Fecal Lipocalin-2 Research Articles

The Emulsifier Carboxymethylcellulose Induces More Aggressive Colitis in Humanized Mice with Inflammatory Bowel Disease Microbiota Than Polysorbate-80.

Commonly used synthetic dietary emulsifiers, including carboxymethylcellulose (CMC) and polysorbate-80 (P80), promote intestinal inflammation. We compared abilities of CMC vs. P80 to potentiate colitis and impact human microbiota in an inflammatory environment using a novel colitis model of ex-germ-free (GF) IL10−/− mice colonized by pooled fecal transplant from three patients with active inflammatory bowel diseases. After three days, mice received 1% CMC or P80 in drinking water or water alone for four weeks. Inflammation was quantified by serial fecal lipocalin 2 (Lcn-2) and after four weeks by blinded colonic histologic scores and colonic inflammatory cytokine gene expression. Microbiota profiles in cecal contents were determined by shotgun metagenomic sequencing. CMC treatment significantly increased fecal Lcn-2 levels compared to P80 and water treatment by one week and throughout the experiment. Likewise, CMC treatment increased histologic inflammatory scores and colonic inflammatory cytokine gene expression compared with P80 and water controls. The two emulsifiers differentially affected specific intestinal microbiota. CMC did not impact bacterial composition but significantly decreased Caudoviricetes (bacteriophages), while P80 exposure non-significantly increased the abundance of both Actinobacteria and Proteobacteria. Commonly used dietary emulsifiers have different abilities to induce colitis in humanized mice. CMC promotes more aggressive inflammation without changing bacterial composition.

The potent and selective RIPK2 inhibitor BI 706039 improves intestinal inflammation in the TRUC mouse model of inflammatory bowel disease.

Mouse and human data implicate the NOD1 and NOD2 sensors of the intestinal microbiome and the associated signal transduction via the receptor interacting protein kinase 2 (RIPK2) as a potential key signaling node for the development of inflammatory bowel disease (IBD) and an attractive target for pharmacological intervention. The TRUC mouse model of IBD was strongly indicated for evaluating RIPK2 antagonism for its effect on intestinal inflammation based on previous knockout studies with NOD1, NOD2, and RIPK2. We identified and profiled the BI 706039 molecule as a potent and specific functional inhibitor of both human and mouse RIPK2 and with favorable pharmacokinetic properties. We dosed BI 706039 in the spontaneous TRUC mouse model from age 28 to 56 days. Oral, daily administration of BI 706039 caused dose-responsive and significant improvement in colonic histopathological inflammation, colon weight, and terminal levels of protein-normalized fecal lipocalin (all P values <0.001). These observations correlated with dose responsively increasing systemic levels of the BI 706039 compound, splenic molecular target engagement of RIPK2, and modulation of inflammatory genes in the colon. This demonstrates that a relatively low oral dose of a potent and selective RIPK2 inhibitor can modulate signaling in the intestinal immune system and significantly improve disease associated intestinal inflammation.NEW & NOTEWORTHY The RIPK2 kinase at the apex of microbiome immunosensing is an attractive target for pharmacological intervention. A low oral dose of a RIPK2 inhibitor leads to significantly improved intestinal inflammation in the murine TRUC model of colitis. A selective and potent inhibitor of the RIPK2 kinase may represent a new class of therapeutics that target microbiome-driven signaling for the treatment of IBD.

E-cadherin activating antibodies limit barrier dysfunction and inflammation in mouse inflammatory bowel disease

ABSTRACT Deficits in gastrointestinal (GI) paracellular permeability has been implicated in etiology of Inflammatory Bowel Disease (IBD), and E-cadherin, a key component of the epithelial junctional complex, has been implicated in both barrier function and IBD. We have previously described antibodies against E-cadherin that activate cell adhesion, and in this study, we show that they increase transepithelial electrical resistance in epithelial cell monolayers in vitro. We therefore tested the hypothesis that adhesion activating E-cadherin mAbs will enhance epithelial barrier function in vivo and limit progression of inflammation in IBD. Activating mAbs to mouse E-cadherin were tested in different mouse models of IBD including the IL10-/- and adoptive T cell transfer models of colitis. Previously established histological and biomarker measures of inflammation were evaluated to monitor disease progression. Mouse E-cadherin activating mAb treatment reduced total colitis score, individual histological measures of inflammation, and other hallmarks of inflammation compared to control treatment. Activating mAbs also reduced the fecal accumulation lipocalin2 and albumin content, consistent with enhanced barrier function. Therefore, E-cadherin activation could be a potential strategy for limiting inflammation in UC.

Beneficial Effects of Natural Mineral Waters on Intestinal Inflammation and the Mucosa-Associated Microbiota.

Natural mineral water (NMWs) intake has been traditionally used in the treatment of various gastrointestinal diseases. We investigated the effect of two French NMWs, one a calcium and magnesium sulphate, sodium chloride, carbonic, and ferruginous water (NMW1), the other a mainly bicarbonate water (NMW2) on the prevention of intestinal inflammation. Intestinal epithelial cells stimulated with heat inactivated Escherichia coli or H2O2 were treated with NMWs to evaluate the anti-inflammatory effects. Moderate colitis was induced by 1% dextran sulfate sodium (DSS) in Balbc/J mice drinking NMW1, NWW2, or control water. General signs and histological features of colitis, fecal lipocalin-2 and pro-inflammatory KC cytokine levels, global mucosa-associated microbiota, were analyzed. We demonstrated that both NMW1 and NMW2 exhibited anti-inflammatory effects using intestinal cells. In induced-colitis mice, NMW1 was effective in dampening intestinal inflammation, with significant reductions in disease activity scores, fecal lipocalin-2 levels, pro-inflammatory KC cytokine release, and intestinal epithelial lesion sizes. Moreover, NMW1 was sufficient to prevent alterations in the mucosa-associated microbiota. These observations, through mechanisms involving modulation of the mucosa-associated microbiota, emphasize the need of investigation of the potential clinical efficiency of such NMWs to contribute, in human beings, to a state of low inflammation in inflammatory bowel disease.

A32 EMPAGLIFOZIN IMPROVES GASTROINTESTINAL INFLAMMATION IN A MOUSE MODEL OF COLITIS

Abstract Background Empagliflozin (EMPA) is a highly selective sodium glucose cotransporter-2 (SGLT2) inhibitor and is increasingly being utilized as an antihyperglycemic agent in the management of type 2 diabetes. Interestingly, it has been demonstrated in human trials that EMPA treatment exerts potent cardioprotective effects by reducing cardiac inflammation independently of glycemic control. Further, EMPA has also been shown to suppress LPS-induced renal and systemic inflammation in an animal model. Based on these findings, we hypothesized that EMPA treatment may also be effective in reducing gut inflammation. Aims The aim of this study was to examine the effects of treatment with EMPA on gastrointestinal inflammation in an animal model of inflammatory bowel disease and to determine mechanistic insights regarding its direct effects on gut cytokine secretion. Methods Adult male and female IL-10-/- mice with established colitis were treated with a daily gavage of EMPA (10mg/kg; n=10) or vehicle (n=10) for 14 days. Disease activity was assessed by measurement of mouse weight, colonic weight and length, histological score, cytokine levels in colonic homogenate and lipocalin-2 levels in stool. To examine for possible direct effects of EMPA, colonic explants from wild-type (n=8) and IL-10-/- (n=8) mice were incubated with increasing doses of EMPA (0.1–5 µM) ± LPS (10µg/ml) for 2 hours and tissue levels of IL-1β and TNFα protein measured by ELISA. Results After 14 days EMPA treated IL-10-/- mice had a significant improvement in colonic inflammation as evidenced by decreased colonic weight to length ratio (p=0.019), decreased fecal lipocalin-2 (p=0.03), as well as decreased enterocyte injury (p=0.01), decreased lamina propria neutrophils (p=0.01) and decreased total histological score (p=0.006). EMPA treated mice also maintained their weight over the 14 days while untreated mice continued to lose weight (p=0.04). There were no significant differences in colonic homogenate levels of TNFα, IL-1β, or IL-6 or in blood glucose levels between EMPA-treated mice and controls. In addition, EMPA did not suppress levels of basal or LPS-induced TNFα and IL-1β in colonic explants from either wild-type or IL-10-/- mice suggesting that the beneficial effects in IL-10-/- mice were not due to direct effects of EMPA on colonic TNFα or IL-1β cytokine levels. Conclusions EMPA treatment dramatically improved histologic and fecal inflammatory markers and maintained body weight in adult IL-10-/- mice with established colitis. These findings suggest further investigations into the effects of EMPA in treating gut inflammation are warranted. Funding Agencies CAG, CIHR

A13 A HIGH SALT DIET SYNERGIZES WITH UC MICROBIOTA TO INDUCE A PROINFLAMMATORY IMMUNE TONE IN IMMUNOCOMPETENT GNOTOBIOTIC MICE

Abstract Background The exact cause for inflammatory bowel disease (IBD) is unknown, however, there is consensus that a combination of genetic, environmental, and immune factors, participate in its pathogenesis. Recently, high salt diet (HSD) has been shown to increase the severity of experimental colitis through depletion of lactobacilli in specific pathogen-free mice. However, whether HSD-microbiota interaction occurs in mice colonized with microbiota from patients with IBD, is unknown. Aims Our aim was to determine whether mice colonized with microbiota from patients with active ulcerative colitis (UC), one of two forms of IBD, develop more severe inflammation when fed a HSD. Methods 10–15 week-old germ-free C57BL/6 mice were colonized with fecal microbiota from a healthy control (HC, n=13) or from a patient with UC experiencing a flare (UC, n=13). Colonized mice were housed in ISO positive cages (Techniplast) in a gnotobiotic facility during the experiment. For each group, half of the mice were kept on a control diet (7004, Teklad) and the other half were fed a HSD (7004 supplemented with 4% NaCl) plus 1% NaCl in drinking water for 3 weeks. At sacrifice, colon tissue was collected for histological analysis, RNA sequencing, and flow cytometry. Fecal samples were collected for lipocalin-2 determination. Results Colonic polymorphonuclear (PMN) cells were higher in UC compared with HC mice, regardless of diet (p&amp;lt;0.01). In mice fed HSD, UC mice also had higher PMN cell counts than HC mice (p&amp;lt;0.001). Administration of HSD to UC, but not to HC mice, also increased the proportion of α4+IFNg-producing T cells in the colonic lamina propria, compared with UC mice fed control diet (p&amp;lt;0.01). This was paralleled by higher fecal lipocalin-2 in UC mice fed HSD compared with control diet (p&amp;lt;0.05). Targeted analysis of RNA sequencing data revealed that 7 genes, mainly related to the JAK/STAT pathway (i.e., Stat2, Tyk2, Tlr7) were differentially expressed (p≤0.05) between HC and UC mice fed the control diet. A different gene expression signature was found in mice fed HSD compared with control diet, with an increase in proinflammatory genes in UC colonized mice compared to HC colonized mice, such as Il12a and Il6 (p&amp;lt;0.05). Conclusions Immunocompetent mice colonized with microbiota from a UC patient in flare spontaneously developed a proinflammatory immune tone, which was exacerbated by HSD. The different pattern of gene induction observed in mice colonized with the microbiota from the same UC donor, but fed HSD, suggests independent and synergistic pathways conducive to inflammation. Future studies will explore the preclinical therapeutic effect of novel drug candidates for the inhibition of such pathways. Funding Agencies CCC

