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: 7953158
• 负责人: Franco R D'Alessio
• 金额: $ 13.64万
• 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 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7953158
• 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; capillary; cytokine; human NOS2A protein; improved; in vivo; injured; killings; lung injury; macrophage; monocyte; mortality; mouse model; neutrophil; novel; protein expression; public health relevance; repaired; response

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.

描述（由申请人提供）：虽然已经定义了急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）发病机理的早期事件，但对介导分辨率的机制知之甚少。为了开始寻找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）中，并以第10天的间隔评估了响应，这是损伤解决的时期。 I.T. LPS，其中包括Bal硝酸盐/硝酸盐（NOX）。与NOX的增加一致，我们发现可诱导的一氧化氮合酶（INOS）蛋白表达在肺中显着上调，并在损伤后第4天达到峰值。 i.t.后，iNOS - / - 小鼠的早期肺损伤已减弱。与WT小鼠相比，LPS，但是到第10天的恢复显着受损。 iNOS - / - 小鼠的死亡率增加，并且持续升高的BAL蛋白，白蛋白，细胞，中性粒细胞和促炎性细胞因子。 WT Inos+/+骨髓衍生的单核细胞或将iNOS递送到受伤的iNOS - / - 小鼠（分别为I.T. LPS后1和2天）将ALI分辨率恢复的模式以类似于WT的模式恢复分辨率；相反，iNOS - / - 单核细胞的转移或SHAM腺病毒的递送未达到分辨率。为了开始了解iNOS如何促进ALI的分辨率，我们在I.T.后的间隔进行了肺泡细胞的多色流式细胞仪。 LPS。我们观察到iNOS - / - 小鼠在损伤后的第7天相比，在iNOS - / - 小鼠中，巨噬细胞/单核细胞共同传导分子（例如CD86和CD40）的调节T细胞数量明显减少和持续的表达。在受伤的iNOS - / - 小鼠中，补充WT Tregs或抗体介导的CD86的封锁转移明显恢复了肺损伤的分辨率。我们假设巨噬细胞衍生的INOS通过调节肺中的免疫反应来介导ALI的分辨率起关键作用，从而促进肺泡炎症的清除率并促进肺修复。为了将这些发现转移到临床环境中，我们提出了以下特定目的：1。确定iNOS受伤后iNOS调节肺部肺部免疫反应的机制。使用体内和体外方法，我们将确定iNOS在损伤响应的不同阶段修改巨噬细胞/单核细胞先天免疫反应中的作用。 iNOS介导的对巨噬细胞共同信号分子的影响及其在ALI分辨率方面的相对贡献将通过使用抗体阻断和骨莫罗嵌合体的创造来检查。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中的影响。 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以促进ALI的分辨率。体内和体外研究将用于评估巨噬细胞衍生的iNOS在调节Treg淋巴细胞表型和功能中的作用。还将研究Treg衍生的INOS的潜在影响。 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的直接腺病毒基因递送或没有体内供体的供体来恢复I.T. iNOS - / - 小鼠中ALI的分辨率。 LPS和直接革兰氏活细菌ALI模型。在SA 1和2中鉴定出的靶标（例如共同信号分子）将被操纵，以试图加速“严重”受伤的WT动物的分辨率。 负责解决ALI的机制的定义可能会在重要的临床状况下为治疗提供新的靶标，该临床状况每年杀死75,000多人，目前治疗仍然支持。我们认为，靶向iNOS表达，尤其是在ALI发作后的后期，可能被证明是加速ALI患者分辨率的治疗。 公共卫生相关性：负责解决急性肺损伤（ALI）机制的定义可能会在重要的临床状况下为治疗提供新的靶标，该临床状况每年杀死75,000多人，目前治疗仍然支持。我们认为，靶向iNOS表达，特别是在ALI发作后的后期，可能被证明是加速ALI患者分辨率的治疗。