WISP1 and TLR4 Signaling in ventilator-induced lung injury (VILI)

呼吸机引起的肺损伤 (VILI) 中的 WISP1 和 TLR4 信号转导

• 批准号: 8757083
• 负责人: LI-MING ZHANG
• 金额: $ 29.26万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8757083
• 关键词: Acute Lung Injury; Alveolar; Alveolar Macrophages; Anabolism; Antibodies; Attention; Biochemical; Biological Markers; Biology; Bronchoalveolar Lavage Fluid; CCL4 gene; CD14 gene; Capillary Permeability; Cells; Complex; Coupled; Critical Illness; Data; Disease; Elements; Epithelial; Epithelium; Etiology; Galactosidase; Genes; Genetic; Genetic Determinism; Health; Hepatology; Immune; Immune response; Immune system; In Situ; In Vitro; Individual; Inflammatory; Injection of therapeutic agent; Integrins; Knockout Mice; Lead; Left; Lung; MAPK8 gene; Mechanical Stress; Mechanical ventilation; Mechanics; Mediating; Modeling; Molecular; Morbidity - disease rate; Mouse Strains; Mus; Myeloid Cells; Outcome; Pathogenesis; Pathway interactions; Patients; Peritoneal Macrophages; Permeability; Phenotype; Phosphorylation; Play; Predisposition; Prevention; Production; Protein Family; Proteins; Recombinants; Reporting; Research Personnel; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Source; Stretching; Structure of respiratory epithelium; TLR4 gene; TNF gene; Testing; Transgenic Mice; Ventilator-induced lung injury; WISP1 gene; Work; airway epithelium; biological adaptation to stress; genome wide association study; human WISP1 protein; in vivo; insight; lung injury; macrophage; mortality; neutrophil; novel; public health relevance; research study; respiratory; response; rho; small hairpin RNA; therapeutic target

DESCRIPTION (provided by applicant): Although much of the improvement in outcomes from acute lung injury has been ascribed to lung-protective mechanical ventilation strategies, ventilator-induced lung injury (VILI) remains an important element of morbidity and mortality in the critically ill patient. Using an unbiased genome-wide association study, we (Li et al, Am J Resp Cell Mol Biol 2012) identified a WNT1-inducible signaling pathway protein 1 (WISP1) in murine VILI. Moreover, we and others have identified that innate immune signaling via TLR4 plays a critical role in the pathogenesis of VILI and that stretch-induced WISP1 expression and its pro-inflammatory effect were TLR4- dependent. Accordingly, we propose: Specific Aim 1. To determine the molecular pathway by which mechanical stretch is coupled to respiratory epithelial WISP1 biosynthesis and VILI. We will: a) use pharmacological and genetic approaches to dissect contribution of non-canonical Wnt signaling pathway in WISP1 biosynthesis in cyclic stretched cultured murine respiratory epithelium; b) use biochemical determinants to associate non-canonical pathway in intact lung of mice after HTV (12 ml/kg x 6h); and c) determine the contribution of epithelial derived WISP1 in VILI by changes in alveolar capillary permeability after HTV in wildtype, WISP1 -/- and after silencing WISP1 in alveolar macrophages in situ with intratracheal lentiviral delivery of shRNA (and hence leaving epithelial derived WISP1 as sole source). Specific Aim 2. To determine the role of WISP1 in communicating mechanical stress responses to innate immune system leading to VILI: We will determine the contribution of macrophage (and neutrophil) TLR4 signaling in VILI by comparing the effect of HTV on wildtype, whole body TLR4 and myeloid-cell-specific TLR4 null mice (Lyz-TLR4; Nace et al, Hepatology 2013). Specific Aim 3. To determine the molecular determinants by which WISP1 acts as an accessory molecule transducing stress of mechanical stretch in airway epithelium to pro-inflammatory phenotype of macrophages via TLR4. We will define requisite components for proinflammatory (e.g. TNF¿; NF?B) effect of WISP1 using peritoneal macrophages and confirm accrued information in primary murine alveolar macrophage cultures (that are limiting in number) from wildtype and CD14 and ¿3 and ¿5 null mice. We will then determine the obligatory roles of ¿3 and ¿5 in WISP1 mediated VILI by contrasting the effect of HTV on ¿3 and ¿5 null mice, with and without i.t. injection of WISP1. Collectively, these studies will provide novel insight into the role of matricellular protein, WISP1, and the Wnt pathway, in transducing the effect of mechanical stress on respiratory epithelium to innate immune system and role of TLR4 activation of alveolar macrophages in pathogenesis of VILI. Such mechanistic insight may lead to biomarkers, therapeutic targets in prevention or mitigation of VILI and further understanding of genetic determinants of susceptibility to VILI.

描述（由申请人提供）：尽管急性肺损伤预后的改善大部分归因于肺保护性机械通气策略，但呼吸机诱导的肺损伤（VILI）仍然是危重患者发病率和死亡率的重要因素。通过一项无偏倚的全基因组关联研究，我们（Li et al, Am J Resp Cell Mol Biol 2012）在小鼠VILI中发现了wnt1诱导的信号通路蛋白1 （WISP1）。此外，我们和其他人已经发现通过TLR4的先天免疫信号在VILI的发病机制中起关键作用，并且拉伸诱导的WISP1表达及其促炎作用依赖于TLR4。为此，我们提出：具体目标1。确定机械拉伸与呼吸上皮WISP1生物合成和VILI耦合的分子途径。我们将：a)使用药理学和遗传学方法解剖非规范Wnt信号通路在循环拉伸培养小鼠呼吸上皮WISP1生物合成中的贡献；b)利用生化决定因素在HTV （12 ml/kg x 6h）后小鼠完整肺中关联非典型途径；c)通过野生型、WISP1 -/-和通过气管内慢病毒传递shRNA原位沉默肺泡巨噬细胞中的WISP1后肺泡毛细血管通透性的变化来确定上皮源性WISP1在VILI中的贡献（从而使上皮源性WISP1成为唯一来源）。具体目标2。为了确定WISP1在传递先天免疫系统机械应激反应导致VILI中的作用：我们将通过比较HTV对野生型、全身TLR4和髓细胞特异性TLR4缺失小鼠的影响，确定巨噬细胞（和中性粒细胞）TLR4信号在VILI中的作用（Lyz-TLR4； Nace等，Hepatology 2013）。具体目标3。确定WISP1作为辅助分子通过TLR4将气道上皮机械拉伸应力转导为巨噬细胞促炎表型的分子决定因素。我们将定义促炎的必要成分(如TNF¿；NF？B) WISP1对腹腔巨噬细胞的影响，并证实野生型和CD14以及¿3和¿5小鼠的原代小鼠肺泡巨噬细胞培养（数量有限）中积累的信息。然后，我们将通过对比HTV在注射和不注射WISP1的情况下对¿3和¿5缺失小鼠的影响，确定¿3和¿5在WISP1介导的VILI中的必要作用。总的来说，这些研究将为基质细胞蛋白WISP1和Wnt通路在将呼吸上皮机械应力转导到先天免疫系统中的作用以及TLR4激活肺泡巨噬细胞在VILI发病机制中的作用提供新的见解。这种机制的洞察可能导致生物标志物，治疗靶点在预防或减轻VILI和进一步