Published papers

The intestinal immune system maintains intestinal homeostasis in collaboration with diverse immune cell subsets residing at the epithelial layer, lamina propria and gut-associated lymphoid tissue (GALT). Bacterial components and their metabolites are essential for the establishment of the gut immune system. In addition, nutritional signals contribute to maintaining the mucosal immune response. Specialized epithelial microfold (M) cells in GALT facilitate immune surveillance on the mucosal surface by actively taking up external antigens to transport them into the lymphoid follicles. Because hyperplasia of M cells causes an excessive immune response in GALT, there is a self-regulatory mechanism to control the development of M cells appropriately. In this review, we will discuss the molecular mechanisms of mucosal immune regulation and their biological importance.

AbstractIntestinal microbiota-derived metabolites have biological importance for the host. Polyamines, such as putrescine and spermidine, are produced by the intestinal microbiota and regulate multiple biological processes. Increased colonic luminal polyamines promote longevity in mice. However, no direct evidence has shown that microbial polyamines are incorporated into host cells to regulate cellular responses. Here, we show that microbial polyamines reinforce colonic epithelial proliferation and regulate macrophage differentiation. Colonisation by wild-type, but not polyamine biosynthesis-deficient, Escherichia coli in germ-free mice raises intracellular polyamine levels in colonocytes, accelerating epithelial renewal. Commensal bacterium-derived putrescine increases the abundance of anti-inflammatory macrophages in the colon. The bacterial polyamines ameliorate symptoms of dextran sulfate sodium-induced colitis in mice. These effects mainly result from enhanced hypusination of eukaryotic initiation translation factor. We conclude that bacterial putrescine functions as a substrate for symbiotic metabolism and is further absorbed and metabolised by the host, thus helping maintain mucosal homoeostasis in the intestine.

Retinoid X receptor (RXR) is a nuclear receptor that heterodimerizes with several nuclear receptors, integrating ligand-mediated signals across the heterodimers. Synthetic RXR agonists have been developed to cure certain inflammatory diseases, including inflammatory bowel diseases (IBDs). However, pre-existing RXR agonists, which are lipophilic and readily absorbed in the upper intestine, cause considerable adverse effects such as hepatomegaly, hyperlipidemia, and hypothyroidism. To minimize these adverse effects, we have developed an RXR agonist, NEt-3IB, which has lipophilic and thus poorly absorptive properties. In this study, we evaluated the effects of NEt-3IB in an experimental murine colitis model induced through the adoptive transfer of CD45RBhighCD4+ T cells. Pharmacokinetic studies demonstrated that the major portion of NEt-3IB was successfully delivered to the large intestine after oral administration. Notably, NEt-3IB treatment suppressed the development of T cell-mediated chronic colitis, as indicated by improvement of wasting symptoms, inflammatory infiltration, and mucosal hyperplasia. The protective effect of NEt-3IB was mediated by the suppression of IFN-γ-producing Th1 cell expansion in the colon. In conclusion, NEt-3IB, a large intestine-directed RXR agonist, is a promising drug candidate for IBDs.

Pharmacological inhibition of histone deacetylase 6 (HDAC6) is an effective therapeutic strategy for cancer and immunological diseases. Most of the previously reported HDAC6 inhibitors have a hydroxamate group as a zinc binding group (ZBG), which coordinates to the catalytic zinc ion of HDAC6. The hydroxamate group is liable to metabolically generate mutagenetic hydroxylamine; therefore, non-hydroxamate HDAC6 inhibitors would be advantageous. In this study, to identify novel non-hydroxamate HDAC6-selective inhibitors, screening of a chemical library and the subsequent structural optimization were performed, which led to the identification of HDAC6-selective inhibitors with 3,3,3-trifluorolactic amide (TFLAM) as a novel ZBG. The identified inhibitor showed potent and selective HDAC6-inhibitory activity in cells and induced regulatory T (Treg) cell differentiation.

