and its co‐receptor CD14 reside, we hypothesized that Atx deficiency inhibits TLR4‐mediated innate and adaptive immunity; thereby, accelerating the susceptibility to microbe‐induced intestinal inflammation.To study this hypothesis, we generated myeloid cell lineage‐restricted Atx‐knockout (ko) mice (Atx‐dME/dME) to investigate TLR4‐mediated immune and inflammatory responses and examined LPS‐receptor complex formation through fluorescence resonance energy transfer technique, confocal microscopy, and flow cytometry. Phagocytic activity of macrophages was assessed. Lamina propria CD4+ T cells and bacterial load in the intestinal mucosa were examined. An impact of Atx deficiency in inflammatory bowel diseases (IBD) was investigated using Atx‐dME/dME;Il10‐/‐ mice that have both Atx‐deletion in myeloid cells and a global Il10 deletion. We examined the serum samples from IBD patients.With peritoneal macrophages from Atx‐dME/dME mice, we identified that Atx‐ko disrupted the integrity of lipid rafts at the plasma membrane, resulting in the inhibition of TLR4 complex formation. Accordingly, the recruitment of adaptor molecules to TLR4 was suppressed, and TLR4‐mediated responses were substantially reduced in Atx‐ko macrophages. TLR4‐induced innate immunity such as phagocytosis was attenuated in Atx‐ko macrophages. The activation of CD4+ effector T cells and regulatory T cells was diminished in the lamina propria lymphocytes of Atx‐dME/dME mice compared to that of Atx‐wt littermates. Consequently, Atx‐dME/dME mice had a higher bacterial prevalence in the intestinal mucosa compared to controls. Just like the notion that commensal microbes translocated from the lumen into the intestinal mucosa can elicit spontaneous colitis in Il10‐/‐ mice, combining Atx‐dME/dME with Il10‐/‐ mice (Atx‐dME/dME;Il10‐/‐) did accelerate spontaneous colitis development. Atx‐dME/dME;Il10‐/‐ mice had gross inflammation occurring throughout the colon, massive neutrophil infiltration and necrosis in the colonic mucosa, and increased mortality (Log‐rank P=0.0046), while Atx‐wt;Il10‐/‐ littermates are normal. Notably, ATX serum protein level was lower in UC (n=26) and CD (n=34) patients compared to the level in normal subjects (n=26) (P<0.001), indicating an association of reduced ATX levels with intestinal inflammation.Collectively, we found that Atx deficiency suppresses TLR4‐mediated innate and adaptive immune mechanisms; thereby accelerating the susceptibility to microbe‐induced gut inflammation.

A Mediterranean-like fat blend protects against the development of severe colitis in the mucin-2 deficient murine model

ABSTRACT There is a growing appreciation that the interaction between diet, the gut microbiota and the immune system contribute to the development and progression of inflammatory bowel disease (IBD). A mounting body of scientific evidence suggests that high-fat diets exacerbate IBD; however, there is a lack of information on how specific types of fat impact colitis. The Mediterranean diet (MD) is considered a health-promoting diet containing approximately 40% total fat. It is not known if the blend of fats found in the MD contributes to its beneficial protective effects. Mice deficient in the mucin 2 gene (Muc 2−/−) were weaned to 40% fat, isocaloric, isonitrogenous diets. We compared the MD fat blend (high monounsaturated, 2:1 n-6:n-3 polyunsaturated and moderate saturated fat) to diets composed of corn oil (CO, n-6 polyunsaturated-rich), olive oil (monounsaturated-rich) or milk fat (MF, saturated-rich) on spontaneous colitis development in Muc2−/− mice. The MD resulted in lower clinical and histopathological scores and induced tolerogenic CD103+ CD11b+ dendritic, Th22 and IL-17+ IL-22+ cells necessary for intestinal barrier repair. The MD was associated with beneficial microbes and associated with higher cecal acetic acid levels negatively correlated with colitogenic microbes like Akkermansia muciniphila. In contrast, CO showed a higher prevalence of mucin-degraders including A. muciniphila and Enterobacteriaceae, which have been associated with colitis. A dietary blend of fats mimicking the MD, reduces disease activity, inflammation-related biomarkers and improves metabolic parameters in the Muc2−/− mouse model. Our findings suggest that the MD fat blend could be incorporated into a maintenance diet for colitis.

A228 A FERMENTABLE FIBRE FREE DIET ACCELERATES SPONTANEOUS COLITIS DEVELOPMENT AND ALTERS THE GUT MICROBIOME IN MUC2-/- MICE

Abstract Background A key feature of ulcerative colitis (UC) is a defective colonic mucus layer. As Muc2-/- mice lack intestinal mucus, they’re often used to model UC. In these mice, luminal bacteria translocate from the gut lumen, triggering spontaneous colitis by 12–16 weeks of age. Notably, mucus defects are also seen in wildtype mice fed a fermentable fibre free (FF-free) diet. Aside from thinner mucus, FF-free fed mice show increased susceptibility to bacterial infections and a microbiome shift from fibre-utilizing to mucus-degrading bacteria. Indeed, a recent clinical study found that a microbiome shift towards proteolytic bacteria preceded clinical diagnosis of UC Aims To determine whether feeding newly weaned Muc2-/- mice a FF-free versus chow or inulin-enriched diet leads to - more rapid development of spontaneous colitis - shifts in the gut microbiota towards protein fermentation Methods Newly weaned Muc2-/- mice (3–4 weeks old) were fed 1 of 3 diets: 1) usual chow (Chow; 14.7% fibre mix), 2) FF-free (10% cellulose – poorly fermented) and 3) inulin-enriched (IN; same nutritional content as FF-free, with 10% inulin instead of cellulose). Body weight, diet intake and disease activity were monitored daily. By day 10, mice in the FF-free group developed severe colitis requiring euthanization. At this time, cecal contents (short-chain fatty acid [SCFA] analysis) and colonic tissue from all mice were collected for alcian blue/PAS staining and histopathological scoring. Stool was collected for 16S rRNA sequencing Results FF-free fed mice consumed fewer calories (p=0.0073), experienced less body weight gain (p=0.002), had colonic shortening (p<0.0001) and higher disease activity scores (p<0.0001) than mice fed the IN diet. Histopathological scores were higher in FF-free versus Chow (p<0.0001) and IN (p<0.0001) fed mice. Goblet cell mucin staining was reduced in FF-free compared to Chow and IN fed mice. Beta-diversity analysis showed clear differences in microbiome composition between groups. Acetate, product of fibre fermentation, was higher in the Chow (p<0.0001) and IN (p=0.0195) groups whereas branched-chain fatty acids (BCFA), product of protein fermentation, was higher in the FF-free group (p<0.0001) Conclusions Spontaneous colitis development in FF-free fed Muc2-/- mice was observed within 10 days (5–6 weeks old) of weaning whereas Chow or IN fed mice showed minimal to no signs of colitis, respectively. A diet devoid of fibre also led to less mucin staining and a gut microbiome shift that favoured BCFA over SCFA production, suggesting the microbiota of FF-free fed Muc2-/- mice are targeting mucosal proteins concurrent with disease exacerbation. These findings support future studies examining whether consumption of low fibre diets in UC patients worsen disease outcomes whereas diets rich in fibre and/or inulin prove beneficial Funding Agencies CCC, CIHRMSFHR Trainee Award, CH.I.L.D foundation

