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.

项目总结/摘要 急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）的发病早期事件 （ARDS）已经被定义，关于介导解决的机制知之甚少。开始寻找 潜在的决定因素，我们将野生型（WT）小鼠暴露于脂多糖（i.t. LPS），并在第10天（损伤已经消退的时间）间隔评估反应。肺泡 (BAL)肺介质在i.t. LPS，其中包括BAL亚硝酸盐/硝酸盐（NOx）。符合 增加NOx，我们发现诱导型一氧化氮合酶（iNOS）蛋白表达显著增加， 在肺中上调并在损伤后第4天达到峰值。在iNOS-/-小鼠中， I.T.然而，与WT小鼠相比，第10天的恢复明显受损。iNOS-/-小鼠 死亡率增加以及BAL蛋白、白蛋白、细胞、中性粒细胞和前白细胞持续升高， 炎性细胞因子WT iNOS+/+骨髓来源的单核细胞或直接腺病毒的连续转移 将iNOS基因递送到损伤的iNOS-/-小鼠中（i.t. LPS分别）恢复 与WT相似的ALI消退模式;相比之下，转移iNOS-/-单核细胞或递送假手术组 腺病毒没有达到消退。为了开始了解iNOS如何促进ALI的消退，我们 在i.t. LPS。我们观察到 调节性T细胞（Treg）的数量减少，巨噬细胞/单核细胞共刺激因子的持续表达， 在损伤后第7天，与WT小鼠相比，iNOS-/-小鼠中的CD 86和CD 40信号分子。 在损伤的iNOS-/-小鼠中，补充WT THBG或抗体介导的CD 86阻断剂的转移显著地增加了CD 86的表达。 肺损伤恢复。我们假设巨噬细胞来源的iNOS在 通过调节肺中的免疫应答来介导ALI的消退，从而促进肺泡 炎症和促进肺修复。为了将这些发现应用于临床，我们 提出以下具体目标： 1.为了确定iNOS调节巨噬细胞先天性免疫反应的机制， 肺损伤后。使用体内和体外的方法，我们将确定iNOS在修饰细胞中的作用。 巨噬细胞/单核细胞先天免疫应答在损伤消退应答的不同阶段。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消退中的相对贡献将被 通过使用抗体阻断和骨髓嵌合体的产生来检查。我们将比较 外源性与内源性NO在损伤后调节巨噬细胞免疫应答中的作用。 2.为了确定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- 在调节Treg淋巴细胞表型和功能中的作用。Treg衍生的潜在影响 iNOS也将被研究。 3.确定在不同的ALI模型中，操作iNOS是否可以改善结果。我们将使用 体内直接腺病毒基因递送诱导型一氧化氮合酶或一氧化氮供体，两者都可以恢复诱导型一氧化氮合酶-/-小鼠的ALI缓解 i.t.之后LPS和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的消退。 动物 明确导致ALI消退的机制可能为治疗急性肺损伤提供新的靶点。 每年导致75，000多人死亡的重要临床疾病，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， 证明可作为治疗方法加速ALI患者的消退。