TSLP-activated Dendritic Cells Induce T Helper Type 2 Inflammation In Aspergillus Fumigatus Keratitis

BackgroundFungal keratitis(FK)refers to a kind of infective keratopathy that often occurs in tropical and subtropical regions.It has a high morbidity and has a high rate of blindness.Especially in developing countries,FK is one of the most common blinding eye diseases,and its incidence ranks the first among all varieties of severe infective keratopathy.The etiology of FK is varied.In developed countries,the most common cause of this disease is wearing extended lens.In developing countries,the main cause of this disease is corneal trauma,especially plant corneal trauma.In the world,the pathogenic fungus of FK is more than 70 kinds,and the principle pathogens of FK are Fusarium and Aspergillus.Clinical data show that compared with other types of keratitis,Aspergillus keratitis has more important clinical significance for its lower diagnosis rate,poorer drug treatment effect,worse prognosis and higher recurrence rate.Therefore,this study uses Aspergillus fumigatus to construct the animal model of FK and carries out related research,which has important theoretical value and clinical significance.Since the illness condition of the fungal keratopathy aggregates rapidly,if not diagnosed and treated promptly and effectively,significant damages can occur,such as hypopyon,corneal perforation,malignant glaucoma,fungal endophthalmitis and other symptoms,and even lead to blindness or enucleation.Current research has shown that in the process of FK,the immune function of the body is an important factor in resistance to fungal infection.Therefore,in order to study fungal keratitis,it is necessary to explore the immune defense system of the cornea in the process of disease development.The structure of the cornea is relatively complex,and it is divided into five layers,such as epithelial cell layer,anterior elastic layer,matrix layer,post elastic layer and endothelial cell layer.Corneal epithelial cells become the first biological defense line of the organisms to resist the invasion of pathogenic microorganisms.When pathogenic microorganisms invade the cornea tissue,corneal epithelial cells have the capacity to identify pathogen associated molecular patterns(PAMPs)on the surface of pathogenic microorganisms through important pattern recognition receptors(PRRs):Toll-like receptors(TLRs)and NOD-like receptors(NLRs),thus initiating the downstream immune response and playing a role in resisting the invasion of pathogenic microorganisms.Our previous studies have shown that TLR2 and TLR4 on the surface of corneal epithelial cells can be stimulated by Aspergillus fumigatus and identify them in the process of Aspergillus fumigatus keratitis.They activate nuclear factor kappa B(NF-κB)and downstream signaling pathway,then promoting the release of cytokines interleukin(IL)-6,IL-8,IL-10,IL-1β and TNF-α,as well as antibacterial peptides of hBD2 and LL37.They enhance the innate immune response by interaction with NOD1 and NOD2,whereby play an important role in the resistance to fungal infection.Thymic stromal lymphopoietin(TSLP)is an IL-7 like cytokine derived from thymic stromal cell line.Studies show that TSLP is mainly expressed by various epithelial cells and plays an important role in immune response.Under the stimulation of pathogenic microorganisms,TLRs in the respiratory tract and corneal epithelial cells will up-regulate the expression of NF-κB signaling pathway and further promote the release of TSLP.Thymic stromal lymphopoietin receptor(TSLPR)is a heterodimer composed of TSLPR and IL-7Rα-chain,which is expressed on the surface of dendritic cells(DCs),lymphocytes,mononuclear cells et al.Our previous studies have found that in the process of Aspergillus fumigatus keratitis,corneal epithelial cells can up-regulate the expression of TSLP and enhance the expression of TSLPR through the TLR2/MyD88/NFKB-p65 signaling pathway,and then launche the downstream TSLP/TSLPR/STAT5 signaling pathway,thereby play the role of anti-fungal infection and enhance the innate immunity.This suggests that TSLP is obviously involved