Aspergillus Fumigatus Promotes T Helper Type 2 Responses Through Thymic Stromal Lymphopoietin Production By Human Corneal Epithelial Cells

Background:Fungal keratitis (FK) is a major cause of blindness in developing countries. If not effectively treated, it can lead to blindness or even loss of the eyeball. There is a high incidence of FK in hot and humid area. Normal immune function is the key factor in resistance to fungal infection and control of disease progression. Therefore, deep understanding of corneal immune system, seeking effective strategies against corneal fungal infection are of great theoretical and clinic significance.Corneal epithelial cells are the first innate line of defense against fungi, recognizing pathogen associated molecular pattern (PAMPs) in microbes through pattern recognition receptors (PRRs), which include Toll like receptors (TLRs), NOD like receptors (NLRs) and C-type lectin receptors (CLRs). Crosstalk among TLRs, NLRs and CLRs can enhancenuclear factor κB activation, induce inflammatory factors and antimicrobial peptides release, such as interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor a (TNF a), human betadefensin 2 and LL37.Activated innate immunity subsequently leads to effective adaptive immunity. Recently, Zhang et al. reported that mice that had previously encountered Candida albicans developed milder FK, healed faster and produced more immunoglobulin (Ig) G and IgA in the serum and more Ig deposition and lymphocyte infiltration. Therefore, besides innate immunity, adaptive immunity also participates in fungal infection of the cornea, and interaction between the innate and adaptive immune systems is critical in both acute and chronic inflamatory responses in the eye.TSLP was identified as a critical factor in acquired immunity, which is predominantly expressed by epithelial cells (airway, ocular tissues and gut). Expression of TSLP could be regulated by TLRs and nuclear factor κB signalling pathways. Liu YJ et al. proved that TSLP could induce dendritic cells activation and maturation, promote T and B lymphocytes proliferation and differentiation, and initiate adaptive immunity.At present, researches about TSLP are mostly restricted to allergic disease, such as atopic dermatitis, asthma and allergic conjunctivitis, and few studies focus on fungal infection. Kobayashi et al. also showed that in vitro exposure of human airway epithelial cells to Alternaria extract induced production and extracellular release of TSLP. Here, we further investigated whether and how TSLP participates in the adaptive immune response of corneal fungal infection.Purpose:To confirm expression of TSLP in A. fumigatus challenged by THCEs and determine whether TSLP has the ability to polarize Th2 responses. To explore the potential role of thymic stromal lymphopoietin (TSLP) in adaptive immunity of fungal keratitis and clarify the influence of THCE-derived TSLP on lymphocyte proliferation, activation as well as differentiation.Methods:1. Preparing of A. fumigatus hyphae, using the A. fumigatus strain CCTCC 93024, purchased from the China Centre for Type Culture Collection. The strain was grown on Sabouraud glucose agar for 24h, then heated at 56℃ for 60min. Then, the mycelia were disruptedinto 20-to 40-μm pieces and kept frozen at-80 ℃ until use as the A. fumigatus hyphae antigen.2. THCEs were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 50% defined keratinocyte serum-free medium. THCEs were divided into two groups at random:experimental group and control group. The experimental group was stimulated with A. fumigatus hyphae (106 pieces per millilitre) for various times (1,3,6,12,24 and 48 h). The cell culture supernatants were collected for measurement of TSLP expression.3. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers taken from ethylene diamine tetraacetic acid blood were purified by Ficoll-Hypaque density gradient centrifugation. PBMCs were seeded at 1×106 cells per well in six-well plates in the presence of human recombinant TSLP (1ng/ml) or culture medium only. PBMCs and culture supernatants were collected at different times and prepared for flow cytometry and protein detection.4. THCEs and PBMCs were co-cultured in a transwell system for various periods. To co-culture with PBMCs, four types of THCEs were applied:1 THCEs treated with culture medium (control); 2 THCEs stimulated with A. fumigatus hyphae (A. fumigatus); 3 THCEs preincubated with control siRNA followed with stimulation by A. fumigatus hyphae (control siRNA+A. fumigatus); and 4 TSLP-knockdown THCEs stimulated by A. fumigatus hyphae (TSLP siRNA+A. fumigatus). THCEs in groups 3 and 4 were transfected with control siRNA and TSLP siRNA, respectively. A. fumigatus hyphae were added at 106 pieces per millilitre to the lower chamber of groups 2,3 and 4 at 48 h after transfection. PBMCs and culture supernatants in the upper inserts were collected.5. We detected the proliferation and activation of collected PBMCs as well as T helper type 2 (Th2) differentiation by flow cytometry and quantitative real-time reverse transcription polymerase chain reaction. IgG and IgA levels in supernatants of PBMCs were measured by means of ELISA.Results:Compared with control group, expression of TSLP was increased after 3h of stimulation in THCEs. Compared with PBMCs cultured with medium only, recombinant TSLP induced PBMCs to produce high amounts of Th2 type cytokines, IL-4 and IL-13. The percentage of IL-4-producing and IL-13-producing CD4+T cells also significantly increased after stimulated with TSLP. TSLP siRNA transfected THCEs, the level of mRNA and protein of TSLP were inhibited significantly. Thymic stromal lymphopoietin could induce a Th2 response in vitro, and the expression of TSLP was highly increased in THCEs stimulated with A. fumigatus hyphae. A. fumigatus-infected THCEs were capable of promoting human lymphocyte proliferation and activating human CD4+ T cells, CD8+ T cells and B cells by up-regulating the expression of activation marker CD69. Importantly, Th2 differentiation of CD4+ T cells was induced during co-culture with A. fumigatus-infected THCEs in a transwell system. Interestingly, blockade of TSLP using siRNA prevented the proliferation and activation of lymphocytes as well as Th2 differentiation. We also detected an increased IgG level that was associated with TSLP.Conclusions:Our results suggested that TSLP is highly produced in THCEs stimulated with A. fumigatus hyphae. