Colitis In IL-10 Research Articles

Loss of Acid Ceramidase in Myeloid Cells Impairs MHC II Expression on Dendritic Cells and Macrophages to Alleviate Chronic Colitis in IL10−/− Mice

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the colon and drastically increases the risk in the development of colorectal cancer (CRC). Over expression of acid ceramidase (AC) resulting in accumulation of sphingosine-1-phosphate (S1P), which amplify inflammatory pathways, has been implicated in patients with IBD and CRC. The conditional loss of AC in myeloid cells (ACMYE) has demonstrated promise as a therapeutic target in an acute colitis model. We sought to expand the investigation of AC loss in a physiologically relevant model of IBD using IL-10 deficient mice. IL10−/− mice were crossed with AC conditional knockout mice where AC is deleted in myeloid cells to generate ACMYE/IL10−/−, with ACfl/fl/IL10−/− mice used as control. Male and female animals were collected at 8, 12, and 24 weeks of age and assessed for intestinal inflammation. Upon examination of basic parameters of colitis, ACMYE mice exhibited reduced body weight and enlarged spleens at 12 weeks of age. Histologic assessment revealed no difference between genotypes in colitis severity at 8 or 12 weeks of age. Flow cytometry analysis was utilized to define the role of AC in immune cell recruitment. ACMYE mice exhibit reduced MHC II surface expression on antigen presenting cells (APCs) as well as reductions in neutrophil activation markers in peripheral tissues and the colon at 8 weeks of age. Interestingly, at 12 weeks of age ACfl/fl mice demonstrate significant immunosuppression across various cell types in the colon compared to ACMYE mice, whose colonic immune cells demonstrate little change over time. Furthermore, CD8+ cytotoxic T cell populations were significantly elevated in peripheral tissues despite continued reductions of MHC II surface expression on APCs in ACMYE mice at 12 weeks of age. These data indicate that loss of AC may provide protection in early age while resulting in altered inflammation in later age; however, loss of AC may also disrupt MHC II expression as seen consistently in APCs, and may indicate interference with cell-to-cell cross talk. Realtime qPCR revealed that ACMYE mice exhibited significant ablation in the expression of several pro-inflammatory cytokines at 24 weeks of age. Together these data implicate AC as a potential therapeutic target in reducing inflammation in chronic IBD. This work is supported by the National Institute of Diabetes and Digestive and Kidney Diseases R01 DK132079 (AJS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enteral nutrition promotes the remission of colitis by gut bacteria-mediated histidine biosynthesis

Exclusive enteral nutrition (EEN) is an important alternative strategy for patients with Crohn's disease (CD), and during this process, microbiota alterations have been observed. However, the underlying mechanisms by which EEN reduces intestinal inflammation are currently unclear. The therapeutic potential of enteral nutrition (EN) was assessed using various mouse models. Fecal full-length 16S rDNA sequencing analysis and several CD metagenome datasets were used to identify the candidate therapeutic bacteria Faecalibaculum rodentium (F.rodentium). Whole genome sequencing of F.rodentium and widely-targeted metabolome analysis of the supernatant showed that EN-induced F.rodentium accumulation protected against colitis via histidine biosynthesis. The therapeutic potential of EN therapy was observed in both dextran sulfate sodium (DSS)-induced colitis and Il10-/- spontaneous colitis mouse models. Accumulation of F.rodentium after EN therapy was determined using full-length 16S rDNA sequencing and verified with several metagenome datasets from patients with CD. Colonization of an isolated F.rodentium could reduce colitis in Il10-/- mice. Significant histidine enrichment was observed in the F.rodentium culture supernatant, and a series of histidine biosynthesis genes were observed in the F.rodentium genome. Engineered Escherichia coli Nissle 1917 (EcN), encoding the heterologous hisG of F.rodentium (EcN-hisG), which was a key driver of histidine biosynthesis in F.rodentium, was found to protect against colitis. This study suggests that EN-induced F.rodentium accumulation protects against colitis in mice via gut bacteria-mediated histidine biosynthesis. A full list of funding bodies can be found in the Acknowledgements section.