in the process of anti fungal infection in the cornea.Studies have shown that TSLP acts on DCs in a variety of ways and participates in the acquired immune response.In the process of cornea wound healing,DCs participate,in collaboration with epithelial cells,in the repair and maintenance of corneal tissues,which is closely related to TSLP.TSLP activates CD11c+DCs to promote the synthesis of Th2-attracting chemokines,thymus and activation-regulated chemokine(TARC),macrophage-derived chemokine(MDC)and pulmonary activation-regulated chemokine(PARC),which will further promote the proliferation of CD4+T cells and induce CD4+T cells to differentiate into T helper type 2(Th2)cells,which are capable of secreting cytokines of IL-4,IL-5,IL-13 and TNF-a,finally inducing the Th2 inflammation.The OX40 ligand(L),a member of the TNF superfamily,can combine with the counterreceptor OX40 on the surface of activated T cells to stimulate T cells proliferation and promote the secretion of immunoglobulin.TSLP has the capacity to stimulate DCs to express OX40L,to induce initial T cells differentiation into Th2 cells,and to induce Th2 inflammation.TLR4 ligand can stimulate the corneal epithelial cells secretion of TSLP,which further induce the maturation of CD11c+DCs through the TSLP/TSLPR signaling pathway,then the combination of OX40L with OX40 on the surface of CD4+T cells will induce CD4+T differentiate into Th2 cells,which are capable of secreting cytokines,such as IL-4,IL-5,IL-13,and to induce Th2 inflammation.However,the present study on the involvement of TSLP and DCs in acquired immunity is more common in the area of allergic diseases,while is rare in the area of infectious diseases,especially in the FK.Our previous studies have found that,TSLP,produced by corneal epithelial cells under the stimulation of A.fumigatus,have the ability to propel the proliferation of CD4+T cells,promoting CD4+T cells to express Th2 cytokines such as IL-4 and IL-13,inducing Th2 inflammation to participate in the adaptive immune response.However,it is still unclear in FK,through which way TSLP stimulate CD4+T cells proliferation and differentiation.In that way,whether DCs have also participated in the immune response in FK,and if DCs do,what role they have played in such process,are issues remaining to be researched by further studies.Answering these questions will help us to understand more clearly that how the body starts the acquired immunity in the process of fungal infection of the cornea.This provides a new theoretical basis for the corneal resistance to fungal infection and provides a new direction for the prevention and treatment of fungal infection of the cornea.In present study,we demonstrated that in A.fumigatus keratitis,TSLP promotes the aggregation,maturation and migration of DCs,while mature DCs secrete Th2-attracting chemokines,promoting secretion of proinflammatory factor and Th2 cytokines,and inducing Th2 inflammation,thereby play the role of antifungal infection.This is useful for us to further understand the mechanism of acquired immune response in fungal keratitis and provide a new theoretical basis and direction for the prevention and treatment of fungal keratitis.It has important theoretical significance and clinical value.Part I Mechanism of DCs induce Th2 inflammation in Aspergillus fumigatus keratitis[Purpose]Study how dendritic cells(DCs)participate in the immune response and induce Th2 inflammation in Aspergillus fumigatus keratitis.[Methods]1.The central cornea of wild-type C57BL/6(B6)mouse was wounded with three parallel 1 mm scratches by a needle.Fungal conidia suspension was applied to the surface of the wounded cornea and an artificial molded parafilm contact lens was placed onto the cornea before the eyelid was sewed closed.One day after infection,the artificial contact lenses were removed.We examined the mice by slit lamp at 0.5d,1d,3d,5d,and 7d p.i,and graded the opacification.Mice with phosphate buffer saline(PBS)were used as the negative control.Mice with no treatment were used as the blank control.2.PCR was done to assess the expression of DCs according to CD11c.PCR and Western blot was done to assess the maturation of CD11c+DCs according to OX40L.Immunofluorescent staining was done to show the proliferation,migration,and maturation of DCs.3.PCR and ELISA were done to assess the expression of Th2-attracting chemokines TARC and MDC secreted by DCs.PCR and ELISA were done to assess expression of proinflammatory cytokine TNF-α and Th2 cytokines IL-4,IL-5,and IL-13.