COMMON FOOD ADDITIVES ACCELERATE ONSET OF INFLAMMATORY BOWEL DISEASE IN MICE BY ALTERING MICROBIOME COMPOSITION AND HOST-MICROBE INTERACTION

Multiple factors contribute to inflammatory bowel disease (IBD) pathogenesis, including diet. Although correlations exist between the introduction of processed foods and a rise in IBD prevalence, it is not understood what components of processed foods drive disease pathogenesis or how. Despite a FDA classification of Generally Regarded As Safe (GRAS), food additives significantly impact microbe communities and phenotypes. We hypothesized that common food additives, such as maltodextrin (MDX) and carboxymethyl cellulose (CMC), alter intestinal microbes and increase the onset and progression of IBD. We tested this hypothesis using the interleukin-10 deficient (IL10KO) mouse model of spontaneous colitis. To standardize disease onset, IL10KO mice were pre-conditioned with fecal material from nucleotide-binding oligomerization domain 2 deficient mice (NOD2KO) to promote intestinal inflammation. Pre-conditioned IL10KO mice were randomized to chow diets containing either 1% MDX, 1% CMC, or a control diet. MDX and CMC accelerated the onset of colitis compared to the control diet. This was accompanied by elevated fecal lipocalin-2 (p<0.05) and serum amyloid A (p<0.01), intestinal infiltration of CD3+ cells, and intestinal pathology consistent with chronic inflammation (p<0.05). Although both dietary additives promote disease in this model, the molecular mechanisms involved appear to be distinct. Intestinal mucus modulates interactions of the microbiome and the host. Strikingly, goblet cells in MDX fed mice had reduced mucin granule content (∼50% of control diet) as assessed by Muc2 and Periodic Acid Schiff staining. This mucin depletion did not correlate with degree of disease activity, suggesting this deficiency is not a result of inflammation in MDX fed mice and was not observed in CMC fed mice or control diet mice. Food additives also altered the intestinal microbiome, with MDX fed mice demonstrating a significant decrease in microbiome alpha diversity (p<0.05, ANOVA), and both food additives resulting in a significant shift in beta diversity (R2=0.40, p=0.002, PERMANOVA) as compared to the control diet. Flagella expression by microbes was slightly elevated in CMC-fed mice, but not in MDX-fed mice, suggesting that these food additives have distinct actions on microbes. Magnitude of genus-level changes in microbial abundance correlated with disease severity as assessed by serum amyloid A levels. This data indicates that common food additives are not biologically inert substances, and may drive the progression of inflammatory bowel disease through additive-specific alterations of the microbiome and host-microbe interactions.

BMC322- A RATIONALLY-DESIGNED LIVE BACTERIAL CONSORTIUM BASED ON MICROBIOME FUNCTIONAL GENOMIC ANALYSIS FOR TREATMENT OF IBD

Abstract The mechanisms by which the intestinal microbiome affects intestinal inflammation and disease activity in IBD is not clear. Most of the current data on the association between the intestinal microbiome and IBD is focusing on the intestinal microbial taxa and very little is known about the functionality of the microbiome in this condition. Aim: To construct a live bacterial consortium for reduction of intestinal inflammation in IBD based on a high-resolution functional microbial analysis, and its association with anti-inflammatory processes. Methods: Fecal metagenomics samples (n=581) and corresponding fecal calprotectin (FCP) levels from patients with Crohn’s disease and Ulcerative Colitis were downloaded from the NCBI (SRA SRP057027, http://hmpdacc.org, SRA SRP002163 and SRP056641 (BioProject PRJNA275349)). Computational analysis was performed using PRISM (Biomica Ltd, Rehovot Israel), a proprietary metagenomics analysis platform integrating assembly-based and reference-based analyses for functional genomics profiling and utilizing BioCOGS, Biomica’s internal comprehensive catalogue of microbial gene-orthology groups. We compared microbial functions between subjects based on severity of inflammation (FCP&amp;lt;70 vs FCP &amp;gt;250 and FCP &amp;gt;250 vs FCP &amp;lt;250) and identified microbial functions associated with low inflammation (FDR 0.1). Validation of the anti-inflammatory activity of BMC322 was performed using a DSS-induced colitis mouse model. Results: Out of the dozens of identified functions, 32 were related to host anti-inflammatory processes, intestinal barrier integrity and colonocyte function. Our functional microbiome investigation has pointed to novel metabolic mechanisms by which specific strains may attenuate mucosal inflammation. Using functional annotations, we infer the biological processes governing microbe-host interactions contributing to reduced inflammation, and identify 16 bacteria of which 4 strains comprise the full array of the identified microbial functions associated with lower disease severity. These strains comprise BMC322- a novel live-bacterial consortium for inflammation reduction in IBD. The anti-inflammatory activity of these strains was validated in in-vitro studies and demonstrated efficacy in preliminary a mouse model of DSS-induced colitis; including shortening period of recovery, disease activity index and fecal lipocalin 2. Conclusion: High-resolution functional genomics profiling of the gut microbiome enables identification of microbial-derived functions relevant to inflammatory disease activity, and microbial strains bearing specific inflammation-reduction activity. These strains have provided the basis for the construction of a 4-strain consortium for intervention in IBD. Preliminary work has validated the anti-inflammatory activity of these strains and their potential as novel therapeutic modality for IBD.

