The Roles And Mechanisms Of TSLP And CGAS-STING In Regulating Immunity Of Corneal Fungal Infections

Fungal keratitis(FK)is one of the most destructive corneal infections caused by pathogenic fungi,and it has high morbidity with a global distribution.Extended lens wear and trauma are the important predisposing factors in developed and developing countries,respectively.FK usually develops in people infected with pathogenic fungi such as Aspergillus fumigatus(A.fumigatus)and Fusarium.In China,the most common pathogenic fungus is the plant-associated A.fumigatus.FK has become a major cause of blindness in many developing countries due to the increasing use of antibiotics,poor visual prognosis,and the absence of effective symptomatic treatment.Therefore,clarifying the pathogenesis of FK and developing new therapies are essential.Both innate and adaptive immune responses participate in fungal infections of the cornea.The innate immunity is the first line of host defenses to control infections.After corneal infection,the pattern recognition receptors(PRRs)of the cornea interact with the fungal pathogen-associated molecular patterns(PAMPs),and initiate an innate immune response to defend the cornea against fungal pathogens.This response produces many chemokines and cytokines to recruit inflammatory cells and eliminate the pathogens,which are events that are important for maintaining the normal corneal structure.The activated innate immunity subsequently leads to an effective adaptive immune response.Dendritic cells(DCs)are responsible for sampling antigenic material from the environment,expressing several PRRs that recognize A.fumigatus,shaping T cell responses through the secretion of cytokines,and priming T cell responses via antigen presentation.In conclusion,DCs are key regulators of immunity and the most important cells for initiating adaptive fungal immunity.CD4+T helper cells are crucial to the adaptive immune system.After antigenic stimulation,naive CD4+T cells get activated,proliferate,and differentiate into cells with different effector phenotypes,including Thl,Th2,Th17,and regulatory T(Treg)cell lineages.Several reports have implicated DC-derived Th17-promoting cytokines,such as IL-1β,IL-6,and IL-23,that are thought to promote Th17 responses that develop during bacterial and fungal infections.However,whether an A.fumigatus-induced Th17 inflammatory response takes place during FK is still unknown.How DCs sense A.fumigatus and further induce CD4+T cell responses also need to be explored.Thymic stromal lymphopoietin(TSLP)is an IL-7-related cytokine that mainly expresses in human epithelial cells in response to several stimuli,including bacteria,fungi,parasites,and inflammatory cytokines.Recent studies have revealed that other types of cells such as mast cells,smooth muscle cells,fibroblasts,DCs,trophoblasts,and cancer or cancer-associated cells are also able to produce TSLP under certain inflammatory conditions.TSLP is thought to mediate its biological activity through a heterodimeric receptor complex consisting of the IL-7 receptor subunit α(IL-7RA)and a TSLP-specific receptor(TSLPR).The role of TSLP in the induction of the allergic Th2 immune response by DCs and the pathogenesis of allergic diseases(asthma,AD,and IBS,and others)has been previously clarified.Our initial research demonstrated that TSLP,produced by human corneal epithelial cells(HCECs)under the stimulation of A.fumigatus,could increase the proliferation of CD4+T cells,promote CD4+T cells to express IL-4 and IL-13,and finally induce a Th2 inflammatory response.However,whether DCs secrete TSLP and what the role of this cytokine is in the Thl7 inflammatory response that occurs during FK remains unknown.Cyclic GMP-AMP(cGAMP)synthase(cGAS)is a cytoplasmic DNA sensor that activates the STING-mediated signal cascade by generating a second messenger,cGAMP,to further promote the production of type Ⅰ interferons(IFNs)and initiate the innate immune response.The cGAS-STING signaling pathway can not only mediate the protective immune response against pathogen infection,but also recognize tumor-derived DNA and generate intrinsic anti-tumor immunity.However,abnormal activation of cGAS-STING signaling pathway by self and pathogen DNA can also lead to autoimmune and inflammatory diseases.Therefore,maintaining proper activation of