Modulation of neonatal alveolar macrophage by cftr mutation

描述（由申请人提供）：在本提案中，我们将讨论新生儿肺泡巨噬细胞（AM）作为发展中囊性纤维化（CF）肺中免疫反应受损的重要贡献者的经常争论的作用。AM在抵抗细菌和病毒（包括呼吸道合胞病毒（RSV），一种在CF肺中引起显著发病率的重要病毒病原体）的初始防御中是重要的。作为关注氧化应激对新生儿肺中AM功能的影响的少数实验室之一，我们已经开始使用小鼠cftr敲除肠道校正模型（cftr KO）评估cftr突变对新生儿AM的影响。虽然cftr KO小鼠本身不会自发地发展人类CF肺的特征性肺部病理，但它们在没有肺部感染的情况下充当评价新生AM中的内在cftr改变的极好模型。在cftr KO新生小鼠中，即使在没有感染的情况下，支气管肺泡灌洗液（BALF）中的氧化应激也增加。Cftr KO AM显著增加TGFβ1和M2标记物精氨酸酶1（Arg 1），并抑制吞噬作用。这表明cftr突变使新生儿AM的活化状态从病原体清除（经典活化，M1）向免疫抑制（替代活化，M2）转变。有趣的是，用从患有CF的幼儿获得的BALF处理的对照新生儿AM也模拟cftr KO AM的作用，具有增加的氧化应激、TGFβ1和M2极化。CF相关抗氧化剂谷胱甘肽（GSH）减少的核心作用通过细胞外GSH减弱增加的TGFβ1和Arg 1并改善cftr KO AM和CFBALF暴露AM中的吞噬作用的能力得到证实。这表明氧化剂诱导的TGFβ1上调调节新生cftr KO AM的炎症状态;但增加细胞外GSH减弱CF诱导的TGFβ1信号传导及其对AM极性和功能的下游作用。我们的中心假设是，cftr突变导致GSH池减少，增加AM氧化应激，并通过增加TGFβ1损害吞噬作用，从而使新生儿AM向M2活化转移，并降低新生儿肺对RSV的防御。在目的1中，我们将确定cftr突变对操作细胞系的基线和RSV刺激状态的影响，以及从cftr KO、ΔF508和G551 D突变的新生小鼠中分离的原代新生AM。在目标2中，我们将研究维持GSH池调节新生儿CF AM免疫功能的机制。AM激活状态的改变和随后的炎症信号传导的变化可能会损害RSV清除。AM的这种受损反应可能是促进中性粒细胞持续募集到空气中的信号。通过早期识别CF新生儿，开发潜在的早期治疗干预措施可以在呼吸道症状发生之前改变肺部炎症和感染。该提案将开始解决新生儿AM是否是开发此类潜在治疗策略的适当靶点。

DESCRIPTION (provided by applicant): In this proposal, we will address the often debated role of the neonatal alveolar macrophage (AM) as an important contributor to impaired immune responses in the developing cystic fibrosis (CF) lung. AM are important in the initial defenses against bacteria and viruses, including respiratory syncytial virus (RSV), an important viral pathogen which causes significant morbidity in the CF lung. As one of few laboratories focused on the effects of oxidative stress on the function of AM in the newborn lung, we have begun to evaluate the effects of cftr mutation on neonatal AM using a mouse cftr knockout gut corrected model (cftr KO). While cftr KO mice do not spontaneously develop characteristic pulmonary pathologies of human CF lung per se, they serve as excellent models to evaluate the intrinsic cftr alterations in neonatal AM, in the absence of pulmonary infection. In cftr KO neonatal mice, oxidative stress was increased in the bronchoalveolar lavage fluid (BALF), even in the absence of infection. Cftr KO AM had dramatic increases in TGFβ1 and the M2 marker arginase 1 (Arg1), and depressed phagocytosis. This suggests that the cftr mutation shifts the neonatal AM's activation state away from pathogen clearance (classically activated, M1) towards immunosuppression (alternative activation,M2). Interestingly, control neonatal AM treated with the BALF obtained from young children with CF also mimic the effects of the cftr KO AM with increased oxidant stress, TGFβ1 and M2 polarization. A central role for CF-related decreases in the antioxidant glutathione (GSH) was demonstrated by the ability of extracellular GSH to attenuate the increased TGFβ1 and Arg1 and improve phagocytosis in cftr KO AM and CFBALF-exposed AM. This suggested that oxidant-induced up- regulation of TGFβ1 modulated the neonatal cftr KO AM's inflammatory state; but increasing extracellular GSH blunted CF-induced TGFβ1 signaling and its downstream effects on AM polarity and function. Our central hypothesis is that decreased GSH pools due to cftr mutation increase AM oxidant stress and impair phagocytosis by increasing TGFβ1 thereby shifting neonatal AM towards M2 activation and diminishing the neonatal lung's defenses against RSV. In Aim 1, we will determine the effects of cftr mutations on baseline and RSV-stimulated state of manipulated cells lines, and primary neonatal AM isolated from cftr KO, ΔF508 and G551D mutated neonatal mice. In Aim 2, we will examine the mechanisms by which maintaining GSH pools modulates the immune functions of neonatal CF AM. Alterations in AM activation state and subsequent changes in inflammatory signaling may compromise RSV clearance. This impaired response by AM may then be the signal that promotes sustained neutrophil recruitment into the airspace. With early identification of the CF newborn, the development of potential early therapeutic interventions could modify pulmonary inflammation and infection before respiratory symptoms occur. This proposal will begin to address whether the neonatal AM is an appropriate target for the development of such potential therapeutic strategies.