COMMON FOOD ADDITIVES ACCELERATE ONSET OF INFLAMMATORY BOWEL DISEASE IN MICE BY ALTERING MICROBIOME COMPOSITION AND HOST-MICROBE INTERACTION

Abstract Multiple factors contribute to inflammatory bowel disease (IBD) pathogenesis, including diet. Although correlations exist between the introduction of processed foods and a rise in IBD prevalence, it is not understood what components of processed foods drive disease pathogenesis or how. Despite a FDA classification of Generally Regarded As Safe (GRAS), food additives significantly impact microbe communities and phenotypes. We hypothesized that common food additives, such as maltodextrin (MDX) and carboxymethyl cellulose (CMC), alter intestinal microbes and increase the onset and progression of IBD. We tested this hypothesis using the interleukin-10 deficient (IL10KO) mouse model of spontaneous colitis. To standardize disease onset, IL10KO mice were pre-conditioned with fecal material from nucleotide-binding oligomerization domain 2 deficient mice (NOD2KO) to promote intestinal inflammation. Pre-conditioned IL10KO mice were randomized to chow diets containing either 1% MDX, 1% CMC, or a control diet. MDX and CMC accelerated the onset of colitis compared to the control diet. This was accompanied by elevated fecal lipocalin-2 (p&amp;lt;0.05) and serum amyloid A (p&amp;lt;0.01), intestinal infiltration of CD3+ cells, and intestinal pathology consistent with chronic inflammation (p&amp;lt;0.05). Although both dietary additives promote disease in this model, the molecular mechanisms involved appear to be distinct. Intestinal mucus modulates interactions of the microbiome and the host. Strikingly, goblet cells in MDX fed mice had reduced mucin granule content (~50% of control diet) as assessed by Muc2 and Periodic Acid Schiff staining. This mucin depletion did not correlate with degree of disease activity, suggesting this deficiency is not a result of inflammation in MDX fed mice and was not observed in CMC fed mice or control diet mice. Food additives also altered the intestinal microbiome, with MDX fed mice demonstrating a significant decrease in microbiome alpha diversity (p&amp;lt;0.05, ANOVA), and both food additives resulting in a significant shift in beta diversity (R2=0.40, p=0.002, PERMANOVA) as compared to the control diet. Flagella expression by microbes was slightly elevated in CMC-fed mice, but not in MDX-fed mice, suggesting that these food additives have distinct actions on microbes. Magnitude of genus-level changes in microbial abundance correlated with disease severity as assessed by serum amyloid A levels. This data indicates that common food additives are not biologically inert substances, and may drive the progression of inflammatory bowel disease through additive-specific alterations of the microbiome and host-microbe interactions.

Teasaponin Ameliorates Murine Colitis by Regulating Gut Microbiota and Suppressing the Immune System Response.

Background: Dietary intervention is an exciting topic in current research of inflammatory bowel disease (IBD). The effect of teasaponin (TS) on IBD has not been fully elucidated. Here, we aim to investigate the intestinal anti-inflammatory activity of TS in a dextran sodium sulfate (DSS)-induced colitis mouse model and identify potential mechanisms.Methods: We applied TS to mice with DSS-induced colitis and then monitored the body weight, disease activity index (DAI) daily. When sacrificed, the intestinal permeability was measured. The analysis of mucin and tight junction proteins was conducted. We detected the inflammatory cytokines, the immune cells and related inflammatory signaling pathways. In addition, the gut microbiota were analyzed by 16S rRNA sequencing and we also performed fecal microbiota transplantation (FMT).Results: It showed that TS ameliorated the colonic damage by lowering the DAI, prolonging the colon length, reducing inflammatory cytokines and improving the mucus barrier. Parallel to down-regulation of the inflammatory cytokines, the fecal lipocalin 2, p-P65, p-STAT3, and neutrophil accumulation were also decreased in TS-treated mice. Microbiota characterization showed that Campylobacteria, Proteobacteria, Helicobacter, and Enterobacteriaceae were the key bacteria associated with IBD. In addition, TS could reverse the Firmicutes/Bacteroidetes (F/B) ratio and increase the beneficial bacteria, including Akkermansia and Bacteroides. TS ameliorated DSS-induced colitis by regulating the gut microbiota, and the gut microbiota could regulate gut inflammation.Conclusions: These studies demonstrated that TS ameliorated murine colitis through the modulation of immune response, mucus barrier and gut microbiota, thus improving gut dysbiosis. In addition, the gut microbiota may play an important role in regulating the host's innate immune system, and the two coexist and are mutually beneficial. We provide a promising perspective on the clinical treatment of IBD.

