Shinji Oki

NKT Cell-Dependent Amelioration of a Mouse Model of Multiple Sclerosis by Altering Gut Flora

Improved hygiene has been suggested to influence certain autoimmune disorders, such as multiple sclerosis. In this study, we addressed whether altering the composition of gut flora may affect susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We administered a mixture of non-absorbing antibiotics, kanamycin, colistin, and vancomycin (KCV), orally to mice induced to develop EAE. The antibiotic treatment, beginning 1 week prior to sensitization, altered the composition of gut flora and, intriguingly, also ameliorated the development of EAE. While this result was associated with a reduced production of pro-inflammatory cytokines from the draining lymph node cells, a reduction of mesenteric Th17 cells was found to correlate with disease suppression. In addition, we found that Valpha14 invariant NKT (iNKT) cells were necessary for maintaining the mesenteric Th17 cells. The homologous effects of KCV treatment and iNKT cell depletion led us to speculate that KCV treatment may suppress EAE by altering the function of iNKT cells. Consistent with this hypothesis, KCV treatment did not suppress EAE that was induced in iNKT cell-deficient mice, although it was efficacious in mice that lacked Valpha19 mucosal-associated invariant T cells. Thus, gut flora may influence the development of EAE in a way that is dependent on iNKT cells, which has significant implications for the prevention and treatment of autoimmune diseases.

Invariant NKT Cells Biased for IL-5 Production Act as Crucial Regulators of Inflammation

Although invariant NKT (iNKT) cells play a regulatory role in the pathogenesis of autoimmune diseases and allergy, an initial trigger for their regulatory responses remains elusive. In this study, we report that a proportion of human CD4+ iNKT cell clones produce enormous amounts of IL-5 and IL-13 when cocultured with CD1d+ APC in the presence of IL-2. Such IL-5 bias was never observed when we stimulated the same clones with α-galactosylceramide or anti-CD3 Ab. Suboptimal TCR stimulation by plate-bound anti-CD3 Ab was found to mimic the effect of CD1d+ APC, indicating the role of TCR signaling for selective induction of IL-5. Interestingly, DNA microarray analysis identified IL-5 and IL-13 as the most highly up-regulated genes, whereas other cytokines produced by iNKT cells, such as IL-4 and IL-10, were not significantly induced. Moreover, iNKT cells from BALB/c mice showed similar IL-5 responses after stimulation with IL-2 ex vivo or in vivo. The iNKT cell subset producing IL-5 and IL-13 could play a major role in the development of allergic disease or asthma and also in the immune regulation of Th1 inflammation.

Neuropeptide Y (NPY) Suppresses Experimental Autoimmune Encephalomyelitis: NPY1 Receptor-Specific Inhibition of Autoreactive Th1 Responses In Vivo

Prior studies have revealed that the sympathetic nervous system regulates the clinical and pathological manifestations of experimental autoimmune encephalomyelitis (EAE), an autoimmune disease model mediated by Th1 T cells. Although the regulatory role of catecholamines has been indicated in the previous works, it remained possible that other sympathetic neurotransmitters like neuropeptide Y (NPY) may also be involved in the regulation of EAE. Here we examined the effect of NPY and NPY receptor subtype-specific compounds on EAE, actively induced with myelin oligodendrocyte glycoprotein 35–55 in C57BL/6 mice. Our results revealed that exogenous NPY as well as NPY Y1 receptor agonists significantly inhibited the induction of EAE, whereas a Y5 receptor agonist or a combined treatment of NPY with a Y1 receptor antagonist did not inhibit signs of EAE. These results indicate that the suppression of EAE by NPY is mediated via Y1 receptors. Furthermore, treatment with the Y1 receptor antagonist induced a significantly earlier onset of EAE, indicating a protective role of endogenous NPY in the induction phase of EAE. We also revealed a significant inhibition of myelin oligodendrocyte glycoprotein 35–55-specific Th1 response as well as a Th2 bias of the autoimmune T cells in mice treated with the Y1 receptor agonist. Ex vivo analysis further demonstrated that autoimmune T cells are directly affected by NPY via Y1 receptors. Taken together, we conclude that NPY is a potent immunomodulator involved in the regulation of the Th1-mediated autoimmune disease EAE.

Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

Recent evidence suggests that interleukin-17-producing CD4(+) T cells (Th17 cells) are the dominant pathogenic cellular component in autoimmune inflammatory diseases, including multiple sclerosis. It has recently been demonstrated that all-trans retinoic acid can suppress Th17 differentiation and promote the generation of Foxp3(+) regulatory T cells via retinoic acid receptor signals. Here, we investigated the effects of AM80, a synthetic retinoid with enhanced biological properties to all-trans retinoic acid, on Th17 differentiation and function and evaluated its therapeutic potential in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. AM80 treatment was more effective than all-trans retinoic acid in inhibiting Th17 differentiation in vitro. Oral administration of AM80 was protective for the early development of EAE and the down-modulation of Th17 differentiation and effector functions in vivo. Moreover, AM80 inhibited interleukin-17 production by splenic memory T cells, in vitro-differentiated Th17 cells, and central nervous system-infiltrating effector T cells. Accordingly, AM80 was effective when administered therapeutically after the onset of EAE. Continuous AM80 treatment, however, was ineffective at inhibiting late EAE symptoms despite the maintained suppression of RORgammat and interleukin-17 expression levels by central nervous system-infiltrating T cells. We reveal that continuous AM80 treatment also led to the suppression of interleukin-10 production by a distinct T cell subset that expressed both Foxp3 and RORgammat. These findings suggest that retinoid signaling regulates both inflammatory Th17 cells and Th17-like regulatory cells.

Suppression of Experimental Autoimmune Encephalomyelitis by Ghrelin

Ghrelin is a recently identified gastric hormone that displays strong growth hormone-releasing activity mediated by the growth hormone secretagogue receptor. While this unique endogenous peptide participates in the regulation of energy homeostasis, increases food intake, and decreases energy expenditure, its ability to inhibit the production of proinflammatory cytokines in vitro indicates its role in the regulation of inflammatory process in vivo. Here we examine the effect of exogenous ghrelin on the development of experimental autoimmune encephalomyelitis (EAE), a representative model of multiple sclerosis. In the C57BL/6 mouse model of EAE induced by sensitization to myelin oligodendrocyte glycoprotein 35–55 peptide, we found that alternate-day s.c. injections of ghrelin (5 μg/kg/day) from day 1 to 35 significantly reduced the clinical severity of EAE. The suppression of EAE was accompanied by reduced mRNA levels of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 in the spinal cord cellular infiltrates and microglia from ghrelin-treated mice at the peak of disease, suggesting the role of ghrelin as an antiinflammatory hormone. Consistently, ghrelin significantly suppressed the production of proinflammatory cytokines in LPS-stimulated microglia in vitro. These results shed light on the new role of ghrelin in the regulation of inflammation with possible implications for management of human diseases.

Orphan nuclear receptor NR4A2 expressed in T cells from multiple sclerosis mediates production of inflammatory cytokines

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) mediated by Th17 and Th1 cells. DNA microarray analysis previously showed that NR4A2, an orphan nuclear receptor, is strongly up-regulated in the peripheral blood T cells of MS. Here, we report that NR4A2 plays a pivotal role for mediating cytokine production from pathogenic T cells. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, NR4A2, was selectively up-regulated in the T cells isolated from the CNS. Strikingly, a forced expression of NR4A2 augmented promoter activities of IL-17 and IFN-γ genes, leading to an excessive production of these cytokines. Conversely, treatment with siRNA for NR4A2, resulted in a significant reduction in the production of IL-17 and IFN-γ. Furthermore, treatment with NR4A2 siRNA reduced the ability of encephalitogenic T cells to transfer EAE in recipient mice. Thus, NR4A2 is an essential transcription factor for triggering the inflammatory cascade of MS/EAE and may serve as a therapeutic target.

Nuclear Receptor NR4A2 Orchestrates Th17 Cell-Mediated Autoimmune Inflammation via IL-21 Signalling

IL-17-producing CD4+ T helper 17 (Th17) cells are pathogenic in a range of human autoimmune diseases and corresponding animal models. We now demonstrate that such T cells infiltrating the target organ during the induction of experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveoretinitis (EAU) specifically express NR4A2. Further, we reveal a critical involvement of NR4A2 in Th17 cell functions and Th17 cell-driven autoimmune diseases. When NR4A2 expression was blocked with siRNA, full Th17 differentiation was prevented in vitro: although cells expressed the master Th17 regulator, RORγt, they expressed reduced levels of IL-23R and were unable to produce IL-17 and IL-21. Notably, Th17 differentiation in the absence of NR4A2 was restored by exogenous IL-21, indicating that NR4A2 controls full maturation of Th17 cells via autocrine IL-21 signalling. Preventing NR4A2 expression in vivo by systemic treatment with NR4A2-specific siRNA also reduced Th17 effector responses and furthermore protected mice from EAE induction. In addition, the lack of disease was associated with a reduction in autocrine IL-21 production and IL-23R expression. Similar modulation of NR4A2 expression was also effective as an intervention, reversing established autoimmune responses and ameliorating clinical disease symptoms. Thus, NR4A2 appears to control Th17 differentiation and so plays an essential role in the development of Th17-mediated autoimmune disease. As NR4A2 is also upregulated during human autoimmune disease, targeting NR4A2 may provide a new therapeutic approach in treating autoimmune disease.

