Pathogenesis of Fungal Keratitis

摘要 致病性镰刀菌和曲霉菌霉菌是美国视力丧失的重要原因， 国际吧来自无偏基因阵列分析（Nanostring和RNA测序）的初步数据显示， 从镰刀菌和曲霉菌分离的中性粒细胞中多个基因的预期表达增加 感染的小鼠角膜与来自相同小鼠的骨髓中性粒细胞相比。然而，我们发现了一个 促炎性细胞因子IL-1 α和IL-10的表达意外显著增加， Spp 1/骨桥蛋白（OPN），已知具有促炎活性，并以分泌型（sOPN）存在 和细胞内（iOPN）。通过Q-PCR、ELISA和细胞内流证实IL-1 α和OPN升高 细胞仪目的1将表征膜，分泌和细胞内活动的前和切割 IL-1 α在鼠和人嗜中性粒细胞中的形式，以及在镰刀菌和曲霉菌角膜炎中的形式。目标2将 确定OPN表达的调节因子，并使用OPN-/-和iOPN基因敲入小鼠来检查OPN的作用。 这种蛋白质在人中性粒细胞和真菌性角膜炎中的细胞内与分泌形式。目标3将 描述了中性粒细胞胞外陷阱（NET）的作用，它可以限制菌丝生长，但也具有 可能导致组织损伤。拟议的实验将确定导致 NET的形成，并使用缺乏组蛋白瓜氨酸酶所需的酶的小鼠， 通过使用去凝聚（PAD 4），我们将检查NET在镰孢菌和曲霉菌角膜炎中的作用。总的来说， 这些研究的结果将增加我们对这种致盲的发病机制的理解。 疾病，并有可能确定免疫干预的新靶点，以防止中性粒细胞介导的组织 损害和角膜透明度的丧失。

Abstract Pathogenic Fusarium and Aspergillus molds are an important cause of vision loss in the USA and worldwide. Preliminary data from an unbiased gene array analyses (Nanostring and RNA sequencing) showed an anticipated increase in expression of multiple genes in neutrophils isolated from Fusarium and Aspergillus infected mouse corneas compared with bone marrow neutrophils from the same mice. However, we found an unexpected significant increase in expression of the pro-inflammatory cytokine IL-1 alpha, and of Spp1/osteopontin (OPN), which has known pro-inflammatory activity, and which exists as a secreted (sOPN) and intracellular (iOPN). Elevated IL-1 alpha and OPN was confirmed by Q-PCR, ELISA and intracellular flow cytometry. Aim 1 will characterize the membrane, secreted and intracellular activities the pro- and cleaved forms of IL-1 alpha in murine and human neutrophils, and in Fusarium and Aspergillus keratitis. Aim 2 will identify regulators of OPN expression, and use OPN-/- and iOPN knock-in mice to examine the role of the intracellular versus secreted forms of this protein in human neutrophils and in fungal keratitis. Aim 3 will characterize the role of neutrophil extracellular traps (NETs), which can limit hyphal growth, but also have the potential to contribute to tissue damage. Proposed experiments will identify the molecular pathways leading to NET formation in infected corneas, and using mice that lack the enzyme required for histone citrullinatation and decondensation (PAD4), we will examine the role of NETs in Fusarium and Aspergillus keratitis. Collectively, the results of these proposed studies will increase our understanding of the pathogenesis of this blinding disease, and will potentially identify novel targets for immune intervention to prevent neutrophil mediated tissue damage and loss of corneal clarity.