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。巨噬细胞和粒细胞的脱颗粒以及T细胞产生的细胞因子主要是由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内G蛋白偶联信号转导的机制以及随后发生炎症的途径。 GPCRs激活异三聚体G蛋白，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过敏性炎症部位的募集。趋化因子是作用于白细胞中GPCRs的一类主要化合物，在免疫反应过程中协调细胞的运输。我们发现RGS16富含在激活/效应T淋巴细胞中。我们发现RGS16通过调节趋化因子诱导的T细胞运输来抑制肺部炎症，以响应曼氏血吸虫蠕虫的攻击。幼稚的RGS16缺陷小鼠通过在肺中积累CCR10+T细胞来准备炎症。在病原体暴露后，这些小鼠比野生型小鼠发展出更强大的肉芽肿性肺纤维化。表达CCR4或CCR10的不同的Th2或假定的Th17亚群在Rgs16基因缺失小鼠攻击后在肺中积累得更快，并产生促炎细胞因子IL-13和IL-17B。缺乏RGS16的Th1和Th2细胞分别比野生型趋化因子CXCL9和CCL17迁移更多。RGS16缺陷的Th2细胞在被攻击的肺中异常定位。在Rgs16基因敲除小鼠的肺肉芽肿中，T淋巴细胞部分被排除在外，而是形成了支气管周围/血管周围的聚集体。因此，RGS16介导的T细胞对血吸虫肉芽肿的限制可以减轻广泛存在的细胞因子介导的肺部炎症。 第二个研究领域是过敏反应中控制嗜碱性细胞运输的机制。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。我们研究了包括嗜碱性粒细胞和T细胞在内的免疫细胞对模型蛋白酶变应原木瓜酶的反应。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，但这些粒细胞在变态反应性炎症中的确切作用尚不完全清楚。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。我们研究了木瓜酶如何诱导嗜碱性粒细胞迁移到LNS，以及各种白细胞在木瓜酶诱导的免疫反应中的作用。我们用木瓜酶在足垫免疫小鼠，并研究了引流窝淋巴结中白细胞的募集和炎性细胞因子和趋化因子的产生。木瓜酶通过蛋白水解酶激活受体(PAR)2直接激活幼稚T细胞，启动包括CCL17、CCL22和IL-4在内的趋化因子/细胞因子程序。木瓜酶触发的趋化因子和细胞因子的产生以及嗜碱性粒细胞向LNS的运输都依赖于CD4T细胞和PAR2，在没有CCR4的情况下，这种作用强烈减少。CCR4是CCL17/CCL22的主要受体。

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 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 intracellular G-protein-coupled signal transduction in immune cells and subsequent pathways to inflammation. GPCRs activate 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 allergic inflammation. Chemokines are a major class of compounds acting on GPCRs in leukocytes, which orchestrate cell trafficking during the immune response. We found that RGS16 is enriched in activated/effector T lymphocytes. We showed that RGS16 constrains pulmonary inflammation by regulating chemokine-induced T cell trafficking in response to challenge with the helminth Schistosoma mansoni. Naive RGS16-deficienct mice were primed for inflammation by accumulation of CCR10+ T cells in the lung. Upon pathogen exposure, these mice developed more robust granulomatous lung fibrosis than wild-type counterparts. Distinct Th2 or putative Th17 subsets expressing CCR4 or CCR10 accumulated more rapidly in lungs of Rgs16 gene-deleted mice following challenge and produced proinflammatory cytokines IL-13 and IL-17B. Th1 and Th2 cells lacking RGS16 migrated more than wild-type counterparts to the chemokines CXCL9 and CCL17, respectively. RGS16-deficient Th2 cells localized aberrantly in challenged lungs. T lymphocytes were partially excluded from lung granulomas in Rgs16 knockout mice, instead forming peribronchial/perivascular aggregates. Thus, RGS16-mediated confinement of T cells to Schistosome granulomas mitigates widespread cytokine-mediated pulmonary inflammation. A second research area is the mechanisms controlling basophil trafficking in allergic responses. Many allergens contain intrinsic proteolytic activity and bind protease activated GPCRs. We studied the response of immune cells including basophils and T cells to the model protease allergen papain. Although sensitization to protease allergens, such as papain, helminth infection, chronic allergic skin inflammation, and nasal rhinitis are associated with basophil recruitment to inflammed tissue or to draining lymph nodes (LNs), the precise role of these granulocytes in allergic inflammation 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 studied how papain induces basophil migration to LNs and the contribution of various leukocytes to papain-induced immune responses. We immunized mice in the footpad with papain and studied leukocyte recruitment and inflammatory cytokine and chemokine production in the draining popliteal LNs. Papain directly activated naive T cells through protease-activated receptor (PAR)2 to initiate a chemokine/cytokine program that includes CCL17, CCL22, and IL-4. Papain-triggered chemokine and cytokine production and basophil trafficking to LNs were dependent on both CD4 T cells and PAR2 and were strongly reduced in the absence of CCR4, the primary receptor for CCL17/CCL22.