Pathogenesis of Fungal Keratitis

摘要 在美国，致病的镰刀菌和曲霉菌是导致视力丧失的重要原因 全世界。来自无偏倚基因阵列分析(纳米串和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种基因表达的预期增加 将感染的小鼠角膜与来自同一小鼠的骨髓中性粒细胞进行比较。然而，我们发现了一个 促炎症细胞因子IL-1α的表达意外显著增加，以及 Spp1/骨桥蛋白(OPN)，具有已知的促炎活性，以分泌型(SOPN)形式存在 和细胞内(IOPN)。定量聚合酶链式反应、酶联免疫吸附试验和细胞内血流检测证实IL-1α和OPN升高 细胞学。目标1将表征支持和切割的细胞膜、分泌和细胞内的活性 IL-1α在小鼠和人中性粒细胞中的存在形式，以及在镰刀菌和曲霉菌角膜炎中的存在形式。目标2将 确定OPN表达的调节因子，并使用OPN-/-和iOPN敲入小鼠来检测OPN-/-和iOPN基因敲入小鼠的作用 在人类中性粒细胞和真菌性角膜炎中这种蛋白的细胞内和分泌形式。目标3将 描述中性粒细胞胞外陷阱(Net)的作用，它可以限制菌丝的生长，但也有 有可能导致组织损伤。拟议中的实验将确定导致 Net在感染的角膜中形成，并使用缺乏组蛋白瓜氨酸化所需酶的小鼠和 解凝集(PAD4)，我们将研究Net在镰刀菌和曲霉菌角膜炎中的作用。总而言之， 这些拟议的研究结果将增加我们对这种致盲机制的理解。 疾病，并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 角膜透明度受损和丧失。

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.