Cells In Colonic Lamina Propria Research Articles

P175 HucMSC-Exo ameliorate experimental colitis via modulating gut microbiota and metabolites

Abstract Background Gut microbiota and metabolites have been shown to influence the development of inflammatory bowel disease (IBD). Exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-Exo) have been identified as a promising biological therapy for the treatment of IBD. Previous studies have described that HucMSC-Exo could modulate gut microbiota. However, the mechanism remains unclear. Here, we explored the role of gut microbiota and metabolites in HucMSC-Exo-mediated amelioration of experimental colitis. Methods Dextran sulfate sodium (DSS) was used to induce colitis. The role of HucMSC-Exo were assessed in colitis mice. Fecal microbiota transplantation (FMT) and sterile fecal filtrate gavage were employed to evaluate the effect of gut microbiota and metabolites. Gut microbiota and metabolites were analyzed through 16S rRNA sequencing and metabolomic profiling. The proportion of CD4+ T cells were phenotyped by multicolour flow cytometry. Results HucMSC-Exo treatment alleviated colon inflammation by regulating flora composition, significantly up-regulated the levels of bacteria such as Bacteroides, Parabacteroides distasonis, and Tannerellaceae. Meanwhile, HucMSC-Exo transformed metabolite short-chain fatty acid (SCFA) profiles, particularly increased butyrate level. Additionally, HucMSC-Exo selectively up-regulated the frequencies of regulatory T (Treg) cells as well as down-regulated the ratio of T helper type 17 (Th17) cells in colonic lamina propria to maintain intestinal immune homeostasis and repaired the colonic mucus barrier. FMT and sterile fecal filtrate gavage were conducted to confirm the above mechanism. Microbiota from HucMSC-Exo-treated mice alleviated the colitis over microbiota from DSS-treated mice. Sterile fecal filtrate from HucMSC-Exo-treated mice and butyrate induced similar beneficial outcomes, such as improved the Th17/Treg balance and repaired the colonic mucus barrier. Collectively, HucMSC-Exo ameliorate colitis by directly modulating gut microbiota and metabolites. Conclusion HucMSC-Exo ameliorated experimental colitis via directly modulating gut microbiota and metabolite butyrate, which could regulate CD4+T cells homeostasis and repair colonic mucus barrier. The findings demonstrated the HucMSC-Exo as a potential gut microbiota and metabolites modulator to treat IBD.

Zinc finger protein 189 promotes the differentiation of lamina propria T helper 17.1 cells in dextran sulfate sodium-induced colitis

The factors regulating the heterogeneity of interleukin-17A (IL-17A)-expressing CD4+ T cells in inflammatory bowel diseases remain unclear. In the current study, we characterised the expression and function of zinc finger protein 189 (ZFP189) in a murine colitis model. Mice were given dextran sulphate sodium to induce acute colitis. Flow cytometry was applied to recognise and enrich Th17 and Th17.1 cells based on the expression of IL-17A, interferon-γ (IFN-γ), C-X-C motif chemokine receptor 3 (CXCR3), and C-C motif chemokine receptor 4 (CCR4). The expression of ZFP189 in Th17 and Th17.1 cells was determined by Immunoblotting. Lentivirus-mediated ZFP189 knockdown was conducted to evaluate the effect of ZFP189 on the differentiation of Th17 and Th17.1 cells. The adoptive transfer was performed to analyse the pathogenicity of Th17.1 cells in vivo. We found that ZFP189 was mildly up-regulated in IL-17A-expressing CD4+ T cells in colonic lamina propria. Lamina propria Th17.1 cells expressed higher ZFP189 than Th17 cells. In vitro ZFP189 knockdown in CD4+ T cells did not impact Th17 cell differentiation but suppressed Th17.1 cell differentiation, as evidenced by lower T-box expressed in T cells (T-bet) and IFN-γ. When adoptively transferred into mice, ZFP189-deficient Th17.1 cells produced fewer IFN-γ, tumour necrosis factor-alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) than ZFP189-expressing Th17.1 cells. Moreover, ZFP189-deficient Th17.1 cells induced less severe colitis than ZFP189-expressing Th17.1 cells, as evidenced by less body weight loss, a lower disease activity index, and a lower colon histological score. In summary, ZFP189 acts as a positive regulator of the differentiation and pathogenicity of lamina propria Th17.1 cells in colitis.

