Contribution of Mast Cells in Non-Allergic Ocular Inflammation

肥大细胞在非过敏性眼部炎症中的作用

• 批准号: 10583991
• 负责人: Sunil K Chauhan
• 金额: $ 49.25万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10583991
• 关键词: ANGPT1 gene; ANXA5 gene; Acceleration; Allergic Disease; Antibodies; Antibody Therapy; Attention; Attenuated; Biological Assay; Blood Vessels; Bone Marrow; Brightfield Microscopy; CRISPR/Cas technology; CSF3 gene; CXCL2 gene; Cell Communication; Cell Survival; Cell physiology; Cells; Clinical; Coculture Techniques; Computer software; Confocal Microscopy; Cornea; Corneal Injury; Corneal Neovascularization; Cromoglicic Acid; Data; Disease; Effector Cell; Endothelial Cells; Enzymes; Epithelial Cells; Eye; Eye diseases; Fluorescein; Functional disorder; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Growth; Histologic; ITGAM gene; IgE; Image; Immune; Immunofluorescence Immunologic; Immunologics; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Injury; Interleukin-1 beta; Interleukins; Investigation; Kinetics; Laboratories; Ligands; Mast Cell Stabilizer; Measures; Mediating; Modeling; Mus; Neutrophil Infiltration; Ocular Pathology; PECAM1 gene; Pathogenesis; Pathologic; Pathology; Plug-in; Propidium Diiodide; Proteins; Publications; Reporting; Research; Series; Site; Stains; Standardization; System; TIE-2 Receptor; Testing; Tissues; Topical application; Transcript; Treatment Efficacy; Tube; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; Work; angiogenesis; arm; chemokine; cytokine; design; differential expression; experimental study; first responder; granulocyte; immune function; improved outcome; in vivo; inhibitor; lymphatic vessel; mast cell; mouse model; neovascularization; neutralizing antibody; neutrophil; novel; novel therapeutic intervention; novel therapeutics; ocular surface; receptor; reconstitution; slit lamp imaging; therapeutic target; tissue injury; tissue repair; vessel regression

This is a competitive renewal application to further investigate the mechanisms by which IgE-independent activation of mast cells contributes to ocular inflammation. Mast cells have garnered much attention over the past decade for their diverse IgE-independent effector function in the setting of non-allergic inflammatory diseases. Nevertheless, fundamental questions remain unanswered regarding the function and immune mechanisms of mast cells in mediating ‘non-allergic’ ocular pathologies. Our laboratory has made substantial progress in understanding how ocular mast cells contribute to the non-allergic inflammatory response. Reports from our lab provide evidence that (i) epithelial cell-derived interleukin-33 activates mast cells in an IgE- independent manner and that mast cells (ii) as resident immune cells initiate neutrophil infiltration by releasing CXCL2, and (iii) promote pathological growth of blood and lymph vessels in part by secreting high levels of VEGFs. Moreover, our robust preliminary findings indicate that mast cells augment the tissue-damaging function of neutrophils and that deficiency of mast cells results in faster regression of mature pathological corneal vessels. However, the exact mechanisms by which mast cells interact with neutrophils and vascular cells to promote their pathological functions are not well defined. In accordance with our laboratory’s expertise in immunological studies and our well-established murine models of corneal injury and neovascularization, we propose a series of novel experiments to decipher the function of mast cells as an orchestrator of inflammation and tissue damage. Our pilot investigations show that mast cells express high levels of neutrophil (granulocyte)-stimulating factors, GM-CSF and G-CSF. In Aim 1, we will test the hypothesis that Mast cell-derived granulocyte stimulating factors (GM-CSF and G-CSF) promote the release of tissue-damaging cytokines and enzymes by neutrophils following corneal injury. Specifically, we will assess the effect of GM-CSF versus G-CSF deficient mast cells (using CRISPR-Cas9) and neutralizing antibody treatment on (i) neutrophil effector function and survival using our standardized in vitro co-culture assays and (ii) neutrophil-mediated tissue damage in the corneal injury model. In Aim 2, based on our preliminary data of mast cells expressing high levels of vessel stabilizing Angiopoietin 1 (Ang1), we will test the hypothesis that Mast cells juxtaposing pathological vessels prolong vascular endothelial cell (VEC) survival and maintain vessel integrity by secreting Ang1. Specifically, we will determine the effect of (i) mast cells and VECs interaction on Ang1-Tie2 ligand-receptor axis, (ii) Ang1-deficient mast cells on the stability of pathological blood vessels. We will also evaluate the relative therapeutic efficacy of topical application of Ang1 blocked versus general mast cell inhibitor in accelerating vessel regression. It is anticipated that the completion of these aims will elucidate as-yet-unknown mechanisms of mast cell function in non-allergic inflammation and provide a framework to develop new therapeutics for tissue injury and angiogenesis.

这是一个竞争性的更新申请，以进一步研究IgE非依赖性 肥大细胞的活化导致眼部炎症。肥大细胞已经获得了很多关注， 在过去十年中，它们在非过敏性炎症环境中具有多种IgE非依赖性效应子功能， 疾病然而，关于功能和免疫的基本问题仍然没有答案， 肥大细胞介导“非过敏性”眼部病理的机制。我们的实验室已经做出了大量的 在了解眼部肥大细胞如何促进非过敏性炎症反应方面取得了进展。报告 从我们的实验室提供的证据表明，（i）上皮细胞来源的白细胞介素-33激活肥大细胞在IgE- 肥大细胞（ii）作为常驻免疫细胞通过释放 CXCL 2，和（iii）部分通过分泌高水平的CXCL 2来促进血管和淋巴管的病理性生长。 VEGF。此外，我们强有力的初步研究结果表明，肥大细胞增加了组织损伤功能 嗜中性粒细胞的缺乏和肥大细胞的缺乏导致成熟的病理性角膜血管更快地消退。 然而，肥大细胞与中性粒细胞和血管细胞相互作用以促进其增殖的确切机制尚不清楚。 病理功能没有很好的定义。 根据我们实验室在免疫学研究方面的专业知识和我们完善的小鼠 角膜损伤和新生血管模型，我们提出了一系列新的实验来破译 肥大细胞作为炎症和组织损伤的协调者的功能。我们的试点调查显示， 肥大细胞表达高水平的中性粒细胞（粒细胞）刺激因子、GM-CSF和G-CSF。目标1： 将检验肥大细胞源性粒细胞刺激因子（GM-CSF和G-CSF）促进 角膜损伤后中性粒细胞释放组织损伤性细胞因子和酶。具体来说，我们将 评估GM-CSF相对于G-CSF缺陷型肥大细胞（使用CRISPR-Cas9）和中和抗体的作用 （i）使用我们的标准化体外共培养测定对中性粒细胞效应子功能和存活的治疗，和（ii） 角膜损伤模型中嗜酸性粒细胞介导的组织损伤。在目标2中，根据我们的初步数据， 表达高水平血管稳定性血管生成素1（Ang 1）的细胞，我们将检验肥大细胞 并置病变血管可延长血管内皮细胞（VEC）存活并维持血管完整性 分泌Ang 1。具体而言，我们将确定（i）肥大细胞和VECs相互作用对Ang 1-Tie 2的影响， 配体-受体轴，（ii）Ang 1缺陷型肥大细胞对病理血管的稳定性。我们还将 评价局部应用Ang 1阻断剂与一般肥大细胞抑制剂的相对疗效 加速血管退化预计这些目标的完成将阐明迄今未知的问题。 肥大细胞在非过敏性炎症中的功能机制，并为开发新的 用于组织损伤和血管生成的治疗剂。