Medium-chain triglycerides (MCTs), which consist of medium-chain fatty acids (MCFAs), are unique forms of dietary fat with various health benefits. G protein-coupled 84 (GPR84) acts as a receptor for MCFAs (especially C10:0 and C12:0); however, GPR84 is still considered an orphan receptor, and the nutritional signaling of endogenous and dietary MCFAs via GPR84 remains unclear. Here, we showed that endogenous MCFA-mediated GPR84 signaling protected hepatic functions from diet-induced lipotoxicity. Under high-fat diet (HFD) conditions, GPR84-deficient mice exhibited nonalcoholic steatohepatitis (NASH) and the progression of hepatic fibrosis but not steatosis. With markedly increased hepatic MCFA levels under HFD, GPR84 suppressed lipotoxicity-induced macrophage overactivation. Thus, GPR84 is an immunomodulating receptor that suppresses excessive dietary fat intake-induced toxicity by sensing increases in MCFAs. Additionally, administering MCTs, MCFAs (C10:0 or C12:0, but not C8:0), or GPR84 agonists effectively improved NASH in mouse models. Therefore, exogenous GPR84 stimulation is a potential strategy for treating NASH.

Abstract Commensal bacteria-derived metabolites are critical in regulating the host immune system. Although the impact of gut microbiota-derived hydrophilic metabolites, such as short-chain fatty acids, on immune cell functions and development has been well documented, the immunomodulatory effects of gut microbiota-derived lipids are still of interest. Here, we report that lipid extracts from the feces of specific-pathogen-free (SPF), but not germ-free (GF), mice showed regulatory T (Treg)-cell-inducing activity. We conducted RP-HPLC-based fractionation and liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based lipidome profiling and identified two bioactive lipids, 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) and all-trans retinoic acid (atRA), with Treg-inducing activity in vitro. The luminal abundance of 9,10-DiHOME in the large intestine was significantly decreased by dextran sulfate sodium (DSS)-induced colitis, indicating that 9,10-DiHOME may be a potential biomarker of colitis. These observations implied that commensal bacteria-derived lipophilic metabolites might contribute to Treg development in the large intestine.

Allergen immunotherapy (AIT) is the only curative treatment for allergic diseases. However, AIT has many disadvantages related to efficiency, safety, long-term duration, and patient compliance. Dendritic cells (DCs) have an important role in antigen-specific tolerance induction; thus, DC-targeting strategies to treat allergies such as glutaraldehyde crosslinked antigen to mannoprotein (MAN) have been established. However, glutaraldehyde crosslinking may reduce the antigen presentation efficiency of DCs. To overcome this, we developed a MAN-coated ovalbumin (OVA) nanoparticle (MDO), which uses intermolecular disulfide bond to crosslink OVA and MAN. MDO effectively targeted DCs resulting in tolerogenic DCs, and promoted higher antigen presentation efficiency by DCs compared with OVA or glutaraldehyde crosslinked nanoparticles. In vitro and in vivo experiments showed that DCs exposed to MDO induced Treg cells. Moreover, MDO had low reactivity with anti-OVA antibodies and did not induce anaphylaxis in allergic mice, demonstrating its high safety profile. In a mouse model of allergic asthma, MDO had significant preventative and therapeutic effects when administered orally or subcutaneously. Therefore, MDO represents a promising new approach for the efficient and safe treatment of allergies.