Interleukin 17 B regulates colonic myeloid cell infiltration in a mouse model of DSS-induced colitis.

Cytokines play vital roles in the pathogenesis of inflammatory bowel disease. IL17B is protective in the development of colitis. However, how IL17B regulates intestinal inflammation and what cells are regulated by IL17B is still unknown. Here, we aimed to illustrate the IL17B dependent cellular and molecular changes in colon tissue in a mouse colitis model. The results showed that IL17B expression in colon tissues was elevated in inflamed tissues than non-inflamed tissues of IBD patients. Wild type (WT) and Il17b deficient (Il17b -/-) mice were given 2.5% dextran sodium sulfate (DSS) water, and in some case, Il17b -/- mice were treated with recombinant mouse IL17B. IL17B deficiency resulted in severe DSS-induced colitis with exaggerated weight loss, shorter colon length, and elevated proinflammatory cytokines in colon. Reconstitution of Il17b -/- mice with recombinant IL17B alleviated the severity of DSS-induced colitis. Single cell transcriptional analyses of CD45+ immune cells in colonic lamina propria revealed that loss of IL17B resulted in an increased neutrophil infiltration and enhanced inflammatory cytokines in intestinal macrophages in colitis, which were confirmed by real-time PCR and flow cytometry. IL17B treatment also inhibited lipopolysaccharide-induced inflammation in bone marrow-derived macrophages and mice. IL17B inhibits colitis by regulating colonic myeloid cell response. It might represent a novel potential therapeutic approach to treat the colitis.

Analysis of Peripherally Derived Treg in the Intestine.

Elucidation of the symbiotic relationship between the host and its gut microbiota is critically important for understanding host pathophysiology. Peripherally derived regulatory T cells (pTregs) are recognized as central to immune homeostasis in the intestine. Moreover, the gut microbiota nourishes the intestinal and systemic immune systems, including pTreg, via their metabolites and other components. Therefore, methods to detect pTreg as well as to analyze the interactions between the gut microbiota and pTreg are important for better understanding of the symbiotic relationship with these microorganisms. Here, we describe a protocol to isolate colonic lamina propria cells and analyze pTregs in mice.

Mineralocorticoid receptor deficiency in Treg cells ameliorates DSS‐induced colitis in a gut microbiota‐dependent manner

Mineralocorticoid receptor (MR) is a classic nuclear receptor and an effective drug target in the cardiovascular system. The function of MR in immune cells such as macrophages and T cells has been increasingly appreciated. The aim of this study was to investigate the function of Treg MR in the process of inflammatory bowel disease (IBD). We treated Treg MR-deficient (MRflox/flox Foxp3YFP-Cre , KO) mice and control (Foxp3YFP-Cre , WT) mice with dextran sodium sulphate (DSS) to induce colitis and found that the severity of DSS-induced colitis was markedly alleviated in Treg MR-deficient mice, accompanied by reduced production of inflammatory cytokines, and relieved infiltration of monocytes, neutrophils and interferon γ+ T cells in colon lamina propria. Faecal microbiota of mice with colitis was analysed by 16S rRNA gene sequencing and the composition of gut microbiota was vastly changed in Treg MR-deficient mice. Furthermore, depletion of gut microbiota by antibiotics abolished the protective effects of Treg MR deficiency and resulted in similar severity of DSS-induced colitis in WT and KO mice. Faecal microbiota transplantation from KO mice attenuated DSS-induced colitis characterized by alleviated inflammatory infiltration compared to that from WT mice. Hence, our study demonstrates that Treg MR deficiency protects against DSS-induced colitis by attenuation of colonic inflammatory infiltration. Gut microbiota is both sufficient and necessary for Treg MR deficiency to exert the beneficial effects.

