Heterotrimeric G Protein Signaling In Allergic Inflammation

过敏性炎症中的异三聚体 G 蛋白信号传导

• 批准号: 8336114
• 负责人: Kirk m Druey
• 金额: $ 60.02万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8336114
• 关键词: Adenosine; Adrenergic beta-Agonists; Affect; Allergens; Allergic; Allergic Disease; Allergic inflammation; Anaphylaxis; Antigen Presentation; Antigen Receptors; Antigens; Area; Attenuated; B-Lymphocytes; Basophils; Binding; Biochemical; CCL17 gene; CCL22 gene; CD4 Positive T Lymphocytes; Cell Degranulation; Cells; Clinical; Collaborations; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cysteine Protease; Development; Disease; Effector Cell; Enzymes; Family; Fibrosis; G(q) Alpha; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Gene Deletion; Genetic; Genetic Polymorphism; Goals; Guanosine Diphosphate; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Half-Life; Helminths; Heterotrimeric GTP-Binding Proteins; Human; Human G(i) Alpha Proteins; Hypersensitivity; IgE; Immune; Immune response; Immunity; In Vitro; Infection; Infiltration; Inflammation; Inflammatory; Interleukin-4; Investigation; Knowledge; Laboratories; Leukocytes; Ligands; Lung; Lymphocyte; Maps; Mediating; Metabolism; Modeling; Mus; National Institute of Allergy and Infectious Disease; PAR-2 Receptor; Papain; Parasitic Diseases; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Physiological; Platelet Activating Factor; Process; Production; Protein p53; Proteins; Regulation; Role; Schistosoma; Schistosoma mansoni; Signal Pathway; Signal Transduction; Site; Source; T-Lymphocyte; Time; Translating; Work; allergic response; base; cell growth; chemokine; chemokine receptor; crosslink; cytokine; desensitization; environmental allergen; eosinophil; granulocyte; human MAPK14 protein; insight; lymph nodes; mast cell; migration; novel; promoter; receptor; receptor coupling; release of sequestered calcium ion into cytoplasm; response; therapeutic target; trafficking

Mast cells(MCs), basophils, eosinophils, 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. Compounds acting on GPCRs, such as platelet-activating factor (PAF) and adenosine, induce granulocyte and MC degranulation independently of IgE. In previous years' work, we identified an RGS protein, RGS13, which inhibits IgE-mediated mast cell degranulation and anaphylaxis in mice by binding to and counteracting activation of the critical downstream enzyme phosphoinositide-3 kinase (PI3 kinase). We also found that RGS13 regulates GPCR-induced degranulation and cytokine production by human MCs through its GAP activity. These results uncovered a new physiological function of RGS proteins with broad implications for cell growth, metabolism, and immunity: the direct inhibition of PI3 kinase. We hypothesized that abnormalities in RGS13 activity may exist in patients with anaphylaxis or other disorders that may be associated with increased mast cell reactivity. Current studies of RGS13 have focused on the regulation of its expression in immune cells. We found that the tumor suppressor p53 binds to a functional site in the RGS13 promoter, leading to inhibition of RGS13 expression in MCs and B lymphocytes. During the course of these studies, we also discovered (using MCs from p53-deficient mice) that p53 exerts a negative regulatory effect on MC degranulation but is required for IgE-antigen-induced cytokine production. These results suggest that p53 expression and/or function should also be examined in clinical disorders of MC degranulation such as anaphylaxis. We also found that RGS13 was phosphorylated by protein kinase A (PKA). GPCR ligands such as beta-adrenergic agonists generate intracellular cyclic adenosine monophosphate (cAMP), which in turn activates PKA. We mapped the site of RGS13 phosphorylation by PKA and showed that phosphorylated RGS13 protein had a prolonged half-life inside cells. Collectively, these studies have provided insight into the regulation of RGS13 expression, and we have begun to search for polymorphisms affecting RGS13 expression and/or function in subjects with anaphylaxis. The other major area of investigation in this project is the recruitment of inflammatory cells to sites of allergic inflammation. A major class of compounds acting on GPCRs in leukocytes are chemokines. Chemokines and their receptors orchestrate cell trafficking during the immune response. We found that RGS16 was expressed in activated Th1, Th2, and Th17 CD4+ effector T cells. RGS16-deficient T cells migrated more to Th2-associated chemokines such as CCL17 in vitro. These results translated into abnormal T cell trafficking in Th2-associated pathologies. In collaboration with Dr. Thomas Wynn (Laboratory of Parasitic Diseases, NIAID), we showed that lungs from Rgs16-/- mice infected with the helminth Schistosoma mansoni had more inflammation, fibrosis, and T cell infiltration than wild type counterparts. We concluded that RGS16 attenuates Th2 responses to Schistosoma antigens. We have also embarked on identification of chemokine receptors, G protein, and RGS proteins involved in regulating the function of basophils in allergic inflammation in mice. Sensitization to protease allergens, such as papain, or helminth infection, is associated with basophil recruitment to draining lymph nodes. Basophils may promote Th2 differentiation directly through antigen presentation or indirectly through IL-4 production. How papain induces basophil migration to lymph nodes is unknown. We found that papain directly activates a subset of naive T cells that expresses protease-activated receptor 2 (PAR2). Papain induced PAR2+CD4+ T cell production of CCL17, CCL22, and IL-4 upstream of basophils by activating p38 mitogen activated protein (MAP) kinase and Jak3-STAT signaling pathways. Papain-triggered accumulation of CCL17, CCL22, and basophils in lymph nodes was abolished by the absence of CD4+ T cells or PAR2, and basophil trafficking was strongly diminished by CCR4 deficiency. These results defined a novel innate function of naive T cells in the direct recognition of a cysteine protease allergen through PAR2, which precedes TH2 effector cell development. As many environmental allergens have proteolytic activty, these findings have broad implications for the allergic response and suggest viability of therapeutic targeting of PAR2 in allergic diseases.