Arabinose confers protection against intestinal injury by improving integrity of intestinal mucosal barrier

There is a growing amount of research that highlights the significant involvement of metabolic imbalance and the inflammatory response in the advancement of colitis. Arabinose is a naturally occurring bioactive monosaccharide that plays a crucial role in the metabolic processes and synthesis of many compounds in living organisms. However, the more detailed molecular mechanism by which the administration of arabinose alleviates the progression of colitis and its associated carcinogenesis is still not fully understood. In the present study, arabinose is recognized as a significant and inherent protector of the intestinal mucosal barrier through its role in preserving the integrity of tight junctions within the intestines. Also, it is important to note that there is a positive correlation between the severity of inflammatory bowel disease (IBD) and colorectal cancer (CRC), as well as chemically-induced colitis in mice, and lower levels of arabinose in the bloodstream. In two mouse models of colitis, caused by dextran sodium sulfate (DSS) or by spontaneous colitis in IL-10-/- mice, damage to the intestinal mucosa was reduced by giving the mice arabinose. When arabinose is administrated to model with colitis, it sets off a chain of events that help keep the lysosomes together and stop cathepsin B from being released. During the progression of intestinal epithelial injury, this process blocks myosin light chain kinase (MLCK) from damaging tight junctions and causing mitochondrial dysfunction. In summary, the results of the study have provided evidence supporting the beneficial effects of arabinose in mitigating the progression of colitis. This is achieved through its ability to avoid dysregulation of the intestinal barrier. Consequently, arabinose may hold promise as a therapeutic supplementation for the management of colitis.

SIRT3 Activator Honokiol Inhibits Th17 Cell Differentiation and Alleviates Colitis.

Honokiol (HKL), a natural extract of the bark of the magnolia tree and an activator of the mitochondrial protein sirtuin-3 (SIRT3), has been proposed to possess anti-inflammatory effects. This study investigated the inhibitory effects of HKL on T helper (Th) 17 cell differentiation in colitis. Serum and biopsies from 20 participants with ulcerative colitis (UC) and 18 healthy volunteers were collected for the test of serum cytokines, flow cytometry analysis (FACS), and relative messenger RNA (mRNA) levels of T cell subsets, as well as the expression of SIRT3 and phosphorylated signal transducer and activator of transcription/retinoic acid-related orphan nuclear receptor γt (p-STAT3/RORγt) signal pathway in colon tissues. In vitro, naïve clusters of differentiation (CD) 4 + T cells isolated from the mouse spleen differentiated to subsets including Th1, Th2, Th17, and regulatory T (Treg) cells. Peripheral blood monocytes (PBMCs) from healthy volunteers were induced to the polarization of Th17 cells. After HKL treatment, changes in T cell subsets, related cytokines, and transcription factors were measured. The dextran sulfate sodium (DSS)-induced colitis and interleukin (IL)-10-deficient mice were intraperitoneally injected with HKL. These experiments were conducted to study the effect of HKL on the development, cytokines, and expression of signaling pathway proteins in colitis. Patients with UC had higher serum IL-17 and a higher proportion of Th17 differentiation in blood compared with healthy participants; while IL-10 level and the proportion of Treg cells were lower. Higher relative mRNA levels of RORγt and a lower SIRT3 expression in colon tissues were observed. In vitro, HKL had little effect on the differentiation of naïve CD4+ T cells to Th1, Th2, or Treg cells, but it downregulated IL-17 levels and the Th17 cell ratio in CD4+ T cells from the mouse spleen and human PBMCs under Th17 polarization. Even with a STAT3 activator, HKL still significantly inhibited IL-17 levels. In DSS-induced colitis mice and IL-10 deficient mice treated with HKL, the length of the colon, weight loss, disease activity index, and histopathological scores were improved, IL-17 and IL-21 levels, and the proportion of Th17 cells were decreased. Sirtuin-3 expression was increased, whereas STAT3 phosphorylation and RORγt expression were inhibited in the colon tissue of mice after HKL treatment. Our study demonstrated that HKL could partially protect against colitis by regulating Th17 differentiation through activating SIRT3, leading to inhibition of the STAT3/RORγt signaling pathway. These results provide new insights into the protective effects of HKL against colitis and may facilitate the research of new drugs for inflammatory bowel disease.