Neonatal Antibiotic Exposure Alters Intestinal Macrophage Frequency and Polarization

Abstract More than 75% of premature infants are given antibiotics in the first week of life. This is lifesaving for neonates with serious bacterial infections but is associated with increased risks of necrotizing enterocolitis (NEC). Given the association of intestinal macrophages (Mϕ) in the pathogenesis in NEC, we characterized the effect of antibiotics on this cell type. Pups were exposed to ampicillin and neomycin via the dam from postnatal day 1 (P1) to P7/8. Mϕ were identified as CD45+ CX3CR1+ CD11b+ F480+ using flow cytometry. In the small intestine (SI) there was an increase in the percentage of Mϕ in antibiotic-exposed lamina propria, but no difference in the proportion of F480-myeloid cells to Mϕ. The percentage of Mϕ in exposed colonic lamina propria cells was unchanged, but the proportion of Mϕ to F480-myeloid cells was increased. This suggests that antibiotic exposure expands myeloid cells in both the SI and colon, but likely via different mechanisms. To further characterize the phenotype of the intestinal Mϕ, we assessed polarity by immunoassay of 13 Mϕ associated cytokines. There were no significant differences in the proximal small intestine cytokine expression between control and antibiotic-exposed tissue. In the distal SI, antibiotic exposed mice had higher levels of TGFβ and CCL2, M2 Mϕ markers. In the colon, control tissue had elevated M1 Mϕ markers compared to antibiotic-exposed mice. Antibiotic-exposed colon had significantly increased levels of TGFβ. These data demonstrate that antibiotic exposure alters Mϕ proportion and polarization. Further work will be necessary to determine how this initial shift away from pro-inflammatory polarization leaves antibiotic-exposed infants at risk for intestinal inflammatory diseases. Supported by NIH T32DK077653

Survivin and Programmed Death Ligand-1 as Possible Players in the Pathogenesis of Ulcerative Colitis: An Immunohistochemical Study

BACKGROUND: Ulcerative colitis (UC) is a relapsing chronic inflammatory disorder, with many catastrophic complications. The pathogenesis of UC is not yet well understood. Exploring the exact immunologic pathogenesis of UC may eventually offer new therapeutic options.
 AIM OF THE WORK: In this study, we proposed that survivin and programmed death ligand (PDL1) may have roles in the pathogenesis of UC.
 MATERIALS AND METHODS: The study examined the immunohistochemical expression of both markers in the colonic epithelial cells and lamina propria in 43 cases of UC and 43 cases of non-specific chronic colitis (non-inflammatory bowel disease colitis).
 RESULTS: The results uncovered that both survivin and PDL1 expression were significantly expressed in the colonic lamina propria cells in UC cases in comparison to colitis cases (p < 0.001). On the other hand, the expression of PDL1 was shown to be lost in the colonic mucosal cells in UC cases when compared to cases of the control group (p < 0.05).
 CONCLUSION: The study, therefore, concluded that both survivin and PDL1 may play an important role in the UC pathogenesis and hence may be a novel interest in new therapeutic trends.

The Essential Role of Selenoproteins in the Resolution of Citrobacter rodentium-Induced Intestinal Inflammation.

Enteropathogenic Escherichia coli (EPEC) leads to adverse colonic inflammation associated with poor resolution of inflammation and loss of epithelial integrity. Micronutrient trace element selenium (Se) is incorporated into selenoproteins as the 21st amino acid, selenocysteine (Sec). Previous studies have shown that such an incorporation of Sec into the selenoproteome is key for the anti-inflammatory functions of Se in macrophages and other immune cells. An intriguing mechanism underlying the anti-inflammatory and pro-resolving effects of Se stems from the ability of selenoproteins to skew arachidonic acid metabolism from pro-inflammatory mediators, prostaglandin E2 (PGE2) toward anti-inflammatory mediators derived from PGD2, such as 15-deoxy-Δ12, 14- prostaglandin J2 (15d-PGJ2), via eicosanoid class switching of bioactive lipids. The impact of Se and such an eicosanoid-class switching mechanism was tested in an enteric infection model of gut inflammation by C. rodentium, a murine equivalent of EPEC. C57BL/6 mice deficient in Se (Se-D) experienced higher mortality when compared to those on Se adequate (0.08 ppm Se) and Se supplemented (0.4 ppm Se) diets following infection. Decreased survival was associated with decreased group 3 innate lymphoid cells (ILC3s) and T helper 17 (Th17) cells in colonic lamina propria of Se-D mice along with deceased expression of epithelial barrier protein Zo-1. Inhibition of metabolic inactivation of PGE2 by 15-prostaglandin dehydrogenase blocked the Se-dependent increase in ILC3 and Th17 cells in addition to reducing epithelial barrier integrity, as seen by increased systemic levels of FITC-dextran following oral administration; while 15d-PGJ2 administration in Se-D mice alleviated the effects by increasing ILC3 and Th17 cells. Mice lacking selenoproteins in monocyte/macrophages via the conditional deletion of the tRNA[Sec] showed increased mortality post infection. Our studies indicate a crucial role for dietary Se in the protection against inflammation following enteric infection via immune mechanisms involving epithelial barrier integrity.

