Extracellular matrix proteoglycans regulate toll-like receptors 4 and 9

细胞外基质蛋白聚糖调节 Toll 样受体 4 和 9

基本信息

批准号：
10563132
负责人：
Shukti Chakravarti
金额：
$ 41万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10563132
关键词：
Address Anti-Bacterial Agents Antibiotic Resistance Autoimmunity Bacterial DNA Bacterial Infections Binding Binding Sites C-terminal CAV1 gene CD14 gene Cell Wall Cell membrane Cell surface Cells Chronic Collagen Fibril Cornea DNA Data Dendritic Cells Early Endosome Endocytosis Endosomes Extracellular Matrix Extracellular Matrix Proteins Eye Future Homeostasis Host Defense Infection Inflammation Inflammatory Injections Interferon Type I Keratitis Knockout Mice Leucine-Rich Repeat Ligands Lipopolysaccharides Location Macrophage Mediating Membrane Microdomains Molecular Mus Mutate N-terminal Nuclear Pathway interactions Peritoneal Macrophages Phenylalanine Play Production Property Proteins Proteoglycan Pseudomonas Infections Pseudomonas aeruginosa Recombinants Regulation Role Signal Pathway Signal Transduction Site Sterility Structure TLR4 gene TLR9 gene Testing Therapeutic Intervention Tissues Toll-like receptors Tyrosine Viral Vision Wild Type Mouse Work caveolin 1 conjunctiva corneal regeneration cytokine healing in vivo knowledge base lumican monocyte novel pathogen permissiveness prototype receptor response therapeutic target therapy development trafficking translational impact translational potential treatment strategy viral DNA

项目摘要

Pseudomonas (P.) aeruginosa bacterial infections cause sight-threatening inflammation of the cornea. With antibiotic resistance on the rise, additional treatment strategies are needed. To address this gap, the current proposal focuses on novel interactions of pathogen recognition toll-like receptors (TLRs) with lumican (Lum), an extracellular matrix protein of the cornea. The TLR-4 and TLR-9 receptors recognize P. aeruginosa cell wall-derived lipopolysaccharides (LPS) and nuclear DNA, respectively, to induce pro-inflammatory cytokines and type I interferons. LPS recognition by TLR4 at the cell surface of macrophages and dendritic cells (DCs) leads to MyD88-dependent pro-inflammatory cytokine production, while endosomal TLR4 stimulates an alternative pathway that additionally produces type I interferons. Endolysosomal TLR9, on the other hand, stimulated by bacterial DNA or the synthetic TLR9 ligand CpGDNA, leads to the production of both pro- inflammatory cytokines and type I interferons. Interestingly, Lum has opposing effects on TLR4 and TLR9; it promotes TLR4 but suppresses TLR9 responses. We demonstrated that a tyrosine at the N-terminal end (Y20) of Lum binds with the TLR-adaptor CD14 to promote cell surface TLR4 signals. Our preliminary data suggest a potential caveolin-1 binding site at the C-terminal end (F228) of Lum that may be involved in caveolar trafficking of TLRs. Our central hypothesis is that through binding CD14 and CAV1 Lum regulates TLR4 and TLR9 locations within cells to promote TLR4 but restrict TLR9 signals, and this will impact pro-inflammatory cytokine and type I interferon inductions to modulate inflammation and regain of tissue homeostasis in keratitis. In the following aims we will test our hypothesis using wild type mice, Lum-null mice, and macrophages and dendritic cells from these mice. Aim 1) determine if Lum regulates TLR4 location and degradation, with CD14-binding preferentially promoting the cell surface while CAV1 the endosomal TLR4 pathway, Aim 2) test if Lum increases the inactive TLR9 pool in the cell surface and endosomal compartments to suppress its signals, and Aim 3) determine the course of sterile keratitis mediated by LPS or CpGDNA and P. aeruginosa mediated infectious keratitis in Lum- deficient and wild type mice and the effects of subconjunctival injections of mutated recombinant Lum with loss of CD14 or Cav1 binding activity. Type I interferons have broad antibacterial and tissue protective roles in addition to their well-known antiviral properties. Thus, a provocative translational potential is that by manipulating the CD14 and the CAV1 binding properties of Lum, it will be possible to regulate the pro- inflammatory cytokine and type I interferon induction pathways separately to resolve inflammation and expedite corneal healing keratitis. Our study will develop a strong knowledge base on the role played by the ECM in infection and inflammation for developing novel molecular therapeutic interventions in the future.

铜绿假单胞菌 (P.) 细菌感染会导致威胁视力的角膜炎症。和抗生素耐药性不断上升，需要额外的治疗策略。为了解决这一差距，当前提案重点关注病原体识别 Toll 样受体 (TLR) 与 lumican (Lum) 的新型相互作用，角膜的细胞外基质蛋白。 TLR-4 和 TLR-9 受体识别铜绿假单胞菌细胞壁源性脂多糖 (LPS) 和核 DNA 分别诱导促炎细胞因子和I型干扰素。巨噬细胞和树突状细胞 (DC) 细胞表面 TLR4 识别 LPS 导致 MyD88 依赖性促炎细胞因子的产生，而内体 TLR4 则刺激另外产生 I 型干扰素的替代途径。另一方面，内溶酶体 TLR9，受到细菌 DNA 或合成 TLR9 配体 CpGDNA 的刺激，导致产生两种亲- 炎症细胞因子和 I 型干扰素。有趣的是，Lum 对 TLR4 和 TLR9 有相反的作用；它促进 TLR4 但抑制 TLR9 反应。我们证明了 N 末端的酪氨酸 (Y20) Lum 与 TLR 适配器 CD14 结合，促进细胞表面 TLR4 信号。我们的初步数据表明 Lum C 末端 (F228) 的潜在 Caveolin-1 结合位点可能参与小凹 TLR 的贩运。我们的中心假设是 Lum 通过结合 CD14 和 CAV1 来调节 TLR4 和 TLR9 在细胞内的位置促进 TLR4 但限制 TLR9 信号，这将影响促炎细胞因子和 I 型干扰素诱导以调节炎症以及角膜炎组织稳态的恢复。在以下目标中，我们将使用以下方法检验我们的假设野生型小鼠、Lum-null 小鼠以及来自这些小鼠的巨噬细胞和树突状细胞。目标 1) 确定 Lum 是否调节 TLR4 定位和降解，优先结合 CD14 促进细胞表面，同时 CAV1 内体 TLR4 途径，目标 2) 测试 Lum 是否增加非活性 TLR9 汇集在细胞表面和内体区室中以抑制其信号，目标 3) 确定由 LPS 或 CpGDNA 介导的无菌性角膜炎和铜绿假单胞菌介导的感染性角膜炎的病程缺陷型和野生型小鼠以及结膜下注射突变重组 Lum 的影响 CD14 或 Cav1 结合活性丧失。 I 型干扰素具有广泛的抗菌和组织保护作用除了其众所周知的抗病毒特性。因此，一个具有挑衅性的翻译潜力是操纵 Lum 的 CD14 和 CAV1 结合特性，将有可能调节亲炎症细胞因子和I型干扰素诱导途径分别解决炎症和加速角膜炎的角膜愈合。我们的研究将为以下方面所发挥的作用奠定坚实的知识基础： ECM 在感染和炎症中的应用，用于未来开发新型分子治疗干预措施。