SIMPLE SUGAR TRIGGERS SPONTANEOUS COLITIS DEVELOPMENT INIL10 -/- MICE

Abstract The Western diet characterized by high fat and high sugar is considered a risk factor for inflammatory bowel disease (IBD) such as colitis. However, the precise role of sugar in colitis pathogenesis is poorly understood. We, therefore, investigated the role of glucose, a simple sugar, in colitis pathogenesis using IBD susceptible Il10-/- mice. We fed healthy 14-week old Il10-/- mice with 10% glucose in drinking water for 5 week and monitored body weight changes. Sugar fed Il10-/- mice spontaneously developed severe colitis, characterized by higher body weight loss and increased diarrhea, and shorter colon lengths. In agreement, there was higher expression of pro-inflammatory cytokines and chemokines in glucose-fed mice. Since colitis induction in Il10-/- mice is microbiota dependent, development of spontaneous colitis is often irregular. To induce colitis uniformly in all mice, glucose-fed and control Il10-/- mice were treated with a piroxicam-fortified diet. Alternatively, healthy Il10-/- mice were administered with a piroxicam-fortified diet for 5 days followed by feeding with high-glucose or regular drinking water. In both instances, glucose-fed Il10-/- mice lost more body weight and developed worse colitis. Increased colitis pathogenesis of glucose-fed Il10-/- mice was associated with increased inflammatory response, activation of inflammatory signaling pathways, induction of cell death, and erosion of mucus layer. Consistent to reduced mucus layer, there was increased bacterial colonization on the colonic mucosa and higher activity of bacteria-derived mucolytic enzymes in the gut lumen of sugar-fed Il10-/- mice. Notably, high sugar diets altered the composition of gut microbiota, particularly the abundance of mucus degrading bacteria, such as Akkermansia muciniphila and Bacteroides sp., was increased. Together these data suggest that high-sugar diet predisposes spontaneous colitis and enhances colitis pathogenesis by promoting the growth of mucus degrading bacteria in the gut of Il10-/- mice.

Colonizing Microbes, IL-10 and IL-22: Keeping the Peace at the Mucosal Surface

Our world is filled with microbes. Each multicellular organism has developed ways to interact with this microbial environment. Microbes do not always pose a threat; they can contribute to many processes that benefit the host. Upon colonization both host and microbes adapt resulting in dynamic ecosystems in different host niches. Regulatory processes develop within the host to prevent overt inflammation to beneficial microbes, yet keeping the possibility to respond when pathogens attempt to adhere and invade tissues. This review will focus on microbial colonization and the early (innate) host immune response, with special emphasis on the microbiota-modifying roles of IL-10 and IL-22 in the intestine. IL-10 knock out mice show an altered microbial composition, and spontaneously develop enterocolitis over time. IL-22 knock out mice, although not developing enterocolitis spontaneously, also have an altered microbial composition and increase of epithelial-adherent bacteria, mainly caused by a decrease in mucin and anti-microbial peptide production. Recently interesting links have been found between the IL-10 and IL-22 pathways. While IL-22 can function as a regulatory cytokine at the mucosal surface, it also has inflammatory roles depending on the context. For example, lack of IL-22 in the IL-10–/– mice model prevents spontaneous colitis development. Additionally, the reduced microbial diversity observed in IL-10–/– mice was also reversed in IL-10/IL-22 double mutant mice (Gunasekera et al., 2020). Since in early life, host immunity develops in parallel and in interaction with colonizing microbes, there is a need for future studies that focus on the effect of the timing of colonization in relation to the developmental phase of the host. To illustrate this, examples from zebrafish research will be compared with studies performed in mammals. Since zebrafish develop from eggs and are directly exposed to the outside microbial world, timing of the development of host immunity and subsequent control of microbial composition, is different from mammals that develop in utero and only get exposed after birth. Likewise, colonization studies using adult germfree mice might yield different results from those using neonatal germfree mice. Lastly, special emphasis will be given to the need for host genotype and environmental (co-housing) control of experiments.

A44 FATTY ACID COMPOSITION OF THE MEDITERRANEAN DIET PROTECTS AGAINST SPONTANEOUS COLITIS IN THE MUCIN-2 DEFICIENT MURINE MODEL

