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

• 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
• 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

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.

项目摘要/摘要 而急性肺损伤（ALI）和急性呼吸窘迫综合征发病机理的早期事件 （ARDS）已定义，对介导分辨率的机制知之甚少。开始搜索 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）并以第10天的间隔评估了反应，这是受伤解决的时期。支气管肺泡 I.T.（BAL）和肺介质增加LPS，其中包括Bal硝酸盐/硝酸盐（NOX）。与 NOX的增加，我们发现诱导型一氧化氮合酶（INOS）蛋白表达显着 在肺部上调，在受伤后第4天达到峰值。在iNOS - / - 小鼠中，早期肺损伤在 它。与WT小鼠相比，LPS，但是到第10天的恢复显着受损。 iNOS - / - 鼠有 死亡率增加以及持续升高的BAL蛋白，白蛋白，细胞，中性粒细胞和促脂 炎症细胞因子。 WT Inos+/+骨髓衍生的单核细胞或直接腺病毒的收养转移 iNOS的基因输送到受伤的iNOS - / - 小鼠（分别在I.T. LPS后1和2天）恢复 ALI以类似于WT的模式解决；相反，iNOS - / - 单核细胞的转移或假手术的传递 腺病毒没有达到解决方案。为了开始了解iNOS是如何为Ali解决的贡献，我们 在I.T.进行的肺泡细胞进行多色流式细胞仪。 LPS。我们明显观察到 调节性T细胞数量减少（Tregs）和巨噬细胞/单核细胞共同表达 与损伤后第7天的WT小鼠相比，iNOS - / - 小鼠中的信号分子（例如CD86和CD40）。 受伤的iNOS中的CD86的补充WT Tregs或抗体介导的封锁 - / - 小鼠明显地转移 恢复了肺损伤的分辨率。我们假设巨噬细胞衍生的Inos在 通过调节肺中的免疫反应来介导ALI的分辨率，从而促进肺泡的清除 炎症和促进肺修复。为了将这些发现转向临床背景下的考虑，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 受伤后的肺。使用体内和体外方法，我们将确定iNOS在修改中的作用 巨噬细胞/单核细胞先天免疫反应在损伤响应的不同阶段。 ionos- 介导对巨噬细胞共签名分子的影响及其在ALI分辨率方面的相对贡献将是 通过使用抗体阻断和骨芯嵌合体的创建进行检查。我们将比较 外源性与内源性NO在受伤后调节巨噬细胞免疫反应。 2。确定iNOS在调节肺泡巨噬细胞调节的T细胞相互作用中的作用 促进阿里的解决方案。体内和体外研究将用于评估巨噬细胞的作用 在调节Treg淋巴细胞表型和功能中得出的iNOS。 Treg衍生的潜在影响 iNOS也将对其进行研究。 3。确定iNOS的操纵是否可以改善不同模型的ALI模型。我们将使用 iNOS的直接腺病毒基因在体内或没有供体的供体，均可以恢复iNOS - / - 小鼠的ALI分辨率 I.T. LPS和直接革兰氏活细菌ALI模型。在SA 1和2中确定的目标（例如 信号分子）将被操纵，以试图加速“严重”受伤的WT的分辨率 动物。 负责解决ALI的机制的定义可能会为治疗提供新的目标 重要的临床状况，每年杀死75,000多人，目前为此治疗 仍然支持。我们认为，靶向iNOS表达，尤其是在Ali发作后的后期，可能 被证明是可以加速ALI患者分辨率的治疗。