Intervention of oncostatin M-driven mucosal inflammation by berberine exerts therapeutic property in chronic ulcerative colitis

Ulcerative colitis (UC) is a chronic and etiologically refractory inflammatory gut disorder. Although berberine, an isoquinoline alkaloid, has been revealed to exert protective effects on experimental colitis, the underlying molecular mechanism in chronic intestinal inflammation remains ill-defined. This study was designed to uncover the therapeutic efficacy and immunomodulatory role of berberine in chronic UC. Therapeutic effects of oral administration of berberine were investigated in dextran sodium sulfate (DSS)-induced murine chronic UC and the underlying mechanisms were further identified by si-OSMR transfection in human intestinal stromal cells. Berberine significantly attenuated the experimental symptoms and gut inflammation of chronic UC. Berberine treatment could also maintain the intestinal barrier function and rectify tissue fibrosis. In accordance with infiltrations of antigen-presenting cells (APCs), innate lymphoid cells (ILCs), and activated NK cells in colonic lamina propria, increased expression of OSM and OSMR were observed in the inflamed tissue of chronic UC, which were decreased following berberine treatment. Moreover, berberine inhibited the overactivation of human intestinal stromal cells through OSM-mediated JAK-STAT pathway, which was obviously blocked upon siRNA targeting OSMR. The research provided an infusive mechanism of berberine and illustrated that OSM and OSMR intervention might function as the potential target in chronic UC.

OP37 Fibrogenesis in chronic DSS colitis is driven by an innate lymphoid cell-independent innate immune response

Abstract Background Obtaining insights in the pathogenesis of intestinal fibrosis is a priority for improving outcomes in inflammatory bowel diseases (IBD). Studies in murine models and human organ fibrosis indicate a role for innate immunity pathways in fibrosis. The aim of this study was to dissect the role of innate lymphoid cells (ILC) in chronic intestinal inflammation and fibrosis. Methods A chronic 3-cycles dextran sulphate sodium (DSS) model was induced in wild-type (WT), recombinant activating gene (RAG)-deficient (lacking adaptive immunity), RAG−/− common γ-chain (ɣc)−/− (lacking ILC) and anti-CD90.2 treated (ILC depleted) C57Bl/6 RAG−/− mice. One cycle of DSS comprised 1 week of DSS administration followed by 2 weeks of recovery with normal drinking water. Colonic lamina propria cells were isolated and CD45+Lineage−CD127+CD90.2+ ILC, Ly6C+ monocytes and Ly6G+ neutrophils were identified after staining by flow cytometry. Inflammation and fibrosis were scored by macroscopic and HE and Martius Scarlet Blue staining and fibrosis was evaluated by hydroxyproline quantification. For analysis Kruskal–Wallis testing with multiple Dunn’s comparisons was performed. Results In RAG-1−/− mice chronic inflammation and fibrosis developed similarly as in WT mice, with elevated KLRG-1+ ILC2 (68.90 vs. 48.00% of ILC, p = 0.012) after repeated DSS exposure as compared with control mice (Figure 1). Chronic colitis could also be induced in RAG−/−ɣc−/− or ILC depleted RAG−/− mice (ILC: 0.99 vs. 25.70% of CD45+ cells, p = 0.029), with no attenuation of fibrosis (p > 0.99) as compared with chronic DSS exposed RAG-1−/− mice despite the absence of ILC (Figure 2). Colon length decrease was more pronounced in RAG−/−yc−/− as compared with RAG-1−/− mice after chronic DSS colitis (7.15 vs. 8.30 cm, p = 0.046), while hydroxyproline levels and thickness of mucosa and muscularis propria were not different in RAG−/−yc−/− as compared with RAG-1−/− mice after chronic DSS. Moreover, after the second cycle of DSS a slower recovery of weight was seen in RAG−/−yc−/− mice as compared with RAG-1−/− mice (d31: 88.64 vs. 111.0% of initial weight, p < 0.001; d35 p = 0.001; d39 p = 0.006 and d42 p = 0.002) (Figure 3). In absence of ILC, RAG−/−yc−/− mice increased lamina propria neutrophils (19.10 vs. 5.91% of CD45+ cells, p = 0.004) and monocytes (11.80 vs. 3.25% of CD45+ cells, p = 0.004) may represent an alternative source of inflammation. Conclusion These data argue against a pro-fibrotic role of ILC in the induction of fibrosis in chronic DSS colitis, and suggest a protective and recovery-enhancing role of ILC after repeated intestinal injury.