Mesenteric Adipose Tissue-Derived Klebsiella variicola Disrupts Intestinal Barrier and Promotes Colitis by Type VI Secretion System.

Mesenteric adipose tissue (MAT) in Crohn's disease (CD) is associated with transmural inflammation. Extended mesenteric excision can reduce surgical recurrence and improve long-term outcomes, indicating that MAT plays an important role in the pathogenesis of CD. Bacterial translocation has been reported to occur inthe MATof patientswithCD (CD-MAT), but the mechanisms by which translocated bacteria lead to intestinal colitis remain unclear. Here it is shown that members of Enterobacteriaceae are highly enriched in CD-MAT compared with non-CD controls. Viable Klebsiella variicola in Enterobacteriaceae is isolated exclusively in CD-MAT and can induce a pro-inflammatory response in vitro and exacerbates colitis both in dextran sulfate sodium (DSS)-induced colitis mice model and IL-10-/- spontaneous colitis mice model. Mechanistically, active type VI secretion system (T6SS) is identified in the genome of K. variicola, which can impair the intestinal barrier by inhibiting the zonulaoccludens(ZO-1)expression. Dysfunction of T6SS by CRISPR interference system alleviates the inhibitory effect of K. variicola on ZO-1 expression and attenuated colitis in mice. Overall, these findings demonstrate that a novel colitis-promoting bacteria exist in the mesenteric adipose tissue of CD, opening a new therapeutic avenue for colitis management.

P103 Colitis-associated faecal miRNAs are mediators of host-microbiota interactions and key regulators of intestinal permeability and inflammation

Abstract Background Like in other chronic pathologies, evidence has shown disruption of host immune-microbial interactions and intestinal dysbiosis in the pathogenesis of Inflammatory Bowel Diseases (IBD), also characterized by chronic inflammation and increased intestinal permeability. Faecal microRNAs (miRNAs) have recently emerged as potential inter-kingdom mediators between the host and its microbiota, but the influence of its interaction on chronic intestinal disorders remains unclear. Here, we aimed to study the impact of colitis-associated miRNAs on intestinal inflammation, permeability and microbiota. Methods A panel of faecal miRNAs was profiled in IL10-/- mice, known as a model of spontaneous colitis. Faecal miRNAs and microbiota composition were compared between IBD and healthy controls. Identified colitis-associated miRNAs were then administered with/out antibiotics ad libitum to C57BL6 mice. Parameters of inflammation, intestinal epithelial barrier function, as well as microbiota composition, were analysed. Ex vivo and in silico studies were performed to comprehend the direct effects of the miRNAs on the gut microbiota and the host. Lastly, endogenous miRNAs were inhibited in IL10-/- mice administering miRNA-inhibitors through oral gavage. Results We identified in mice two colitis-associated miRNAs that were also increased in faecal samples of IBD compared to healthy controls (Graphic 1). Notably, the exogenous administration of these two miRNAs was sufficient to drive intestinal inflammation (Graphic 2) and alter the intestinal barrier function in mice. Such effects were directly impacted by the microbiota presence and composition. In humans, the measurement of faecal lipopolysaccharides and the 16S microbiota sequencing further showed strong associations between miRNAs and bacterial species, such as Faecalibacterium prausnitzii and Parabacteroides distasonis. In mice, the treatment with colitis-associated miRNAs altered the microbiota composition with a significant increase of Cyanobacteria and Verrucomicrobiota. Importantly, the inhibition of endogenous miRNAs through anti-miRNA administration prevented the emergence of severe colitis in IL10-/- mice (Graphic 3). Conclusion Overall, our findings identify an inflammatory role played by faecal miRNAs in the gastrointestinal environment, which pathogenic potential appears closely related to dysbiosis. Hence, our data suggest that miRNAs or miRNA inhibitors might be considered therapeutic agents to beneficially alter the microbiota and intestinal inflammation in IBD and other dysbiosis-associated conditions.

