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生过程中不可或缺的一部分。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。 作用于GPCRs的化合物，如血小板激活因子(PAF)和腺苷，不依赖IgE诱导粒细胞和MC脱颗粒。在前些年的工作中，我们发现了一种RGS蛋白，RGS13，它通过与关键的下游酶磷酸肌醇3激酶(PI3激酶)结合并对抗激活来抑制IgE介导的肥大细胞脱颗粒和过敏反应。我们还发现，RGS13通过其GAP活性调节GPCR诱导的人巨噬细胞脱颗粒和细胞因子的产生。这些结果揭示了RGS蛋白的一种新的生理功能，对细胞生长、新陈代谢和免疫具有广泛的意义：直接抑制PI3激酶。我们推测，RGS13活性异常可能存在于过敏反应或其他疾病患者中，这些患者可能与肥大细胞反应性增加有关。 目前对RGS13的研究主要集中在其在免疫细胞中的表达调控上。我们发现抑癌基因P53与RGS13启动子中的一个功能位点结合，导致MCs和B淋巴细胞中RGS13表达的抑制。在这些研究过程中，我们还发现(使用P53缺陷小鼠的MC)P53对MC脱颗粒具有负面调节作用，但对于IgE抗原诱导的细胞因子的产生是必需的。这些结果表明，临床上MC脱颗粒的疾病，如过敏反应，也应检测P53的表达和/或功能。我们还发现RGS13被蛋白激酶A(PKA)磷酸化。GPCR配体，如β-肾上腺素能激动剂，产生细胞内的环磷酸腺苷(CAMP)，进而激活PKA。我们用PKA定位了RGS13的磷酸化位置，结果表明，磷酸化的RGS13蛋白在细胞内有较长的半衰期。总之，这些研究为RGS13的表达调控提供了洞察力，我们已经开始寻找影响RGS13表达和/或功能的多态在过敏反应受试者中。 该项目的另一个主要研究领域是将炎症细胞招募到过敏性炎症部位。作用于白细胞中GPCRs的一类主要化合物是趋化因子。趋化因子及其受体在免疫反应中协调细胞的运输。我们发现RGS16在活化的Th1、Th2和Th17的CD4+效应T细胞中表达。在体外，缺乏RGS16的T细胞更多地迁移到与Th2相关的趋化因子，如CCL17。这些结果转化为Th2相关病理中T细胞的异常运输。在与托马斯·韦恩博士(寄生虫病实验室，NIAID)的合作中，我们发现感染曼氏血吸虫的RGS16-/-小鼠的肺比野生型小鼠的肺部有更多的炎症、纤维化和T细胞渗透。我们的结论是，RGS16减弱了Th2对血吸虫抗原的应答。 我们还开始了趋化因子受体、G蛋白和RGS蛋白的鉴定，这些蛋白参与调节嗜碱性粒细胞在小鼠过敏性炎症中的功能。对蛋白水解酶过敏原，如木瓜酶，或蠕虫感染，与嗜碱性粒细胞募集到引流淋巴结有关。嗜碱性粒细胞可直接通过抗原提呈或间接通过产生IL-4促进Th2分化。木瓜酶是如何诱导嗜碱性粒细胞向淋巴结迁移的尚不清楚。我们发现，木瓜酶直接激活表达蛋白酶激活受体2(PAR2)的幼稚T细胞的一个亚群。木瓜酶通过激活p38丝裂原活化蛋白(MAP)激酶和JAK3-STAT信号通路，诱导嗜碱性粒细胞上游的PAR2+CD4+T细胞产生CCL17、CCL22和IL-4。在缺乏CD4+T细胞或PAR2的情况下，木瓜酶引发的CCL17、CCL22和嗜碱性粒细胞在淋巴结中的积聚被取消，而CCR4缺乏则使嗜碱性粒细胞的运输显著减少。这些结果定义了幼稚T细胞通过PAR2直接识别半胱氨酸蛋白酶过敏原的一种新的固有功能，该功能先于TH2效应细胞的发育。由于许多环境过敏原具有蛋白分解活性，这些发现对过敏反应具有广泛的影响，并提示PAR2在变态反应性疾病中的治疗靶点的可行性。