Modulation of neonatal alveolar macrophage by cftr mutation

描述（由申请人提供）：在本提案中，我们将解决新生儿肺泡巨噬细胞（AM）作为发展中的囊性纤维化（CF）肺免疫反应受损的重要贡献者的经常争论的角色。AM在对细菌和病毒的初始防御中很重要，包括呼吸道合胞病毒（RSV），这是一种重要的病毒性病原体，可导致CF肺的显著发病率。作为少数几个专注于氧化应激对新生儿肺中AM功能影响的实验室之一，我们已经开始使用小鼠cftr敲除肠道校正模型（cftr KO）来评估cftr突变对新生儿AM的影响。虽然cftr KO小鼠本身不会自发地出现人类CF肺的特征性肺部病理，但在没有肺部感染的情况下，它们可以作为评估新生儿AM内在cftr改变的优秀模型。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。中药复方可显著增加tgf - β1和M2标记物精氨酸酶1 (Arg1)，抑制吞噬功能。这表明cftr突变将新生儿AM的激活状态从病原体清除（经典激活，M1）转变为免疫抑制（替代激活，M2）。有趣的是，用幼年CF患儿获得的BALF治疗对照新生儿AM也模仿cftr KO AM的效果，增加氧化应激、TGFβ1和M2极化。细胞外谷胱甘肽（GSH）能够减弱增加的TGFβ1和Arg1，并改善cftr KO AM和cfalf暴露AM的吞噬能力，这证明了抗氧化剂谷胱甘肽（GSH）在cf相关降低中的核心作用。这表明氧化诱导的TGFβ1上调调节了新生儿cftr KO AM的炎症状态；但细胞外谷胱甘肽的增加减弱了cf诱导的tgf - β1信号及其对AM极性和功能的下游影响。我们的中心假设是，由于cftr突变导致的GSH池减少增加了AM氧化应激，并通过增加TGFβ1来损害吞噬作用，从而使新生儿AM向M2激活方向转移，降低新生儿肺部对RSV的防御能力。在Aim 1中，我们将确定cftr突变对操作细胞系基线和rsv刺激状态的影响，以及从cftr KO、ΔF508和G551D突变新生小鼠中分离的原发性新生儿AM。在目的2中，我们将研究维持谷胱甘肽池调节新生儿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的改变可能损害RSV的清除。AM的这种受损反应可能是促进中性粒细胞持续招募进入空域的信号。随着CF新生儿的早期识别，潜在的早期治疗干预措施的发展可以在呼吸道症状出现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合适目标。

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.