Fungal keratitis causes devastating corneal ulcers which can result in significant visual impairment and even blindness. As a ligand that activates the non-canonical Wnt signaling pathways, Wnt5a triggers the production of important inflammatory chemokines and the chemotactic migration of neutrophils. In this study we aimed to characterize the role of Wnt5a production, in situ, in vivo and in vitro in response to fungal keratitis. Wnt5a expression in corneas of Aspergillus fumigatus (A. fumigatus) keratitis patients was determined by quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence. In vivo and in vitro experiments were then performed in mouse models and THP-1 macrophages cell cultures infected with A. fumigatus, respectively. C57BL/6 mice were pretreated with siRNAs or neutralizing antibodies for dectin-1, LOX-1 and Wnt5a, or inhibitors of erk1/2 and JNK. Changes in Wnt5a expression were assessed by clinical evaluation, qRT-PCR, immunofluorescence, western blot and bioluminescence imaging system image acquisition. We confirmed that corneal Wnt5a expression increased with A. fumigatus keratitis in patients and a murine model. Wnt5a production was dependent on dectin-1 and LOX-1 expression with contributions by Erk1/2 and JNK pathways. Additionally, Wnt5a knockdown revealed decreased levels of MPO, lower neutrophil recruitment, and a higher fungal load in mouse models. Compared with controls, Wnt5a knockdown impaired pro-inflammatory cytokine IL-1β production in response to A. fumigatus exposure. Wnt5a also produces dectin-1 and LOX-1 induced inflammatory signature via effective neutrophil recruitment and inflammatory cytokine production in response to A. fumigatus keratitis. These findings demonstrate that Wnt5a is a critical component of the antifungal immune response.

真菌性角膜炎可导致破坏性角膜溃疡，可导致严重的视力障碍甚至失明。作为激活非典型Wnt信号通路的配体，Wnt5a触发重要炎症趋化因子的产生和嗜中性粒细胞的趋化性迁移。在这项研究中，我们旨在表征Wnt5a生产对真菌性角膜炎的原位，体内和体外作用。通过定量聚合酶链反应（qRT-PCR）和免疫荧光测定烟曲霉（A.fumigatus）角膜炎患者角膜中Wnt5a的表达。体内和体外然后分别在感染了烟曲霉的小鼠模型和THP-1巨噬细胞细胞培养物中进行了实验。用siRNA或dectin-1，LOX-1和Wnt5a的中和抗体或erk1 / 2和JNK抑制剂对C57BL / 6小鼠进行预处理。通过临床评估，qRT-PCR，免疫荧光，蛋白质印迹和生物发光成像系统图像采集来评估Wnt5a表达的变化。我们证实，烟曲霉的角膜Wnt5a表达增加患者的角膜炎和小鼠模型。Wnt5a的生产取决于dectin-1和LOX-1的表达，并通过Erk1 / 2和JNK途径发挥作用。此外，Wnt5a敲低表明小鼠模型中MPO含量降低，中性粒细胞募集降低和真菌负荷增加。与对照组相比，Wnt5a敲低了针对烟曲霉暴露的促炎性细胞因子IL-1β的产生。Wnt5a还通过有效的嗜中性粒细胞募集和对烟曲霉性角膜炎的炎性细胞因子产生而产生dectin-1和LOX-1诱导的炎性信号。这些发现表明，Wnt5a是抗真菌免疫反应的关键组成部分。