Plasmablasts as Migratory IgG-Producing Cells in the Pathogenesis of Neuromyelitis Optica

Neuromyelitis optica (NMO) is an inflammatory disease characterized by recurrent attacks of optic neuritis and myelitis. It is generally accepted that autoantibodies against aquaporin 4 water channel protein play a pathogenic role in neuromyelitis optica. We have recently reported that plasmablasts are increased in the peripheral blood of this autoimmune disease, and are capable of producing autoantibodies against aquaporin 4. Here, we demonstrate that CD138+HLA-DR+ plasmablasts, a subset of IgG-producing cells, are increased in the peripheral blood and are enriched among the cerebrospinal fluid (CSF) lymphocytes during the relapse of neuromyelitis optica. Notably, these CD138+HLA-DR+ plasmablasts overexpress CXCR3, whose ligands are present in the cerebrospinal fluid during the relapse of neuromyelitis optica. These results led us to speculate that plasmablasts producing anti-aquaporin 4 autoantibodies might traffic toward the central nervous system (CNS). Furthermore, we performed single-cell sorting of plasmablasts from peripheral blood and CSF samples from NMO and sequenced the complementarity-determining regions (CDRs) of the IgG heavy chain expressed by the sorted plasmablast clones. There were high frequencies of mutations in the CDRs compared with framework regions, indicating that these plasmablast clones would represent a post-germinal center B-cell lineage. Consistent with the preceding results, the plasmablast clones from the peripheral blood shared the same CDR sequences with the clones from the CSF. These results indicate that IgG-producing plasmablasts, which are guided by helper T-cells, may migrate from the peripheral blood preferentially to the CSF. Since migratory plasmablasts could be involved in the inflammatory pathology of NMO, the B-cell subset and their migration might be an attractive therapeutic target.

Eomesodermin-expressing T-helper cells are essential for chronic neuroinflammation

Development of acute experimental autoimmune encephalomyelitis (EAE) depends on Th17 cells expressing the nuclear factor NR4A2. However, in mice lacking NR4A2 in T cells, a late-onset disease is still inducible, despite a great reduction in acute inflammation. We here reveal that development of this late onset disease depends on cytotoxic T-cell-like CD4+ T cells expressing the T-box transcription factor Eomesodermin (Eomes). T-cell-specific deletion of the Eomes gene remarkably ameliorates the late-onset EAE. Strikingly, similar Eomes+ CD4+ T cells are increased in the peripheral blood and cerebrospinal fluid from patients in a progressive state of multiple sclerosis. Collective data indicate an involvement of granzyme B and protease-activated receptor-1 in the neuroinflammation mediated by Eomes+ CD4+ T cells.

Stat6 activation and Th2 cell proliferation driven by CD28 signals

CD28 engagement by specific monoclonal antibody (mAb) or binding of the natural ligands, CD80 and CD86, induces tyrosine phosphorylation of CD28, which in turn recruits and activates the signal transducer and activator of transcription 6 (Stat6). The Stat6 association with CD28 is specifically induced by CD80 or CD86 ligand binding and is not dependent upon the secretion of IL‐4 or IL‐13. Activated Stat6 translocates to the nucleus and binds to a Stat6‐responsive element on the human IL‐4 promoter. CD28 ligation induces Stat6‐dependent transcriptional activation of a reporter gene under the control of a multimerized Stat6‐responsive element fused to an essential part of the IL‐4 promoter. Primary stimulation of naive CD4+ T cells with anti‐CD28 mAb in the presence of IL‐2, but in the absence of anti‐CD3 mAb induces preferential production of IL‐4 and expression of CCR4 mRNA after secondary stimulation with anti‐CD3, indicating the preferential differentiation of Th2 cells. These findings suggest that initial IL‐4 production required for commitment of naive T cells toward Th2 cells may be provided in response to signals delivered via CD28 by antigen‐presenting cells.