Abstract Background A Mediterranean diet (MD) has been proven efficacious in reducing inflammation in many chronic conditions, mediated by the interaction between diet, the gut microbiota and the immune system. The role of the MD as a dietary management strategy in the management of colitis requires further elucidation. While high fat diets have been shown to result in dysbiosis, our lab has clarified that the type of fat, not total calories derived from fat, predict gut dysbiosis and immunity in murine models of colitis. The n-6 polyunsaturated fatty acids (PUFA) promote colitis, however monounsaturated fatty acids (MUFA) protect against colitis. We hypothesize that a blend of fats, similar to the MD (high MUFA, low n-6 PUFA) will promote gut health beneficial to colitis. Aims Using a murine model of chronic intestinal inflammation, the aim of this study was to investigate the effects of dietary fatty acids on colitis by studying dietary fats in isolation from each other, as well as in a fatty acid profile similar to the blend contained in the MD. Methods Mice lacking the mucin 2 gene (Muc 2-/-) were weaned on to a 9-week, high fat (41%), isocaloric, isonitrogenous diet where the fat was derived from corn oil, olive oil, milk fat or a MD fat blend (28% MUFA, 8% SFA, 4% n-6 PUFA, 1% n-3 PUFA). The MD fat blend mimicked the fat profile consumed in the human diet. Disease activity, colon histology, cytokines (serum and colonic expression), cecal short chain fatty acids, intestinal permeability, glucose tolerance and gut microbiota (16S rRNA) were analyzed. Results Muc 2-/- fed the MD were protected from developing the most severe form of colitis, showing significantly lower disease activity and the absence of rectal prolapse. Histological damage was more severe in the corn oil and milk fat groups which coincided with an increase in infiltrating inflammatory cells and increased mucosal ulcerations. MD and milk fat diets exhibited enhanced intestinal permeability, glucose tolerance, intestinal alkaline phosphatase compared to the corn oil diet. Lower colonic mRNA levels of pro-inflammatory RELM-ß and IL-6 were also seen in the MD and MF diet in comparison to corn oil diet with the milk fat eliciting unique protective immune responses as evidenced by increased expression of IL-22 and Reg3-γ. Differences in alpha-diversity were seen between the MD and milk fat diets, beta-diversity revealed differences in taxa between diet groups. Conclusions The fatty acid profile of the MD protects against the development of spontaneous colitis in the Muc2-/- mouse model. In summary, not all dietary fats aggravate colitis, and some may be beneficial during colitis. A diet low in n-6 PUFA and high in MUFA is recommended. Funding Agencies CCCCFDR

P162 CRITICAL ROLES OF DIETARY SIMPLE SUGARS IN COLITIS PATHOGENESIS

Abstract The incidence of inflammatory bowel disease (IBD) is strikingly high in Western countries, implicating the role of Western diet in its etiology and pathogenesis. Western diet is characterized by high fat, low fiber, and high sugar. Despite clinical evidence of an association between high sugar diet and IBD susceptibility, the precise role of dietary simple sugars such as glucose, fructose, and sucrose in colitis pathogenesis is unknown. Using dextran sodium sulfate (DSS) and IL10-deficient mouse models of colitis, we studied the effect of simple sugars in colitis susceptibility. Mice were given high glucose, fructose or sucrose in their drinking water or left untreated before and during colitis induced by DSS. Sugar-fed mice exhibited increased colitis susceptibility evidenced by higher body weight loss, diarrhea, rectal bleeding, and severe histopathological changes in the colon as compared to those of sugar-untreated colitic mice. Pre-colitis dietary habit of sugar consumption was critical since sugar pretreated mice were susceptible to DSS-induced colitis even without high sugar diet intake during DSS administration. Consistent with these findings, there were higher incidence of spontaneous colitis development in Il10-/- mice following consumption of high sugar. To understand the underlying mechanism, we evaluated the effect of high sugar diet on intestinal epithelial cell death, inflammation, epithelial barrier permeability, and gut microbiota composition in healthy mice. We did not observe any major pathological changes and apoptosis in the colon of sugar-fed mice. Inflammatory responses, activation of inflammatory signaling pathways, and the expression of tight junction proteins were comparable between control and sugar-fed mice. Interestingly, gut microbiota composition of sugar-fed mice was altered as measured by 16S rRNA gene sequencing of DNA isolated from feces. Microbial species richness was reduced and relative abundance of several bacterial species was either increased or decreased in sugar-fed mice. We further confirmed that sugar-induced alteration of gut microbiota is responsible for exacerbated colitis by using antibiotics or germ-free mice. Mice receiving antibiotics during high-sugar intake did not show increased DSS-colitis susceptibility. Similarly, high-sugar diet did not induce overt colitis pathogenesis in germ-free mice. These findings demonstrate a critical role of dietary caloric sugars in the predisposition and promotion of colitis and could be implicated in the treatment and management of IBD.

