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.)铜绿假单胞菌细菌感染会导致危及视力的角膜发炎。使用 抗生素耐药性正在上升，因此需要更多的治疗策略。为了解决这一差距，目前的 建议集中在病原体识别Toll样受体(TLRs)与Lum(Lum)的新相互作用， 角膜的一种细胞外基质蛋白。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表面TLR4对内毒素的识别导致 MyD88依赖的促炎细胞因子的产生，而内体TLR4刺激另一种选择 另外产生I型干扰素的途径。另一方面，内吞小体TLR9被刺激 细菌DNA或合成的TLR9配体CpGDNA，导致两者产生促炎作用 细胞因子和I型干扰素。有趣的是，Lum对TLR4和TLR9有相反的影响；它促进 TLR4，但抑制TLR9响应。我们证明了Lum N-末端(Y20)的一个酪氨酸 与TLR-Adaptor CD14结合，促进细胞表面TLR4信号。我们的初步数据显示， 位于Lum C末端(F228)的Caveolin-1结合位点，可能参与TLR的腔隙运输。 我们的中心假设是Lum通过结合CD14和CAV1来调节TLR4和TLR9 细胞内的位置促进TLR4，但限制TLR9信号，这将影响促进 炎性细胞因子和I型干扰素诱导调节炎症和再发 角膜炎的组织动态平衡。在以下目标中，我们将使用野生型小鼠Lum- Null小鼠及其巨噬细胞和树突状细胞；目的1)确定Lum是否调节TLR4位置和 降解，CD14结合优先促进细胞表面，而CAV1促进内体TLR4 途径，目的2)测试Lum是否增加细胞表面和内体隔室中的非活性TLR9池以 目的3)确定内毒素或CpGDNA和P。 铜绿假单胞菌介导的Lum缺乏和野生型小鼠感染性角膜炎及结膜下注射的影响 注射CD14或Cav1结合活性丧失的突变重组Lum。I型干扰素具有广泛的 除了众所周知的抗病毒特性外，还具有抗菌和保护组织的作用。因此，挑衅性的 翻译潜力是，通过操纵CD14和Lum的CAV1结合属性，它将被 可能分别调节促炎细胞因子和I型干扰素诱导途径来解决 炎症和加速角膜愈合的角膜炎。