A37 ENTERIC MICROBIOTA CONTRIBUTE TO BEHAVIORAL ALTERATIONS OBSERVED IN MICE WITH COLITIS

Abstract Background The enteric microbiota has been recognized as an essential regulator of both gut and brain physiology, a complex interaction generally termed the microbiota-gut-brain axis. Disturbances to gastrointestinal physiology lead to alterations in the composition of the enteric microbiota, whereas dysbiosis can also contribute to pathophysiology. Inflammatory bowel diseases (IBD) are chronic, relapsing inflammatory conditions of the gastrointestinal tract, associated with microbial dysbiosis. Interestingly, IBD patients exhibit an increased incidence of mental illness (i.e. anxiety and depression), often termed “sickness behavior”, even during the remitting phase of their disease. Nevertheless, it is unclear if alterations in the enteric microbiota associated with IBD are responsible for the observed modification in brain function and behavior. Here, we hypothesized that sickness behavior is driven by alterations in microbial composition, which occur in the context of intestinal inflammation. Aims We sought to determine whether transfer of the microbiota from colitic mice, exhibiting sickness behaviour, into healthy counterparts would induce behavioral changes. Methods Male mice (C57Bl/6J; 8 weeks old) were used in all experiments. Colitis was induced by administration of 2.5% dextran sodium sulfate (DSS) in the drinking water for 5 days. Colonic inflammation was assessed by measuring fecal lipocalin-2 and the expression of pro-inflammatory mediators via qPCR. Cecal matter from donor mice (control or DSS treated) were collected for fecal microbiota transplant (FMT). FMT was performed via oral gavage in antibiotic-treated recipient mice. Gut bacteria were evaluated by 16S rRNA sequencing in cecal samples. Anxiety- and depression-like behavior were assessed by elevated plus maze and tail suspension test, respectively. Results DSS-treated mice exhibited clinical disease, reflected by body weight loss, increased fecal lipocalin-2 and elevated colonic pro-inflammatory cytokine transcripts. An increase in anxiety-like behavior was observed in mice with colitis, although no alterations in depression-like behavior were detected. Colitic mice exhibited a unique microbial community. Transferring cecal material from colitic mice into recipient, antibiotic-treated mice, recapitulated alterations in behavior seen in colitic donors, as shown by increased anxiety-like behavior and unexpectedly, increased depression-like behavior. These behavioral changes occurred in the absence of colonic or brain inflammation in the recipient mice, but were associated with changes in stress-related gene expression (i.e. Crh). Conclusions Colitis-associated sickness behavior can be transmitted to antibiotic-treated recipient mice via FMT, which occurs in the absence of overt intestinal or brain inflammation. Funding Agencies CIHRNational Council for Scientific and Technological Development (CNPq-Brazil)

A36 THE ROLE OF MICROBIAL INDOLE METABOLITES IN CONTROLLING INFLAMMATORY RESPONSES AND HEALING IN RESPONSE TO DSS-INDUCED COLITIS

Abstract Background Failure to resolve inflammation is often associate with the complications of Crohn’s Disease (CD). The pregnane X receptor (PXR), a xenobiotic receptor, is recognized for its role in suppressing inflammation and has recently been shown to influence fibrogenesis in the liver. In the intestine, PXR-signaling can be influenced by the microbial tryptophan metabolite indole-3- propionic acid (IPA), which can modulate intestinal inflammation, in turn influencing fibrogenesis, resolution and healing. This suggests that the gut microbiota could modulate mucosal homeostasis and resolution of inflammation via microbial metabolites Aims To understand and characterize the interplay between microbial complexity and the regulation of host inflammatory and healing responses, specifically focusing on the PXR and its microbial metabolite ligand IPA. Methods Intestinal inflammation was induced using DSS (1%, 1.5%, 2% and 3.5%) for 5 days followed by healing for 25 days in C57Bl/6 stably derived moderately diverse mouse microbiota 2 (sDMDMm2) colonized gnotobiotic and C57Bl/6 specific pathogen free (SPF) mice. Inflammation, architectural changes and fibrosis were assessed using Haemotoxylin and Eosin and Masson-Trichrome histological stains. Weight was recorded daily for the first 10 days and every other day after for 25 days, for a total of 30 days. Fecal lipocalin was quantified in samples collected throughout the study to assess inflammation. Innate immune cell influx was measured by flow cytometry, and the microbiota assessed via 16S rRNA sequencing. Results The gnotobiotic sDMDMm2 mice were exquisitely sensitive to DSS-induced colitis, exhibiting significantly increased mortality and morbidity at 2% and 3.5% w/v DSS compared to the SPF group. To elicit the same degree of disease to assess recovery, sDMDMm2 mice were exposed to 1.5% DSS and SPF mice to 3.5% DSS. Following 25 days recovery, sDMDMm2 colonized mice showed increased levels of fecal lipocalin 2, as compared to the SPF mice. DSS-treated sDMDMm2 mice supplemented with IPA during their recovery presented lower levels of fecal lipocalin, similar to colitic SPF mice. IPA supplemented sDMDMm2 mice also exhibited greater overall survival, with no significant differences in neutrophil count compared to mice given H20 during recovery. Conclusions A model system with a less complex microbiota (sDMDMm2) has a higher susceptibility to acute inflammation and a diminished capacity to resolve said inflammation. Addition of the microbial metabolite IPA normalized the recovery of the sDMDMm2 colonized mice, to a response indistinguishable from SPF mice, while also increasing survival. These data highlight the importance of microbial complexity in the regulation of intestinal mucosal homeostasis. Funding Agencies CAG, CCC, CIHR