A high salt diet protects interleukin 10-deficient mice against chronic colitis by improving the mucosal barrier function

A high salt diet (HSD) is often associated with a high risk for a variety of diseases, such as obesity and cardiovascular disease. Previous studies have demonstrated that an HSD enhances Th17 responses and increases the severity of autoimmune diseases. In this study, we investigated the effects of HSD (4% NaCl w/w) on colitis in IL-10-/- mice by comparing it with IL-10-/- mice on a normal salt diet (NSD, 1% NaCl w/w). The colonic epithelial barrier integrity in IL-10-/- mice, as well as differentiated Caco-2 cells exposed to high NaCl and proinflammatory cytokines, was also evaluated. Surprisingly, an HSD significantly ameliorated macroscopic colitis, improved the intestinal permeability of FITC-dextran, and decreased multiple proinflammatory cytokines in the colonic mucosa of IL-10-/- mice. While occludin and claudin-1, two major tight-junction proteins, were markedly down-regulated in IL-10-/- mice, HSD effectively restored their expressions. In Caco-2 cells, proinflammatory cytokines (TNF-α and IL-1β) potently decreased the expression of occludin and claudin-1 regardless of salt conditions [0.9% (standard), 1.2%, or 1.5% NaCl]. Under high salt conditions (1.5% NaCl), transepithelial electrical resistance (TEER) was elevated, while the addition of IL-10 further downregulated occludin and claudin-1 expressions by ~50% and lowered TEER. These findings suggest that, in the absence of IL-10, HSD promotes intestinal epithelial integrity and exerts an anti-inflammatory role as demonstrated by alleviated colitis in IL-10-/- mice. Moreover, Caco-2 data indicate that, in an inflammatory environment and under high NaCl conditions, IL-10 may play a proinflammatory role by disrupting colonic epithelial integrity and thus further promoting inflammation.

Patient-derived Enterococcus faecium with inflammatory genotypes promote colitis.

Dysbiosis of gut microbiota promotes colitis in ulcerative colitis (UC). Enterococcus faecium is an important constituent of dysbiotic microbiota. However, the mechanisms underlying E. faecium-induced colitis remain unclear. Overall, 23 E. faecium strains isolated from human feces and 3 commercial strains were inoculated into Il10-/- mice. Mouse colons were histologically evaluated and analyzed using real-time PCR analysis of cytokines. Genes in 26 E. faecium strains were identified by whole-genome shotgun sequencing of genomic DNA. The production of reactive oxygen species (ROS) from each strain was measured. An antioxidant, lipoic acid, was orally administered to the colitis mouse model. Inoculation of E. faecium derived from patients with UC resulted in colitis in Il10-/- mice. The genotypes of 26 strains were characterized by identifying 1893 known genes; clustering all the strains based on the genotypes showed two major groups-inflammatory and probiotic clusters. Additionally, linear discriminant analysis clarified that lipoic acid metabolism was a significantly abundant pathway in the probiotic cluster compared to the inflammatory cluster. Further, the production of ROS was greater in inflammatory than in probiotic strains. Administration of lipoic acid in E. faecium-inoculated mice ameliorated colitis. Enterococcus faecium strains in the inflammatory cluster promoted colitis with higher production of ROS than the strains in the probiotic cluster.

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.

P104 Icariin Attenuates Intestinal Inflammation through Inhibition of the Nuclear Factor-ĸB signaling

Abstract Background Icariin is a natural flavonoid glucoside from plants in Epimedium family, known to have anti-oxidative and anti-inflammatory activities. This study investigated the effects of Icariin on nuclear factor-kappa B (NF-ĸB) signaling pathway in intestinal epithelial cells (IEC) and macrophages, and in acute and chronic murine colitis models. Methods Human IEC line COLO 205 and murine macrophage cell line RAW 264.7 were pretreated with Icariin and then stimulated with tumor necrosis factor-α (TNF-α) and lipopolysaccharide respectively for in vitro studies. The expression of pro- and anti-inflammatory cytokines were evaluated by real-time RT-PCR (TNF- α, IL-6, IL-8, IL-12, and IL-10), and the effect of Icariin on NF-ĸB signaling pathway was examined by immunoblot analysis to detect IĸBα phosphorylation and degradation. Dextran sulfate sodium (DSS)-induced acute colitis in C57BL/6 wild-type mice and chronic colitis in IL-10-/- mice were treated with or without Icariin for in vivo studies. The severity of colitis was evaluated by daily body weight change, colon length, and colonic tissue histopathology. Results Icariin significantly attenuated the production of pro-inflammatory cytokines and degeneration of anti-inflammatory cytokines, and alleviated IĸBα phosphorylation and degradation in both IEC and murine macrophages stimulated by TNF-α and LPS, respectively. Icariin also significantly reduced the severity of colitis in both DSS-induced acute colitis model and chronic colitis in IL-10-/- mice models. Conclusion Icariin inhibits NF-ĸB signaling in both IECs and macrophages, and attenuates experimental murine colitis. These results suggest that Icariin may be a potential novel therapeutic agent for inflammatory bowel disease.