Background & Aims In epithelial cells, protein sorting mechanisms regulate localization of plasma membrane proteins that generate and maintain cell polarity. The clathrin-adaptor protein (AP) complex AP-1B is expressed specifically in polarized epithelial cells, where it regulates basolateral sorting of membrane proteins. However, little is known about its physiological significance. Methods We analyzed the intestinal epithelia of mice deficient in Ap1m2 (Ap1m2-/- mice), which encodes the AP-1B μ1B subunit, and compared it with 129/B6/CD1 littermates (controls). Notch signaling was inhibited by intraperitoneal injection of dibenzazepine, and β-catenin signaling was inhibited by injection of IWR1. Intestinal tissue samples were collected and analyzed by immunofluorescence analysis. Results Ap1m2 -/- mice developed intestinal epithelial cell hyperplasia. The polarity of intestinal epithelial cells was disrupted, as indicated by the appearance of ectopic microvilli-like structures on the lateral plasma membrane and mislocalization of basolateral membrane proteins, including the low-density lipoprotein receptor and E-cadherin. The E-cadherin-β-catenin complex therefore was disrupted at the adherens junction, resulting in nuclear translocation of β-catenin. This resulted in up-regulation of genes regulated by β-catenin/transcription factor 4 (Tcf4) complex, and increased the proliferation of intestinal epithelial cells. Conclusions AP-1B is required for protein sorting and polarization of intestinal cells in mice. Loss of AP-1B in the intestinal epithelia results in mislocalization of E-cadherin, activation of β-catenin/Tcf4 complex, proliferation, and hyperplasia. © 2013 by the AGA Institute.

Activation-induced cytidine deaminase (AID) expressed by germinal center B cells is a central regulator of somatic hypermutation (SHM) and class switch recombination (CSR). Humans with AID mutations develop not only the autosomal recessive form of hyper-IgM syndrome (HIGM2) associated with B cell hyperplasia, but also autoimmune disorders by unknown mechanisms. We report here that AID(-/-) mice spontaneously develop tertiary lymphoid organs (TLOs) in non-lymphoid tissues including the stomach at around 6 months of age. At a later stage, AID(-/-) mice develop a severe gastritis characterized by loss of gastric glands and epithelial hyperplasia. The disease development was not attenuated even under germ-free (GF) conditions. Gastric autoantigen -specific serum IgM was elevated in AID(-/-) mice, and the serum levels correlated with the gastritis pathological score. Adoptive transfer experiments suggest that autoimmune CD4(+) T cells mediate gastritis development as terminal effector cells. These results suggest that abnormal B-cell expansion due to AID deficiency can drive B-cell autoimmunity, and in turn promote TLO formation, which ultimately leads to the propagation of organ-specific autoimmune effector CD4(+) T cells. Thus, AID plays an important role in the containment of autoimmune diseases by negative regulation of autoreactive B cells.

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is an intractable disease of the gastrointestinal tract. Multiple environmental factors, including food ingredients, have been implicated in the development of these diseases. For example, animal fat-rich diets are predisposing factors for ulcerative colitis, whereas n-3 unsaturated fatty acids such as docosahexaenoic acid (DHA) show protective effects in experimental colitis and are negatively correlated with the incidence of ulcerative colitis and Crohn's disease. Given that DHA exhibits agonistic activity on retinoid X receptor (RXR), activation of RXR could be a therapeutic strategy for IBD. However, conventional full RXR agonists are known to show considerable adverse effects. We therefore took advantage of a partial RXR agonist, CBt-PMN, to minimize the adverse effects, and evaluated its efficacy in dextran sodium sulfate-induced colitis. Administration of CBt-PMN efficiently ameliorated the symptoms of colitis. This effect was attributed to the down-regulation of pro-inflammatory cytokines such as Tnf and Il6 in colon-infiltrating monocytes. Down-regulation of pro-inflammatory cytokines by CBt-PMN was also evident in lipopolysaccharide-stimulated bone marrow-derived macrophages (BMDMs). Among many RXR-associated nuclear receptors, activation of peroxisome proliferator-activated receptor δ (PPARδ) and nuclear hormone receptor 77 (Nur77) suppressed cytokine production by BMDMs. These observations suggest that the activation of PPARδ/RXR and Nur77/RXR heterodimers by CBt-PMN through the permissive mechanism is responsible for diminishing the monocyte-mediated inflammatory response in the gut. Our data highlight the importance of RXR activation in the regulation of colitis.