P162 CRITICAL ROLES OF DIETARY SIMPLE SUGARS IN COLITIS PATHOGENESIS

The incidence of inflammatory bowel disease (IBD) is strikingly high in Western countries, implicating the role of Western diet in its etiology and pathogenesis. Western diet is characterized by high fat, low fiber, and high sugar. Despite clinical evidence of an association between high sugar diet and IBD susceptibility, the precise role of dietary simple sugars such as glucose, fructose, and sucrose in colitis pathogenesis is unknown. Using dextran sodium sulfate (DSS) and IL10-deficient mouse models of colitis, we studied the effect of simple sugars in colitis susceptibility. Mice were given high glucose, fructose or sucrose in their drinking water or left untreated before and during colitis induced by DSS. Sugar-fed mice exhibited increased colitis susceptibility evidenced by higher body weight loss, diarrhea, rectal bleeding, and severe histopathological changes in the colon as compared to those of sugar-untreated colitic mice. Pre-colitis dietary habit of sugar consumption was critical since sugar pretreated mice were susceptible to DSS-induced colitis even without high sugar diet intake during DSS administration. Consistent with these findings, there were higher incidence of spontaneous colitis development in Il10-/- mice following consumption of high sugar. To understand the underlying mechanism, we evaluated the effect of high sugar diet on intestinal epithelial cell death, inflammation, epithelial barrier permeability, and gut microbiota composition in healthy mice. We did not observe any major pathological changes and apoptosis in the colon of sugar-fed mice. Inflammatory responses, activation of inflammatory signaling pathways, and the expression of tight junction proteins were comparable between control and sugar-fed mice. Interestingly, gut microbiota composition of sugar-fed mice was altered as measured by 16S rRNA gene sequencing of DNA isolated from feces. Microbial species richness was reduced and relative abundance of several bacterial species was either increased or decreased in sugar-fed mice. We further confirmed that sugar-induced alteration of gut microbiota is responsible for exacerbated colitis by using antibiotics or germ-free mice. Mice receiving antibiotics during high-sugar intake did not show increased DSS-colitis susceptibility. Similarly, high-sugar diet did not induce overt colitis pathogenesis in germ-free mice. These findings demonstrate a critical role of dietary caloric sugars in the predisposition and promotion of colitis and could be implicated in the treatment and management of IBD.

