B cell-derived GABA elicits IL-10+ macrophages to limit anti-tumour immunity.

Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu1-3. Here we identify the metabolite and neurotransmitter GABA as a candidate signalling molecule synthesized and secreted by activated B cells and plasma cells. We show that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8+ T cell killer function. In mice, B cell deficiency or B cell-specific inactivation of the GABA-generating enzyme GAD67 enhances anti-tumour responses. Our study reveals that, in addition to cytokines and membrane proteins, small metabolites derived from B-lineage cells have immunoregulatory functions, which may be pharmaceutical targets allowing fine-tuning of immune responses.

TH1 cell-inducing Escherichia coli strain identified from the small intestinal mucosa of patients with Crohn's disease.

Dysbiotic microbiota contributes to the pathogenesis of Crohn's disease (CD) by regulating the immune system. Although pro-inflammatory microbes are probably enriched in the small intestinal (SI) mucosa, most studies have focused on fecal microbiota. This study aimed to examine jejunal and ileal mucosal specimens from patients with CD via double-balloon enteroscopy. Comparative microbiome analysis revealed that the microbiota composition of CD SI mucosa differs from that of non-CD controls, with an increased population of several families, including Enterobacteriaceae, Ruminococcaceae, and Bacteroidaceae. Upon anaerobic culturing of the CD SI mucosa, 80 bacterial strains were isolated, from which 9 strains representing 9 distinct species (Escherichia coli, Ruminococcus gnavus, Klebsiella pneumoniae, Erysipelatoclostridium ramosum, Bacteroides dorei, B. fragilis, B. uniformis, Parabacteroides distasonis, and Streptococcus pasteurianus) were selected on the basis of their significant association with CD. The colonization of germ-free (GF) mice with the 9 strains enhanced the accumulation of TH1 cells and, to a lesser extent, TH17 cells in the intestine, among which an E. coli strain displayed high potential to induce TH1 cells and intestinal inflammation in a strain-specific manner. The present results indicate that the CD SI mucosa harbors unique pro-inflammatory microbiota, including TH1 cell-inducing E. coli, which could be a potential therapeutic target.

Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and its frequent complication with ulcerative colitis highlights the pathogenic role of epithelial barrier dysfunction. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC, yet its underlying mechanism remains unknown. Here, we identify Klebsiella pneumonia in the microbiota of patients with PSC and demonstrate that K. pneumoniae disrupts the epithelial barrier to initiate bacterial translocation and liver inflammatory responses. Gnotobiotic mice inoculated with PSC-derived microbiota exhibited T helper 17 (TH17) cell responses in the liver and increased susceptibility to hepatobiliary injuries. Bacterial culture of mesenteric lymph nodes in these mice isolated K. pneumoniae, Proteus mirabilis and Enterococcus gallinarum, which were prevalently detected in patients with PSC. A bacterial-organoid co-culture system visualized the epithelial-damaging effect of PSC-derived K. pneumoniae that was associated with bacterial translocation and susceptibility to TH17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the TH17 immune response induced by PSC-derived microbiota. These results highlight the role of pathobionts in intestinal barrier dysfunction and liver inflammation, providing insights into therapeutic strategies for PSC.

A defined commensal consortium elicits CD8 T cells and anti-cancer immunity.

There is a growing appreciation for the importance of the gut microbiota as a therapeutic target in various diseases. However, there are only a handful of known commensal strains that can potentially be used to manipulate host physiological functions. Here we isolate a consortium of 11 bacterial strains from healthy human donor faeces that is capable of robustly inducing interferon-γ-producing CD8 T cells in the intestine. These 11 strains act together to mediate the induction without causing inflammation in a manner that is dependent on CD103+ dendritic cells and major histocompatibility (MHC) class Ia molecules. Colonization of mice with the 11-strain mixture enhances both host resistance against Listeria monocytogenes infection and the therapeutic efficacy of immune checkpoint inhibitors in syngeneic tumour models. The 11 strains primarily represent rare, low-abundance components of the human microbiome, and thus have great potential as broadly effective biotherapeutics.

Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells.

Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.

An Interleukin-33-Mast Cell-Interleukin-2 Axis Suppresses Papain-Induced Allergic Inflammation by Promoting Regulatory T Cell Numbers.

