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

巨噬细胞衍生的 iNOS 解决和修复急性肺损伤

基本信息

批准号：
8527234
负责人：
Franco R D'Alessio
金额：
$ 24.9万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-09 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8527234
关键词：
Acute Acute Lung Injury Adenoviruses Adoptive Transfer Adult Respiratory Distress Syndrome Albumins Alveolar Alveolar Cell Alveolar Macrophages Animals Antibodies Attenuated Blood capillaries Bone Marrow Cell Communication Cells Chimera organism Clinical Coupled Edema Epithelial Event Extravasation Flow Cytometry Gene Delivery Generations Gram-Negative Bacteria Healthcare Systems ITGAM gene Immune response In Vitro Inflammation Inflammation Mediators Inflammatory Injury Life Lipopolysaccharides Lung Lung Inflammation Lymphocyte Mediating Mediator of activation protein Modeling Molecular Mus Nitrates Nitric Oxide Nitric Oxide Donors Nitric Oxide Synthase Nitrites Outcome Pathogenesis Patients Pattern Permeability Phenotype Play Process Production Protein Isoforms Proteins Recovery Regulation Regulatory T-Lymphocyte Relative (related person)Reporting Resolution Role Signaling Molecule Staging Stimulus TNF gene TNFRSF5 gene Time Wild Type Mouse abstracting capillary cytokine human NOS2A protein improved in vivo injured killings lung injury lung repair macrophage monocyte mortality mouse model neutrophil novel protein expression repaired response

项目摘要

Project Summary/Abstract 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- signalling 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-signalling 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- signalling 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.

项目概要/摘要急性肺损伤（ALI）和急性呼吸窘迫综合征发病机制的早期事件（ARDS）已经被定义，但人们对调解解决机制知之甚少。开始寻找为了解决分辨率的潜在决定因素，我们将野生型（WT）小鼠暴露于气管内脂多糖（i.t. LPS）并每隔一段时间评估反应，直至第 10 天（损伤痊愈时）。支气管肺泡 (BAL) 和肺介质在 i.t. 后增加。 LPS，其中包括 BAL 亚硝酸盐/硝酸盐 (NOx)。符合 NOx 增加时，我们发现诱导型一氧化氮合酶 (iNOS) 蛋白表达显着增加肺部表达上调，并在受伤后第 4 天达到峰值。 iNOS-/- 小鼠的早期肺损伤在它。然而，与 WT 小鼠相比，LPS 在第 10 天的恢复明显受损。 iNOS-/- 小鼠有死亡率增加以及 BAL 蛋白、白蛋白、细胞、中性粒细胞和亲细胞持续升高炎症细胞因子。 WT iNOS+/+ 骨髓来源的单核细胞或直接腺病毒的过继转移将 iNOS 基因递送至受伤的 iNOS-/- 小鼠（分别在 i.t. LPS 后 1 和 2 天给予）恢复 ALI 的分辨率与 WT 类似；相反，iNOS-/-单核细胞的转移或假手术的递送腺病毒没有达到分辨率。为了开始了解 iNOS 如何帮助解决 ALI，我们在 i.t. 后每隔一定时间对肺泡细胞进行多色流式细胞术。脂多糖。我们明显观察到调节性 T 细胞 (Treg) 数量减少，巨噬细胞/单核细胞共表达持续表达损伤后第 7 天，iNOS-/- 小鼠与 WT 小鼠中的信号分子（例如 CD86 和 CD40）相比。在受伤的 iNOS-/- 小鼠中显着转移补充的 WT Tregs 或抗体介导的 CD86 阻断肺损伤恢复恢复。我们假设巨噬细胞衍生的 iNOS 在通过调节肺部的免疫反应来介导 ALI 的缓解，从而促进肺泡的清除炎症并促进肺部修复。为了将这些发现推向临床背景，我们提出以下具体目标： 1. 确定 iNOS 调节巨噬细胞先天免疫反应的机制肺部受伤后。使用体内和体外方法，我们将确定 iNOS 在修饰中的作用损伤缓解反应不同阶段的巨噬细胞/单核细胞先天免疫反应。诱导型一氧化氮合酶- 对巨噬细胞协同信号分子的介导作用及其在 ALI 解决中的相对贡献将是通过使用抗体阻断和骨髓嵌合体的创建进行检查。我们将比较效果外源性与内源性 NO 在调节损伤后巨噬细胞免疫反应中的作用。 2. 确定 iNOS 在调节肺泡巨噬细胞-调节性 T 细胞相互作用中的作用促进 ALI 的解决。体内和体外研究将用于评估巨噬细胞的作用衍生的 iNOS 调节 Treg 淋巴细胞表型和功能。 Treg 衍生的潜在影响 iNOS 也将受到调查。 3. 确定 iNOS 的操纵是否可以改善不同 ALI 模型的结果。我们将使用体内直接递送 iNOS 或 NO 供体的腺病毒基因，均可恢复 iNOS-/- 小鼠中 ALI 的缓解在它之后LPS 和 ALI 的直接活革兰氏阴性菌模型。 SA 1 和 2 中确定的目标（例如 Co- 信号分子）将被操纵，以试图加速“严重”损伤的WT的解决动物。解决 ALI 的机制的定义可能为治疗提供新的靶点每年导致 75,000 多人死亡的重要临床病症以及目前的治疗方法仍然支持。我们相信，针对 iNOS 表达，特别是在 ALI 发作后的后期，可能会被证明可作为加速 ALI 患者康复的疗法。