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

• 批准号: 10329965
• 负责人: Shukti Chakravarti
• 金额: $ 39.77万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10329965
• 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; Mediating; Membrane Microdomains; Molecular; Mus; Mutate; N-terminal; Nuclear; Pathway interactions; Peritoneal Macrophages; Phenylalanine; Play; Production; Property; Proteins; Proteoglycan; Pseudomonas; Pseudomonas Infections; Recombinants; Regulation; Role; Signal Pathway; Signal Transduction; Site; Sterility; Structure; TLR4 gene; TLR9 gene; Testing; Therapeutic Intervention; Tissues; Toll-like receptors; Tyrosine; Vision; Wild Type Mouse; Work; caveolin 1; conjunctiva; corneal regeneration; cytokine; healing; in vivo; knowledge base; lumican; macrophage; monocyte; novel; pathogen; prototype; receptor; response; therapeutic target; therapy development; trafficking; translational impact; translational potential; treatment strategy; viral DNA

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.

假单胞菌（P.）绿脓杆菌感染引起危及视力的角膜炎症。与 抗生素耐药性上升，需要额外的治疗策略。为了弥补这一差距，目前 提案集中于病原体识别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TLR 4对LPS的识别 导致MyD 88依赖性促炎细胞因子的产生，而内体TLR 4刺激MyD 88依赖性促炎细胞因子的产生。 另外产生I型干扰素的替代途径。另一方面，内溶酶体TLR 9， 由细菌DNA或合成的TLR 9配体CpGDNA刺激，导致产生两种原- 炎性细胞因子和I型干扰素。有趣的是，Lum对TLR 4和TLR 9有相反的作用;它 促进TLR 4但抑制TLR 9应答。我们证明了N-末端的酪氨酸（Y20） 的Lum与TLR-接头CD 14结合以促进细胞表面TLR 4信号。我们的初步数据显示 在Lum的C-末端（F228）可能参与小窝蛋白-1结合位点， TLR的贩运。我们的中心假设是Lum通过结合CD 14和CAV 1调节 TLR 4和TLR 9在细胞内的位置，以促进TLR 4但限制TLR 9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 和恢复角膜炎中的组织稳态。在下面的目标中，我们将使用 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细胞。 目的1）确定Lum是否调节TLR 4的定位和降解，并优先与CD 14结合 促进细胞表面，而CAV 1促进内体TLR 4途径，目的2）测试Lum是否增加非活性的 TLR 9池在细胞表面和内体区室抑制其信号，目的3）确定TLR 9池的细胞内信号。 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在Lum- 缺陷型和野生型小鼠以及结膜下注射突变的重组Lum与 CD 14或Cav 1结合活性的丧失。I型干扰素具有广泛的抗菌和组织保护作用， 除了它们众所周知的抗病毒特性之外。因此，一个挑衅性的翻译潜力是， 操纵Lum的CD 14和CAV 1结合特性，将有可能调节Lum的前 炎性细胞因子和I型干扰素诱导途径分别解决炎症， 加速角膜炎愈合。我们的研究将建立一个强大的知识基础， ECM在感染和炎症中的应用，用于未来开发新的分子治疗干预。