Extracellular matrix proteoglycans regulate toll-like receptors 4 and 9 - Equipment Supplement

细胞外基质蛋白聚糖调节 Toll 样受体 4 和 9 - 设备补充

• 批准号: 10848823
• 负责人: Shukti Chakravarti
• 金额: $ 4.52万
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10848823
• 关键词: Address; Anti-Bacterial Agents; Antibiotic Resistance; Bacterial DNA; Bacterial Infections; Binding; Binding Sites; C-terminal; CAV1 gene; CD14 gene; Cell Wall; Cell surface; Cells; Cornea; DNA; Data; Dendritic Cells; Endosomes; Equipment; Extracellular Matrix; Extracellular Matrix Proteins; Eye; Future; Homeostasis; Inflammation; Inflammatory; Injections; Interferon Type I; Keratitis; Knockout Mice; Ligands; Lipopolysaccharides; Location; Macrophage; Mediating; Mutate; N-terminal; Nuclear; Pathway interactions; Production; Property; Proteoglycan; Pseudomonas Infections; Pseudomonas aeruginosa; Recombinants; Role; Signal Transduction; Sterility; TLR4 gene; TLR9 gene; Testing; Tissues; Toll-like receptors; Tyrosine; Viral; Vision; Wild Type Mouse; caveolin 1; corneal regeneration; cytokine; lumican; novel; pathogen; receptor; response; therapy development; trafficking; translational potential; treatment strategy

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 the 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 their macrophages and dendritic cells; 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 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.

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， 衍生的脂多糖（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）的细胞表面上的TLR 4识别LPS导致 MyD 88依赖性促炎细胞因子的产生，而内体TLR 4刺激另一种 另外产生I型干扰素的途径。另一方面，内溶酶体TLR 9被 细菌DNA或合成的TLR 9配体CpGDNA，导致产生促炎性和 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;它促进了TLR 4和TLR 9的表达。 TLR 4但抑制TLR 9应答。我们证明Lum的N-末端（Y20）的酪氨酸 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 Caveolin-1结合位点位于Lum的C-末端（F228），其可能参与TLR的小窝运输。 我们的中心假设是，通过结合CD 14和CAV 1，Lum调节TLR 4和TLR 9 细胞内的位置，以促进TLR 4，但限制TLR 9信号，这将影响亲， 炎性细胞因子和I型干扰素诱导以调节炎症并恢复 角膜炎的组织内稳态。在下面的目标中，我们将使用野生型小鼠Lum来测试我们的假设。 裸小鼠及其巨噬细胞和树突状细胞;目的1）确定Lum是否调节TLR 4定位， 降解，CD 14结合优先促进细胞表面，而CAV 1促进内体TLR 4 目的2）测试Lum是否增加细胞表面和内体区室中的非活性TLR 9库， 目的3）确定LPS或CpGDNA和P介导的无菌性角膜炎的病程。 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中的作用以及结膜下 注射具有CD 14或Cav 1结合活性损失的突变重组Lum。I型干扰素具有广泛的 除了它们众所周知的抗病毒特性之外，还具有抗菌和组织保护作用。因此，一个挑衅 翻译潜力是通过操纵Lum的CD 14和CAV 1结合特性， 可能分别调节促炎细胞因子和I型干扰素诱导途径， 促进角膜炎愈合。