| Objective:To investigate the role of epidermal growth factor receptor(EGFR)/mitogenactivated protein kinase(MAPK)signaling pathway in the activation of innate epithelial immune responses during vulvovaginal candidiasis(VVC).Methods:(1)The vaginal epithelial cells(VK2/E6E7 cells)were co-cultured with the standard strain of Candida albicans(C.albicans).To determine the activation of EGFR and MAPK signaling pathways in the interaction between VK2/E6E7 cells and C.albicans,RNA sequencing,Western blot(WB)and cellular immunofluorescence were used.Then,the relationship between EGFR and MAPK signaling pathway proteins was verified by using EGFR inhibitors and constructing EGFR-overexpressing cells(EGFR-Ad-VK2 cells).Enzyme-linked immunosorbent assay(ELISA)and Real Time Cellular Analysis(RTCA)techniques were used to detect the effect of EGFR-MAPK signaling pathway in regulating the secretion of inflammatory cytokines and cell damage induced by C.albicans.The mouse model of VVC infected by C.albicans was constructed,and the function of EGFR signaling pathway on local inflammation in the mice vagina was determined with EGFR inhibitors,histopathological sections and immunofluorescence.(2)Vaginal epithelial cells were co-cultured with sensitive C.albicans strain SC5314 and clinical drug-resistant isolate 1052 respectively,and the two strains were compared in terms of the activation of EGFR-MAPK signaling pathway,inflammatory response and cell damage via Western blotting,ELISA and RTCA.Through the mouse model of VVC,the differences in EGFR signaling pathway activation,inflammatory response and cell damage induced by the two strains were further compared in vivo.The morphological differences between the sensitive C.albicans strain SC5314 and the clinical drug-resistant isolate 1052 were directly compared under microscope.(3)Vaginal epithelial cells were co-cultured with 4 strains of C.albicans and 5 strains of non-albicans Candida(NAC)species,including Candida glabrata,Candida parapsilosis and Candida tropicalis and 2 strains of Candida auris.WB,ELISA,RTCA technology were used to compare the differences between C.albicans and NAC species in inducing EGFR-MAPK signaling pathway activation,inflammatory response and cell damage.The morphological differences between C.albicans and 4 strains of NAC species were compared under microscope.Result:(1)The results of RNA-Seq showed that the EGFR and MAPK signaling pathways were significantly upregulated in VK2/E6E7 cells under the stimulation of C.albicans;WB and immunofluorescence results showed that the phosphorylation of EGFR,ERK1/2,p38,c-Fos and p65 was significantly and differentially activated by SC5314(P<0.05);EGFR inhibitors significantly reduced the levels of EGFR,ERK1/2 and p38 phosphorylation,but overexpression of EGFR only led to a significant increase of EGFR and p38 phosphorylation(P<0.05).ELISA results showed that EGFR,ERK1/2 and p38 inhibitors significantly reduced C.albicans-induced cytokine secretion(CCL20,G-CSF,GM-CSF,IL-1β,IL-6 and IL-17A),among which p38 inhibitor was the most effective;yet the overexpression of EGFR led to a significant increase of GCSF,GM-CSF,IL-1β,IL-6 and IL-17A levels,except CCL20(P<0.05).RTCA results showed that only EGFR and p38 inhibitor suppressed C.albicans-induced vaginal epithelial cell damage.In the mouse model of VVC,EGFR inhibitors effectively improved vaginal inflammation and epithelial damage induced by SC5314.(2)With respect to the activation of EGFR-MAPK signaling pathway,clinical drug-resistant isolate 1052 induced higher levels of EGFR and p38 phosphorylation and lower levels of ERK1/2 and c-Fos phosphorylation compared to SC5314.In terms of cytokine release,1052 induced higher levels of CCL20,G-CSF,GM-CSF,IL-1β,IL-6 and IL-17A.In addition,EGFR,ERK1/2 and p38 inhibitors significantly reduced the secretion of these cytokines(P<0.05).RTCA results showed that only EGFR and p38 inhibitors significantly reduced vaginal epithelial cell damage induced by the two strains(P<0.05).In the mouse model of VVC,the levels of EGFR phosphorylation induced by 1052 in the vagina was significantly higher than that induced by SC5314,and EGFR inhibitors significantly reduced the phosphorylation of EGFR(P<0.05).Moreover,the number of neutrophil infiltration and apoptotic cells induced by 1052 were significantly greater than those of SC5314,and EGFR inhibitor also significantly reduced the inflammatory response and apoptosis induced by the two strain(P<0.05).By directly observing the morphological difference between SC5314 and 1052 under the microscope,we found that 1052 was more filamentous.(3)In terms of the activation of EGFR-MAPK signaling pathway,all NAC species activated higher levels of ERK1/2,c-Fos and p65 phosphorylation,and much lower levels of EGFR and p38 phosphorylation compared to C.albicans.The levels of cytokines(CCL20,G-CSF,GM-CSF,IL-1β,IL-6 and IL-17A)induced by SC5314 were higher than that by all NAC species(C.glabrata,C.parapsilosis,C.tropicalis and C.auris).Inhibition of EGFR,ERK1/2 and p38 resulted in significant reduction of CCL20,G-CSF,GM-CSF,IL-1β,IL-6 and IL-17A induced by these NAC species.RTCA results showed that the cell damage curve induced by each Candida strain was species-specific,and the cell damage induced by CBS 14918 and C.parapsilosis was significantly stronger than that of C.albicans,C.glabrata and C.tropicalis.Only p38 inhibitor significantly reduced vaginal epithelial cell damage induced by all the NAC species.By directly observing the differences of these NAC species under the microscope,it was found that only C.tropicalis occasionally form pseudohyphae,while other NAC species grew only in the form of yeast.ConclusionThe EGFR-MAPK signaling pathway plays a key role in the activation of innate epithelial immune responses and inhibition of EGFR can significantly ameliorate the inflammatory response and epithelial damage in experimental VVC. |
