掲載種別：研究論文（学術雑誌）  

Background and Aims: To examine the roles of microRNAs in the development of colitis, we conducted the RNA-sequencing studies using RNA derived from normal and colitogenic CD4+ T cells. Colitogenic CD4+ T cells demonstrated the increased expression of miR-150. We focused on the involvement of miR-150 in the colitis. Methods: We crossed miR-150 knockout mice and T-cell–specific Rap1KO mice, which is colitis model mice and spontaneously develop the colitis with tubular adenomas in microbiota-dependent manner. Results: MiR-150 silencing completely inhibited the expansion of pathogenic Th17 cells and the development of colitis. Conclusion: MiR-150 is a potential therapeutic target of inflammatory bowel diseases.

DOI： 10.1016/j.gastha.2023.01.021

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掲載種別：研究論文（学術雑誌）  

T-cell-specific Rap1 deletion causes spontaneous colitis in mice. In the present study, we revealed that Rap1 deficiency in T cells impaired the preceding induction of intestinal RORγt+ Treg cells. In the large intestinal lamina propria (LILP) of T-cell-specific Rap1-knockout mice (Rap1KO mice), Th17 cells were found to increase in a microbiota-dependent manner, and the inhibition of IL-17A production prevented the development of colitis. In the LILP of Rap1KO mice, RORγt+ Treg cells were scarcely induced by 4 weeks of age. The expression of CTLA-4 on Rap1-deficient Treg cells was reduced and the expression of CD80 and CD86 on dendritic cells was consequently elevated in Rap1KO mice. When cultured under each polarizing condition, Rap1-deficient naïve CD4+ T cells did not show biased differentiation into Th17 cells; their differentiation into Treg cells as well as Th1 and Th2 cells was lesser than that of wild-type cells. Rap1-deficient naïve CD4+ T cells were found to exhibit the defective nuclear translocation of NFAT and formation of actin foci in response to TCR engagement. These data suggest that Rap1 amplifies the TCR signaling required for Treg-mediated control of intestinal colitogenic Th17 responses.

DOI： 10.1038/s42003-022-03129-x

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掲載種別：研究論文（学術雑誌）  

Integrin regulation by Rap1 is indispensable for lymphocyte recirculation. In mice having B-cell-specific Rap1a/b double knockouts (DKO), the number of B cells in lymph nodes decreased to approximately 4% of that of control mice, and B cells were present in the spleen and blood. Upon the immunization with NP-CGG, DKO mice demonstrated the defective GC formation in the spleen, and the reduced NP-specific antibody production. In vitro, Rap1 deficiency impaired the movement of activated B cells along the gradients of chemoattractants known to be critical for their localization in the follicles. Furthermore, B-1a cells were almost completely absent in the peritoneal cavity, spleen and blood of adult DKO mice, and the number of B-cell progenitor/precursor (B-p) were reduced in neonatal and fetal livers. However, DKO B-ps normally proliferated, and differentiated into IgM+ cells in the presence of IL-7. CXCL12-dependent migration of B-ps on the VCAM-1 was severely impaired by Rap1 deficiency. Immunostaining study of fetal livers revealed defects in the co-localization of DKO B-ps and IL-7-producing stromal cells. This study proposes that the profound effects of Rap1-deficiency on humoral responses and B-1a cell generation may be due to or in part caused by impairments of the chemoattractant-dependent positioning and the contact with stromal cells.

DOI： 10.3389/fimmu.2021.624419

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掲載種別：研究論文（学術雑誌）  

Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in α4β7 integrin. We first inserted the PA tag into the plexin-semaphorin-integrin (PSI) domain of β7 chain, which reacted with an anti-PA tag antibody (NZ-1) in an Mn2+-dependent manner. The small GTPase Rap1 deficiency, as well as chemokine stimulation and the introduction of the active form of Rap1, Rap1V12, enhanced the binding of NZ-1 to the PA-tagged mutant integrin, and increased the binding affinity to mucosal addressing cell adhesion molecule-1 (MAdCAM-1). Furthermore, we generated two kinds of hybridomas producing monoclonal antibodies (mAbs) that recognized Mn2+-dependent epitopes of β7. Both epitopes were exposed to bind to mAbs on the cells by the introduction of Rap1V12. Although one epitope in the PSI domain of β7 was exposed on Rap1-deficienct cells, the other epitope in the hybrid domain of β7 was not. These data indicate that the conversion of Rap1-GDP to GTP exerts two distinct effects stepwise on the conformation of α4β7. The induction of colitis by Rap1-deficient CD4+ effector/memory T cells suggests that the removal of constraining effect by Rap1-GDP on α4β7 is sufficient for homing of these pathogenic T cells into colon lamina propria (LP).

DOI： 10.1038/s41598-020-70111-0

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掲載種別：研究論文（学術雑誌）  

Background: Lymphocytes circulate between peripheral lymphoid tissues via blood and lymphatic systems, and chemokine-induced migration is important in trafficking lymphocytes to distant sites. The small GTPase Rap1 is important in mediating lymphocyte motility, and Rap1-GEFs are involved in chemokine-mediated Rap1 activation. Here, we describe the roles and mechanisms of Rap1-GEFs in lymphocyte trafficking. Results: In this study, we show that RA-GEF-1 and 2 (also known as Rapgef2 and 6) are key guanine nucleotide exchange factors (GEF) for Rap1 in lymphocyte trafficking. Mice harboring T cell-specific knockouts of Rapgef2/6 demonstrate defective homing and egress of T cells. Sphingosine-1-phosphate (S1P) as well as chemokines activates Rap1 in a RA-GEF-1/2-dependent manner, and their deficiency in T cells impairs Mst1 phosphorylation, cell polarization, and chemotaxis toward S1P gradient. On the other hand, B cell-specific knockouts of Rapgef2/6 impair chemokine-dependent retention of B cells in the bone marrow and passively facilitate egress. Phospholipase D2-dependent production of phosphatidic acid by these chemotactic factors determines spatial distribution of Rap1-GTP subsequent to membrane localization of RA-GEFs and induces the development of front membrane. On the other hand, basal de-phosphorylation of RA-GEFs is necessary for chemotactic factor-dependent increase in GEF activity for Rap1. Conclusions: We demonstrate here that subcellular distribution and activation of RA-GEFs are key factors for a directional movement of lymphocytes and that phosphatidic acid is critical for membrane translocation of RA-GEFs with chemokine stimulation.

DOI： 10.1186/s12915-020-00809-0

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