Mucin O-glycans facilitate symbiosynthesis to maintain gut immune homeostasis.

BackgroundThe dysbiosis of gut microbiota has been implicated in the pathogenesis of inflammatory bowel diseases; however, the underlying mechanisms have not yet been elucidated. Heavily glycosylated mucin establishes a first-line barrier against pathogens and serves as a niche for microbial growth.MethodsTo elucidate relationships among dysbiosis, abnormal mucin utilisation, and microbial metabolic dysfunction, we analysed short-chain fatty acids (SCFAs) and mucin components in stool samples of 40 healthy subjects, 49 ulcerative colitis (UC) patients, and 44 Crohn's disease (CD) patients from Japan.FindingsLevels of n-butyrate were significantly lower in stools of both CD and UC patients than in stools of healthy subjects. Correlation analysis identified seven bacterial species positively correlated with n-butyrate levels; the major n-butyrate producer, Faecalibacterium prausnitzii, was particularly underrepresented in CD patients, but not in UC patients. In UC patients, there were inverse correlations between mucin O-glycan levels and the production of SCFAs, such as n-butyrate, suggesting that mucin O-glycans serve as an endogenous fermentation substrate for n-butyrate production. Indeed, mucin-fed rodents exhibited enhanced n-butyrate production, leading to the expansion of RORgt+Treg cells and IgA-producing cells in colonic lamina propria. Microbial utilisation of mucin-associated O-glycans was significantly reduced in n-butyrate-deficient UC patients.InterpretationMucin O-glycans facilitate symbiosynthesis of n-butyrate by gut microbiota. Abnormal mucin utilisation may lead to reduced n-butyrate production in UC patients.FundJapan Society for the Promotion of Science, Health Labour Sciences Research Grant, AMED-Crest, AMED, Yakult Foundation, Keio Gijuku Academic Development Funds, The Aashi Grass Foundation, and The Canon Foundation.

β-Glucans in food modify colonic microflora by inducing antimicrobial protein, calprotectin, in a Dectin-1-induced-IL-17F-dependent manner.

Dectin-1 (gene symbol: Clec7a) is a receptor for β-glucans that play an important role for the host defense against fungi. Recently, we showed that Clec7a-/- mice are resistant against dextran sodium sulfate (DSS)-induced colitis because of regulatory T-cell population expansion in the colon. The regulatory T-cell expansion is caused by expansion of commensal Lactobacillus murinus whose growth is suppressed by an antimicrobial protein, calprotectin S100A8/A9. In this report, we showed that S100A8 was mainly produced by mouse colonic epithelial cells. S100A8 was not induced directly by Dectin-1 but by Dectin-1-induced cytokines, especially interleukin-17F (IL-17F), that were produced by several types of innate immune cells including CD11c+/CD11b+ myeloid cells in colonic lamina propria. S100A8/A9 heterodimer preferentially suppressed the growth of L. murinus that was increased in both Clec7a-/- and Il17f-/- mice. Furthermore, similar expansion of L. murinus and DSS-colitis resistance were observed in mice fed with β-glucan-free food. These observations suggest that food-derived β-glucans control the specific commensal microbiota via the Dectin-1-IL-17F-calprotectin axis to maintain the intestinal homeostasis.

