2014 Fiscal Year Research-status Report
Impact of the active lipid-transporter Spinster-homoloque-2 on S1P-mediated immune cell dynamics
Project/Area Number |
26860329
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Research Institution | Osaka University |
Principal Investigator |
シモンス シャンドゥア 大阪大学, 免疫学フロンティア研究センター, 特任助教(常勤) (60598176)
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Project Period (FY) |
2014-04-01 – 2016-03-31
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Keywords | Spns2 / lymphocyte migration / S1P / gut-immunity |
Outline of Annual Research Achievements |
We revealed that Lyve1-CRE/Spns2Δ/Δ (KO) mice have an impaired distribution of B- and T-cells in peripheral lymphoid organs. We found mature rec. B- and T-cells to be strongly reduced in pLN, mLN, spleen, BM, blood and lymph. Short-term homing assays showed that lymphocyte entry in KO mice was impaired in pLN and mLN. IHC analyses of KO mice showed significantly smaller HEVs then the controls. Analyses of the PPs showed an elevated lymphocyte entry rate and a strong accumulation of B- and T-cells in PPs of KO mice. We could show that egress of lymphocytes from pLNs, mLN and PPs of KO mice is impaired. Strikingly, lymphocyte egress in PP is about 20-fold reduced in comparison to pLN and mLN. FACS/IHC analyses of the lymph and lymph vessels showed almost complete absence of lymphocytes in KO mice. Masspectrometric analyses of blood and lymph revealed that S1P is strongly reduced in lymph but not in blood whereas other lysophospholipids were not affected. We could show that IgG1 and IgG3 immunoglobulin levels are significantly reduced but IgA levels are enhanced in blood of KO mice. Furthermore, IgA levels were strongly reduced in feces. FACS analyses revealed that B220-/IgA+ plasmablast were elevated in blood, spleen, pLN, BM and PC but where reduced in PPs. 16s-pyrosequencing of feces detected bacterial dysbiosis in KO mice represented by expansion of phyla like firmicutes, proteobacteria, tenericutes, actinobacteria, TM7, deferribacteria and other. PPs of KO mice showed impaired integrity of their stromal cell microenvironment and appearance of gp38hi expressing cells.
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Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We were able to generate and characterize Lyve1-CRE/Spns2Δ/Δ. As described under “research achievements” we were able to successfully perform and answer the working questions 1.-3. of the research plan for FY2014 of the original application. In addition, we got indication that HEVs of pLNs and PP are differently regulated, possibly by DCs. Furthermore, we could describe the consequences of impaired egress of effector cells into lymphatic endothelium on gut-associated lymphoid tissues and the integrity of intestinal commensal bacteria populations. In addition, we discovered an unexpected dependency of stromal cells in the PPs on accumulated lymphocytes and/or lymphatic endothelium derived S1P. Hence, we generated Lyve1-CRE/Spns2Δ/Δ mice on a CXCL12-EGFP background in order to characterize specifically this phenomenon in the future. In addition, we were able to successfully breed Lyve1-CRE/Spns2Δ/Δ mice on an R26r-tdTomato background in order to be able to visualize lymphocyte migration in lymph vessels in the future.
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Strategy for Future Research Activity |
From our recent results the focus of our study has changed from the working questions 4.-5. of the research plan for FY2014 of the original application to the following questions and hypotheses: 1. Moussion C & Girard JP, Nature, 2011 as well as Wendland et al., Immunity, 2011 have shown that activated CCR7+ dendritic cells (DC) are important regulators of HEV function, thereby regulating lymphocyte immigration into pLNs. Since we observed the phenomenon of hypotrophic pLNs and hypertrohic PPs as well as impaired immigration of lymphocytes in pLN and mLN, but not into PPs, we will focus on the role of DCs in regulating HEV function in KO mice. Therefore we will analyze DC subsets in lymphoid organs of KO mice and their interaction with HEVs using FACS and IHC. Since in our observation regulation of HEV function and integrity in PPs seems to be different to pLNs we would like to take advantage of CD11c-DTR mice in order to analyze the effect of DCs deletion on lymphocyte immigration into PPs in comparison to pLN. 2. The appearance of an unusual gp38hi expressing stromal cell in hypertrophic PPs of KO animals caught our intention in order to analyze the dependency of PP stromal microenvironment on lymphocyte accumulation and/or S1P deficiency. Schmidt TH et al., JEM, 2013, has reported an unique organization of the stromal cell architecture in PPs which shows CXCL12+ perilymphatic zones in comparison to that of pLNs. Hence, we would like to analyze the stromal cells in PP of Lyve1-CRE/Spns2Δ/Δ mice on a CXCL12-EGFP background.
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Research Products
(3 results)