FUNGAL PATHOGENESIS OF MODERATE TO SEVERE ASTHMA

这项建议的广泛、长期的目标是了解慢性哮喘的原因，从而 提高这一常见疾病的诊断和治疗水平。我们之前展示了先天的 对呼吸道真菌感染的免疫激活与T辅助细胞2(TH2)的发展有关。 偏向的过敏性呼吸道炎症和相关疾病(哮喘和慢性鼻窦炎)。真菌是 在人类环境中普遍存在，并通过不断的变化很容易进入呼吸道粘膜 吸入分生孢子(孢子)。我们已经证明，从人类呼吸道分离出的真菌可以引起呼吸道 小鼠的高反应性，表明呼吸道真菌的生长，即呼吸道真菌病，激活了先天和 获得性免疫反应可能导致易感人群哮喘。这一点进一步得到了 我们在分泌真菌蛋白酶的小鼠身上发现，它会在呼吸道中裂解纤维蛋白原，形成裂解 激活Toll样受体4(TLR4)以诱导抗真菌天然免疫反应的产物(FCP)。 然而，这些因素如何调节肺部的过敏性炎症仍不清楚。我们的中央 假说认为，真菌蛋白酶介导的纤维蛋白原裂解引发过敏性呼吸道疾病和 呼吸道内的抗真菌先天免疫反应。我们将通过以下目标来检验这一假设：1) 确定FCPS启动变态反应性炎症和抗真菌免疫的分子机制 至TLR4。假说：真菌蛋白酶裂解纤维蛋白原产生FCP，通过TLR4传递信号 CD18-CD11b整合素异源二聚体(Mac-1)激活STAT6和NF-κB。 STAT6、NF-κB和mac-1的结构性和靶向性缺失以及携带该基因突变的小鼠 纤维蛋白原伽马链阻止与Mac-1结合来验证我们的假设，证实了我们的发现 人单核细胞来源的巨噬细胞。2)确定FCPS如何引发过敏性炎症和抗真菌 哮喘患者的呼吸道上皮细胞免疫功能。假设：1)上皮细胞分泌凝血因子 对真菌蛋白水解酶的应答2)FCP由增强型蛋白介导的过敏和抗真菌反应 呼吸道上皮细胞分泌呼吸道凝血因子。我们将确定其生理意义 FCP介导的凝血因子(如纤维蛋白原、凝血酶原)诱导的抗真菌免疫和 使用哮喘动物模型和人类呼吸道上皮细胞的慢性过敏性炎症。3)确定 哮喘合并呼吸道真菌病患者先天抗真菌免疫功能障碍的机制。假设： 来自中到重度哮喘和呼吸道真菌病患者的免疫细胞无法 在体外抑制真菌生长。我们将检测人类单核细胞来源的巨噬细胞的抗真菌能力。 (HMDM)抗真菌分生孢子。解决先天免疫有效和无效的标志 哮喘患者对真菌的反应，我们将研究极端表型，进行差异转录组 用RNA测序法分析HMDM对真菌分生孢子的反应

The broad, long-term objective of this proposal is to understand the causes of chronic asthma and thereby improve diagnosis and therapy of this common and debilitating ailment. We showed previously how innate immune activation in response to fungal infection of the airway is linked to the development of T helper 2 (TH2)- biased allergic airway inflammation and associated diseases (asthma and chronic rhinosinusitis). Fungi are ubiquitous in human environments and readily gain access to the airway mucosal membrane through constant inhalation of conidia (spores). We have shown that fungi isolated from the human airway can cause airway hyperreactivity in mice, suggesting that airway fungal growth, i.e., airway mycosis, activates innate and acquired immune responses that could cause asthma in susceptible individuals. This is further supported by our discovery in mice that secreted fungal proteinases cleave fibrinogen in the airways to form cleavage products (FCPs) that activate Toll like receptor 4 (TLR4) to induce fungistatic innate immune responses. However, how these factors mediate allergic inflammation in the lungs remain unknown. Our central hypothesis states that fungal proteinase-mediated cleavage of fibrinogen initiates allergic airway disease and fungistatic innate immune responses in the airways. We will test this hypothesis through the following Aims: 1) Determine the molecular mechanism by which FCPs initiate allergic inflammation and antifungal immunity through TLR4. Hypotheses: Fungal proteinases cleave fibrinogen to yield FCPs that 1) signal through TLR4 via the CD18-CD11b integrin heterodimer (Mac-1) to 2) activate STAT6 and NF-κB. We will use mice with constitutive and targeted deletions of STAT6, NF-κB, and Mac-1 as well as mice harboring a mutation in the fibrinogen gamma chain that prevents binding to Mac-1 to test our hypothesis, confirming our findings using human monocyte derived macrophages. 2) Determine how FCPs initiate allergic inflammation and antifungal immunity through airway epithelia of asthmatics. Hypotheses: 1) Epithelial cells secrete coagulant factors in response to fungal proteinases 2) FCPs initiate allergic and anti-fungal responses mediated by enhanced secretion of airway coagulant factors by airway epithelial cells. We will determine the physiological significance of FCP-mediated induction of clotting factors (e.g., fibrinogen, prothrombin) regarding antifungal immunity and chronic allergic inflammation using animal models of asthma and human airway epithelial cells. 3) Determine the mechanism of innate antifungal immune dysfunction in asthmatics with airway mycosis. Hypothesis: Immune cells from a subset of patients with moderate to severe asthma and airway mycosis are unable to restrain fungal growth in vitro. We will examine the fungistatic ability of human monocyte-derived macrophages (HMDM) against fungal conidia. To resolve the signatures of effective and ineffective innate immune responses to fungi in asthmatics, we will study extreme phenotypes, performing differential transcriptome analyses of HMDM in response to fungal conidia by RNA sequencing.