Masayuki Mizui

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.

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.

Exploring RNA interference as a therapeutic strategy for renal disease.

The short synthetic interfering RNA duplexes (siRNAs) can selectively suppress gene expression in somatic mammalian cells without nonselective toxic effects of double-stranded RNA (dsRNA). However, a selective in vivo delivery of siRNA transfer has not been reported in kidney. Here, we investigated whether injection of synthetic siRNAs via renal artery followed by electroporation could be effective and therapeutic in silencing specific gene in glomerulus. We investigated the effect of siRNA in rat cultured mesangial cells (MCs) and showed that siRNA sequence-specific suppression of transgene expression was over a 1000-fold more potent than that by antisense oligodeoxynucleotide (ASODN). Transfection of siRNA targeting luciferase into rat kidneys significantly inhibited expression of a cotransfected luciferase expression vector in vivo. The delivery of siRNA targeting enhanced green fluorescent protein (EGFP) in the transgenic ‘green’ rat reduced endogenous EGFP expression, mainly in glomerular MCs. Furthermore, RNAi targeting against TGF-β1 significantly suppressed TGF-β1 mRNA and protein expression, thereby ameliorated the progression of matrix expansion in experimental glomerulonephritis. In addition, vector-based RNAi also inhibited TGF-β1 expression in vitro and in vivo. In conclusion, siRNA-directed TGF-β1 silencing may be of therapeutic value in the prevention and treatment of fibrotic diseases.

CaMK4-dependent activation of AKT/mTOR and CREM-α underlies autoimmunity-associated Th17 imbalance

Tissue inflammation in several autoimmune diseases, including SLE and MS, has been linked to an imbalance of IL-17-producing Th (Th17) cells and Tregs; however, the factors that promote Th17-driven autoimmunity are unclear. Here, we present evidence that the calcium/calmodulin-dependent protein kinase IV (CaMK4) is increased and required during Th17 cell differentiation. Isolation of naive T cells from a murine model of lupus revealed increased levels of CaMK4 following stimulation with Th17-inducing cytokines but not following Treg, Th1, or Th2 induction. Furthermore, naive T cells from mice lacking CaMK4 did not produce IL-17. Genetic or pharmacologic inhibition of CaMK4 decreased the frequency of IL-17-producing T cells and ameliorated EAE and lupus-like disease in murine models. Inhibition of CaMK4 reduced Il17 transcription through decreased activation of the cAMP response element modulator α (CREM-α) and reduced activation of the AKT/mTOR pathway, which is known to enhance Th17 differentiation. Importantly, silencing CaMK4 in T cells from patients with SLE and healthy individuals inhibited Th17 differentiation through reduction of IL17A and IL17F mRNA. Collectively, our results suggest that CaMK4 inhibition has potential as a therapeutic strategy for Th17-driven autoimmune diseases.

B cell-intrinsic deficiency of the Wiskott-Aldrich syndrome protein (WASp) causes severe abnormalities of the peripheral B-cell compartment in mice

Wiskott Aldrich syndrome (WAS) is caused by mutations in the WAS gene that encodes for a protein (WASp) involved in cytoskeleton organization in hematopoietic cells. Several distinctive abnormalities of T, B, and natural killer lymphocytes; dendritic cells; and phagocytes have been found in WASp-deficient patients and mice; however, the in vivo consequence of WASp deficiency within individual blood cell lineages has not been definitively evaluated. By conditional gene deletion we have generated mice with selective deficiency of WASp in the B-cell lineage (B/WcKO mice). We show that this is sufficient to cause a severe reduction of marginal zone B cells and inability to respond to type II T-independent Ags, thereby recapitulating phenotypic features of complete WASp deficiency. In addition, B/WcKO mice showed prominent signs of B-cell dysregulation, as indicated by an increase in serum IgM levels, expansion of germinal center B cells and plasma cells, and elevated autoantibody production. These findings are accompanied by hyperproliferation of WASp-deficient follicular and germinal center B cells in heterozygous B/WcKO mice in vivo and excessive differentiation of WASp-deficient B cells into class-switched plasmablasts in vitro, suggesting that WASp-dependent B cell-intrinsic mechanisms critically contribute to WAS-associated autoimmunity.

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.

