Makoto Kinoshita

Neuromyelitis optica: Passive transfer to rats by human immunoglobulin.

Recurrent attacks of optic neuritis and myelitis are the hallmarks of both neuromyelitis optica (NMO) and multiple sclerosis (MS). NMO immunoglobulin G (NMO-IgG), which recognizes astrocytic aquaporin-4 (AQP4) water channels, is a specific serum autoantibody that distinguishes NMO from MS. The pathogenic role of the anti-AQP4 antibody (AQP4-Ab, NMO-IgG) in NMO has been speculated based on several studies in vitro. The aim of this study was to demonstrate the pathogenicity of AQP4-Ab in vivo. We obtained IgG from patients who underwent therapeutic plasmapheresis, and developed an animal model by passive transfer of IgG to rats. The active lesions of the rats exhibited pathological characteristics strikingly similar to those of NMO, marked by astrocytic loss and perivascular deposition of immunoglobulin and complements. These findings provide the first evidence of the pathogenicity of AQP4-Ab in vivo and support the therapeutic efficacy of eliminating the antibodies by plasmapheresis.

Commensal Bacteria-Dependent Indole Production Enhances Epithelial Barrier Function in the Colon

Microbiota have been shown to have a great influence on functions of intestinal epithelial cells (ECs). The role of indole as a quorum-sensing (QS) molecule mediating intercellular signals in bacteria has been well appreciated. However, it remains unknown whether indole has beneficial effects on maintaining intestinal barriers in vivo. In this study, we analyzed the effect of indole on ECs using a germ free (GF) mouse model. GF mice showed decreased expression of junctional complex molecules in colonic ECs. The feces of specific pathogen-free (SPF) mice contained a high amount of indole; however the amount was significantly decreased in the feces of GF mice by 27-fold. Oral administration of indole-containing capsules resulted in increased expression of both tight junction (TJ)- and adherens junction (AJ)-associated molecules in colonic ECs in GF mice. In accordance with the increased expression of these junctional complex molecules, GF mice given indole-containing capsules showed higher resistance to dextran sodium sulfate (DSS)-induced colitis. A similar protective effect of indole on DSS-induced epithelial damage was also observed in mice bred in SPF conditions. These findings highlight the beneficial role of indole in establishing an epithelial barrier in vivo.

Anti-aquaporin-4 antibody induces astrocytic cytotoxicity in the absence of CNS antigen-specific T cells

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system (CNS). Anti-aquaporin-4 antibody (AQP4-Ab) is a highly specific serum autoantibody that is detected in patients with NMO. Several lines of evidence indicate that AQP4-Ab not only serves as a disease marker but also plays a pivotal role in the pathogenesis of NMO. Although the pathogenicity of AQP4-Ab in vivo has recently been demonstrated, the presence of CNS antigen-specific T cells is recognized as a prerequisite for the antibody to exert pathogenic effects. Thus, it remains unclear whether AQP4-Ab is the primary cause of the disease or a disease-modifying factor in NMO. Here we report that pre-treatment with complete Freunds adjuvant (CFA) alone is sufficient for AQP4-Ab to induce astrocytic damage in vivo. Our results show the primary pathogenic role of AQP4-Ab in the absence of CNS antigen-specific T cells, and suggest that danger signals provided by nonspecific inflammation can be a trigger for those who harbor the autoantibody to develop NMO.

Dietary Folic Acid Promotes Survival of Foxp3+ Regulatory T Cells in the Colon

Dietary compounds as well as commensal microbiota contribute to the generation of a unique gut environment. In this study, we report that dietary folic acid (FA) is required for the maintenance of Foxp3+ regulatory T cells (Tregs) in the colon. Deficiency of FA in the diet resulted in marked reduction of Foxp3+ Tregs selectively in the colon. Blockade of folate receptor 4 and treatment with methotrexate, which inhibits folate metabolic pathways, decreased colonic Foxp3+ Tregs. Compared with splenic Tregs, colonic Tregs were more activated to proliferate vigorously and were highly sensitive to apoptosis. In colonic Tregs derived from mice fed with a FA-deficient diet, expression of anti-apoptotic molecules Bcl-2 and Bcl-xL was severely decreased. A general reduction of peripheral Tregs was induced by a neutralizing Ab against IL-2, but a further decrease by additional FA deficiency was observed exclusively in the colon. Mice fed with an FA-deficient diet exhibited higher susceptibility to intestinal inflammation. These findings reveal the previously unappreciated role of dietary FA in promotion of survival of Foxp3+ Tregs that are in a highly activated state in the colon.

