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）：虽然急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）发病机制的早期事件已被确定，但对介导缓解的机制知之甚少。为了开始寻找解决的潜在决定因素，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。支气管肺泡（BAL）和肺介质增加后，i. LPS，其中包括BAL亚硝酸盐/硝酸盐（NOx）。与NOx的增加一致，我们发现诱导型一氧化氮合酶（iNOS）蛋白表达在肺中显著上调，并在损伤后第4天达到峰值。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，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连续转移或将iNOS的直接腺病毒基因递送到损伤的iNOS-/-小鼠中（i.t. LPS）以类似于WT的模式恢复了ALI的消退;相反，转移iNOS-/-单核细胞或递送假腺病毒没有达到消退。为了开始了解iNOS是如何促进ALI的消退的，我们在i.t. LPS。我们观察到与WT小鼠相比，iNOS-/-小鼠中的调节性T细胞（TCRs）的数量显著减少，并且巨噬细胞/单核细胞共信号传导分子（例如CD 86和CD 40）的表达持续到损伤后第7天。在损伤的iNOS-/-小鼠中转移补充的WT THBG或抗体介导的CD 86阻断显著恢复肺损伤的消退。我们推测巨噬细胞来源的iNOS通过调节肺内的免疫反应，从而促进肺泡炎症的清除和促进肺修复，在介导ALI的消退中起关键作用。为了将这些发现纳入临床考虑，我们提出了以下具体目标：1。目的：探讨肺损伤后诱导型一氧化氮合酶（iNOS）调节巨噬细胞先天性免疫反应的机制。使用在体内和体外的方法，我们将确定的作用，在修改巨噬细胞/单核细胞的先天性免疫反应在不同阶段的损伤解决反应的诱导型一氧化氮合酶。iNOS介导的对巨噬细胞共信号分子的作用及其在ALI消退中的相对贡献将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细胞相互作用促进急性肺损伤缓解中的作用。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的潜在作用。3.确定在不同的ALI模型中，操作iNOS是否可以改善结果。我们将在体内使用iNOS或NO供体的直接腺病毒基因递送，两者都在i.t. LPS和直接活革兰氏阴性菌模型。在SA 1和2中鉴定的靶标（例如共信号传导分子）将被操纵以试图加速“严重”损伤的WT动物的消退。 确定解决ALI的机制可能会为治疗一种重要的临床疾病提供新的靶点，这种疾病每年导致超过75，000人死亡，目前的治疗仍然是支持性的。我们认为，靶向iNOS表达，特别是在ALI发作后的后期，可能被证明是有效的治疗，以加速ALI患者的解决。 公共卫生相关性：急性肺损伤（ALI）是一种重要的临床疾病，每年有超过75，000人死于这种疾病，目前的治疗方法仍然是支持性的，对这种疾病的机制的定义可能为治疗提供新的靶点。我们认为，靶向iNOS表达，特别是在ALI发作后的后期，可能被证明是有效的治疗，以加速ALI患者的解决。