A235 THE INFLUENCE OF IL10 ON COLITIS DEVELOPMENT IN NOD2 DEFICIENT MICE

Abstract Background Inflammatory bowel disease (IBD) risk genes include interleukin-10 (IL10) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2). Although their specific roles in intestinal inflammation are unclear, these proteins are involved in modulating host-microbe interactions and intestinal homeostasis. Aims Since IL10 is an anti-inflammatory cytokine that maintains intestinal homeostasis, we hypothesized that mice deficient for both IL10 and NOD2 would be more susceptible to colitis than mice with only the NOD2 deletion. Methods In-house generated NOD2-/- x IL10-/- (double knockout) and NOD2-/- x IL10+/+ littermate mice of varying ages (3–20 weeks of age) were weighed and stools samples were collected at weekly intervals. Lipocalin-2 (LCN-2) levels were quantified in stool by ELISA. A subset of mice from each genotype received 3.5% dextran sulfate sodium (DSS) supplemented water for 5 days starting at eight weeks of age. Mice were sacrificed after two days of regular water, on day 8. Results Untreated NOD2-/- x IL10+/+ mice showed higher fecal LCN-2 compared to NOD2-/- x IL10-/- mice at all ages, although this trend was not significant. Within DSS-treated mice, LCN-2 levels in stool collected at sacrifice (on day 8) were significantly higher in NOD2-/- x IL10+/+ mice, compared to NOD2-/- x IL10-/- mice. Moreover, only 50% (2 of 4) of the NOD2-/- x IL10+/+ DSS-treated mice survived to day 8, whereas all four NOD2-/- x IL10-/- DSS-treated mice survived. Conclusions Fecal LCN-2 indicates the extent of neutrophil infiltration, which is reflective of the severity of intestinal inflammation. As such, LCN-2 levels suggest that NOD2-/- x IL10+/+ mice have higher baseline inflammation and are more susceptible to DSS-induced colitis than double knockout NOD2-/- x IL10-/- mice. These results are contrary to our hypothesis that combining IL10 and NOD2 deficiency would increase colitis susceptibility as compared to NOD2 deficiency alone. This suggests that the absence of IL10 may actually reduce the severity of DSS-induced colitis in NOD2 deficient mice. The role of IL10 in modulating the gut microbiome using this model remains to be assessed. Funding Agencies CAG

P074 HUMAN-DERIVED CLOSTRIDIUM VE202 STRAINS REDUCE ENTEROBACTERIACEAE AND FUSOBACTERIA AND REVERSE EXPERIMENTAL COLITIS INDUCED BY HUMAN GUT MICROBIOTA

Abstract Background 17 human-derived Clostridium strains (VE202), belonging to a bacterial cluster under-represented in active IBD, induce colonic IL-10-producing FOXP3+ regulatory T cells and prevent colitis in murine IBD models (Nature 2013). The VE202 consortium is a promising live biotherapeutic product for clinical application. However, its 1) therapeutic benefit on established colitis and 2) effect on intestinal microbiota composition remain unclear. Methods Models: We established colitis in ex-germ-free (exGF) Rag2−/− mice with naïve T cell transfer (Hu-nT) and Il10−/− (Hu-Il10−/−) mice colonized with healthy human fecal microbiota, using stool with low abundance of Clostridiales. In Hu-Il10−/−, we tested two other healthy donor stools (one with low Clostridiales and another with Fusobacteria, which is a potential pathobiont in IBD). For a selected bacteria-colonized model, we selected three aggressive Enterobacteriaceae strains cultured from the human donor stool used for the previous studies, based on colonic Th1 response and colitis in monoassociated exGF-Il10−/−. We then established an aggressive Klebsiella (K), Enterobacter (E) and E. coli (E) strain -colonized exGF Il10−/− colitis model (KEE-colitis). In addition, we established a human IBD-derived E.coli and Fusobacterium varium consortium-colonized Il10−/− (EF-colitis) model. In vitro, we anaerobically co-cultured human stools with VE202 and analyzed bacterial composition. Therapy: We treated mice by oral administration of VE202 or vehicle twice weekly for 2–4 weeks. Evaluation: Colitis severity was assessed by blinded histological score, fecal lipocalin-2, stool consistency score and colonic IFNγ +CD4+ T cells. Bacteria profiles in cecal contents were analyzed by metagenomics or qPCR. Results Hu-nT and Hu-Il10−/− (Fig.1A, B): VE202 significantly reversed histological colitis and other inflammatory endpoints compared to vehicle. Consistently VE202 attenuated colitis induced by three different donor stools. Bacteria profiling revealed that VE202 attenuated the colitis-associated bloom of Enterobacteriaceae and Fusobacteria. In KEE-colitis (Fig.1C) VE202 reversed colitis with reduction of all Enterobacteriaceae strains. In EF-colitis (Fig.1D) VE202 reversed colitis with reduction of E. coli and F. varium. Co-culture (Fig.1E): VE202 reduced abundance of Enterobacteriaceae in a dose dependent manner. Conclusion Our findings suggest a novel IL-10-independent protective mechanism for human Clostridium VE202 strains, i.e. correction of dysbiosis with reduction of levels of Enterobacteriaceae and Fusobacteria. In addition, VE202 treatment is consistently effective for different human donor microbiota in vivo and in vitro. These results provide a rationale and target for therapeutic use of rationally selected resident protective bacterial cocktails in IBD patients.