the cGAS-STING signaling pathway has great significance.HCECs,distributed in the outermost layer of human corneal tissue,are the first barrier of the cornea against pathogen infection.After the cornea is infected by pathogens,it activates PRRs in HCECs and initiates an innate immune response.However,whether A.fumigatus could activate the cGAS-STING signaling pathway in HCECs and the role of this pathway in fungal keratitis remains unclear.In the present study,we try to understand the role of TSLP secreted by A.fumigatus-stimulated DCs during the Th17 immune response.We also aim to clarify the mechanisms contributing to the Th17 immune response of FK in vitro and in vivo.In addition,we will explore the role and mechanism of cGAS-STING signaling pathway in fungal keratitis.The content of this study is mainly divided into the following three parts:Part ⅠStudy on the role of TSLP in the Th17 inflammatory response induced by A.fumigatus-stimulated DCs1.ObjectThis part aims to study whether A.fumigatus-stimulated DCs can induce the differentiation of CD4+T cells into Th17 cells,and to clarify the role of DCs in inducing Th17-type acquired immunity.This part also tries to study whether A.fumigatus promotes the secretion of TSLP in DCs,and to clarify the role of TSLP in the Th17 inflammatory response induced by A.fumigatus-stimulated DCs.2.Methods2.1 Bone marrow-derived dendritic cells(BMDCs)were isolated from the femur and tibia of C57BL/6 mice.CD4+T cell purification kit was used to extract CD4+ T cells from the mouse spleen.Flow cytometry was used to identify the purity and activation level of the isolated DCs and CD4+ T cells.2.2 A.fumigatus-stimulated DCs were co-cultured with CD4+T cells for 3 or 4 days,and the proliferation level of CD4+T cells was detected by CFSE assay.qRT-PCR and flow cytometry were used to detect the expression levels of Th17 cytokines IL-17A,IL-17F,and IL-22 in CD4+T cells.2.3 DCs were stimulated with A.fumigatus for 1,3,6,12,24,and 48 h.qRT-PCR,ELISA,and Western blot were used to detect the expression level of TSLP in DCs.2.4 Mouse corneas were scratched with a sterile needle to establish the model of fungal keratitis.Slit-lamp examination,fluorescein staining,and H&E staining were used to evaluate the severities of mouse keratitis.Clinical scores were calculated to assess the clinical manifestation.qRT-PCR and immunofluorescence were used to detect the expression of TSLP and CD11c in mouse corneal tissues.2.5 Naive CD4+T cells were treated with rmTSLP for 3 or 4 days,CFSE was used to detect the proliferation level of CD4+T cells.qRT-PCR,ELISA,and flow cytometry were used to detect the expression level of Th17 cytokines.2.6 TSLP siRNA was used to knockdown TSLP in DCs,TSLPR siRNA was used to knockdown TSLPR in CD4+T cells,and Western blot was used to detect the knockdown efficiency of TSLP and TSLPR.CFSE was used to detect CD4+T cell proliferation levels.qRT-PCR and flow cytometry were used to detect the expression level of Th17 cytokines.2.7 The mouse cornea was subconjunctival injected with TSLP siRNA or treated with exogenous rmTSLP,and then infected with A.fumigatus for 1 day.Slit-lamp examination and fluorescein staining were used to evaluate the severities of mouse keratitis.Clinical scores were calculated to assess the clinical manifestation.qRT-PCR and ELISA were used to detect the expression of TSLP,IL-17A,IL-17F,and IL-22.3.Results3.1 The purity of the isolated CD4+T cells was more than 95%.Flow cytometry assay showed that the expressions of CD80,CD86,and MHC-II on the cell surfaces were increased A.fumigatus stimulation in DCs.3.2 The CFSE assay showed that A.fumigatus-stimulated DCs could significantly promote the proliferation of CD4+T cells.A.fumigatus-stimulated DCs induced CD4+T cell differentiation into Th cells that expressed IL-17A,IL-17F,and IL-22.Flow cytometry assay showed that the level of IL-17A in CD4+ T cells infected with A.fumigatus-stimulated DCs was markedly increased.3.3 qRT-PCR,ELISA,and Western blot showed that A.treatment promoted the expression of TSLP in DCs.The immunofluorescence results also showed that A.fumigatus increased the level of TSLP in DCs.3.4 These corneal injuries in mice were most serious one day after infection with A.fumigatus.The expression of TSLP and CD11c in mouse corneal tissues were significantly increased