Kyoko Inagaki-Ohara

Microbiota-derived lactate accelerates colon epithelial cell turnover in starvation-refed mice

Oral food intake influences the morphology and function of intestinal epithelial cells and maintains gastrointestinal cell turnover. However, how exactly these processes are regulated, particularly in the large intestine, remains unclear. Here we identify microbiota-derived lactate as a major factor inducing enterocyte hyperproliferation in starvation-refed mice. Using bromodeoxyuridine staining, we show that colonic epithelial cell turnover arrests during a 12- to 36-h period of starvation and increases 12-24 h after refeeding. Enhanced epithelial cell proliferation depends on the increase in live Lactobacillus murinus, lactate production and dietary fibre content. In the model of colon tumorigenesis, mice exposed to a carcinogen during refeeding develop more aberrant crypt foci than mice fed ad libitum. Furthermore, starvation after carcinogen exposure greatly reduced the incidence of aberrant crypt foci. Our results indicate that the content of food used for refeeding as well as the timing of carcinogen exposure influence the incidence of colon tumorigenesis in mice.

Interleukin-17A is required to suppress invasion of Salmonella enterica serovar Typhimurium to enteric mucosa

Salmonella enterica serovar Typhimurium (S. typhimurium) causes a localized enteric infection and its elimination is dependent on a T helper type 1 immune response. However, the mechanism of the protective immune response against the pathogen in gut‐associated lymphoid tissue (GALT) at an early stage of the infection is not yet clarified. Here, we show that interleukin‐17A (IL‐17A) was constitutively expressed in GALT; it was also detected on crypt and epithelial cells of the small intestine. Neutralization of the IL‐17A in the intestinal lumen exacerbated epithelial damage induced by intestinal S. typhimurium infection at an early stage of the infection. The result suggests that IL‐17A has a pivotal role in the immediate early stage of protection against bacterial infection at the intestinal mucosa. As IL‐17A neutralization also suppressed the constitutive localization of β‐defensin 3 (BD3), an IL‐17A‐induced antimicrobial peptide, at the apical site of the intestinal mucosa, it is estimated that IL‐17A constitutively induces the expression of the antimicrobial peptide to kill invading pathogens at the epithelial surface immediately after the infection. In contrast, interferon‐γ is induced around 3 days after S. typhimurium infection, and its expression level increases thereafter. Taken together, the findings lead to the hypothesis that IL‐17A participates in the immediate early stage of protection against S. typhimurium intestinal infection whereas interferon‐γ is important at a later stage of the infection.

Enhancement of leptin receptor signaling by SOCS3 deficiency induces development of gastric tumors in mice

Leptin acts on its receptor (ObR) in the hypothalamus to inhibit food intake and energy expenditure. Leptin and ObR are also expressed in the gastrointestinal tract; however, the physiological significance of leptin signaling in the gut remains uncertain. Suppressor of cytokine signaling 3 (SOCS3) is a key negative feedback regulator of ObR-mediated signaling in the hypothalamus. We now show that gastrointestinal epithelial cell-specific SOCS3 conditional knockout (T3b-SOCS3 cKO) mice developed gastric tumors by enhancing leptin production and the ObRb/signal transducer and activator of transcription 3 (STAT3) signaling pathway. All T3b-SOCS3 cKO mice developed tumors in the stomach but not in the bowels by 2 months of age, even though the SOCS3 deletion occurred in both the epithelium of stomach and bowels. The tumors developed in the absence of the inflammatory response and all cKO mice died within 6 months. These tumors displayed pathology and molecular alterations, such as an increase in MUC2 (Mucin 2, oligomeric mucus/gel-forming) and TFF3 (trefoil factor 3), resembling human intestinal-type gastric tumors. Administration of antileptin antibody to T3b-SOCS3 cKO mice reduced hyperplasia of gastric mucosa, which is the step of the initiation of gastric tumor. These data suggest that SOCS3 is an antigastric tumor gene that suppresses leptin overexpression and ObRb/STAT3 hyperactivation, supporting the hypothesis that the leptin/ObRb/STAT3 axis accelerates tumorigenesis and that it may represent a new therapeutic target for the treatment of gastric cancer.

Adrenomedullin treatment reduces intestinal inflammation and maintains epithelial barrier function in mice administered dextran sulphate sodium.

Hyperactivation and hyperpermeability of the intestinal epithelium is a hallmark of IBD. AM has been shown to reduce the severity of colitis in the acetic acid and TNBS‐induced colitis model, however the mechanism of the therapeutic effect of AM against the colitis has not been clarified. Here, we show that the protective capability of AM is associated with suppression of inflammation and maintenance of the intestinal epithelial barrier function. In the DSS‐induced colitis model, intra‐rectal AM‐treated mice showed a reduction in loss of body weight and severity of colitis. AM‐treatment suppressed phosphorylation of STAT1 and STAT3 in the colonic epithelium, and altered the cytokine balance in the intestinal T cells, with lower levels of IFN‐γ and TNF‐α but higher levels of TGF‐β. Expression of the epithelial intercellular junctions such as tight and adherence junctions were sustained in the AM‐treated mice. In contrast, the epithelial junctions were down‐regulated in the control mice, leading to loss of epithelial barrier integrity and enhanced permeability. Collectively, these data indicate a broad spectrum of AM‐induced effects with respect to protection against DSS‐induced colitis, and suggest a potential therapeutic value of this treatment for IBD.