Resveratrol Treatment Prevents Increase of Mast Cells in Both Murine OVA Enteritis and IL-10-/- Colitis.

Mast cells are involved in allergic and other inflammatory diseases. The polyphenol resveratrol is known for its anti-inflammatory properties and may be used as nutraceutical in mast cell associated diseases. We analyzed the effect of resveratrol on mast cells in vivo in ovalbumin-induced allergic enteritis as well as experimental colitis in IL-10−/− mice which received resveratrol via drinking water. Treatment with resveratrol prevented the increase in mast cells in both allergic enteritis and chronic colitis in duodenum as well as in colon. Further, it delayed the onset of diseases symptoms and ameliorated diseases associated parameters such as tissue damage as well as inflammatory cell infiltration in affected colon sections. In addition to the findings in vivo, resveratrol inhibited IgE-dependent degranulation and expression of pro-inflammatory cytokines such as TNF-α in IgE/DNP-activated as well as in LPS-activated bone marrow-derived mast cells. These results indicate that resveratrol may be considered as an anti-allergic and anti-inflammatory plant-derived component for the prevention or treatment of mast cell-associated disorders of the gastrointestinal tract.

Epac-2 ameliorates spontaneous colitis in Il-10-/- mice by protecting the intestinal barrier and suppressing NF-κB/MAPK signalling.

Intestinal barrier dysfunction and intestinal inflammation interact in the progression of Crohn's disease (CD). A recent study indicated that Epac‐2 protected the intestinal barrier and had anti‐inflammatory effects. The present study examined the function of Epac‐2 in CD‐like colitis. Interleukin‐10 gene knockout (Il‐10 −/−) mice exhibit significant spontaneous enteritis and were used as the CD model. These mice were treated with Epac‐2 agonists (Me‐cAMP) or Epac‐2 antagonists (HJC‐0350) or were fed normally (control), and colitis and intestinal barrier structure and function were compared. A Caco‐2 and RAW 264.7 cell co‐culture system were used to analyse the effects of Epac‐2 on the cross‐talk between intestinal epithelial cells and inflammatory cells. Epac‐2 activation significantly ameliorated colitis in mice, which was indicated by reductions in the colitis inflammation score, the expression of inflammatory factors and intestinal permeability. Epac‐2 activation also decreased Caco‐2 cell permeability in an LPS‐induced cell co‐culture system. Epac‐2 activation significantly suppressed nuclear factor (NF)‐κB/mitogen‐activated protein kinase (MAPK) signalling in vivo and in vitro. Epac‐2 may be a therapeutic target for CD based on its anti‐inflammatory functions and protective effects on the intestinal barrier.

Cereal Vinegar Sediment Alleviates Spontaneous Ulcerative Colitis in Il-10 Deficient Mice.

Cereal vinegar sediment (CVS) is precipitation generated during the preservation of vinegar. It has various functions such as anti-inflammatory, anti-tumor, hypoglycemic, and hypolipidemic. This study evaluates the effects of CVS on spontaneous colitis in Il-10-/- mice. CVS (1 g kg-1 body weight) is administered to mice for 42 days. CVS alleviated epithelium damage, inhibited myeloperoxidase (MPO) activity and malondialdehyde (MDA) level, decreased gene expression of tumor necrosis factor (Tnf )-a, inducible nitric oxide synthase (Inos), Interleukin(Il-23) in colon tissues is found. CVS also inhibited secretion of IL-2, IL-6, IL-13, Granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), Interferon (IFN)-γ, and Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES) in serum. While CVS enhanced Regenerating Family Member 3 Gamma (Reg3γ), Mucin (Muc2, Muc3, and Muc4 gene expression, promoted intestinal epithelial cells to secrete Muc-2, and increased the content of acetic acid in intestinal tract of Il-10-/- mice. Additionally, CVS altered the composition of the gut microbiota by promoting the abundance of Akkermansia, Alistipes, and Lactobacillus, while inhibiting Desulfovibrio and Clostridium sensu stricto 1. These changes may be related to the regulation of steroid, fatty acids, and bile acid biosynthesis. This study demonstrated that CVS ameliorates spontaneous ulcerative colitis in Il-10-/- mice, which suggests CVS supplementation may serve as a protective dietary nutrient against colitis.

