Jing Lin1, Kun He2, Guiqiu Zhao3, Cui Li1, Liting Hu1, Guoqiang Zhu1, Yawen Niu1, Guipei Hao1. 1. The Affiliated Hospital of Qingdao University Medical College, PR China. 2. The Affiliated Hospital of Qingdao University Medical College, PR China; Ophthalmic Center of QuHua Hospital, QuHua 324004, PR China. 3. The Affiliated Hospital of Qingdao University Medical College, PR China. Electronic address: zhaoguiqiu_good@126.com.
Abstract
PURPOSE: To determine whether macrophage inducible C-type lectin (Mincle) regulates neutrophils and macrophages apoptosis in A. fumigatus keratitis. MATERIALS AND METHODS: A murine model (C57BL/6) of fungal keratitis (AF) was established by gently scraping corneal central epithelium, smearing A. fumigatus on the epithelium surface and covering the eye with contact lenses. AF cell model was established by extracting neutrophils (PMN) and macrophages, and then infecting cells with A. fumigatus. Animals and cells were randomly divided into control and A. fumigatus keratitis group, which were treated with Mincle ligand Trehalose-6,6-dibehenate (TDB), Mincle neutralizing antibody (MincleAb) or PBS before infection. The cornea infection was monitored using a slit lamp and further analyzed using H&E assay. PCR, Western blot, immunostaining, TUNEL staining and flow cytometry were used to examine the expression of Mincle and apoptosis factors, PMN infiltration and cell apoptosis, respectively. RESULTS: Higher levels of Mincle mRNA and protein, as well as epithelial thickness and presence of inflammatory cells in the stroma, were observed in the AF group compared to control. In addition, higher Mincle mRNA levels were observed in normal and stimulated neutrophils and macrophages. Furthermore, Fas, FasL and CASP3 mRNA levels, neutrophils infiltration rate and TUNEL-positive cells were significantly increased in AF+MincleAb mice compared with the control. Similar results, as well as significantly higher neutrophils and macrophages apoptosis, were observed by treating cells with MincleAb in vitro. Most importantly, opposite results i.e. lower mRNA levels, neutrophils infiltration rate and TUNEL-positive cells, as well as lower cell apoptosis in vitro, were observed in mice and cells treated with TDB. CONCLUSION: Mincle-participated in inflammatory process which inhibits neutrophils and macrophages apoptosis induced by A. fumigatus involved in Fas-dependent apoptotic pathways.
PURPOSE: To determine whether macrophage inducible C-type lectin (Mincle) regulates neutrophils and macrophages apoptosis in A. fumigatuskeratitis. MATERIALS AND METHODS: A murine model (C57BL/6) of fungal keratitis (AF) was established by gently scraping corneal central epithelium, smearing A. fumigatus on the epithelium surface and covering the eye with contact lenses. AF cell model was established by extracting neutrophils (PMN) and macrophages, and then infecting cells with A. fumigatus. Animals and cells were randomly divided into control and A. fumigatuskeratitis group, which were treated with Mincle ligand Trehalose-6,6-dibehenate (TDB), Mincle neutralizing antibody (MincleAb) or PBS before infection. The cornea infection was monitored using a slit lamp and further analyzed using H&E assay. PCR, Western blot, immunostaining, TUNEL staining and flow cytometry were used to examine the expression of Mincle and apoptosis factors, PMN infiltration and cell apoptosis, respectively. RESULTS: Higher levels of Mincle mRNA and protein, as well as epithelial thickness and presence of inflammatory cells in the stroma, were observed in the AF group compared to control. In addition, higher Mincle mRNA levels were observed in normal and stimulated neutrophils and macrophages. Furthermore, Fas, FasL and CASP3 mRNA levels, neutrophils infiltration rate and TUNEL-positive cells were significantly increased in AF+MincleAb mice compared with the control. Similar results, as well as significantly higher neutrophils and macrophages apoptosis, were observed by treating cells with MincleAb in vitro. Most importantly, opposite results i.e. lower mRNA levels, neutrophils infiltration rate and TUNEL-positive cells, as well as lower cell apoptosis in vitro, were observed in mice and cells treated with TDB. CONCLUSION: Mincle-participated in inflammatory process which inhibits neutrophils and macrophages apoptosis induced by A. fumigatus involved in Fas-dependent apoptotic pathways.
Authors: Matthias Kelm; Sylvain Lehoux; Veronica Azcutia; Richard D Cummings; Asma Nusrat; Charles A Parkos; Jennifer C Brazil Journal: FASEB J Date: 2019-12-23 Impact factor: 5.191