Upregulation of Intestinal Barrier Function in Mice with DSS-Induced Colitis by a Defined Bacterial Consortium Is Associated with Expansion of IL-17A Producing Gamma Delta T Cells.

Bacterial consortium transplantation (BCT) is a promising alternative to fecal microbiota transplantation in treating inflammatory bowel disease (IBD). Here, we showed that a defined bacterial consortium derived from healthy mice was able to enhance the intestinal barrier function of mice with dextran sulfate sodium (DSS)-induced colitis. Interestingly, we found that the bacterial consortium significantly promoted the expansion of IL-17A-producing γδT (γδT17) cells in colonic lamina propria, which was closely associated with changing of intestinal microbial composition. The increased IL-17A secretion upon treatment with microbial products derived from the bacterial consortium was accompanied with upregulation of TLR2 expression by γδT cells, and it might be responsible for the upregulation of mucosal barrier function through IL-17R-ACT1-mediated recovery of the disrupted occludin subcellular location. Changing of some specific microbial groups such as Bifidobacterium and Bacillus spp. was closely correlated with the promotion of TLR2+ γδT cells. Our results support that BCT can restore the alliance between commensal microbiota and intestinal γδT cells, which contributes to the improvement of intestinal barrier function. This study provides new insight into the development of bacteria transplantation therapy for the treatment of IBD.

Cigarette smoke attenuated colon inflammation through the induction of regulatory T cells in ulcerative colitis model

Abstract Background Ulcerative colitis (UC) is a chronic inflammatory disease induced by dysregulation of immune response in intestinal mucosa. Several cohort studies have shown that cigarette smoke (CS) is the protective factor for UC, but the underlying mechanisms are not fully elucidated. Here, we evaluated the influence of CS in the experimental UC model. Methods UC was induced by dextran sodium sulfate (DSS) during three weeks of CS exposure, and mice were sacrificed at day 7 following UC induction. Colon pathology, lymphocytes recruitment into intestinal mucosa, and gut microbiota were analyzed. Results CS exposure suppressed colon inflammation and disease activity in DSS-induced colitis model. CS exposed mice displayed significant reduction of activated CD8+ T cells in colon lamina propria, whereas regulatory T cells (Tregs) were significantly increased. In addition, Ruminococcus, which belongs to Clostridium cluster IV, increased in fecal microbiota of CS exposed mice, suggesting that Tregs are induced by CS exposure through the alteration of microbiota composition. Conclusions CS might suppress colon inflammation in UC through the alteration of microbiota composition and immune system including Tregs.

SEW2871 protects from experimental colitis through reduced epithelial cell apoptosis and improved barrier function in interleukin-10 gene-deficient mice.

Loss of intestinal epithelial barrier function including typical tight junction changes and epithelial cell apoptosis plays an important role in Crohn's disease. SEW2871, a selective sphingosine-1-phosphate type-1 receptor agonist, has been proven to be efficient in protecting against colitis in IL-10(-/-) mice in our previous study. Here we performed additional studies to investigate whether treatment with SEW2871 was associated with an improved epithelial barrier function in IL-10(-/-) mice. SEW2871 was administered by gavage at a dose of 20 mg/kg/day for 2 weeks to IL-10(-/-) mice. Severity of colitis, CD4+ T cells in colon lamina propria and proinflammatory cytokine productions were evaluated. Furthermore, intestinal permeability, tight junction (occludin and ZO-1) expressions and distributions, as well as epithelial cell apoptosis, were also assessed. SEW2871 treatment attenuated established colitis associated with decreased CD4+ T cells in colon lamina propria and reduced TNF-α and IFN-γ levels. Moreover, enhanced barrier function, which resulted from ameliorated tight junction (occludin and ZO-1) expressions and suppressed epithelial cell apoptosis, was found to contribute to the therapeutic effects. SEW2871 treatment protects from colitis in IL-10(-/-) mice through reduced epithelial cell apoptosis and improved barrier function. Thus, targeting sphingosine-1-phosphate may represent a new therapeutic approach in Crohn's disease.

