Impact of versican deficiency on the innate immune response to influenza virus

项目摘要 我们发表的工作和使用小鼠模型和体外细胞培养的初步数据表明， 细胞外基质（ECM）分子如多功能蛋白聚糖在暴露于病毒后在肺中积累 和细菌。一个专门的versican丰富ECM的创建与入侵和保留相一致 在对流感病毒的免疫反应中肺中白细胞的减少。我们发现两个细胞 响应Toll样受体（TLR）激动剂合成多功能蛋白聚糖的来源：巨噬细胞和 成纤维细胞研究确定了在这两种细胞中指导多功能蛋白聚糖合成的两种不同的信号通路 类型-基质细胞如平滑肌细胞中的β-连环蛋白/T细胞因子（TCF）途径， 成纤维细胞和TLR/Trif/I型干扰素（IFN）受体途径。但 这些信号传导途径的细胞特异性是未知的。利用两种新的基因 我们的研究人员发表了一种基因工程小鼠，其中多功能蛋白聚糖基因以细胞和时间特异性的方式被删除， 初步数据显示多功能蛋白聚糖对白细胞表型有显著影响。处理时 使用聚肌胞苷酸（poly（I：C）），多功能蛋白聚糖全面缺乏的小鼠减少了 支气管肺泡（BAL）液中白细胞的恢复表明多功能蛋白聚糖是促炎性的， 白细胞迁移到肺部所必需的。相反，巨噬细胞中缺乏多能蛋白聚糖的小鼠， BAL液中白细胞回收率增加，表明巨噬细胞衍生的多功能蛋白聚糖是抗- 炎症和抑制白细胞迁移到肺部。两种versican缺陷小鼠都有 与对照组相比， 对照我们的体外研究将其归因于这些抗炎细胞因子的产生减少， 多功能蛋白聚糖缺陷型巨噬细胞在这些新的versican缺陷小鼠中观察到的差异是： 我们的中心假设的基础是，巨噬细胞和巨噬细胞形成富含versican的ECM， 基质细胞提供了先天性免疫应答的关键背景线索和细胞外控制， 病毒性肺部感染我们提出了三个目标来检验这一假设。目标1定义了全能运动员的角色 来源于巨噬细胞和/或I型IFN信号传导，在提供先天性巨噬细胞的细胞外控制中， 暴露于流感病毒小鼠肺中的免疫应答。目的2确定是否基质细胞和/或β- 连环蛋白/TCF信号传导促进了多功能蛋白聚糖富集的ECM的产生，该ECM提供了对 对流感病毒的先天免疫反应。目的3确定机制和信号通路 由此巨噬细胞-相对于成纤维细胞-衍生的多功能蛋白聚糖提供了细胞外环境对细胞的控制， 先天免疫反应了解versican提供精细控制的上下文设置 对流感病毒的先天免疫应答对于设计治疗策略是至关重要的， 提高对病毒性肺部感染的免疫反应。

PROJECT SUMMARY Our published work and preliminary data using mouse models and in vitro cell cultures show that extracellular matrix (ECM) molecules such as versican accumulate in lungs following exposure to viruses and bacteria. The creation of a specialized versican-enriched ECM coincides with invasion and retention of leukocytes in lungs during the immune response to influenza virus. We have found that two cellular sources synthesize versican in response to Toll-like receptor (TLR) agonists: macrophages and fibroblasts. Studies identify two distinct signaling pathways directing versican synthesis in these two cell types - the β-catenin/T-cell factor (TCF) pathway in stromal cells such as smooth muscle cells and fibroblasts and the TLR/Trif/Type I interferon (IFN) receptor pathway in macrophages. However, the cellular specificity of these signaling pathways is not known. Using two novel strains of genetically engineered mice in which the versican gene is deleted in a cell- and time-specific manner, our published and preliminary data shows that versican has a dramatic effect on leukocyte phenotype. When treated with polyinosinic:polycytidylic acid (poly(I:C)), mice with a global deficiency in versican have decreased recovery of leukocytes in bronchoalveolar (BAL) fluid suggesting that versican is pro-inflammatory and required for leukocyte migration into lungs. In contrast, mice lacking versican in macrophages have increased recovery of leukocytes in BAL fluid suggesting that macrophage-derived versican is anti- inflammatory and suppresses leukocyte migration into lungs. Both strains of versican-deficient mice had significantly decreased recovery of Type I IFNs and IL-10 in lung tissue or BAL fluid when compared to controls. Our in vitro studies attribute this to decreased production of these anti-inflammatory cytokines by versican-deficient macrophages. The differences observed in these novel versican-deficient mice are the basis of our central hypothesis that the formation of a versican-enriched ECM by macrophages and stromal cells provides critical contextual cues and extracellular-control of the innate immune response to viral lung infection. We propose three aims to test this hypothesis. Aim 1 defines the role of versican derived from macrophages and/or Type I IFN signaling in providing extracellular-control of the innate immune response in lungs of mice exposed to influenza virus. Aim 2 determines if stromal cells and/or β- catenin/TCF signaling promote the generation of a versican-enriched ECM that provides fine-control of the innate immune response to influenza virus. Aim 3 identifies the mechanisms and signaling pathways whereby macrophage- versus fibroblast-derived versican provide contextual extracellular-control of the innate immune response. Understanding the contextual settings in which versican provides fine-control of the innate immune response to influenza virus is critical for the design of therapeutic strategies for improving the immune response to viral lung infection.