Aspergillus Fumigatus Promotes Th2 Immune Response Through Thymic Stromal Lymphopoietin In Fungal Keratitis

Background:Fungal keratitis(fungal keratitis,FK)is a serious and destructive role of blinding eye disease.If not treatment or misdiagnosed,it will lead to blindness.At present,the study suggests that different immune states may affect the development of fungal keratitis in fungal infections.Therefore,the study of the role of acquired immune response in fungal keratitis may provide a new theoretical for the treatment of diseases.The pathologic changes of keratitis can be divided into infiltration,stage,regression and healing.Studies have shown that the immune system plays an important role in fungal keratitis.First of all,the innate immune(congenital immune)is the defense system's first line,fungal infection after by pattern recognition receptors(PRRs)is to identify pathogenic microorganisms on the surface of the pathogen associated molecular patterns(PAMPs)so as to play a role of immune defense.Among them,the pathogen-associated molecular pattern(PAMPs)mainly consists of the c-type agglutinin receptor(CLRs),toll-like receptor(TLR)and nucleotide binding oligomerization domain sample receptor(NLRs)and the removal of the receptor(SR)family.The specific immune response(acquired immune response)is the second line of immune system.In the acquired immune response,T cells play an important role and the dynamic changes between Th1,Th2,Th17 and Treg cells may affect the pathogenesis of fungal keratitis.Therefore,the study of acquired immune response is of great significance in fungal keratitis.The findings of Thymic stromal lymphopoietin(TSLP)revealed the pathogenesis and mechanism of many allergic diseases,such as allergic conjunctivitis and allergic rhinitis.At present,the study found the epithelial cells can secrete TSLP and through stimulation of dendritic cells(DC)secrete Th2 type transcription factor(GATA-3)and Th2 type chemokines(TARC,MDC),promote the differentiation of CD4+T cells into Th2 cells and induce the Th2 immune response.We found that the TSLP initiates the acquired immune response after stimulating the corneal epithelial cells.However,the role of foreign aid TSLP in the development of fungal keratitis and how to induce the acquired immune response remain unclear.Purpose:To study the expression of Thymic stromal lymphopoietin(TSLP)after aspergillus fumigatus infection mice and to explore the potential role of TSLP in adaptive immunity of aspergillus fumigatus.Methods:1.Preparation of aspergillus fumigatus hyphae:used aspergillus fumigatus strains CCTCC93024,form China center for Type Culture Collection.And the strain was grown on Sabouraud glucose agr,shake at room temperature for 24 hours.2.Aspergillus infection in mice model was established,scratched corneal and inoculated aspergillus fumigatus hyphae and stitching eyelid for 24 hours,observe corneal infection through slit lamp microscope at different time points.3.TSLPsiRNA?scramble siRNA?the human recombinant TSLP protein and IgG were injected under the conjunctivium,treated with microinjection needle 24 hours and 4 hours before the fungal infection,and 5ul each time.4.The experimental animals were divided into six groups:empty group,PBS group,AF group,TSLPsiRNA+AF group,scramble siRNA+AF group,human recombinant TSLP+AF group and IgG+AF group,and taken picture by slit lamp also given clinical evaluation.HE staining to observe the pathological change,and to measure Th1 cytokines(IFN-?),Th2 cytokines(IL-4,1L-13),Th17 cytokines(IL-17 a,IL-17 f),Treg cells factor(TGF-beta 1),Th1 type transcription factor(T-bet),Th2 type transcription factor(GATA-3),Th17 transcription factor(STAT-3)and the expression of Treg transcription factors(Foxp3)by RT-PCR and ELISA.Results:The corneal infection in mice can be observed in 24 hours with clear corneal infiltration,severe corneal ulcer in 72 hours,and corneal scarring in the 7th day.RT-PCR,ELISA:normal infection group compared with control group and PBS group,arrives at a peak on the first day,a few days later gradually decline,the fifth day increased again.Th2 cells secreted cytokines(IL-4,IL-13)and Th17 cytokines(IL-17A and IL-17F)were highest in the fifth day of infection.IFN-?,T-bet,GATA-3 and STAT-3 highest expression in the early stages after infection.However,there was no significant effect on the expression of Treg cytokines(TGF-beta)and transcription factor(Foxp3).At TSLPsiRNA+AF group and Human recombinant TSLP protein+AFgroup,IL 4,IL-13 and GATA-3 show the tendency of lower and higher respectively compared with AF group.IFN-?,IL-17 A,IL-17 F,T-bet and STAT-3 did not change significantly.Conclusions:In fungal keratitis,our results suggested that TSLP is highly and it could induce the Th2 immune response,promote T cell differentiation towards Th2.There was an imbalance between Th1,Th2 and Th17,but no effect on Treg.This can provide the theoretical basis for the clinical treatment of fungal keratitis.