P158 LOSS OF AUTOTAXIN ALTERS INNATE AND ADAPTIVE IMMUNE MECHANISM AND PROMOTES SPONTANEOUS COLITIS IN IL10-KO MICE

Abstract Background & Aims Autotaxin (Atx) is a secreted enzyme converting lysophosphatidylcholine (LPC) and sphingosyl-phosphorylcholine into lysophosphatidic acid and sphingosine 1-phosphate, respectively. Given the high affinity of LPC to cholesterol and the enrichment of cholesterol and sphingolipids in lipid rafts wherein LPS sensor Toll-like receptor 4 (TLR4) and its co-receptor CD14 reside, we hypothesized that Atx deficiency inhibits TLR4-mediated innate and adaptive immunity; thereby, accelerating the susceptibility to microbe-induced intestinal inflammation. Method We generated myeloid cell lineage-restricted Atx-knockout (ko) mice (AtxdME/dME) to study TLR4-mediated immune and inflammatory responses and investigated LPS-receptor complex formation through fluorescence resonance energy transfer technique, confocal microscopy, and flow cytometry. Lamina propria CD4+ T cells and bacterial load in the intestinal mucosa were examined. An impact of Atx deficiency in inflammatory bowel diseases (IBD) was investigated using AtxdME/dME;Il10-/- mice that have both Atx-deletion in myeloid cells and a global Il10 deletion. We examined the serum samples from IBD patients. Results With peritoneal macrophages from AtxdME/dME mice, we identified that Atx-ko disrupted the integrity of lipid rafts at the plasma membrane, resulting in the inhibition of TLR4 complex formation. Accordingly, the recruitment of adaptor molecules to TLR4 was suppressed, and TLR4-mediated responses were substantially reduced in Atx-ko macrophages. TLR4-induced innate immunity such as phagocytosis was attenuated in Atx-ko macrophages. The activation of CD4+ effector T cells and regulatory T cells was diminished in the lamina propria lymphocytes of AtxdME/dME mice compared to that of Atx+/+ littermates. Consequently, AtxdME/dME mice had a higher bacterial prevalence in the intestinal mucosa compared to controls. Just like the notion that commensal microbes translocated from the lumen into the intestinal mucosa can elicit spontaneous colitis in Il10-/- mice, combining AtxdME/dME with Il10-/- mice (AtxdME/dME; Il10-/-) did accelerate spontaneous colitis development. AtxdME/dME; Il10-/- mice had gross inflammation occurring throughout the colon, massive neutrophil infiltration and necrosis in the colonic mucosa, and increased mortality (Log-rank P=0.0046), while Atx+/+;Il10-/- littermates are normal. Notably, ATX serum protein level was lower in UC (n=26) and CD (n=34) patients compared to the level in normal subjects (n=26) (P<0.001), indicating an association of reduced ATX levels with the intestinal inflammation. Conclusions Collectively, we found that Atx deficiency suppresses TLR4-mediated innate and adaptive immune mechanisms; thereby accelerating the susceptibility to microbe-induced gut inflammation.

Epithelial RABGEF1 deficiency promotes intestinal inflammation by dysregulating intrinsic MYD88-dependent innate signaling.

Intestinal epithelial cells (IECs) contribute to the regulation of intestinal homeostasis and inflammation through their interactions with the environment and host immune responses. Yet our understanding of IEC-intrinsic regulatory pathways remains incomplete. Here, we identify the guanine nucleotide exchange factor RABGEF1 as a regulator of intestinal homeostasis and innate pathways dependent on IECs. Mice with IEC-specific Rabgef1 deletion (called Rabgef1IEC-KO mice) developed a delayed spontaneous colitis associated with the local upregulation of IEC chemokine expression. In mouse models of colitis based on Interleukin-10 deficiency or dextran sodium sulfate (DSS) exposure, we found that IEC-intrinsic RABGEF1 deficiency exacerbated development of intestinal pathology and dysregulated IEC innate pathways and chemokine expression. Mechanistically, we showed that RABGEF1 deficiency in mouse IECs in vitro was associated with an impairment of early endocytic events, an increased activation of the p38 mitogen-activated protein kinase (MAPK)-dependent pathway, and increased chemokine secretion. Moreover, we provided evidence that the development of spontaneous colitis was dependent on microbiota-derived signals and intrinsic MYD88-dependent pathways in vivo. Our study identifies mouse RABGEF1 as an important regulator of intestinal inflammation, MYD88-dependent IEC-intrinsic signaling, and chemokine production. This suggests that RABGEF1-dependent pathways represent interesting therapeutic targets for inflammatory conditions in the gut.

The Dynamic Changes of Gut Microbiota in Muc2 Deficient Mice.