The lactic acid bacterium Pediococcus acidilactici suppresses autoimmune encephalomyelitis by inducing IL-10-producing regulatory T cells.

Background Certain intestinal microflora are thought to regulate the systemic immune response. Lactic acid bacteria are one of the most studied bacteria in terms of their beneficial effects on health and autoimmune diseases; one of which is Multiple sclerosis (MS) which affects the central nervous system. We investigated whether the lactic acid bacterium Pediococcus acidilactici, which comprises human commensal bacteria, has beneficial effects on experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Methodology/Principal Findings P. acidilactici R037 was orally administered to EAE mice to investigate the effects of R037. R037 treatment suppressed clinical EAE severity as prophylaxis and therapy. The antigen-specific production of inflammatory cytokines was inhibited in R037-treated mice. A significant increase in the number of CD4+ Interleukin (IL)-10-producing cells was observed in the mesenteric lymph nodes (MLNs) and spleens isolated from R037-treated naive mice, while no increase was observed in the number of these cells in the lamina propria. Because only a slight increase in the CD4+Foxp3+ cells was observed in MLNs, R037 may primarily induce Foxp3− IL10-producing T regulatory type 1 (Tr1) cells in MLNs, which contribute to the beneficial effect of R037 on EAE. Conclusions/Significance An orally administered single strain of P. acidilactici R037 ameliorates EAE by inducing IL10-producing Tr1 cells. Our findings indicate the therapeutic potential of the oral administration of R037 for treating multiple sclerosis.

Ecto-Nucleoside Triphosphate Diphosphohydrolase 7 Controls Th17 Cell Responses through Regulation of Luminal ATP in the Small Intestine

Extracellular ATP is released from live cells in controlled conditions, as well as dying cells in inflammatory conditions, and, thereby, regulates T cell responses, including Th17 cell induction. The level of extracellular ATP is closely regulated by ATP hydrolyzing enzymes, such as ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases). ENTPDase1/CD39, which is expressed in immune cells, was shown to regulate immune responses by downregulating the ATP level. In this study, we analyzed the immunomodulatory function of ENTPDase7, which is preferentially expressed in epithelial cells in the small intestine. The targeted deletion of Entpd7 encoding ENTPDase7 in mice resulted in increased ATP levels in the small intestinal lumen. The number of Th17 cells was selectively increased in the small intestinal lamina propria in Entpd7−/− mice. Th17 cells were decreased by oral administration of antibiotics or the ATP antagonist in Entpd7−/− mice, indicating that commensal microbiota-dependent ATP release mediates the enhanced Th17 cell development in the small intestinal lamina propria of Entpd7−/− mice. In accordance with the increased number of small intestinal Th17 cells, Entpd7−/− mice were resistant to oral infection with Citrobacter rodentium. Entpd7−/− mice suffered from severe experimental autoimmune encephalomyelitis, which was associated with increased numbers of CD4+ T cells producing both IL-17 and IFN-γ. Taken together, these findings demonstrate that ENTPDase7 controls the luminal ATP level and, thereby, regulates Th17 cell development in the small intestine.

Generation of colonic IgA-secreting cells in the caecal patch

Gut-associated lymphoid tissues are responsible for the generation of IgA-secreting cells. However, the function of the caecal patch, a lymphoid tissue in the appendix, remains unknown. Here we analyse the role of the caecal patch using germ-free mice colonized with intestinal bacteria after appendectomy. Appendectomized mice show delayed accumulation of IgA(+) cells in the large intestine, but not the small intestine, after colonization. Decreased colonic IgA(+) cells correlate with altered faecal microbiota composition. Experiments using photoconvertible Kaede-expressing mice or adoptive transfer show that the caecal patch IgA(+) cells migrate to the large and small intestines, whereas Peyers patch cells are preferentially recruited to the small intestine. IgA(+) cells in the caecal patch express higher levels of CCR10. Dendritic cells in the caecal patch, but not Peyers patches, induce CCR10 on cocultured B cells. Thus, the caecal patch is a major site for generation of IgA-secreting cells that migrate to the large intestine.