Probiotics alleviate inflammatory bowel disease in mice by regulating intestinal microorganisms-bile acid-NLRP3 inflammasome pathway.

The objective of the present study was to elucidate the mechanism of intestinal microorganisms- bile acid- NLRP3 inflammasome regulation in mice with inflammatory bowel disease treated with probiotics. The abnormal activation of NLRP3 inflammasome is the main pathogenic factor that leads to the development of chronic colitis in IL-10-/- mice. In this study, we divided the IL-10-/- and wild-type mice on a C57BL/6 background into 3 groups: control group (wt mice, n=10), IBD group (IL-10-/- mice, n=10), and probiotic group (IL-10-/- mice treated with probiotics, n=10). The analyses included mRNA levels of cytokines and protein expression of NLRP3 inflammasome and NOD2, as well as colorimetric determination of Wnt, Notch and BMP activity in colon tissue and fresh colon mass. The fresh colon mass was increased in the IBD mice when compared with the control and the probiotic groups (P<0.05). The histological score of the proximal colon in the IBD group was higher than in two other groups (P<0.05). The probiotic group showed lower levels of IFN-γ, IL-17F, IL-1α and IL-25 mRNA compared to the IBD group (P<0.05). The main components of NLRP3 inflammasome (NLRP3, ASC, caspase-1 and IL-1β) and NOD2 were increased in IBD group compared to the control, and decreased after probiotic treatment (P<0.05). FXR, TGR5, vitamin D, and CAR were all increased in IBD group compared to the control and probiotic groups (P<0.05). In conclusion, probiotics modulated the intestinal microbial-bile acid-NLRP3 inflammation in IBD mice.

Early-Life Microbial Restitution Reduces Colitis Risk Promoted by Antibiotic-Induced Gut Dysbiosis in Interleukin 10–/– Mice

Perturbations in the early-life gut microbiome are associated with increased risk for complex immune disorders like inflammatory bowel diseases. We previously showed that maternal antibiotic-induced gut dysbiosis vertically transmitted to offspring increases experimental colitis risk in interleukin (IL) 10 gene deficient (IL10-/-) mice, a finding that may result from the loss/lack of essential microbes needed for appropriate immunologic education early in life. Here, we aimed to identify key microbes required for proper development of the early-life gut microbiome that decrease colitis risk in genetically susceptible animals. Metagenomic sequencing followed by reconstruction of metagenome-assembled genomes was performed on fecal samples of IL10-/- mice with and without antibiotic-induced dysbiosis to identify potential missing microbial members needed for immunologic education. One high-value target strain was then engrafted early and/or late into the gut microbiomes of IL10-/- mice with antibiotic-induced dysbiosis. Early-, but not late-, life engraftment of a single dominant Bacteroides strain of non-antibiotic-treated IL10-/- mice was sufficient to restore the development of the gut microbiome, promote immune tolerance, and prevent colitis in IL10-/- mice that had antibiotic-induced dysbiosis. Restitution of a keystone microbial strain missing in the early-life antibiotic-induced gut dysbiosis results in recovery of the microbiome, proper development of immune tolerance, and reduced risk for colitis in genetically prone hosts.

Loss of PTPN22 Promotes Intestinal Inflammation by Compromising Granulocyte-mediated Antibacterial Defence.