Gut dysbiosis is associated with colitis-associated colorectal carcinogenesis, and the genetic deficiency of the Muc2 gene causes spontaneous development of colitis and colorectal cancer. Whether there are changes of gut microbiota and a linkage between the changes of microbiota and intestinal pathology in Muc2−/− mice are unclear. Muc2−/− and Muc2+/+ mice were generated by backcrossing from Muc2+/− mice, and the fecal samples were collected at different dates (48th, 98th, 118th, 138th, and 178th day). Gut microbiota were analyzed by high-throughput sequencing with the universal 16S rRNA primers (V3–V5 region). All mice were sacrificed at day 178 to collect colonic tissue and epithelial cells for the analysis of histopathology and inflammatory cytokines. On the 178th day, Muc2−/− mice developed colorectal chronic colitis, hyperplasia, adenomas and adenocarcinomas, and inflammatory cytokines (e.g., cyclooxygenase 2 (COX-2), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin 1 β (IL-1β), i-kappa-B-kinase β (IKKβ)) were significantly increased in colonic epithelial cells of Muc2−/− mice. In general, structural segregation of gut microbiota was observed throughout the experimental time points between the Muc2−/− and Muc2+/+ mice. Impressively, in Muc2−/− mice, Alpha diversities reflected by Shannon and Chao indexes were higher, the phylum of Firmicutes was enriched and Bacteroidetes was decreased, and Desulfovibrio, Escherichia, Akkermansia, Turicibacter, and Erysipelotrichaceae were significantly increased, but Lactobacilli and Lachnospiraceae were significantly decreased. Moreover, the abundance of Ruminococcaceae and butyrate-producing bacteria was significantly higher in the Muc2−/− mice. There were significant differences of gut microbiota between Muc2−/− and Muc2+/+ mice. The dynamic changes of microbiota might contribute to the development of colitis and colitis-associated colorectal carcinogenesis. Therefore, this study revealed specific functional bacteria in the development of colitis and colitis-associated colorectal carcinogenesis, which will benefit the development of preventive and therapeutic strategies for chronic inflammation and its malignant transformation.

The Proton-activated Receptor GPR4 Modulates Intestinal Inflammation.

During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/-/Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+/Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.

Increased NCR negative ILC 3 may be indicative for the early development of spontaneous colitis in the IL 10 knockout mouse

Abstract The IL10 knockout (KO) mouse is a model for environmentally and genetically triggered spontaneous colitis. The response of the adaptive immune system to these changes has been studied extensively in this model. However, the response of the innate immune system, specifically the response of innate lymphoid cell (ILCs) populations to the genetic background of the mouse and the environmental stimulus of introducing microbiota has not been well studied. Herein, we evaluated the response of ILCs and Lin marker positive lymphocytes to changes in genotype (wild type Mice B6 in SPF to IL10 KO B6 mice in SPF) and also compared IL10KO mice who were either germ free or SPF exposed. To accomplish this, IL10 knockout mice were exposed to SPF conditions for three weeks. LPMCs were isolated from the colon and small bowel. Innate lymphoid cells were identified as Lin− CD90.2+CD127+cells, and within total ILC, ILC2s were ST2+ cells, ILC1s were ST2−CD117− cells, and ILC3s were ST2−CD117+NKp46+/−cells. As expected, we saw a less developed adaptive immune response in GF IL10 KO mice, with up regulation in this compartment following SPF exposure (Lin+ population increase 1.87 fold). Upon comparison of WT SPF versus IL10 KO SPF, we observed a 5 fold increase in total ILCs and an 18% increase in NCR-ILC3 in the SPF. Comparison of IL10 KO GF versus SPF revealed a 1.8 fold increase in total ILC and a 3% increase in NCR-ILC3s. In conclusion, we show that both genetic and environmental factors play a role in the innate immune changes that occur prior to the development of spontaneous colitis in the IL10 KO mouse and that the shift towards NCR-ILC 3 maybe an early marker for the development of spontaneous colitis.

NLRP6 Protects Il10−/− Mice from Colitis by Limiting Colonization of Akkermansia muciniphila

Dysfunction in host immune responses and pathologic alterations in the gut microbiota, referred to as dysbiosis, can both contribute to the development of inflammatory bowel disease (IBD). However, it remains unclear how specific changes in host immunity or the microbiota cause disease. We previously demonstrated that the loss of the innate immune receptor NLRP6 in mice resulted in impaired production of interleukin-18 (IL-18) and increased susceptibility to epithelial-induced injury. Here, we show that NLRP6 is important for suppressing the development of spontaneous colitis in the Il10-/- mice model of IBD and that NLRP6 deficiency results in the enrichment of Akkermansia muciniphila. A. muciniphila was sufficient for promoting intestinal inflammation in both specific-pathogen-free and germ-free Il10-/- mice. Our results demonstrate that A. muciniphila can act as a pathobiont to promote colitis in a genetically susceptible host and that NLRP6 is a key regulator of its abundance.