House dust mite-derived proteases contribute to allergic disorders in part by disrupting epithelial barrier function. Interleukin-33 (IL-33), produced by lung cells after exposure to protease allergens, can induce innate-type airway eosinophilia by activating natural helper (NH) cells, a member of group 2 innate lymphoid cells (ILC2), to secrete Th2 type-cytokines. Because IL-33 also can induce mast cells (MCs) to secrete Th2 type-cytokines, MCs are thought to cooperate with NH cells in enhancing protease or IL-33-mediated innate-type airway eosinophilia. However, we found that MC-deficient Kit(W-sh/W-sh) mice exhibited exacerbated protease-induced lung inflammation associated with reduced numbers of regulatory T (Treg) cells. Moreover, IL-2 produced by IL-33-stimulated MCs promoted expansion of numbers of Treg cells, thereby suppressing development of papain- or IL-33-induced airway eosinophilia. We have thus identified a unique anti-inflammatory pathway that can limit induction of innate-type allergic airway inflammation mediated by NH cells.

Potential role of myeloid cell/eosinophil-derived IL-17 in LPS-induced endotoxin shock.

IL-17RA is a shared receptor subunit for several cytokines of the IL-17 family, including IL-17A, IL-17C, IL-17E (also called IL-25) and IL-17F. It has been shown that mice deficient in IL-17RA are more susceptible to sepsis than wild-type mice, suggesting that IL-17RA is important for host defense against sepsis. However, it is unclear which ligands for IL-17RA, such as IL-17A, IL-17C, IL-17E/IL-25 and/or IL-17F, are involved in the pathogenesis of sepsis. Therefore, we examined IL-17A, IL-17E/IL-25 and IL-17F for possible involvement in LPS-induced endotoxin shock. IL-17A-deficient mice, but not IL-25- or IL-17F-deficient mice, were resistant to LPS-induced endotoxin shock, as compared with wild-type mice. Nevertheless, studies using IL-6-deficient, IL-21Rα-deficient and Rag-2-deficient mice, revealed that neither IL-6 and IL-21, both of which are important for Th17 cell differentiation, nor Th17 cells were essential for the development of LPS-induced endotoxin shock, suggesting that IL-17A-producing cells other than Th17 cells were important in the setting. In this connection, IL-17A was produced by macrophages, DCs and eosinophils after LPS injection. Taken together, these findings indicate that IL-17A, but not IL-17F or IL-25, is crucial for LPS-induced endotoxin shock. In addition, macrophages, DCs and eosinophils, but not Th17 cells or γδ T cells, may be sources of IL-17A during LPS-induced endotoxin shock.

Inhibitory effect of yogurt on aberrant crypt foci formation in the rat colon and colorectal tumorigenesis in RasH2 mice.

The inhibitory effects of yogurt consisting of milk fermented by Lactobacillus delbrueckii subsp. bulgaricus strain 2038 and Streptococcus salivarius subsp. thermophilus strain 1131 on formation of colonic aberrant crypt foci (ACF) in rats and also on development of colorectal tumors in transgenic mice harboring human prototype c-Ha-ras genes (rasH2 mice) were examined. F344 rats and rasH2 mice were fed commercial diet containing freeze-dried yogurt or starter medium (non-fermented milk). Rats were inoculated orally with heterocyclic amine 2-amino-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP) for two weeks. The rats were necropsied 14 days after the PhIP treatment, and ACF in the colon and rectum were counted. RasH2 mice were injected with 1,2-dimethylhydrazine dihydrochloride (DMH) for 20 weeks. Three weeks after the last injection of DMH, rasH2 mice were necropsied to determine the number and the size of colorectal tumors. Yogurt supplementation in diet significantly reduced the number of ACF and aberrant crypts (ACs) in rats fed control diet (P<0.01), but not in rats fed non-fermented milk diet. On the other hand, rasH2 mice receiving the yogurt-supplemented diet had significantly reduced numbers of tumors induced by DMH compared with those fed the non-fermented milk-supplemented diet (P<0.05). These results demonstrate that the yogurt used in this study appears to have tumor-suppressing properties, and rasH2 mice are a useful model for the evaluation of antitumor activities of foods.

Increased interleukin-10 production and Th2 skewing in the absence of 5-lipoxygenase.

Eicosanoids (prostaglandins and leukotrienes) are important mediators of inflammatory responses. These lipid mediators may also regulate the production of peptide mediators of the immune system. In this study, we investigated the effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on interleukin (IL)-10 production. IL-10 is a key regulator of immune and inflammatory responses, and previous studies have suggested that prostaglandins effect their immunosuppressive functions in part by stimulation of IL-10 production. We therefore investigated whether leukotriene production would have a similar role in regulation of IL-10 production. We have made the striking observation that absence of 5-LO-derived leukotrienes results in increased IL-10 production with a concomitant decrease in the production of pro-inflammatory cytokines, including tumour necrosis factor (TNF)-alpha and IL-12. Moreover, T-cell cytokine production in the absence of 5-LO-derived leukotrienes results in increased IL-4 production and decreased interferon (IFN)-gamma production. This may be in part secondary to increased IL-10 production and its effects on dendritic cell function resulting in altered T-cell differentiation. These findings indicate that, in addition to the central role leukotrienes play in the acute inflammatory response, endogenous leukotrienes are also important regulators of inflammatory cytokine production, via regulation of IL-10 production and in vivo differentiation of T cells.