A single nucleotide polymorphism in protein tyrosine phosphatase non-receptor type 22 [PTPN22] has been associated with the onset of autoimmune disorders, but protects from Crohn's disease. PTPN22 deficiency in mice promotes intestinal inflammation by modulating lymphocyte function. However, the impact of myeloid PTPN22 in colitis development remains unclear. The aim of this study was to investigate the role of PTPN2 in the IL-10 and the T cell transfer colitis models. PTPN22-deficient mice were crossed with IL-10-/- and RAG2-/- mice. Naïve T cells were injected in RAG-/- mice to induce T-cell transfer colitis. Spontaneous colitis in IL-10-/- mice was monitored for up to 200 days. Here, we demonstrate that PTPN22 in non-lymphoid immune cells is required to protect against T cell transfer-mediated and IL-10 knock-out colitis. Analysis of the intestinal immune landscape demonstrated a marked reduction of granulocyte influx into the inflamed colon in PTPN22-deficient mice. On a molecular level, granulocytes were not only reduced by numbers, but also revealed a defective function. In particular, granulocyte activation and granulocyte-mediated bacteria killing was impaired upon loss of PTPN22, resulting in elevated bacterial burden and translocation beyond the intestinal epithelial barrier in PTPN22-deficient mice. Consistently, antibiotic-induced depletion of bacteria reverted the increased colitis susceptibility in PTPN22-deficient mice, whereas granulocyte depletion induced acolitis phenotype in wild-type mice similar to that observed in PTPN22-deficient mice. In conclusion, our data demonstrate that PTPN22 is essential for adequate granulocyte activation and antimicrobial defence to protect the inflamed intestine from bacterial invasion and exacerbated colitis.

Alleviation of Intestinal Inflammation by Dietary Pomegranate Extract Supplementation in Mice

ObjectivesInflammatory bowel disease (IBD) results from a complex interaction among host, microbial and environmental factors. Among these, diet has emerged as an important and actionable modifier of IBD activity. Pomegranate major bioactive metabolites, ellagic acid and urolithins, have been shown to improve symptoms of IBD in chemically induced mouse models of colitis. Here, we aim to test the hypothesis that dietary pomegranate extract (PomX) supplementation will reduce the development of colitis in IL-10 knock out (IL-10–/–) mice, which spontaneously develop chronic colitis after birth. Methods4 week old male IL-10–/– mice were randomly assigned to a high fat high sucrose (HFHS) diet, or to a HFHS diet supplemented with 0.25% PomX for 8 weeks. At the end of the experiment, the mice were euthanized and intestinal tissues were isolated and frozen for RNA extraction, or fixed for histologic analysis. Plasma samples were collected and processed for lipocalin 2 assay. ResultsHistomorphological analysis of colonic tissues revealed lower histological scores in HFHS-PomX fed mice, 3.9 ± 1.0 vs. 2.6 ± 0.5 in HFHS fed mice (n = 8, P = 0.02). In addition, signs of rectal prolapse were observed in 62.5% of IL-10–/– mice in the HFHS group vs. 12.5% in the HFHS/PomX group (P = 0.04). RNAseq and bioinformatic analysis, obtained from the colonic tissues (n = 4 mice/each group), showed in PomX treated mice a downregulation of 483 genes and an upregulation of 263 genes. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome analyses showed that PomX downregulated genes which are mainly associated with immune inflammatory responses, defenses, and neutrophil degranulation, including IL1α, IL6 and TNFα pro-inflammatory cytokines. Spleen weights were lower in HFHS-PomX treated mice as compared to HFHS fed control mice (P = 0.04). In addition, PomX treated mice showed a trend of higher body weights (∼13%) and lower lipocalin 2 plasma levels (∼46%) as compared to HFHS fed mice. ConclusionsOur data demonstrated that PomX supplementation decreased colitis symptoms and lowered the inflammatory parameters of colitis in HFHS fed IL10–/– mice. These data support the anti-inflammatory effects of dietary PomX supplementation that were previously observed in the DSS colitis murine model. Funding SourcesThis project was supported by UCLA Center for Human Nutrition.