[Results]Corneal ulcers were aggravated with the progression of infection and healed with a scar.DCs aggregated,maturated,and migrated from the basement membrane to the corneal epithelium,as well as migrated from the corneal limbus to the central cornea in A.fumigatus-infected corneas.Expression of TARC and MDC was increased at early stage of infection.IL-4,IL-5,IL-13,and TNF-α was increased at late stage of infection.[Conclusions]In the process of A.fumigatus keratitis,DCs progressively aggregate and maturate during the process of migration.On one hand,they activated CD4+T cells by expressing OX40L;while on the other hand,they had a Th2-attracting effect by secreting TARC and MDC.Under the collaboration of these activities,expression of proinflammatory cytokine TNF-α and Th2 cytokines IL-4,IL-5,and IL-13 obviously increased in the later phase of the infection,and Th2 inflammation was induced.Part Ⅱ Mechanism of TSLP-activated DCs induce Th2 inflammation in Aspergillus fumigatus keratitis[Purpose]Study the effects of TSLP on the immune response that DCs participate in and the Th2 inflammation that DCs induced in Aspergillus fumigatus keratitis.[Methods]1.The mouse eyes were subconjunctivally injected with TSLP siRNA,scramble siRNA,rTSLP,and IgG,and infected with a fungal conidia suspension.The central cornea of wild-type C57BL/6(B6)mouse was wounded with three parallel lmm scratches by a needle.Fungal conidia suspension was applied to the surface of the wounded cornea and an artificial molded parafilm contact lens was placed onto the cornea before the eyelid was sewed closed.One day after infection,the artificial contact lenses were removed.We examined the mice by slit lamp and graded the opacification.A.fumigatus-infected mice were used as control.2.PCR and Western blot was done to assess the expression of mature DCs affected by TSLP.Immunofluorescent staining was performed to show the distribution of mature DCs affected by TSLP vertically and horizontally.3.PCR and ELISA were done to assess expression of TARC and MDC.PCR and ELISA were done to assess expression of IL-4,IL-5,IL-13,and TNF-a.To exclude the affect that TSLP made on Thl or Th17 inflammation,we evaluated expression of Th1 cytokine IFN-y and Th17 cytokine IL-17.[Results]Compared with A.fumigatus-infected corneas,expression of TSLP was evidently reduced in TSLP siRNA-treated corneas and increased in rTSLP-treated corneas.The corneal inflammation was alleviated in the TSLP siRNA injected group and increased with rTSLP injection.Mature DCs can express TSLPR,and migrated from the basement membrane to the corneal epithelium,the place where TSLP was expressed.Expression of the TSLPR and OX40L were decreased in TSLP siRNA-treated corneas and increased in rTSLP-treated corneas.Mature DCs were fewer and migrated slower in TSLP siRNA-treated corneas,while in rTSLP-treated corneas mature DCs not only expressed more but also migrated faster.Expression of mature DCs decreased both in the paralimbus,the middle region,and the central cornea in TSLP siRNA-treated corneas,while in rTSLP-treated corneas expression of mature DCs increased in both regions of the cornea.Expression of TARC and MDC was decreased in TSLP siRNA-treated corneas and increased in rTSLP-treated corneas.Expression of IL-4,IL-5,IL-13,and TNF-α was decreased in TSLP siRNA-treated corneas and increased in rTSLP-treated corneas.The change of TSLP had no significant effect on expression of IFN-γ and IL-17.[Conclusions]In A.fumigatus keratitis,TSLP promotes the proliferation,maturation and migration of DCs,while mature DCs secrete Th2-attracting chemokines TARC and MDC,promoting the secretion of proinflammatory cytokine TNF-α and Th2 cytokines IL-4,IL-5 and IL-13 to trigger Th2-dominant inflammation.