Tatsusada Okuno

Plexin-A1 and its interaction with DAP12 in immune responses and bone homeostasis

Semaphorins and their receptors have diverse functions in axon guidance, organogenesis, vascularization and/or angiogenesis, oncogenesis and regulation of immune responses. The primary receptors for semaphorins are members of the plexin family. In particular, plexin-A1, together with ligand-binding neuropilins, transduces repulsive axon guidance signals for soluble class III semaphorins, whereas plexin-A1 has multiple functions in chick cardiogenesis as a receptor for the transmembrane semaphorin, Sema6D, independent of neuropilins. Additionally, plexin-A1 has been implicated in dendritic cell function in the immune system. However, the role of plexin-A1 in vivo, and the mechanisms underlying its pleiotropic functions, remain unclear. Here, we generated plexin-A1-deficient (plexin-A1−/−) mice and identified its important roles, not only in immune responses, but also in bone homeostasis. Furthermore, we show that plexin-A1 associates with the triggering receptor expressed on myeloid cells-2 (Trem-2), linking semaphorin-signalling to the immuno-receptor tyrosine-based activation motif (ITAM)-bearing adaptor protein, DAP12. These findings reveal an unexpected role for plexin-A1 and present a novel signalling mechanism for exerting the pleiotropic functions of semaphorins.

Semaphorin 7A initiates T-cell-mediated inflammatory responses through α1β1 integrin

Semaphorins are axon guidance factors that assist growing axons in finding appropriate targets and forming synapses. Emerging evidence suggests that semaphorins are involved not only in embryonic development but also in immune responses. Semaphorin 7A (Sema7A; also known as CD108), which is a glycosylphosphatidylinositol-anchored semaphorin, promotes axon outgrowth through β1-integrin receptors and contributes to the formation of the lateral olfactory tract. Although Sema7A has been shown to stimulate human monocytes, its function as a negative regulator of T-cell responses has also been reported. Thus, the precise function of Sema7A in the immune system remains unclear. Here we show that Sema7A, which is expressed on activated T cells, stimulates cytokine production in monocytes and macrophages through α1β1 integrin (also known as very late antigen-1) as a component of the immunological synapse, and is critical for the effector phase of the inflammatory immune response. Sema7A-deficient (Sema7a-/-) mice are defective in cell-mediated immune responses such as contact hypersensitivity and experimental autoimmune encephalomyelitis. Although antigen-specific and cytokine-producing effector T cells can develop and migrate into antigen-challenged sites in Sema7a-/- mice, Sema7a-/- T cells fail to induce contact hypersensitivity even when directly injected into the antigen-challenged sites. Thus, the interaction between Sema7A and α1β1 integrin is crucial at the site of inflammation. These findings not only identify a function of Sema7A as an effector molecule in T-cell-mediated inflammation, but also reveal a mechanism of integrin-mediated immune regulation.

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.

Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II

The recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate the entry of leukocytes into the lymphatics remain unclear. Here we show that plexin-A1, a principal receptor component for class III and class VI semaphorins, was crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that the semaphorin Sema3A, but not Sema6C or Sema6D, was required for DC transmigration and that Sema3A produced by the lymphatics promoted actomyosin contraction at the trailing edge of migrating DCs. Our findings not only demonstrate that semaphorin signals are involved in DC trafficking but also identify a previously unknown mechanism that induces actomyosin contraction as these cells pass through narrow gaps.

A midline switch of receptor processing regulates commissural axon guidance in vertebrates

Commissural axon guidance requires complex modulations of growth cone sensitivity to midline-derived cues, but underlying mechanisms in vertebrates remain largely unknown. By using combinations of ex vivo and in vivo approaches, we uncovered a molecular pathway controlling the gain of response to a midline repellent, Semaphorin3B (Sema3B). First, we provide evidence that Semaphorin3B/Plexin-A1 signaling participates in the guidance of commissural projections at the vertebrate ventral midline. Second, we show that, at the precrossing stage, commissural neurons synthesize the Neuropilin-2 and Plexin-A1 Semaphorin3B receptor subunits, but Plexin-A1 expression is prevented by a calpain1-mediated processing, resulting in silencing commissural responsiveness. Third, we report that, during floor plate (FP) in-growth, calpain1 activity is suppressed by local signals, allowing Plexin-A1 accumulation in the growth cone and sensitization to Sema3B. Finally, we show that the FP cue NrCAM mediates the switch of Plexin-A1 processing underlying growth cone sensitization to Sema3B. This reveals pathway-dependent modulation of guidance receptor processing as a novel mechanism for regulating guidance decisions at intermediate targets.