IL-33 is produced by colon fibroblasts and differentially regulated in acute and chronic murine colitis

IL-33 is upregulated in ulcerative colitis and has a protective role in chemically-induced acute murine colitis. We aimed to determine whether IL-33 influences Il10−/− chronic colitis and its cellular source in health and during colitis. Il10−/−Il33−/− and Il10−/−Il33+/+ littermates developed colitis of similar severity. Colon Il33 was induced in WT and Il10−/− mice exposed to DSS, but not in unchallenged Il10−/− mice with colitis. Il33-citrine reporter mice showed that Il33-citrine colocalized with α-smooth muscle actin+ myofibroblasts and vimentin+ fibroblasts in WT mice. Citrine+CD74+CD90hi inflammatory fibroblasts were increased with DSS treatment. IL-1β induced Il33 expression in colon myofibroblasts, but colon Il33 expression did not differ between DSS-treated WT and Il1r1−/− mice. In conclusion, deficiency of IL-33 does not alter the severity of chronic colitis in Il10−/− mice. Induction of Il33 upon DSS exposure in WT and Il10−/− mice, but not in unchallenged Il10−/− mice, suggests epithelial injury induces colon IL-33. Fibroblasts are the primary colonic source of IL-33 and IL-33-expressing CD90hiCD74+ fibroblasts are increased during DSS-induced colitis. IL-1β induces Il33 in colon myofibroblasts in vitro, but signaling through the IL-1R1 is not necessary for induction of IL-33 in DSS-induced colitis.

The oxysterol receptor GPR183 in inflammatory bowel diseases.

Immune cell trafficking is an important mechanism for the pathogenesis of inflammatory bowel disease (IBD). The oxysterol receptor GPR183 and its ligands, dihydroxylated oxysterols, can mediate positioning of immune cells including innate lymphoid cells. GPR183 has been mapped to an IBD risk locus, however another gene, Ubac2 is encoded on the reverse strand and associated with Behçet's disease, therefore the role of GPR183 as a genetic risk factor requires validation. GPR183 and production of its oxysterol ligands are up-regulated in human IBD and murine colitis. Gpr183 inactivation reduced severity of colitis in group 3 innate lymphoid cells-dependent colitis and in IL-10 colitis but not in dextran sodium sulphate colitis. Irrespectively, Gpr183 knockout strongly reduced accumulation of intestinal lymphoid tissue in health and all colitis models. In conclusion, genetic, translational and experimental studies implicate GPR183 in IBD pathogenesis and GPR183-dependent cell migration might be a therapeutic drug target for IBD. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Enterococcus faecalis Glucosamine Metabolism Exacerbates Experimental Colitis.

Background & AimsThe inflammatory bowel diseases (IBDs), Crohn’s disease and ulcerative colitis, are caused in part by aberrant immune responses to resident intestinal bacteria. Certain dietary components, including carbohydrates, are associated with IBDs and alter intestinal bacterial composition. However, the effects of luminal carbohydrates on the composition and colitogenic potential of intestinal bacteria are incompletely understood. We hypothesize that carbohydrate metabolism by resident proinflammatory intestinal bacteria enhances their growth and worsens intestinal inflammation.MethodsWe colonized germ-free, wild-type, and colitis-susceptible interleukin-10 knockout mice (Il10-/-) with a consortium of resident intestinal bacterial strains and quantified colon inflammation using blinded histologic scoring and spontaneous secretion of IL12/23p40 by colon explants. We measured luminal bacterial composition using real-time 16S polymerase chain reaction, bacterial gene expression using RNA sequencing and real-time polymerase chain reaction, and luminal glucosamine levels using gas chromatography–mass spectrometry.ResultsWe show that a consortium of 8 bacterial strains induces severe colitis in Il10-/- mice and up-regulates genes associated with carbohydrate metabolism during colitis. Specifically, Enterococcus faecalis strain OG1RF is proinflammatory and strongly up-regulates OG1RF_11616-11610, an operon that encodes genes of a previously undescribed phosphotransferase system that we show imports glucosamine. Experimental colitis is associated with increased levels of luminal glucosamine and OG1RF_11616 causes worse colitis, not by increasing E faecalis numbers, but rather by mechanisms that require the presence of complex microbiota.ConclusionsFurther studies of luminal carbohydrate levels and bacterial carbohydrate metabolism during intestinal inflammation will improve our understanding of the pathogenesis of IBDs and may lead to the development of novel therapies for these diseases.