Contribution of Mast Cells in Non-Allergic Ocular Inflammation

这是一项竞争性更新申请，旨在进一步研究IgE非依赖性的机制 肥大细胞的激活与眼部炎症有关。肥大细胞引起了人们的极大关注 过去十年在非变态反应性炎症环境中不同的IgE非依赖性效应功能 疾病。然而，关于其功能和免疫力的基本问题仍未得到解答。 肥大细胞在非过敏性眼部病变中的作用机制。我们的实验室已经做了大量的 眼部肥大细胞如何参与非过敏性炎症反应的研究进展。报告 来自我们实验室的证据表明：(I)上皮细胞来源的白介素33激活了IgE中的肥大细胞- 肥大细胞(II)作为常驻免疫细胞通过释放启动中性粒细胞浸润 CXCL2，和(Iii)促进血管和淋巴管的病理性生长，部分是通过分泌高水平的 血管内皮生长因子。此外，我们强有力的初步发现表明，肥大细胞增强了组织破坏功能。 中性粒细胞的减少和肥大细胞的缺乏会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的确切机制 病理功能没有很好地定义。 根据我们实验室在免疫学研究方面的专业知识和我们成熟的小鼠 角膜损伤和新生血管的模型，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。在目标1中，我们 将检验肥大细胞衍生的粒细胞刺激因子(GM-CSF和G-CSF)促进 角膜损伤后中性粒细胞释放损伤组织的细胞因子和酶。具体来说，我们将 用CRISPR-Cas9和中和抗体评价GM-CSF对G-CSF缺陷的肥大细胞的作用 用我们标准化的体外共培养试验治疗(I)中性粒细胞效应器功能和存活 中性粒细胞介导的角膜损伤模型中的组织损伤。在目标2中，基于我们对桅杆的初步数据 高水平表达血管稳定血管生成素1(Ang1)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 通过分泌Ang1。具体地说，我们将确定(I)肥大细胞和血管内皮细胞相互作用对Ang1-Tie2的影响 配体-受体轴，(Ii)Ang1缺陷的肥大细胞对病理性血管的稳定性。我们还将 局部应用血管紧张素转换酶1阻滞剂与一般肥大细胞抑制剂的疗效比较 在加速血管退缩方面。预计这些目标的完成将阐明尚不清楚的 肥大细胞在非过敏性炎症中的作用机制并为开发新的 组织损伤和血管生成的治疗。

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.