Heterotrimeric G Protein Signaling In Allergic Inflammation

肥大细胞(MC)、嗜碱性粒细胞和淋巴细胞是过敏反应发生过程中不可或缺的部分。巨噬细胞和粒细胞脱颗粒，T细胞产生细胞因子，B细胞产生抗体，主要是通过抗原受体的交联性诱导的。然而，过敏性炎症也可能通过激活与异三聚体G蛋白(GPCRs)偶联的受体而产生。本研究的目的是了解免疫细胞中G蛋白介导的信号转导机制以及随后发生炎症的途径。 GPCRs激活异源三聚体G蛋白的核心途径，与鸟苷三磷酸(GTP)结合，以换取鸟苷二磷酸(GDP)。G蛋白α亚单位的GTP结合形式诱导下游信号级联反应，包括负责MC/嗜碱性粒细胞脱颗粒的细胞内钙离子通量。该项目关注于G蛋白信号转导调节蛋白家族(RGS蛋白)，它们通过增加GTP酶的活性来抑制Gα-I和Gα-Q蛋白的功能，但不抑制Gα-S蛋白的功能。Gα亚基根据相关受体的配基占有率在GDP结合形式(不活跃)和GTP结合形式(活跃)之间振荡。RGS蛋白的GTP酶加速(GAP)活性限制了活性G-α与其效应分子相互作用的时间，导致GCPR信号失敏。尽管关于RGS作用的生化机制的知识越来越多，但对这些蛋白质在过敏性炎症中的生理作用知之甚少。 一个主要的研究领域是炎症细胞在炎症部位的募集。趋化因子是作用于白细胞GPCRs的一类主要化合物，它协调免疫细胞的运输，而RGS蛋白包括RGS13和RGS16通过脱敏GPCRs信号来抑制趋化因子信号。在高亲和力抗体反应过程中，CXCL12和CXCL13在淋巴器官如脾和淋巴结形成生发中心(GC)。GCS提供了一个促进和调节B细胞与滤泡Th(TFH)细胞相互作用的微环境。我们发现IL-17和IL-21在自身免疫性Bxd2小鼠自发GCs的形成和致病性自身抗体的产生中都起着重要作用。Bxd2-Il17ra-/-小鼠的CXCR5+TFH细胞大部分错配于GC光区(LZ)。IL-17信号的急性中断破坏了TFH-B的相互作用，并阻止了Bxd2小鼠体内形成自身抗体的B细胞的产生。IL-17上调RGS16的表达，促进TFH与B细胞形成结合的能力，这种作用在Bxd2-RGS16-/-小鼠的TFH细胞中被取消。结果提示，IL-17诱导趋化因子停止信号(RGS16)作用于分化后的IL-17RA+TFH，使其能够与LZ的反应性B细胞相互作用。 用Bxd2-RGS13-/-小鼠研究了RGS13促进GCs产生致病自身抗体的机制。在Bxd2小鼠的脾中，RGS13主要由GC B细胞表达，受IL-17刺激，但不受IL-21刺激。IL-17上调A20 GC细胞Rgs13表达，但不上调70z/3非GC B细胞Rgs13表达。Bxd2-RGS13-/-小鼠的GC较小，腺苷诱导脱氨酶(AID)水平较低，表明抗体应答过程中体细胞超突变和亲和力成熟程度较低。而Bxd2-Rgs13-/-小鼠Gc B细胞中IgMbright浆母细胞增多，血浆程序基因IRF4、Blimp1、XBP1和pCREB靶基因FosB和Obf1上调，GC程序基因Aicda、Pax5和Bach2下调。Bxd2-RGS13-/-小鼠表现出较低的免疫球蛋白自身抗体滴度和肾小球中的免疫球蛋白沉积，这表明自身抗体的致病性降低。这些结果表明，RGS13缺陷与Bxd2小鼠GC程序基因的减少和较低致病性的IgM浆母细胞的存在有关。一个延长的GC程序，通过上调RGS13，增强了AID的表达，并能够在自身反应性GC中产生致病自身抗体。 第二个研究领域是控制肥大细胞和嗜碱性细胞在过敏反应中运输的机制。许多变应原具有固有的蛋白分解活性，并与蛋白水解酶激活的GPCRs结合。尽管对木瓜酶、蠕虫感染、慢性过敏性皮肤炎和鼻炎等蛋白酶变应原的致敏作用与嗜碱性粒细胞聚集到炎症组织或引流淋巴结(LNS)有关，但嗜碱性粒细胞的确切作用及其募集机制尚不完全清楚。嗜碱性细胞具有向初始T细胞递呈抗原的能力，并通过产生IL-4直接或间接地促进TH2的分化。我们目前正在研究嗜碱性粒细胞对各种趋化剂的体外趋化作用，目的是确定在上述过敏过程中，哪些嗜碱性粒细胞会重新聚集到组织中。我们正在培育含有肥大细胞或嗜碱性粒细胞对趋化因子高或低反应的小鼠品系，以研究这些细胞对各种过敏反应的贡献。

Mast cells (MCs), basophils, and lymphocytes are integral to the development of an allergic response. Degranulation of MCs and granulocytes, and cytokine production by T cells and antibody production in B cells is induced primarily by cross-linking of the receptor for antigen. However, allergic inflammation may also be generated through activation of receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of G protein-mediated signal transduction in immune cells and subsequent pathways to inflammation. GPCRs activate a core pathway of heterotrimeric G proteins, which bind guanosine triphosphate (GTP) in exchange for guanosine diphosphate (GDP). The GTP-bound form of the G protein alpha subunit induces downstream signaling cascades, including intracellular calcium flux responsible for MC/basophil degranulation. This project focuses on a family of regulators of G protein signaling (RGS proteins), which inhibit the function of G alpha-i and G alpha-q, but not G alpha-s, proteins by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. The GTPase accelerating (GAP) activity of RGS proteins limits the time of interaction of active G-alpha and its effectors, resulting in desensitization of GCPR signaling. Despite a growing body of knowledge concerning the biochemical mechanisms of RGS action, relatively little is known about the physiological role of these proteins in allergic inflammation. A major area of investigation is the recruitment of inflammatory cells to sites