Gut microbial short-chain fatty acids in host defense and immune regulation

Our guts are inhabited by an enormous number of commensal bacteria, commonly referred to as the gut microbiota. Complex host-gut microbiota interactions are involved in the formation of the unique “gut ecosystem”. This ecosystem is thought to play a variety of roles in host physiology and pathology, including the modification and shaping of the host immune system. We have proposed an integrated omics approach for a comprehensive understanding of the gut ecosystem, where different levels of cyclopedic analyses, such as (meta)transcriptome and metabolome, are combined with the metagenome. Using this approach, we have discovered the mechanism by which the gut microbiota-derived short-chain fatty acid acetate protects mice from enterohemorrhagic Escherichia coli O157-infectious death. We have also shown that butyrate produced by the gut microbiota promotes differentiation of peripherally derived regulatory T cells in colonic lamina propria through epigenetic modification. In this minireview, I will discuss recent advances in our understanding of the role of gut microbial short-chain fatty acids in host defense and immune regulation, focusing mainly on results from our laboratory. Rec.2/9/2015, ACC.2/23/2015, pp114-121 Correspondence should be addressed to: Hiroshi Ohno, M.D., Ph.D., Laboratory for Intestinal Ecosystem, RIKEN Center for Integrative Medical Sciences (IMS), 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan. Phone: +81-45-503-7031, Fax: +81-45-503-7068, E-mail: hiroshi.ohno@riken.jp

Stress response protein cirp links inflammation and tumorigenesis in colitis-associated cancer.

Colitis-associated cancer (CAC) is caused by chronic intestinal inflammation and is reported to be associated with refractory inflammatory bowel disease (IBD). Defective apoptosis of inflammatory cell populations seems to be a relevant pathogenetic mechanism in refractory IBD. We assessed the involvement of stress response protein cold-inducible RNA-binding protein (Cirp) in the development of intestinal inflammation and CAC. In the colonic mucosa of patients with ulcerative colitis, expression of Cirp correlated significantly with the expression of TNFα, IL23/IL17, antiapoptotic proteins Bcl-2 and Bcl-xL, and stem cell markers such as Sox2, Bmi1, and Lgr5. The expression of Cirp and Sox2 was enhanced in the colonic mucosae of refractory ulcerative colitis, suggesting that Cirp expression might be related to increased cancer risk. In human CAC specimens, inflammatory cells expressed Cirp protein. Cirp(-/-) mice given dextran sodium sulfate exhibited decreased susceptibility to colonic inflammation through decreased expression of TNFα, IL23, Bcl-2, and Bcl-xL in colonic lamina propria cells compared with similarly treated wild-type (WT) mice. In the murine CAC model, Cirp deficiency decreased the expression of TNFα, IL23/IL17, Bcl-2, Bcl-xL, and Sox2 and the number of Dclk1(+) cells, leading to attenuated tumorigenic potential. Transplantation of Cirp(-/-) bone marrow into WT mice reduced tumorigenesis, indicating the importance of Cirp in hematopoietic cells. Cirp promotes the development of intestinal inflammation and colorectal tumors through regulating apoptosis and production of TNFα and IL23 in inflammatory cells.

Gut microbiota, host defense and immunity: analysis with integrative omics approach

Complex host-gut microbiota interaction is involved in the formation of a unique ecosystem in our body, the "gut ecosystem". In order to understand the complex gut ecosystem, we propose integrated multi-omics approach, where multiple layers of unbiased cyclopedic analyses such as genomics, transcriptomics and metabolomics are combined. Applying this approach, we have revealed the mechanism that gut microbiota-derived acetate, a short-chain fatty acid, protects mice from enterohemorrhagic Escherichia coli O157-infectious death. We have also shown that butyrate produced by gut microbiota such as order Clostridiales promotes differentiation of regulatory T cells from naïve T cells in colonic lamina propria, through epigenetic modification. Epigenetic modification by butyrate also acts on colonic macrophages to confer anti-inflammatory phenotype by rendering them hyporesponsive to Toll-like receptor signaling. Short-chain fatty acids also signal via their G protein-coupled receptors. For example, it has been suggested that gut microbiota-derived short-chain fatty acids absorbed in the blood play a role in regulation of systemic inflammation by inducing apoptosis of neutrophils as well as chemotaxis of regulatory T cells.