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.

Roles of Sema4D-Plexin-B1 Interactions in the Central Nervous System for Pathogenesis of Experimental Autoimmune Encephalomyelitis

Although semaphorins were originally identified as axonal guidance molecules during neuronal development, it is emerging that several semaphorins play crucial roles in various phases of immune responses. Sema4D/CD100, a class IV semaphorin, has been shown to be involved in the nervous and immune systems through its receptors plexin-B1 and CD72, respectively. However, the involvement of Sema4D in neuroinflammation still remains unclear. We found that Sema4D promoted inducible NO synthase expression by primary mouse microglia, the effects of which were abolished in plexin-B1–deficient but not in CD72-deficient microglia. In addition, during the development of experimental autoimmune encephalomyelitis (EAE), which was induced by immunization with myelin oligodendrocyte glycoprotein-derived peptides, we observed that the expression of Sema4D and plexin-B1 was induced in infiltrating mononuclear cells and microglia, respectively. Consistent with these expression profiles, when myelin oligodendrocyte glycoprotein-specific T cells derived from wild-type mice were adoptively transferred into plexin-B1–deficient mice or bone marrow chimera mice with plexin-B1–deficient CNS resident cells, the development of EAE was considerably attenuated. Furthermore, blocking Abs against Sema4D significantly inhibited neuroinflammation during EAE development. Collectively, our findings demonstrate the role of Sema4D–plexin-B1 interactions in the activation of microglia and provide their pathologic significance in neuroinflammation.

Plexin-A4 negatively regulates T lymphocyte responses

Semaphorins and their receptors play crucial roles not only in axon guidance during neuronal development but also in the regulation of immune responses. Plexin-A4, a member of the plexin-A subfamily, forms a receptor complex with neuropilins and transduces signals for class III semaphorins in the nervous system. Although plexin-A4 is also expressed in the lymphoid tissues, the involvement of plexin-A4 in immune responses remains unknown. To explore the role of plexin-A4 in the immune system, we analyzed immune responses in plexin-A4-deficient (plexin-A4-/-) mice. Among immune cells, plexin-A4 mRNA was detected in T cells, dendritic cells and macrophages but not in B cells and NK cells. Plexin-A4-/- mice had normal numbers and cell surface markers for each lymphocyte subset, suggesting that plexin-A4 is not essential for lymphocyte development. However, plexin-A4-/- mice exhibited enhanced antigen-specific T cell responses and heightened sensitivity to experimental autoimmune encephalomyelitis. Plexin-A4-/- T cells exhibited hyperproliferative responses to anti-CD3 stimulation and to allogeneic dendritic cells in vitro. Furthermore, this hyperproliferation was also observed in both T cells from neuropilin-1 mutant (npn-1(Sema-)) mice, in which the binding site of class III semaphorins is disrupted, and T cells from Sema3A-deficient (Sema-3A-/-) mice. Collectively, these results suggest that plexin-A4, as a component of the receptor complex for class III semaphorins, negatively regulates T cell-mediated immune responses.

RGMa modulates T cell responses and is involved in autoimmune encephalomyelitis

In multiple sclerosis, activated CD4+ T cells initiate an immune response in the brain and spinal cord, resulting in demyelination, degeneration and progressive paralysis. Repulsive guidance molecule-a (RGMa) is an axon guidance molecule that has a role in the visual system and in neural tube closure. Our study shows that RGMa is expressed in bone marrow–derived dendritic cells (BMDCs) and that CD4+ T cells express neogenin, a receptor for RGMa. Binding of RGMa to CD4+ T cells led to activation of the small GTPase Rap1 and increased adhesion of T cells to intracellular adhesion molecule-1 (ICAM-1). Neutralizing antibodies to RGMa attenuated clinical symptoms of mouse myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) and reduced invasion of inflammatory cells into the CNS. Silencing of RGMa in MOG-pulsed BMDCs reduced their capacity to induce EAE following adoptive transfer to naive C57BL/6 mice. CD4+ T cells isolated from mice treated with an RGMa-specific antibody showed diminished proliferative responses and reduced interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4 and IL-17 secretion. Incubation of PBMCs from patients with multiple sclerosis with an RGMa-specific antibody reduced proliferative responses and pro-inflammatory cytokine expression. These results demonstrate that an RGMa-specific antibody suppresses T cell responses, and suggest that RGMa could be a promising molecular target for the treatment of multiple sclerosis.

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.