of inflammation. Chemokines are a major class of compounds acting on leukocyte GPCRs, which orchestrate immune cell trafficking, and RGS proteins including RGS13 and RGS16 inhibit chemokine signaling by desensitizing GPCR signals. CXCL12 and CXCL13 shape the formation of germinal centers (GCs) in lymphoid organs such as spleen and lymph nodes during a high affinity antibody response. GCs provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. We showed that IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies in autoimmune BXD2 mice. The majority of CXCR5+ TFH cells from BXD2-Il17ra-/- mice were mis-localized in the GC light zone (LZ). Acute interruption of IL-17 signaling disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of RGS16 to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16-/- mice. The results suggests that IL-17 induces a chemokine stop signal (RGS16) that acts on postdifferentiated IL-17RA+ TFH to enable its interaction with responder B cells in the LZ. The mechanisms by which RGS13 promotes the generation of pathogenic autoantibodies in GCs were determined using BXD2-Rgs13-/- mice. In spleens of BXD2 mice, RGS13 was mainly expressed by GC B cells and was stimulated by IL-17 but not IL-21. IL-17 upregulated Rgs13 in A20 GC but not 70Z/3 non-GC B cells. BXD2-Rgs13-/- mice had smaller GCs and lower adenosine induced deaminase (AID) levels, suggesting lower somatic hypermutation and affinity maturation during the antibody response. There were, however, increased IgMbright plasmablasts, upregulation of plasma program genes Irf4, Blimp1, Xbp1 and pCREB target genes Fosb and Obf1, with downregulation of GC program genes Aicda, Pax5 and Bach2 in GC B cells of BXD2-Rgs13-/- mice. BXD2-Rgs13-/- mice showed lower titers of IgG autoantibodies and IgG deposits in the kidney glomeruli, suggesting reduced autoantibody pathogenicity. These results indicate that RGS13 deficiency is associated with reduction in GC program genes and exit of less pathogenic IgM plasmablasts in BXD2 mice. A prolonged GC program, mediated by upregulation of RGS13, enhanced AID expression and enabled generation of pathogenic autoantibodies in autoreactive GCs. A second research area is the mechanisms controlling mast cell and basophil trafficking in allergic responses. Many allergens contain intrinsic proteolytic activity and bind protease activated GPCRs. Although sensitization to protease allergens, such as papain, helminth infection, chronic allergic skin inflammation, and nasal rhinitis are associated with basophil recruitment to inflamed tissue or to draining lymph nodes (LNs), the precise role of basophils and mechanisms involved in their recruitment is incompletely understood. Basophils have the capacity to present antigen to naive T cells and promote TH2 differentiation directly or indirectly through IL-4 production. We are currently examining the chemotaxis of basophils to various chemoattractants in vitro with the goal of determining which are responsible for basophil recruitment to tissues during the aforementioned allergic processes. We are generating mouse strains containing mast cells or basophils hyper- or hyporesponsive to chemokines in order to study the contribution of these cells to various allergic responses.