R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice.

BACKGROUND & AIMS: R-spondin 1 (Rspo1) is a novel epithelial mitogen that stimulates the growth of mucosa in both the small and large intestine. METHODS: We investigated the therapeutic potential of Rspo1 in ameliorating experimental colitis induced by dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) as well as nonsteroidal anti-inflammatory drug-induced colitis in interleukin (IL)-10-deficient mice. RESULTS: Therapeutic administration of recombinant Rspo1 protein reduced the loss of body weight, diarrhea, and rectal bleeding in a mouse model of acute or chronic DSS-induced colitis. Histologic evaluation revealed that Rspo1 improved mucosal integrity in both villus and/or crypt compartments in the small intestine and colon by stimulating crypt cell growth and mucosal regeneration in DSS-treated mice. Moreover, Rspo1 significantly reduced DSS-induced myeloperoxidase activity and inhibited the overproduction of proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1alpha, IL-6, interferon-gamma, and granulocyte-macrophage colony-stimulating factor, in mouse intestinal tissue, indicating that Rspo1 may reduce DSS-induced inflammation by preserving the mucosal barrier function. Likewise, Rspo1 therapy also alleviated TNBS-induced interstitial inflammation and mucosal erosion in the mouse colon. Furthermore, Rspo1 substantially decreased the histopathologic severity of chronic enterocolitis by repairing crypt epithelium and simultaneously suppressing inflammatory infiltration in piroxicam-exposed IL-10(-/-) mice. Endogenous Rspo1 protein was localized to villus epithelium and crypt Paneth cells in mouse small intestine. CONCLUSIONS: Our results show that Rspo1 may be clinically useful in the therapeutic treatment of inflammatory bowel disease by stimulating crypt cell growth, accelerating mucosal regeneration, and restoring intestinal architecture.

Evidence for oxidative stress in NSAID-induced colitis in IL10-/- mice.

The goal of this study was to evaluate for evidence of oxidative stress in colonic inflammation in a novel model of inflammatory bowel disease, nonsteroidal anti-inflammatory drug- (NSAID-) treated interleukin-10-deficient (IL10(-/-)) mice. IL10(-/-) and wild-type (wt) mice were treated with a nonselective NSAID (piroxicam, 200 ppm in the diet) for 2 weeks to induce colitis, and parameters for oxidative stress in the colonic tissues were evaluated. Mean chemiluminescence enhanced with lucigenin in the colons from IL10(-/-) mice treated with piroxicam was more than 5-fold higher than that of the control wt group. Chemiluminescence was inhibited with diphenylethylene iodinium, but not allopurinol, indomethacin, or N-omega-nitro-L-arginine, indicating that flavin-containing enzymes were the source of the reactive oxygen species. Colonic aconitase activity in NSAID-treated IL10(-/-) mice decreased to 50% of the activity of control mice. There was no difference in the total glutathione levels in the colonic mucosa among the groups; however, glutathione disulfide levels were approximately 2-fold greater in the colon of NSAID-treated IL10(-/-) mice as compared with control groups. Immunohistochemistry studies of colons from NSAID-treated IL10(-/-) mice demonstrated intense staining with two antibodies that recognize advanced glycation endproducts formed through glycation and oxidation: anticarboxymethylysine and antipentosidine. The epithelial cells and lamina propria cells in the colons of NSAID-treated IL10(-/-) mice showed immunostaining with antinitrotyrosine, indicating the presence of reactive nitrogen species. Colonic epithelium of IL10(-/-) mice with colitis showed moderate immunostaining for 8-hydroxy-2'-deoxyguanosine in the nuclei. NSAID-treated IL10(-/-) mice treated with diphenylene idodonium chloride (DPI), an irreversible inhibitor of flavoprotein enzymes, experienced significantly reduced inflammation. Taken together, these results strongly indicate the presence of oxidative stress in the inflammatory bowel disease in NSAID-treated IL10(-/-) mice and suggests a role for oxidative stress in the pathophysiology of this model of inflammatory bowel disease.