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.

假单胞菌（P.）铜绿细菌感染引起角膜的视力威胁炎症。和抗生素耐药性随着增加，需要其他治疗策略。为了解决这个差距，当前提案重点是病原体识别收费受体（TLR）与Lumican（Lum），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衍生的脂多糖（LP）和核DNA分别诱导促炎性细胞因子和类型I干扰素。 TLR4在巨噬细胞和树突状细胞的细胞表面识别LPS（DCS）导致MyD88依赖性促炎细胞因子产生，而内体TLR4刺激另外产生I型干扰素的替代途径。另一方面被细菌DNA或合成TLR9配体CPGDNA刺激，导致两者的产生炎性细胞因子和I型干扰素。有趣的是，Lum对TLR4和TLR9具有相反的影响；它促进TLR4，但抑制TLR9响应。我们证明了N末端的酪氨酸（Y20） LUM与TLR-ADAPTOR CD14结合以促进细胞表面TLR4信号。我们的初步数据表明可能与Caveolol有关贩运TLR。我们的中心假设是通过结合CD14和Cav1 lum调节 TLR4和TLR9位置在细胞内促进TLR4但限制TLR9信号，这将影响促炎性细胞因子和I型干扰素诱导以调节炎症并在角膜炎中恢复组织稳态。在以下目的中，我们将使用这些小鼠的野生型小鼠，Lum-null小鼠以及巨噬细胞和树突状细胞。目标1）确定LUM是否调节TLR4位置和降解，并优先使用CD14结合 CAV1促进细胞表面，而内体TLR4途径，AIM 2）测试LUM是否增加了非活性细胞表面和内体隔室中的TLR9池以抑制其信号，目标3）确定由LPS或CPGDNA和铜绿假单胞菌介导的无菌角膜炎病程不足和野生型小鼠，以及突变重组LUM的结界注射的影响 CD14或CAV1结合活性的丧失。 I型干扰素在除了其众所周知的抗病毒特性。因此，挑衅的转化潜力是操纵CD14和LUM的CAV1结合特性，可以调节炎性细胞因子和I型干扰素诱导途径分别解决炎症和加快角膜愈合的角膜炎。我们的研究将在知识基础上建立强大的知识基础，以了解未来开发新的分子治疗干预措施的感染和炎症中的ECM。