γδ T cells play a protective role during infection with Nippostrongylus brasiliensis by promoting goblet cell function in the small intestine.

The intestinal epithelium is rich in γδ T cells and the gut is a site of residence for a wide variety of pathogens, including nematodes. Although CD4+ T‐cell receptor (TCR) ‐αβ+ T helper type 2 T cells are essential for the expulsion of intestinal nematodes, little information is available on the function of γδ T cells in this type of infection. Here, we demonstrate two major functions of γδ T cells as a potently protective T‐cell population against Nippostrongylus brasiliensis infection using γδ T‐cell‐deficient (TCR‐δ−/−) mice. First, γδ T cells are required to initiate rapid expulsion of adult worms from the intestine and to limit egg production. Second, γδ T cells prevent the pathological intestinal damage associated with nematode infection, evident by increased clinical disease and more severe microscopic lesions in infected TCR‐δ−/− mice. γδ T‐cell deficiency led to delayed goblet cell hyperplasia in association with reduced expression of phosphorylated STAT6, MUC2, Trefoil factor‐3 (TFF3) and T helper type 2 cytokines including interleukin‐13 (IL‐13). TCR‐δ−/− mice also produced more interferon‐γ than wild‐type mice. Within the intraepithelial lymphocyte compartment, γδ T cells produced IL‐13. Adoptive transfer of γδ T cells or administration of recombinant IL‐13 to TCR‐δ−/− mice successfully reduced the egg production by N. brasiliensis. Collectively, these data provide strong evidence that γδ T cells play an important role in controlling infection with intestinal nematodes and limiting infection‐induced pathology.

TWEAK/Fn14 pathway promotes a T helper 2-type chronic colitis with fibrosis in mice.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a TNF superfamily member, induces damage of the epithelial cells (ECs) and production of inflammatory mediaters through its receptor Fn14 in a model of acute colitis. In our current study of chronic colitis induced by repeated rectal injection of a hapten, we found that inflammation, fibrosis, and T helper 2 (Th2)-type immunity were significantly reduced in Fn14 gene knockout (KO) mice when compared with wild-type (WT) control mice. Expression of thymic stromal lymphopoietin (TSLP) was lower in Fn14 KO colon ECs than in WT ECs. TWEAK potentiates the induction of TSLP by interleukin-13 (IL-13) in colon explants from WT but not in Fn14 KO tissue. TSLP receptor KO mice exhibit milder chronic colitis, similar to that in Fn14 KO mice. TWEAK and IL-13 synergistically promote fibroblast proliferation. Thus we propose an IL-13-TWEAK/Fn14-TSLP axis as a key mechanism underlying chronic colitis with fibrosis.

Adrenomedullin as a Potential Therapeutic Agent for Inflammatory Bowel Disease

Adrenomedullin (AM) was originally isolated from human pheochromocytoma as a biologically active peptide with potent vasodilating action but is now known to exert a wide range of physiological effects, including cardiovascular protection, neovascularization, and apoptosis suppression. A variety of tissues, including the gastrointestinal tract, have been shown to constitutively produce AM. Pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1, and lipopolysaccharides, induce the production and secretion of AM. Conversely, AM induces the downregulation of inflammatory cytokines in cultured cells. Furthermore, AM downregulates inflammatory processes in a variety of different colitis models, including acetic acid-induced colitis and dextran sulfate sodium-induced colitis. AM exerts antiinflammatory and antibacterial effects and stimulates mucosal regeneration for the maintenance of the colonic epithelial barrier. Here, we describe the first use of AM to treat patients with refractory ulcerative colitis. The results strongly suggest that AM has potential as a new therapeutic agent for the treatment of refractory ulcerative colitis.

Interleukin-17A is involved in enhancement of tumor progression in murine intestine

Interleukin (IL)-17A is a cytokine involved in neutrophilic inflammation but the role of IL-17A in anti-tumor immunity is controversial because both pro- and anti-tumor activities of IL-17A have been reported. We hypothesized that constitutive expression of IL-17A in intestinal environment modifies tumor growth. To address the issue, mice were inoculated into subserosa of cecum (i.c.) with murine EL4 lymphoma expressing a model tumor antigen, and tumor growth was monitored. IL-17A-producing cells were detected both in tumor mass and in normal intestinal tissue of i.c. tumor-bearing wild type mice. Tumor size in the wild-type mice was significantly higher than that in the cecum of IL-17A gene-knockout mice. Furthermore, anti-IL-17A monoclonal antibody treatment of wild-type mice resulted in decreased tumor size in the cecum. Model tumor-antigen-specific interferon-γ production was not modified in draining mesenteric lymph node cells in the absence or after neutralization of IL-17A. All the results suggest that constitutive expression of IL-17A in intestine enhances tumor growth, and anti-IL-17A antibody treatment is a candidate of a new anti-tumor immunotherapy against intestinal tumors.

Chemokine receptor CCR8 is required for lipopolysaccharide-triggered cytokine production in mouse peritoneal macrophages

Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8- /-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.