Epigenetic Mechanism Reprogramming Mucosal Anti-viral Immunity in Allergic Asthma

项目摘要/摘要 鼻病毒(RV)感染是成人过敏性哮喘(AA)加重的最常见原因。 再生障碍性贫血患者的III型干扰素(IFNL)和T细胞对感染的反应失调，损害了病毒 通行证。我们发现，过敏原暴露使上皮干扰素调节因子(IRF)1-IFNL沉默 抗病毒应答和激活T细胞共抑制物程序性死亡配体(PDL)-1/B7H1的表达。 我们的数据表明锌指E盒(ZEB1)转录因子参与了这种表观遗传 结合组蛋白甲基转移酶(EZH2)和组蛋白乙酰转移酶(p300/CBP)的重编程 特定于启动者的上下文。我们将验证上皮细胞ZEB1是由转化生长因子β信号诱导的假设 由先天诱导的重塑产生，并由免疫调节嗜酸性粒细胞作用维持。这个 ZEB1的表观遗传作用沉默IRF1-IFNL反应但上调PDL1抑制CD8 T细胞 通过在特定启动子环境中招募不同的组蛋白乙酰转移酶来激活。我们的目标 目的：1.确定ZEB1诱导IRF1-IFNL抗病毒基因表观遗传沉默的机制 通过过敏原和嗜酸性粒细胞免疫调节的途径。我们将检查ZEB1-EZH2的效果 抑制正常和再生障碍性贫血上皮细胞中IRF1增强子/启动子的表观遗传修饰 通过精密核连续(PRO-SEQ)和染色质免疫沉淀。我们将研究Zeb的角色- EZH2在嗜酸性粒细胞调节抑制原代嗜酸性粒细胞共培养中的IFNL反应。这些 人ICAM1转基因小鼠对RV CDE重塑或不重塑的体内研究 感染。我们预计有缺陷的IRF/IFNL反应将通过ZEB1-EZH2沉默而逆转。2.澄清 ZEB1上调粘膜PD-L1表达的机制及其对CD8T的影响 细胞耐受性、RV清除和AHR。RV诱导的原代hAECs中PD-L1的表达 沉默ZEB1-p300/CBP通路后。PDL1对CD8-T细胞抑制的影响将在联合实验中进行测试 使用原代人类T细胞的培养系统。我们将在hICAM1中测试PD-L1在RV清除中的作用 封闭抗体转基因小鼠模型的建立及对PD-L1在支气管中表达上调的验证 腹主动脉硬化与正常对照的活检结果。我们预计，抑制PD-L1将增强CD8 T细胞的激活和 阻止AHR。3.检测RV16感染对人粘膜IFNL和PD-L1表达的影响 有或没有过敏原的志愿者都会诱发重塑。我们会招募嗜酸性粒细胞增多症的志愿者， 变应性鼻炎(AR)和正常对照组。我们将测试IRF1/IFNL诱导的重塑之间的关系 重组RV16感染后PD-L1的表达。我们希望IRF1-IFNL响应 主动重塑的AA患者CD8T细胞反应减弱。这项工程将大大推进 我们对粘膜免疫的表观遗传控制的理解，并为恢复正常提供了新的策略 再生障碍性贫血患者的粘膜先天防御。

PROJECT SUMMARY/ABSTRACT Rhinovirus (RV) infections are the most common causes of exacerbations in adults with allergic asthma (AA). Patients with AA have dysregulated type III interferon (IFNL) and T cell responses to infection, impairing viral clearance. We have found that allergen exposures silence the epithelial IFN regulatory factor (IRF)1- IFNL antiviral response and activate expression of the T cell co-inhibitor programmed death ligand (PDL)-1/B7H1. Our data implicate the Zinc Finger E box (ZEB1) transcription factor in mediating this epigenetic reprogramming by binding histone methyltransferase (EZH2) and histone acetyltransferase (p300/CBP) in a promoter-specific context. We will test the hypothesis that epithelial ZEB1 is induced by TGFβ signals produced by innate-induced remodeling and maintained by immunomodulatory eosinophil action. The epigenetic actions of ZEB1 silence the IRF1-IFNL response yet upregulate PDL1 to suppress CD8 T cell activation by recruitment of distinct histone acetyltransferases in specific promoter contexts. Our aims are to: 1. Determine the mechanism how ZEB1 induces epigenetic silencing of the IRF1-IFNL anti-viral pathway by allergen-and eosinophil-immunomodulation. We will examine the effect of ZEB1-EZH2 silencing on epigenetic modifications of the IRF1 enhancer/promoter in normal and AA epithelial cells (hAECs) by precision nuclear run-on (PRO-Seq) and chromatin immunoprecipitation. We will examine the role of ZEB- EZH2 in eosinophil-modulated suppression of IFNL responses in primary eosinophil co-cultures. These pathways will be probed in vivo by human ICAM1 transgenic mice with or without CDE remodeling upon RV infection. We expect the defective IRF/IFNL response will be reversed with ZEB1-EZH2 silencing. 2. Elucidate the mechanism how ZEB1 upregulates mucosal PD-L1 expression and determine its effects on CD8 T cell tolerance, RV clearance and AHR. RV-induced expression of PD-L1 will be measured in primary hAECs after silencing ZEB1-p300/CBP pathway. The effect of PDL1 on CD8-T cell suppression will be tested in co- culture systems using primary human T cells. We will test the role of PD-L1 in RV clearance in the hICAM1 transgenic mouse model using blocking antibodies (Abs) and validate the upregulation of PD-L1 in bronchial biopsies of AAs vs normal controls. We expect that PD-L1 inhibition will enhance CD8+ T cell activation and block AHR. 3. Test the effects of RV16 infection on mucosal IFNL and PD-L1 expression in human volunteers with or without allergen induced remodeling. We will recruit volunteers with eosinophilic AAs, allergic rhinitis (AR) and normal controls. We will test the relationship between remodeling induced IRF1/IFNL and PD-L1 expression in response to infection with recombinant RV16. We expect that IRF1-IFNL response and CD8+T cell response will be blunted in AAs with active remodeling. This project will significantly advance our understanding of the epigenetic control of mucosal immunity and provide new strategies to restore normal mucosal innate defenses in AA.