Resolution and repair of acute lung injury by macrophage-derived iNOS

描述(申请人提供)：虽然在急性肺损伤(ALI)和急性呼吸窘迫综合征(ARDS)的发病机制中的早期事件已经被定义，但对调解解决的机制知之甚少。为了开始寻找潜在的解决决定因素，我们将野生型(WT)小鼠暴露于气管内脂多糖(I.T.LPS)，并每隔10天评估一次反应，这一时间段伤情已痊愈。肺泡灌洗液(BAL)和肺组织介质在I.T后明显升高。LP，其中BAL亚硝酸盐/硝酸盐(NOx)。与NOx增加相一致，我们发现肺组织诱导型一氧化氮合酶(INOS)蛋白表达显著上调，并在伤后第4天达到高峰。INOS-/-小鼠腹腔注射后早期肺损伤减轻。然而，与WT小鼠相比，第10天的恢复明显受损。INOS-/-小鼠死亡率增加，BAL蛋白、白蛋白、细胞、中性粒细胞和促炎细胞因子持续升高。将WT iNOS+/+骨髓来源的单核细胞过继转移或直接将iNOS腺病毒基因转移到受损的iNOS-/-小鼠(在I.T.后1天和2天给予)。脂多糖)可使ALI以类似于WT的模式恢复溶解；相反，iNOS-/-单核细胞转移或假腺病毒转染组不能恢复ALI的溶解。为了开始了解iNOS如何有助于ALI的缓解，我们在I.T后每隔一段时间对肺泡细胞进行多色流式细胞术。LP。我们观察到，与WT小鼠相比，iNOS-/-小鼠的调节性T细胞(Tregs)数量明显减少，巨噬细胞/单核细胞共信号分子(如CD86和CD40)的持续表达到伤后第7天。在受损的iNOS-/-小鼠体内转移补充的WT Tregs或抗体介导的CD86阻断可显著恢复肺损伤的缓解。我们推测，巨噬细胞来源的iNOS通过调节肺内的免疫反应，从而促进肺泡炎的清除，促进肺修复，在介导ALI的消退中发挥关键作用。为了推动这些发现在临床上得到考虑，我们提出了以下具体目标：1.确定iNOS调节肺损伤后巨噬细胞天然免疫反应的机制。利用体内和体外方法，我们将确定iNOS在改变巨噬细胞/单核细胞天然免疫反应中的作用，在损伤解决反应的不同阶段。通过抗体阻断和骨髓嵌合体的建立，将检测iNOS对巨噬细胞共信号分子的影响及其在ALI解决中的相对贡献。我们将比较外源性和内源性NO在调节创伤后巨噬细胞免疫反应中的作用。2.探讨诱导型一氧化氮合酶在调节肺泡巨噬细胞-调节性T细胞相互作用促进ALI消退中的作用。体内和体外研究将用于评估巨噬细胞来源的iNOS在调节Treg淋巴细胞表型和功能中的作用。还将研究Treg来源的iNOS的潜在影响。3.确定iNOS能否改善不同ALI模型的预后。我们将使用体内直接携带iNOS或NO供体的腺病毒基因，两者都可以在iNOS-/-小鼠体内恢复对ALI的分辨。脂多糖和直接活革兰氏阴性菌ALI模型。在SA 1和SA 2中确定的靶点(例如，共信号分子)将被操纵，以试图加速WT严重损伤动物的分解。对ALI解决机制的定义可能为每年导致75,000多人死亡的重要临床疾病的治疗提供新的靶点，目前治疗仍处于支持状态。我们相信，靶向iNOS的表达，特别是在ALI发病的后期，可能被证明是有效的治疗方法，以加速ALI患者的缓解。 公共卫生相关性：急性肺损伤(ALI)的解决机制的定义可能为每年导致75,000多人死亡的重要临床情况的治疗提供新的靶点，目前的治疗仍是支持性的。我们相信，靶向iNOS的表达，特别是在ALI发病的后期，可能被证明是有效的治疗方法，以加速ALI患者的缓解。

DESCRIPTION (provided by applicant): While early events in the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) have been defined, little is known about mechanisms mediating resolution. To begin to search for potential determinants of resolution, we exposed wild type (WT) mice to intratracheal lipopolysaccharide (i.t. LPS) and assessed the response at intervals out to day 10, a time when injury had resolved. Bronchoalveolar (BAL) and lung mediators increased after i.t. LPS, among them BAL nitrites/nitrates (NOx). Consistent with the increase in NOx, we found that inducible nitric oxide synthase (iNOS) protein expression was significantly upregulated in the lung and peaked at day 4 after injury. Early lung injury was attenuated in iNOS-/- mice after i.t. LPS, however recovery by day 10 was markedly impaired in comparison to WT mice. iNOS-/- mice had increased mortality as well as persistently elevated BAL protein, albumin, cells, neutrophils and pro- inflammatory cytokines. Adoptive transfer of WT iNOS+/+ bone marrow-derived monocytes or direct adenoviral gene delivery of iNOS into injured iNOS-/- mice (given 1 and 2 days after i.t. LPS respectively) restored resolution of ALI in a pattern similar to WT; in contrast, transfer of iNOS-/- monocytes or delivery of sham adenovirus did not achieve resolution. To begin to understand how iNOS contributed to resolution of ALI, we performed multicolor flow cytometry of alveolar cells at intervals