Abstract Secretory immunoglobulin A (SIgA), the most abundant antibody isotype in the body, maintains a mutual relationship with commensal bacteria and acts as a primary barrier at the mucosal surface. Colonization by commensal bacteria induces an IgA response, at least partly through a T-cell-independent process. However, the mechanism underlying the commensal-bacteria-induced T-cell-independent IgA response has yet to be fully clarified. Here, we show that commensal-bacteria-derived butyrate promotes T-cell-independent IgA class switching recombination (CSR) in the mouse colon. Notably, the butyrate concentration in human stools correlated positively with the amount of IgA. Butyrate up-regulated the production of transforming growth factor β1 and all-trans retinoic acid by CD103+CD11b+ dendritic cells, both of which are critical for T-cell-independent IgA CSR. This effect was mediated by G-protein-coupled receptor 41 (GPR41/FFA3) and GPR109a/HCA2, and the inhibition of histone deacetylase. The butyrate-induced IgA response reinforced the colonic barrier function, preventing systemic bacterial dissemination under inflammatory conditions. These observations demonstrate that commensal-bacteria-derived butyrate contributes to the maintenance of the gut immune homeostasis by facilitating the T-cell-independent IgA response in the colon.

Rheumatoid arthritis (RA) is systemic autoimmune arthritis that causes joint inflammation and destruction. Accumulating evidence has shown that inhibitors of class I histone deacetylases (HDACs) (i.e., HDAC1, 2, 3, and 8) are potential therapeutic candidates as targeted synthetic disease-modifying antirheumatic drugs (tsDMARDs). Nevertheless, the inhibition of class I HDACs has severe adverse effects because of their broad spectrum. We evaluated the therapeutic effect of a novel selective HDAC1 inhibitor TTA03-107 for collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) models in mice. We also examined the effect of TTA03-107 in bone marrow-derived macrophages (BMDMs) and T helper 17 (Th17) cells in vitro. Here, we delineate that TTA03-107 reduced the severity of autoimmune arthritis without obvious adverse effects in CIA and CAIA models. Moreover, TTA03-107 suppressed the production of inflammatory cytokines, such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-17A, in serum and joint tissue. In vitro treatment of BMDMs with TTA03-107 dampened the M1 differentiation and inflammatory cytokine production. TTA03-107 also suppressed the differentiation of Th17 cells. These results demonstrate that TTA03-107 can attenuate the development of arthritis in experimental RA models by inhibiting the differentiation and activation of macrophages and Th17 cells. Therefore, TTA03-107 is a potential tsDMARD candidate.

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.

Peyer’s patches (PPs) are specialized gut-associated lymphoid tissues that initiate follicular helper T (Tfh)-mediated immunoglobulin A (IgA) response to luminal antigens derived from commensal symbionts, pathobionts, and dietary sources. IgA-producing B cells migrate from PPs to the small intestinal lamina propria and secrete IgA across the epithelium, modulating the ecological balance of the commensal microbiota and neutralizing pathogenic microorganisms. α-glucosidase inhibitors (α-GIs) are antidiabetic drugs that inhibit carbohydrate digestion in the small intestinal epithelium, leading to alterations in the commensal microbiota composition and metabolic activity. The commensal microbiota and IgA responses exhibit bidirectional interactions that modulate intestinal homeostasis and immunity. However, the effect of α-GIs on the intestinal IgA response remains unclear. We investigated whether α-GIs affect IgA responses by administering voglibose and acarbose to mice via drinking water. We analyzed Tfh cells, germinal center (GC) B cells, and IgA-producing B cells in PPs by flow cytometry. We also assessed pathogen-specific IgA responses. We discovered that voglibose and acarbose induced Tfh cells, GCB cells, and IgA-producing B cells in the PPs of the proximal small intestine in mice. This effect was attributed to the modification of the microbiota rather than a shortage of monosaccharides. Furthermore, voglibose enhanced secretory IgA (S-IgA) production against attenuated Salmonella Typhimurium. Our findings reveal a novel mechanism by which α-GIs augment antigen-specific IgA responses by stimulating Tfh-GCB responses in PPs, and suggest a potential therapeutic application as an adjuvant for augmenting mucosal vaccines.