Mechanisms of IL-33 secretion in allergic diseases

项目摘要/摘要 这个项目的长期目标是研究基本的免疫学机制。 参与哮喘和过敏性疾病的发展。越来越清楚的是，细胞因子 由上皮细胞产生，包括胸腺基质淋巴生成素、IL-25和IL-33，在 形成2型免疫和过敏性疾病的病理生理学。然而，我们的主要差距仍然存在 关于分子和细胞控制这些细胞因子的产生和分泌的知识。在这 应用后，我们将研究IL-33的分泌机制。 IL-33主要存储在非血液病细胞的细胞核中；核定位是 这种细胞因子。我们之前发现，人类呼吸道上皮细胞暴露于真菌过敏原， 交链孢霉(Alternaria Alternata)引起细胞外快速释放三磷酸腺苷(ATP)，从而引发 细胞内钙浓度和IL-33分泌到细胞外环境。我们还发现，呼吸道 链格孢菌或屋尘螨变应原后上皮细胞释放乙酰胆碱(ACh) 曝光。重要的是，阻断上皮细胞上的M3 M受体(M3R)可抑制ATP的释放和释放 IL-33的分泌，提示ACh具有自分泌作用。因此，我们假设过敏原诱导的ACh 呼吸道上皮细胞的分泌在触发一系列细胞内事件中起着关键作用，从而导致 与IL-33的细胞外释放有关。 本提案中描述的实验将详细研究这一过程的关键步骤。在目标1中， 我们将确定暴露在空气中的过敏原是如何触发呼吸道ACh分泌和早期细胞反应的 体外培养上皮细胞。在目标2中，我们将研究上皮源性ACh如何通过以下方式促进IL-33的分泌 重点研究KEY的自分泌ACh/M3R信号通路和蛋白激酶C依赖的磷酸化 调节蛋白。在目标3中，我们将研究上皮性ACh/M3R信号在变应原诱导的IL-1中的作用。 33利用小鼠变态反应性炎症模型，在肺内分泌和启动2型免疫。 我们将利用分子和细胞生物学、药理学等方面的互补专业知识 奥格雷迪博士和基塔博士的实验室里。新颖而稳健的体外和体内模型 都是为这个项目开发的。这些研究将提供更好的了解上皮细胞是如何 对环境过敏原做出反应，并将确定关键的细胞和分子机制 IL-33的分泌。最终，这些研究将描述与变应原诱导有关的关键机制(S 免疫反应，允许识别新的治疗靶点(S)以治疗和理想地预防 免疫介导的呼吸道疾病，如哮喘、慢性鼻窦炎和其他过敏性疾病。

PROJECT SUMMARY/ABSTRACT The long-term objective of this project is to investigate the fundamental immunological mechanisms involved in the development of asthma and allergic diseases. It is becoming increasingly clear that cytokines produced by epithelial cells, including thymic stromal lymphopoietin, IL-25, and IL-33, play an important role in shaping type 2 immunity and in the pathophysiology of allergic diseases. However, major gaps remain in our knowledge concerning the molecular and cellular control of production and secretion of these cytokines. In this application, we will investigate the mechanisms involved in IL-33 secretion. IL-33 is primarily stored in the nucleus of non-hematologic cells; nuclear localization is a unique feature of this cytokine. We previously found that exposure of human airway epithelial cells to a fungal allergen, Alternaria alternata, evokes rapid extracellular release of ATP, which triggers sustained increases in intracellular calcium concentration and IL-33 secretion into the extracellular milieu. We also found that airway epithelial cells release acetylcholine (ACh) extracellularly following Alternaria or house dust mite allergen exposure. Importantly, blocking M3 muscarinc receptors (M3R) on epithelial cells suppressed ATP release and IL-33 secretion, suggesting an autocrine role for ACh. Therefore, we hypothesize that allergen-induced ACh secretion by the airway epithelium plays a pivotal role in triggering a sequence of intracellular events that lead to extracellular release of IL-33. The experiments described in this proposal will investigate key steps of this process in detail. In Aim 1, we will deterimne how airborne allergen exposure triggers ACh secretion and early cellular responses in airway epithelial cells in vitro. In Aim 2, we will investigate how epithelium-derived ACh facilitates IL-33 secretion by focusing on the autocrine ACh/M3R signaling pathway and protein kinase C-dependent phosphorylation of key regulatory proteins. In Aim 3, we will investigate the role of epithelial ACh/M3R signaling in allergen-induced IL- 33 secretion and initiation of type 2 immunity in the lung by using mouse models of allergic inflammation. We will employ a combination of complementary expertise in molecular and cell biology, pharmacology and immunology in the laboratories of Dr. O’Grady and Dr. Kita. Novel and robust in vitro and in vivo models have been developed for this project. These studies will provide a better understanding of how the epithelium responds to environmental allergens and will define key cellular and molecular mechanisms responsible for secretion of IL-33. Ultimately, these studies will characterize critical mechanism(s) involved in allergen-induced immune responses, allowing for identification of novel therapeutic target(s) to treat and ideally prevent immune-mediated airway diseases, such as asthma, chronic rhinosinusitis, and other allergic disorders.