2 EFFECTS OF AN IBD-ASSOCIATED MICROBIAL COMMUNITY STATE ON INTESTINAL INFLAMMATION IN HUMANIZED GNOTOBIOTIC MICE

Abstract Background and Aims We previously reported that 20% of unaffected first-degree relatives of pediatric IBD patients share the same microbial community structure (OTU-type) as IBD patients. This suggests that a preexisting dysbiosis can predispose to the development of IBD. The aims of this study were to establish that microbial community types identified in the family cohort could be reconstituted in gnotobiotic mice for further translational studies and to assess for any direct effects of this microbiota on intestinal inflammation. Methods Over 100 germ-free wild-type C57/BL6 mice were colonized for 4 weeks in the UCLA gnotobiotic facility with fecal microbiota from 30 human donors belonging to each of four groups from the pediatric family cohort: unaffected first-degree relatives with OTU-type 1, unaffected first-degree relatives with the IBD-associated OTU-type 2, Crohn’s disease (CD) with OTU-type 1, CD with OTU-type 2. Each group was represented by 8 human donors (n=3–4 mice/donor) with the exception of CD OTU-type 1 (only 5 donors available in the cohort). In addition, 35 eight week old germ-free IL-10-/- C57/BL6 mice were colonized for 12 weeks with fecal microbiota from 4 CD OTU-type 2 donors and 4 unaffected OTU-type 1 donors (n=4–5/donor). Luminal and mucosal microbial composition in the colon and small intestine was evaluated by 16S rRNA sequencing. Intestinal inflammation was assessed in the small intestine and colon by semiquantitative scoring of H&amp;E stained sections. Results Donor-specific microbial composition was observed in the humanized gnotobiotic mice with clear separation between recipients of donor OTU-type 1 vs. OTU-type 2 stool. Parallel differences in differentially abundant microbes and microbial diversity were seen in these gnotobiotic mice as in the original human donors. No histologic evidence was found for colitis, enteritis, or fibrosis in wild-type colonized mice. However, fecal lipocalin was increased two-fold increase in recipients of OTU-type 2 microbiota from CD patients relative to the other three groups. CD humanized IL-10-/- mice exhibited lower microbial diversity and distinct microbial composition compared to non-IBD humanized IL-10-/- mice, mirroring differences in the human donors. Mice colonized with CD microbiota had increased histological disease severity in the colon and cecum (p=0.05) compared to mice colonized with non-IBD microbiota. Conclusion Our results demonstrate that human microbial community states identified in the pediatric IBD cohort could be reconstituted in gnotobiotic mice for translational studies. Moreover, CD-associated dysbiosis exacerbated colitis severity. These data support the concept that dysbiosis plays a direct role in promoting intestinal inflammation and provide an experimental framework for further mechanistic studies of IBD-associated microbial communities.

Rhein modulates host purine metabolism in intestine through gut microbiota and ameliorates experimental colitis.

Background: Gut microbiota, which plays a crucial role in inflammatory bowel diseases (IBD), might have therapeutic benefits for ulcerative colitis or Crohn's disease. Targeting gut microbiota represents a new treatment strategy for IBD patients. Rhein is one of the main components of rhubarb and exhibits poor oral bioavailability but still exerts anti-inflammatory effects in some diseases. Therefore, we investigated the effect of rhein on colitis and studied its possible mechanisms.Methods: The chronic mouse colitis model was induced by four rounds of 2% dextran sulfate sodium (DSS) treatment. The mice were treated with 50 mg/kg and 100 mg/kg rhein daily, body weight, colon length, histological score, inflammatory cytokines in serum or intestine, and fecal lipocalin 2 concentration were determined. Th17 cell, Th1 cell and Th2 cell infiltration in the mesenteric lymph node were analyzed by flow cytometry. Metabolic profiles were collected by non-targeted metabolomics and key metabolic pathways were identified using MetaboAnalyst 4.0. We also assessed intestinal barrier permeability and performed 16s rDNA sequencing. Lactobacillus sp. was cultured, and fecal microbiota transplantation (FMT) was employed to evaluate the contribution of gut microbiota.Results: Rhein could significantly alleviate DSS-induced chronic colitis. Uric acid was identified as a crucial modulator of colitis and rhein treatment led to decreased uric acid levels. We determined that rhein changed purine metabolism indirectly, while the probiotic Lactobacillus was involved in the regulation of host metabolism. Uric acid resulted in a worsened intestinal barrier, which could be rescued by rhein. We further confirmed that rhein-treated gut microbiota was sufficient to relieve DSS-induced colitis by FMT.Conclusion: We showed that rhein could modulate gut microbiota, which indirectly changed purine metabolism in the intestine and subsequently alleviated colitis. Our study has identified a new approach to the clinical treatment of colitis.

370 Dietary soluble fiber increases the intestinal butyrate-producing bacteria, reduces intestinal permeability, and improves metabolic syndrome in sows during perinatal period

Abstract In humans, metabolic and immune changes during the perinatal period also describe metabolic syndrome. Gut microbiota can cause symptoms of metabolic syndrome in pregnant women. Gut microbiota modulations caused by pregnancy nutrition constitute an interesting target in the metabolic status of pregnant individual. However, studies investigating the changes of the metabolic characteristics and gut microbiota in sows throughout pregnancy and lactation are lacking, and the effects of soluble fiber diet during pregnancy on them are still underexplored. Sows were allocated to either control or 2.0% pregelatinized waxy maize starch plus guar gum (SF) dietary treatment during gestation. The metabolic characteristics, gut permeability and inflammation status, and gut microbiota composition and metabolism of sows during pregnancy and lactation were analysed. The plasma levels of hs-CRP and HOMA-IR values were increased in days 109 of gestation (G109) and days 3 of lactation (L3), while dietary SF reduced plasma hs-CRP concentrations and the HOMA-IR values. The levels of plasma zonulin and fecal endotoxin and lipocalin-2 were increased in G109 and L3, while dietary SF reduced elevation of plasma zonulin and endotoxin and fecal lipocalin-2. Next-generation sequencing analysis revealed that dietary SF increased levels of butyrate-producing Ruminococcaceae, Lachnospiraceae, and Oscillospira. The fecal and plasma levels of butyrate were also increased. Additionally, the increased relative abundances of Lachnospiraceae in SF sows negatively correlated with plasma hs-CRP and HOMA-IR while Oscillospira was negatively fecal lipocalin-2. Our data reveal that the sow exhibits a metabolic syndrome and dramatical changes in gut microbiota during perinatal period. Besides, dietary SF has beneficial effects on insulin sensitivity, intestinal permeability and inflammation maybe via increasing butyrate-producing bacteria and butyrate production.