255 : Inhibition of Dectin-1 signaling suppresses colitis by regulating the balance of intestinal microbiota and cellular immunity

The intestinal immune system has to take dual tasks; the system should fight against invading pathogens and, at the same time, the system should be tolerant toward commensal microbiota or food components. Dectin-1 is the receptor for beta-glucans expressed in fungal cell walls and activates the immune system to prevent infection. Because beta-glucans are contained in wide variety of foods and commensal microbiota, we investigated the effects of Dectin-1 signaling on the intestinal immunity. We found that Dectin-1-deficient mice were refractory to dextran sodium sulfate-induced colitis, a model for inflammatory bowel diseases, due to expansion of one genus of Gram-positive commensal bacteria in the colon associated with remodification of T cell subpopulations. Furthermore, oral administration of a seaweed-derived Dectin-1 antagonist, inhibited the proinflammatory cytokine production in colonic lamina propria cells and suppressed the development of colitis. These observations suggest that inhibition of Dectin-1 signaling in the colon regulates the balance of intestinal microbiota and cellular immunity, and further suppresses the development of inflammatory bowel diseases.

Mo1747 Intestinal IL-10-Producing B Cells Induced by Commensal Bacteria May Contribute to Maintenance of Mucosal Homeostasis

Background We demonstrated that wild-type B cells produce Interleukin (IL)-10 following Toll-like receptor (TLR) stimulation and inhibit the production of pro-inflammatory cytokines by T cells and macrophages (MΦ) In Vitro (DDW 2011). However, the In Vivo development and roles of intestinal IL-10-producing B cells in response to gut bacteria are poorly understood. In addition, commensal bacteria have been implicated in the pathogenesis of the inflammatory bowel diseases (IBD) and the regulation of mucosal homeostasis. We hypothesize that commensal microbiota are essential for IL-10 secretion by intestinal B cells.Methods Germ-free (GF) IL-10EGFP reporter (Vert-X) mice were transferred to specific pathogen-free (SPF) conditions and colonized with SPF feces. Mice were studied on days 0, 3 and 7 postcolonization, and populations of IL-10-producing GFP+ cells were analyzed in the colon lamina propria (LP), mesenteric lymph nodes (MLN) and spleen using flow cytometry (FACS) and immunohistochemistry (IHC). To induce chronic colitis, SPF Vert-X mice were cyclically administered 2% dextran sulfate sodium (DSS) solution for 7 day periods. Colonic LP IL10-producing GFP+ cells were identified by FACS.Results 1.5 ± 0.7% of total colonic LP cells in GFVert-Xmicewere GFP+, with the number expanding following bacteria colonization and with chronic inflammation (Table), while those in the spleen and MLN did not significantly change. Of note, the number of GFP+ B cells in the colonic LP greatly expanded following bacterial colonization (1.7-fold from baseline on day 3, and 3.3-fold on day 7, vs. 11.2fold in SPF mice). IHC demonstrated that GFP+ B cells were concentrated in the colonic patches rather than the LP. The ratio of GFP+ colonic Foxp3+CD4+ cells /total GFP+ cells did not change significantly after bacterial colonization (32.6 ± 9.6 % on GF, 32.4 ± 18.9 % on day 3, and 36.8 ± 8.6 % on day 7, vs. 19.7 ± 1.7 % in SPF mice), nor did the numbers of GFP+ MΦ and dendritic cells change. Following chronic DSS administration, the percentage of intestinal GFP+ cells increased (Table). Although CD4+ T cells were predominant among enteric IL-10-producing cells in chronic colitis, the ratio of B cells to total intestinal GFP+ cell increased during chronic inflammation (Table). Summary Commensal bacteria and chronic intestinal inflammation stimulate IL-10 secretion by intestinal B cells, whichmay contribute tomaintainingmucosal homeostasis and help regulate intestinal inflammation. IL-10 producing GFP+ cells in the colonic lymphocytes following bacterial colonization and chronic mucosal inflammation