| Objective:In this study,with subcutaneous and tail-vein injection of Cladosporium cladoporioides spores infected healthy mice,aim to explore the changes of pattern recognition receptors(PRRs)and cytokines expression in skin and lung,as well as the pathogenicity of C.cladoporioides to lungs and skins.Then research innate immune responses induced by different infective methods.Method:Based on the infection by subcutaneous injection of C.cladoporioides spore suspension(2×10~7CFU/m L)in C57BL/6 mice.Skin tissue samples were collected for histopathological examination on 7 d and 14 d post-infection(PI);skin tissues samples were collected for immunofluorescence staining on control group and 7 d PI;skin tissue samples were collected for RT-q PCR on control group and 12 h,1 d,3 d,7 d and 14 d PI to determine the expression of cytokines and PRRs.Based on the systemic infection by tail-vein injecting C.cladoporioides spore suspension(2×10~7CFU/m L)into the of KM mice.Lung tissue samples were collected for histopathological examination on 1 d,2 d,3 d,5 d,9 d,14 d and 21 d PI;lung tissues samples were collected for CFU assay on 2 d,5 d,9 d and 14 d PI;lung tissues samples were collected for immunofluorescence staining on control group and 5 d PI;lung tissue samples were collected for RT-q PCR on control group and 2 d,5 d,9 d and 14 d PI to determine the expression of PRRs.Results:Subcutaneous abscess was formed in mice after subcutaneous injection of C.cladoporioides.A large number of inflammatory cells infiltrated in the abscess,but with less inflammatory cells infiltration in the epidermal and dermal layers of the skin,as well as a little recruitment of dendritic cells,macrophages and neutrophil,and the expression of ICAM-1.The gene expression of PRRs Dectin-1,TLR-2 and TLR-4 significantly increased(p<0.01)on 14 d,3 d and 12 h PI,respectively.The gene expression of pro-inflammatory cytokines significantly increased(p<0.01)happened on 12 h PI that IFN-?,IL-12,IL-23,IL-6,IL-4,IL-17F included,happened on 7 d PI that IL-1?and IL-17A included.Among these pro-inflammatory cytokines,the relative expression multiples of IL-6,IL-1?and IL-17A was higher.The gene expression of anti-inflammatory cytokine TGF-?significantly increased on 7 d PI(p<0.01).After tail intravenous injection of C.cladoporioides,a large number of fungal spores were colonized in the lungs of mice,and serious congestion and hemorrhage,a large number of inflammatory cells infiltrated in the lung tissues.There were abundant recruition of neutrophils,macrophages and dendritic cells,and high expression of ICAM-1.The gene expression of PRRs Dectin-1 significantly decreased(p<0.001)after infection,the gene expression of TLR-2 and TLR-4 significantly increased(p<0.01)on 5 d and 14 d PI,respectively.The gene expression of pro-inflammatory cytokines significantly increased(p<0.01)happened on 2 d and 5 d PI that IFN-?,IL-13 and IL-1?included,happened on 5d PI that IL-4,IL-23 and IL-22 included,happened on 14 d PI that IL-12,TNF-?and IL-17F included,happened on 2 d PI that IL-17A and TNF-?.Among these pro-inflammatory cytokines,the relative expression multiples of IL-4,IL-13,IL-12,IL-23,IL-1?and IL-22 was higher.The gene expression of anti-inflammatory cytokine IL-10 and TGF-?significantly increased on 5 d PI(p<0.01).Conclusion:Abscesses were formed in the skin lesions of mice after subcutaneous injection of C.cladoporioides spore suspension,fungal spores and hyphae only presented in the abscess,Toll-like receptors recognized fungal spores and induce innate and adaptive immune responses,Th17 signaling pathway involved in the host immune responses.The tail intravenous injection of C.cladoporioides spore suspension serious infected the lungs of mice,a mass of fungal spores colonized the lungs and been largely eliminated on5 d PI,Toll-like receptors recognize fungal spores and induce innate and adaptive immune responses,Th2 and Th17 signaling pathways involved in the host immune responses. |
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