after i.t. LPS. We observed markedly decreased numbers of Regulatory T cells (Tregs) and a sustained expression of macrophage/monocyte co- signaling molecules (e.g. CD86 and CD40) in iNOS-/- mice compared to WT mice out to day 7 after injury. Transfer of supplemental WT Tregs or antibody-mediated blockade of CD86 in injured iNOS-/- mice remarkably restored resolution of lung injury. We hypothesize that macrophage-derived iNOS plays a pivotal role in mediating resolution of ALI by modulating immune responses in the lung, thus facilitating clearance of alveolar inflammation and promoting lung repair. To move these findings towards consideration in a clinical context, we propose the following specific aims: 1. To identify mechanisms by which iNOS modulates macrophage innate immune responses in the lung after injury. Using in vivo and in vitro approaches, we will determine the role of iNOS in modifying macrophage/monocyte innate immune responses at different stages of the injury resolution response. iNOS- mediated effects on macrophage co-signaling molecules and its relative contribution in resolution of ALI will be examined by using antibody blockade and creation of bone-marrow chimeras. We will compare the effects of exogenous vs. endogenous NO in modulating macrophage immune responses after injury. 2. To determine the role of iNOS in modulating alveolar macrophage-Regulatory T cell interactions to promote resolution of ALI. In vivo and in vitro studies will be used to evaluate the role of macrophage- derived iNOS in modulating Treg lymphocyte phenotype and function. The potential effects of Treg-derived iNOS will be also be investigated. 3. To determine if manipulation of iNOS can improve outcomes in different models of ALI. We will use direct adenoviral gene delivery of iNOS or NO donors in vivo, both to restore resolution of ALI in iNOS-/- mice after i.t. LPS and direct live gram negative bacteria model of ALI. Targets identified in SA 1 and 2 (e.g. Co- signaling molecules) will be manipulated in an attempt to accelerate resolution in 'severely' injured WT animals. Definition of mechanisms responsible for resolution of ALI may provide novel targets for therapy in an important clinical condition which kills more than 75,000 people annually, and for which therapy at present remains supportive. We believe targeting iNOS expression, particularly at later stages after onset of ALI, may prove useful as therapy to accelerate resolution in patients with ALI. PUBLIC HEALTH RELEVANCE: Definition of mechanisms responsible for resolution of Acute Lung Injury (ALI) may provide novel targets for therapy in an important clinical condition which kills more than 75,000 people annually, and for which therapy at present remains supportive. We believe targeting iNOS expression, particularly at later stages after onset of ALI, may prove useful as therapy to accelerate resolution in patients with ALI.