in fungal keratitis model.3.5 Exogenous rmTSLP treatment increased the proliferation of CD4+T cells.The results of qRT-PCR and ELISA showed that rmTSLP increased the expression levels of IL-17A,IL-17F,and IL-22 in CD4+T cells.3.6 TSLP was deleted about 80%by TSLP siRNA compared to the TSLP amount in NC siRNA-treated cells.The increased proliferative response of CD4+T cells induced by A.fumigatus-stimulated DCs was interrupted by knockdown of TSLP in the DCs.A.fumigatus also failed to promote the expression of IL-17A,IL-17F,and IL-22 in CD4+T cells.Similarly,after knocking down TSLPR in CD4+T cells,A.fumigatus-stimulated DCs could not induce the differentiation of CD4+T cells into Th17 cells.3.7 Exogenous rmTSLP promoted the progression of fungal keratitis in mice and increased the expression levels of IL-17A;,IL-17F,and IL-22 in mouse corneal tissues.Knockdown of TSLP significantly inhibited the progression of mouse fungal keratitis and decreased Th17 cytokine expression in mouse corneal tissues infected with A.fumigatus.4.Conclusion4.1 A.fumigatus-stimulated DCs promoted the proliferation of CD4+ T cells and induced the differentiation of CD4+T cells into Th17 cells.4.2 A.fumigatus increased the expression of TSLP in DCs and in mouse fungal keratitis tissues.4.3 A.fumigatus-stimulated DCs promoted the proliferation of CD4+T cells and induced Th17 inflammatory response through TSLP.4.4 A.fumigatus promoted Th17 inflammatory response through TSLP in a mouse keratitis model.Part ⅡStudy on the mechanism of TSLP in inducing Th17 inflammatory response through JAK/STAT pathway1.ObjectThis part aims to study the signaling pathways involved in the differentiation of CD4+T cells into Th17 cells induced by A.fumigatus-stimulated DCs.We also try to explore the molecular mechanisms of this signaling pathway in mediating the progression of Th17 inflammation and fungal keratitis in the cellular level and in the mouse fungal keratitis model.2.Methods2.1 A.fumigatus-stimulated DCs were co-cultured with CD4+T cells for 4 days.Total RNA was extracted for high-throughput sequencing to identify signaling pathways involved in the differentiation of CD4+T cells into Th17 cells.qRT-PCR was performed to verify the mRNA expression profile involved in the JAK/STAT signaling pathway obtained by high-throughput sequencing.2.2 CD4+T cells were co-cultured with A.fumigatus-stimulated DCs or treated with exogenous rmTSLP,and the expression levels of RORyt and JAK/STAT signaling pathway proteins p-JAKI,p-JAK2,and p-STAT3 were detected by Western blot.After knocking down TSLP or TSLPR,CD4+T cells were co-cultured with A.fumigatus-stimulated DCs for 4 days,and the expression levels of JAK/STAT signaling pathway proteins were detected by Western blot.2.3 CD4+T cells were co-cultured with A.fumigatus-stimulated DCs in the presence or absence of ruxolitinib or BBI608 for 4 days.Flow cytometry and qRT-PCR were used to detect the expression levels of IL-17A,IL-17F,and IL-22 in CD4+T cells.CD4+T cells exposed to rmTSLP were treated with or without ruxolitinib and BBI608 for 3 days.Flow cytometry and qRT-PCR were used to detect the expression levels of Th17 cytokines in CD4+T cells.2.4 The mouse cornea was subconjunctival injected with ruxolitinib or BBI608,and then infected with A.fumigatus for 1 day.Slit-lamp examination and fluorescein staining were used to evaluate the severities of mouse keratitis.Clinical scores were calculated to assess the clinical manifestation.qRT-PCR and ELISA were used to detect the expression of TSLP,IL-17A,IL-17F,and IL-22 in mouse corneal tissues.3.Results3.1 High-throughput sequencing showed that the JAK/STAT signal pathway was the most obvious signaling pathway involved in the Th17 inflammatory response induced by A.fumigatus.qRT-PCR was used to further verify the main molecules in the JAK/STAT signaling pathway obtained by sequencing.3.2 A.fumigatus-stimulated DCs and rmTSLP both could increase the expression levels of RORγt,p-JAK1,p-JAK2,and p-STAT3 in CD4+T cells.The above results could be abrogated by knocking down TSLP or TSLPR in DCs or CD4+ T cells,respectively.3.3 A.fumigatus-stimulated DCs and rmTSLP failed to increase the expression levels of RORγt,p-JAK1,p-JAK2,and p-STAT3 in CD4+T cells in presence of ruxolitinib ord BBI608.3.4 Subconjunctival injection of ruxolitinib or