Seaweed natural products modify the host inflammatory response via Nrf2 signaling and alter colon microbiota composition and gene expression

Seaweeds are an important component of human diets, especially in Asia and the Pacific islands, and have shown chemopreventive as well as anti-inflammatory properties. However, structural characterization and mechanistic insight of seaweed components responsible for their biological activities are lacking. We isolated cymopol and related natural products from the marine green alga Cymopolia barbata and demonstrated their function as activators of transcription factor Nrf2-mediated antioxidant response to increase the cellular antioxidant status. We probed the reactivity of the bioactivation product of cymopol, cymopol quinone, which was able to modify various cysteine residues of Nrf2's cytoplasmic repressor protein Keap1. The observed adducts are reflective of the polypharmacology at the level of natural product, due to multiple electrophilic centers, and at the amino acid level of the cysteine-rich target protein Keap1. The non-polar C. barbata extract and its major active component cymopol, reduced inflammatory gene transcription in vitro in macrophages and mouse embryonic fibroblasts in an Nrf2-dependent manner. Cymopol-containing extracts attenuated neutrophil migration in a zebrafish tail wound model. RNA-seq analysis of colonic tissues of mice exposed to non-polar extract or cymopol showed an antioxidant and anti-inflammatory response, with more pronounced effects exhibited by the extract. Cymopolia extract reduced DSS-induced colitis as measured by fecal lipocalin concentration. RNA-seq showed that mucosal-associated bacterial composition and transcriptional profile in large intestines were beneficially altered to varying degrees in mice treated with either the extract or cymopol. We conclude that seaweed-derived compounds, especially cymopol, alter Nrf2-mediated host and microbial gene expression, thereby providing polypharmacological effects.

Strain-Specific Anti-inflammatory Properties of Two Akkermansia muciniphila Strains on Chronic Colitis in Mice.

Akkermansia muciniphila is potential probiotic in that its type strain ATCC BAA-835 has beneficial effects upon obesity and diabetes. However, whether A. muciniphila can improve inflammatory bowel diseases (IBD), which is a form of chronic intestinal dysbiosis, is unknown. Hence, we used an isolated murine A. muciniphila strain (designated 139) and A. muciniphila type strain ATCC, to investigate their anti-inflammatory properties in cell models and in Dextran Sulfate Sodium (DSS)-induced chronic colitis of mice. In vitro, the two A. muciniphila strains exerted similar anti-inflammatory properties as they both reduced IL-8 production by TNF-α-stimulated HT-29 cells. However, neither of the strains showed capacity to increase the differentiation of regulatory T (Treg)-cells from CD4+ T cell populations significantly. In vivo, both A. muciniphila strains exerted anti-inflammatory effects on chronic colitis as they improved clinical parameters including spleen weight, colon inflammation index, and colon histological score. They also down-regulated the expression of the pro-inflammatory cytokines including TNF-α and IFN-γ in the colon of mice. However, the anti-inflammatory effects of strain ATCC were stronger than strain 139 in that ATCC significantly reduced spleen weight, colon inflammation index, and fecal lipocalin-2 content in mice with chronic colitis, while strain 139 was not. Dysbiosis of the gut microbiota was observed in mice with chronic colitis. Both A. muciniphila strains facilitated the normalization of the gut microbiota. The specific capacity of strain ATCC to modulate the differentiation of Tregs as well as increase production of short chain fatty acids, demonstrated strain-specific characteristics for these two A. muciniphila strains. This study suggests the potential beneficial effect of A. muciniphila on IBD and the importance of the future study of the function of A. muciniphila at the strain-level.

Persistent IL-1b Signaling Aggravates Murine Enteropathogen, Citrobacter rodentium Infection in Mice

Abstract The inflammasome cytokine IL-1b is an important mediator of the inflammatory responses against invading pathogens. However, it is not clear whether sustained IL-1b signaling following the loss of its endogenous inhibitor, secretory IL-1 receptor antagonist (sIL-1Ra), could improve mucosal immunity against the enteropathogen, Citrobacter rodentium (CR). In light of increased prescription of rec-sIL-1Ra (aka Anakinra) to treat inflammatory bowel disease, we undertook this study. At basal levels, sIL-1Ra-deficient (sIL-1RaKO) mice displayed leukocytosis and elevated inflammatory marker, i.e. serum and fecal lipocalin 2. Furthermore, bone marrow-derived macrophages and neutrophils from sIL-1RaKO mice generated higher levels of iNOS and nitrite, and ROS and NETs, respectively. These results collectively suggest sIL-1RaKO mice have low-grade chronic inflammation. Based on these results, we hypothesized that sIL-1RaKO mice with persistent IL1-b signaling could efficiently clear CR infection than their WT littermates. Oral challenge of CR (1×109 CFU/mouse) resulted in luminal colonization, which peaked at day 7 post-infection, in both groups; however, sIL-1RaKO mice displayed a higher CR burden and an exacerbated infection. The aggravated course of infection was further visualized by inoculating sIL-1RaKO mice with bioluminescent CR. Histologic analysis revealed that transmissible colonic hyperplasia was more pronounced in sIL-1RaKO mice. Interestingly, basal ileal Paneth’s cell-specific antimicrobial proteins (Ang4 and Reg3g) were significantly reduced in sIL-1RaKO mice. Collectively, our results demonstrate that a balanced mucosal IL-1b signaling is required to counter and clear enteropathogen infection.