Clinical immune response to rhinovirus challenge in human asthmatics

描述(申请人提供)：大多数儿童和年轻人的哮喘恶化是由鼻病毒(RV)感染引起的。作为哮喘相关发病率的最重要原因，在人类身上进行临床调查以确定潜在的发病机制是至关重要的。我们假设，哮喘患者鼻子中产生的免疫反应是随后免疫系统的系统调节的基础，而在易感人群(既往患有哮喘的人)中，这种改变的鼻部环境是哮喘恶化的原因。具体地说，我们认为这种修饰在哮喘患者的上呼吸道对RV产生了一种独特的免疫应答模式，这触发了Th2细胞因子“签名”状态的发展，从而驱动了哮喘患者下呼吸道RV感染的不良后果。此外，我们认为，在这些过敏性哮喘患者中，由于有效的抗RV免疫反应的发展和表达存在缺陷，导致对RV的更大易感性，这种Th2诱导的鼻呼吸道环境的强度进一步被夸大。确定鼻病毒引起的哮喘加重的病因将确定预防和治疗这些发作的具体目标。具体目标1将解决这一假设，即在过敏性呼吸道疾病的演变过程中，鼻部上皮细胞(EC)发生表观遗传变化，从而导致鼻部EC 被编程为产生细胞因子，以协调过敏性炎症免疫反应。我们主要将确定过敏性哮喘患者的鼻黏膜上皮在感染RV时是否被编程为分泌促进Th2细胞因子“签名”的细胞因子(IL-25、IL-33和TSLP)。特定目标2将询问补充假设，即哮喘患者对RV的易感性增加和鼻部感染的程度会放大这种Th2诱导偏向的后果。具体地说，我们认为随着时间的推移，哮喘患者的鼻黏膜上皮被表观遗传学重新编程，导致EC对轮状病毒感染的易感性更高。首先，我们将在体外分析鼻腔内皮细胞，以检验这一假设，即哮喘患者的鼻腔内皮细胞更容易受到RV感染，表现为RV复制的数量更大，感染节奏更快，RV感染细胞总体上死亡更多。然后，我们将通过用RV感染哮喘患者和对照组的体内研究来证实这一体外分析，监测一段时间内的病毒载量，作为感染速度的衡量标准。最重要的是，我们将在感染高峰期进行鼻部活检，以确定病毒感染的程度，以及这是否代表细胞病变(坏死性)或凋亡性细胞死亡。最后，特殊目标3将解决哮喘患者抗病毒免疫缺陷的分子和细胞基础。我们将在RV感染前建立对照组和哮喘患者EC的原代培养，并检测基线和RV感染诱导的抗病毒免疫中心细胞因子(IFNS-α、-？、λ和IL-15)的表达。我们预计，抗病毒介体的表达将与RV感染的易感性、节奏和严重程度呈负相关。

DESCRIPTION (provided by applicant): Most asthma exacerbations in children and young adults result from rhinovirus (RV) infections. As the most important cause of asthma-related morbidity it is essential that clinical investigations be performed in humans to define the underlying mechanisms. We hypothesize that the immune responses generated in the nose of asthmatics underlie subsequent systemic modulation of the immune system, and that - in susceptible individuals (those with pre-existing asthma) - this modified nasal milieu is responsible for the asthma exacerbation. Specifically, we propose that this modification produces a distinct pattern of immune responsiveness to RV in the upper airway of asthmatics, which triggers the development of a Th2 cytokine "signature" state that drives the adverse outcome of RV infection in the lower airway of asthmatics. In addition, we propose that the intensity of this Th2-inducing nasal airway milieu is further exaggerated in these allergic asthmatics, by a concomitant defect in the development and expression of effective anti-RV immune responses, leading to greater susceptibility to RV. Determining the etiology of rhinovirus-induced asthma exacerbations will identify specific targets to prevent and treat these episodes. Specific Aim 1 will address the hypothesis that epigenetic changes develop in nasal epithelial cells (EC) during the evolution of allergic airway disease as a result of which nasal EC are programmed to produce cytokines central to orchestrating an allergic inflammatory immune response. We will primarily determine whether nasal epithelium from allergic asthmatics, when infected with RV, is programmed to secrete cytokines that promote a Th2 cytokine "signature" (IL-25, IL- 33, and TSLP). Specific Aim 2 will interrogate the complementary hypothesis that increased susceptibility to RV and extent of nasal infections in asthmatics amplifies the consequences of this Th2-inducing bias. Specifically we propose that over time nasal epithelium in asthmatics is epigenetically re-programmed, resulting in the greater susceptibility of EC to RV infection. Initially we will analyze nasal EC ex vivo to test the hypothesis that EC from asthmatics will be more susceptible to RV infection as manifested by a greater magnitude of RV replication, a more rapid tempo of infection and overall greater death of RV-infected cells. We will then corroborate this in vitro analysis with in vivo studies by infecting asthmatics and controls with RV, monitoring viral load over time as a measure of the pace of infection. Most importantly, we will perform nasal biopsies at the peak of infection to determine the extent of viral infection and whether this represents cytopathic (necrotic) or apoptotic cell death. And, finally, Specific Aim 3 will address the molecular and cellular basis for the defect in anti-viral immunity in asthma. We will establish primary cultures of EC from control and asthmatic individuals prior to RV infection and examine them for baseline- and RV infection- induced expression of cytokines central to anti-viral immunity (IFNs-α, -ß, and λ and IL-15). We expect that anti-viral mediator expression will correlate inversely with the susceptibility, tempo, and severity of RV infection.