BBI608 significantly inhibited the progression of mouse fungal keratitis and decreased Th17 cytokines expression in mouse corneal tissues infected with A.fumigatus.4.Conclusion4.1 A.fumigatus-stimulated DCs activated the JAK/STAT signaling pathway through TSLP secretion.4.2 A.fumigatus-stimulated DCs promoted a Th17 response in CD4+T cells via the JAK/STAT signaling pathway.4.3 A.fumigatus promoted fungal keratitis progression through JAK/STAT signaling pathway.Part ⅢStudy on the mechanism of cGAS-STING in promoting the inflammatory response of fungal keratitis1.ObjectThis part aims to clarify the role and mechanism of cGAS-STING signaling pathway in HCECs infected with A.fumigatus and in the mouse fungal keratitis model.2.Methods2.1 HCECs were infected with A.fumigatus for 3,6,12,and 24 h,and the expression levels of TNF-α,IL-1β,IL-6,and IFN-β in HCECs were detected by qRT-PCR and ELISA.2.2 HCECs were infected with A.fumigatus for 3,6,and 12 h.Western blot was used to detect the protein levels of cGAS,STING,p-STING,TBK1,and p-TBK1 in HCECs.The level of cGAMP in the culture supernatant of HCECs was measured by ELISA.2.3 HCECs with cGAS stably knockdown was constructed using lentiviral transfection of cGAS shRNA plasmid.HCECs with cGAS knockdown or not were infected with A.fumigatus for 12 h.Western blot was used to detect the protein levels of cGAS,STING,p-STING,TBK1,and p-TBK1 in HCECs.qRT-PCR and ELISA were used to detect the levels of TNF-α,IL-1β,IL-6,and IFN-β in HCECs.HCECs with cGAS knockdown was transfected with PCMV-cGAS again,followed by treatment with A.fumigatus for 12 h.The levels of TNF-α,IL-1β,IL-6,and IFN-β in HCECs were detected.2.4 HCECs were infected with A.fumigatus for 12 h in the presence of cGAS specific inhibitor RU.521,and then Western blot was used to detect the protein levels of cGAS,STING,p-STING,TBK1,and p-TBK1 in HCECs.qRT-PCR and ELISA were used to detect the levels of TNF-α,IL-1β,IL-6,and IFN-β in HCECs.2.5 The mouse cornea was subconjunctival injected with RU.521or cGAS siRNA,and then infected with A.fumigatus for 1 day.Slit-lamp examination,fluorescein staining,and H&E staining were used to evaluate the severities of mouse keratitis.Clinical scores were calculated to assess the clinical manifestation.ELISA was used to detect the protein level of cGAMP in the corneal tissues.Immunofluorescence was performed to detect the protein levels of p-STING and p-TBKl in mouse corneal tissues.qRT-PCR and ELISA were used to detect the levels of TNF-α,IL-1β,IL-6,and IFN-β in mouse corneal tissues.3.Results3.1 The results of qRT-PCR and ELISA showed that A.fumigatus promoted the expression of TNF-α,IL-1β,IL-6,and IFN-β in HCECs.3.2 A.fumigatus increased the protein level of cGAMP in HCECs.Western blot showed that A.fumigatus increased the protein levels of cGAS,p-STING,and p-TBK1 in HCECs time-dependently.3.3 Western blot results showed that the levels of cGAMP,cGAS,p-STING,and p-TBK1 were reduced after knockdown of cGAS in HCECs infected with A.fumigatus.knockdown of cGAS significantly reduced the expression of pro-inflammatory cytokines in HCECs infected with A.fumigatus.However,re-expression of cGAS restored the increased expression of TNF-α,IL-1β,IL-6,and IFN-β in HCECs induced by A.fumigatus infection.3.4 Western blot results showed that A.fumigatus failed to increase the levels of cGAS,p-STING,and p-TBK1 in the presence of cGAS inhibitor RU.521.qRT-PCR and ELISA showed that RU.521 treatment significantly inhibited the increase in the expression levels of TNF-α,IL-1β,IL-6,and IFN-β in HCECs induced by A.fumigatus infection.3.5 Subconjunctival injection of RU.521 or cGAS siRNA significantly alleviated the progression of mouse fungal keratitis and inhibited the increase in the protein levels of cGAMP,p-STING,and p-TBK1 in mouse corneal tissue induced by A.fumigatus infection.qRT-PCR and ELISA results also showed that RU.521 or cGAS siRNA could inhibit the expression of TNF-α,IL-1β,IL-6 and IFN-β in mouse corneal tissue induced by A.fumigatus infection.4.Conclusion4.1 The cGAS-STING signaling pathway is activated upon A.fumigatus infection in HCECs and in mouse keratitis model.4.2 A.fumigatus accelerates inflammatory cytokine production through cGAS-STING in vitro and in vivo4.3 Inhibition of cGAS alleviates the severity of A.fumigatus keratitis in the mouse model.