Antisense Oligonucleotides Against Thrombospondin-1 Inhibit Activation of TGF-β in Fibrotic Renal Disease in the Rat in Vivo

Specific treatment of chronic progressive renal disease is very limited. TGF-beta, considered as the major cytokine causing tissue scarring, must be activated extracellularly before it can bind to its receptors. Thrombospondin-1 (TSP1) has been identified as an activator of latent TGF-beta in in vitro systems and in pancreas and lung homeostasis in mouse pups in vivo, but whether this is also true in inflammatory fibrotic disease is unknown. We examined a rat model of mesangial proliferative glomerulonephritis, where TGF-beta has been demonstrated to mediate renal fibrosis. In this study, antisense phosphorothioate oligonucleotides against TSP1 were successfully transferred into almost all glomeruli of perfused diseased kidneys and markedly inhibited de novo synthesis of TSP1. This effect was accompanied by decreased activation but not expression of TGF-beta and by the inhibition of the TGF-beta-dependent smad-signaling pathway, as well as transcription of TGF-beta target genes such as EDA-fibronectin, resulting in a markedly suppressed accumulation of extracellular matrix. In sharp contrast, neither glomerular cell proliferation nor influx of macrophages was affected by this therapy in experimental mesangial proliferative glomerulonephritis. These results demonstrate that TSP1 is the major endogenous activator of TGF-beta in experimental inflammatory kidney disease.

IL-2 Protects Lupus-Prone Mice from Multiple End-Organ Damage by Limiting CD4−CD8− IL-17–Producing T Cells

IL-2, a cytokine with pleiotropic effects, is critical for immune cell activation and peripheral tolerance. Although the therapeutic potential of IL-2 has been previously suggested in autoimmune diseases, the mechanisms whereby IL-2 mitigates autoimmunity and prevents organ damage remain unclear. Using an inducible recombinant adeno-associated virus vector, we investigated the effect of low systemic levels of IL-2 in lupus-prone MRL/Faslpr/lpr (MRL/lpr) mice. Treatment of mice after the onset of disease with IL-2-recombinant adeno-associated virus resulted in reduced mononuclear cell infiltration and pathology of various tissues, including skin, lungs, and kidneys. In parallel, we noted a significant decrease of IL-17–producing CD3+CD4−CD8− double-negative T cells and an increase in CD4+CD25+Foxp3+ immunoregulatory T cells (Treg) in the periphery. We also show that IL-2 can drive double-negative (DN) T cell death through an indirect mechanism. Notably, targeted delivery of IL-2 to CD122+ cytotoxic lymphocytes effectively reduced the number of DN T cells and lymphadenopathy, whereas selective expansion of Treg by IL-2 had no effect on DN T cells. Collectively, our data suggest that administration of IL-2 to lupus-prone mice protects against end-organ damage and suppresses inflammation by dually limiting IL-17–producing DN T cells and expanding Treg.

Bimodal regulation of T cell-mediated immune responses by TIM-4

T cell Ig and mucin domain (TIM)-4 is preferentially expressed on antigen-presenting cells, and its counter-ligand, TIM-1, is thought to deliver co-stimulating signals to T cells. However, the physiological functions of TIM-4 remain unclear. Here, we demonstrate that TIM-4 inhibits naive T cell activation through a ligand other than TIM-1. The inhibitory effect of TIM-4 was specific to naive T cells which do not express TIM-1, and the effect disappeared in pre-activated T cells. Conversely, antibody-mediated blockade of TIM-4 in vivo substantially suppressed T cell-mediated inflammatory responses despite enhanced generation of antigen-specific T cells. Furthermore, treatment with anti-TIM-4 reduced the inflammatory responses developed in mice that were adoptively transferred with antigen-primed T cells. These results suggest that TIM-4 exerts bimodal functions depending on the activation status of T cells.

Calcium/Calmodulin-Dependent Protein Kinase IV Suppresses IL-2 Production and Regulatory T Cell Activity in Lupus

The activity of calcium/calmodulin-dependent protein kinase IV (CaMK4) is increased in T cells from patients with systemic lupus erythematosus (SLE) and has been shown to reduce IL-2 production by promoting the effect of the transcriptional repressor cAMP responsive element modulator-α on the IL2 promoter. In this article, we demonstrate that T cells from MRL/lpr mice display increased levels of CaMK4 in the nucleus, and that genetic deletion of Camk4 results in improved survival. We demonstrate that absence of CaMK4 restores IL-2 production, curbs increased T cell activation, and augments the number and activity of regulatory T cells. Analogously, silencing of CaMK4 in T cells from patients with SLE increases the expression of FoxP3 on stimulation in the presence of TGF-β. Our results demonstrate the importance of the serine/threonine kinase CaMK4 in the generation and function of regulatory T cells in patients with SLE and lupus-prone mice, and its potential to serve as a therapeutic target.