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: 8122310
• 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/8122310
• 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定义，但对调解机制知之甚少。为了开始寻找解决的潜在决定因素，我们将野生型（WT）小鼠暴露于气管内脂多糖（简称LPS），并在间隔至第10天的时间间隔内评估反应，此时损伤已经解决。LPS作用后，支气管肺泡（BAL）和肺介质浓度升高，其中亚硝酸盐/硝酸盐（NOx）浓度升高。与NOx浓度升高一致，我们发现诱导型一氧化氮合酶（iNOS）蛋白在损伤后第4天表达显著上调，并达到峰值。iNOS-/-小鼠在注射LPS后早期肺损伤减轻，但与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+/+骨髓源单核细胞过继转移或将iNOS直接腺病毒基因递送至损伤的iNOS-/-小鼠（分别在注射LPS后1天和2天），以与WT相似的模式恢复ALI的分辨率；相比之下，iNOS-/-单核细胞的转移或假腺病毒的递送都没有达到解决的效果。为了开始了解iNOS如何促进ALI的消退，我们在注射LPS后每隔一段时间对肺泡细胞进行多色流式细胞术。我们观察到，与WT小鼠相比，iNOS-/-小鼠的调节性T细胞（Tregs）数量明显减少，巨噬细胞/单核细胞共信号分子（如CD86和CD40）在损伤后第7天持续表达。在iNOS-/-损伤小鼠中添加WT Tregs或抗体介导的CD86阻断，可显著恢复肺损伤的消退。我们假设巨噬细胞来源的iNOS通过调节肺中的免疫反应在介导ALI的解决中起关键作用，从而促进肺泡炎症的清除和肺修复。为了将这些发现推向临床研究，我们提出以下具体目标：1。目的：探讨iNOS调节损伤后肺巨噬细胞先天免疫反应的机制。采用体内和体外方法，我们将确定iNOS在损伤消退反应的不同阶段对巨噬细胞/单核细胞先天免疫反应的影响。iNOS介导对巨噬细胞共信号分子的影响及其在ALI解决中的相对贡献将通过抗体阻断和骨髓嵌合体的创建来研究。我们将比较外源性NO和内源性NO对损伤后巨噬细胞免疫反应的调节作用。2. 目的：探讨iNOS在调节肺泡巨噬细胞-调节性T细胞相互作用中促进ALI消退的作用。体内和体外研究将用于评估巨噬细胞来源的iNOS在调节Treg淋巴细胞表型和功能中的作用。treg衍生的iNOS的潜在作用也将被研究。3. 目的：确定操作iNOS是否可以改善不同ALI模型的预后。我们将使用直接腺病毒基因在体内递送iNOS或NO供体，既可以恢复iNOS-/-小鼠在注射LPS后的ALI的分辨率，也可以恢复ALI的直接革兰氏阴性菌活体模型。在sa1和sa2中确定的靶标（例如Co-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。对急性脑损伤解决机制的定义可能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。我们相信靶向iNOS表达，特别是在ALI发病后的后期，可能被证明是加速ALI患者消退的有效治疗方法。