肥大细胞（MC）、嗜碱性粒细胞、嗜酸性粒细胞和淋巴细胞是过敏反应发展的组成部分。 MC和粒细胞的脱粒以及T细胞的细胞因子产生主要由抗原受体的交联诱导。 然而，过敏性炎症也可能通过激活与异源三聚体G蛋白（GPCR）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的炎症途径。 GPCR激活异源三聚体G蛋白，其结合三磷酸鸟苷（GTP）以交换二磷酸鸟苷（GDP）。G蛋白α亚基的GTP结合形式诱导下游信号级联，包括负责MC/嗜碱性粒细胞脱粒的细胞内钙流。该项目的重点是G蛋白信号传导调节因子（RGS蛋白）家族，其通过增加GT3活性抑制G α-i和G α-q蛋白的功能，但不抑制G α-s蛋白的功能。G α亚基基于相关受体的配体占有率在GDP-（非活性）和GTP-（活性）结合形式之间振荡。RGS蛋白的GTP酶加速（GAP）活性限制了活性G-α及其效应物相互作用的时间，导致GCPR信号转导的脱敏。尽管关于RGS作用的生化机制的知识越来越多，但关于这些蛋白质在过敏性炎症中的生理作用的知识相对较少。 作用于GPCR的化合物，如血小板活化因子（PAF）和腺苷，诱导粒细胞和MC脱粒，而不依赖于IgE。 在前几年的工作中，我们鉴定了RGS蛋白RGS 13，其通过结合并抵消关键下游酶磷酸肌醇-3激酶（PI 3激酶）的活化来抑制小鼠中IgE介导的肥大细胞脱粒和过敏反应。 我们还发现RGS 13通过其GAP活性调节GPCR诱导的人MC的脱粒和细胞因子产生。这些结果揭示了RGS蛋白的一种新的生理功能，对细胞生长、代谢和免疫具有广泛的意义：直接抑制PI 3激酶。我们假设RGS 13活性异常可能存在于过敏反应或其他可能与肥大细胞反应性增加相关的疾病患者中。 目前对RGS 13的研究主要集中在其在免疫细胞中表达的调控上。 我们发现肿瘤抑制基因p53与RGS 13启动子中的一个功能位点结合，导致MCs和B淋巴细胞中RGS 13表达的抑制。 在这些研究的过程中，我们还发现（使用来自p53缺陷小鼠的MC），p53对MC脱粒发挥负调节作用，但需要IgE抗原诱导的细胞因子产生。 这些结果表明，p53的表达和/或功能也应该检查临床疾病的MC脱粒，如过敏反应。我们还发现RGS 13被蛋白激酶A（PKA）磷酸化。 GPCR配体如β-肾上腺素能激动剂产生细胞内环磷酸腺苷（cAMP），其继而激活PKA。 我们通过PKA定位了RGS 13磷酸化的位点，并显示磷酸化的RGS 13蛋白在细胞内具有延长的半衰期。 总的来说，这些研究提供了对RGS 13表达调控的深入了解，我们已经开始寻找影响过敏反应受试者RGS 13表达和/或功能的多态性。 本项目的另一个主要研究领域是炎症细胞在过敏性炎症部位的募集。作用于白细胞中的GPCR的主要类别的化合物是趋化因子。 趋化因子及其受体在免疫应答期间协调细胞运输。我们发现RGS 16在活化的Th 1、Th 2和Th 17 CD 4+效应T细胞中表达。在体外，RGS 16缺陷型T细胞更多地迁移到Th 2相关趋化因子（例如CCL 17）。 这些结果转化为Th 2相关病理中的异常T细胞运输。 与托马斯韦恩博士（寄生虫病实验室，NIAID）合作，我们发现感染曼氏血吸虫蠕虫的Rgs 16-/-小鼠的肺比野生型小鼠有更多的炎症、纤维化和T细胞浸润。我们的结论是RGS 16减弱了Th 2对血吸虫抗原的反应。 我们还开始鉴定参与调节小鼠过敏性炎症中嗜碱性粒细胞功能的趋化因子受体、G蛋白和RGS蛋白。对蛋白酶过敏原（如木瓜蛋白酶）或蠕虫感染的致敏作用与嗜碱性粒细胞向引流淋巴结的募集有关。嗜碱性粒细胞可以直接通过抗原呈递或间接通过IL-4产生促进Th 2分化。木瓜蛋白酶如何诱导嗜碱性粒细胞迁移到淋巴结尚不清楚。我们发现木瓜蛋白酶直接激活表达蛋白酶激活受体2（PAR 2）的幼稚T细胞亚群。木瓜蛋白酶通过激活p38丝裂原活化蛋白（MAP）激酶和Jak 3-STAT信号通路诱导PAR 2 + CD 4 + T细胞产生CCL 17、CCL 22和嗜碱性粒细胞上游的IL-4。 木瓜蛋白酶触发的CCL 17，CCL 22和嗜碱性粒细胞在淋巴结中的积累被取消的情况下，CD 4 + T细胞或PAR 2，嗜碱性粒细胞的运输强烈减少CCR 4缺陷。这些结果定义了幼稚T细胞在通过PAR 2直接识别半胱氨酸蛋白酶过敏原中的新的先天功能，其先于TH 2效应细胞发育。 由于许多环境过敏原具有蛋白水解活性，这些发现对过敏反应具有广泛意义，并表明在过敏性疾病中治疗靶向PAR 2的可行性。