The Ectoenzyme E-NPP3 Negatively Regulates ATP-Dependent Chronic Allergic Responses by Basophils and Mast Cells

Crosslinking of the immunoglobulin receptor FcεRI activates basophils and mast cells to induce immediate and chronic allergic inflammation. However, it remains unclear how the chronic allergic inflammation is regulated. Here, we showed that ecto-nucleotide pyrophosphatase-phosphodiesterase 3 (E-NPP3), also known as CD203c, rapidly induced by FcεRI crosslinking, negatively regulated chronic allergic inflammation. Basophil and mast cell numbers increased in Enpp3(-/-) mice with augmented serum ATP concentrations. Enpp3(-/-) mice were highly sensitive to chronic allergic pathologies, which was reduced by ATP blockade. FcεRI crosslinking induced ATP secretion from basophils and mast cells, and ATP activated both cells. ATP clearance was impaired in Enpp3(-/-) cells. Enpp3(-/-)P2rx7(-/-) mice showed decreased responses to FcεRI crosslinking. Thus, ATP released by FcεRI crosslinking stimulates basophils and mast cells for further activation causing allergic inflammation. E-NPP3 decreases ATP concentration and suppresses basophil and mast cell activity.

Elevation of Sema4A Implicates Th Cell Skewing and the Efficacy of IFN-β Therapy in Multiple Sclerosis

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the CNS and a leading cause of lasting neurologic disabilities in young adults. Although the precise mechanism remains incompletely understood, Ag presentation and subsequent myelin-reactive CD4+ T cell activation/differentiation are essential for the pathogenesis of MS. Although semaphorins were initially identified as axon guidance cues during neural development, several semaphorins are crucially involved in various phases of immune responses. Sema4A is one of the membrane-type class IV semaphorins, which we originally identified from the cDNA library of dendritic cell (DC). Sema4A plays critical roles in T cell activation and Th1 differentiation during the course of experimental autoimmune encephalomyelitis, an animal model of MS; however, its pathological involvement in human MS has not been determined. In this study, we report that Sema4A is increased in the sera of patients with MS. The expression of Sema4A is increased on DCs in MS patients and shed from these cells in a metalloproteinase-dependent manner. DC-derived Sema4A is not only critical for Th1 but also for Th17 cell differentiation, and MS patients with high Sema4A levels exhibit Th17 skewing. Furthermore, patients with high Sema4A levels have more severe disabilities and are unresponsive to IFN-β treatment. Taken together, our results suggest that Sema4A is involved in the pathogenesis of MS by promoting Th17 skewing.

Disease-Association Analysis of an Inflammation-Related Feedback Loop

The IL-6-triggered positive feedback loop for NFκB signaling (or the IL-6 amplifier/Inflammation amplifier) was originally discovered as a synergistic-activation signal that follows IL-17/IL-6 stimulation in nonimmune cells. Subsequent results from animal models have shown that the amplifier is activated by stimulation of NFκB and STAT3 and induces chemokines and inflammation via an NFκB loop. However, its role in human diseases is unclear. Here, we combined two genome-wide mouse screens with SNP-based disease association studies, revealing 1,700 genes related to the IL-6 amplifier, 202 of which showed 492 indications of association with ailments beyond autoimmune diseases. We followed up on ErbB1 from our list. Blocking ErbB1 signaling suppressed the IL-6 amplifier, whereas the expression of epiregulin, an ErbB1 ligand, was higher in patients with inflammatory diseases. These results indicate that the IL-6 amplifier is indeed associated with human diseases and disorders and that the identified genes may make for potential therapeutic targets.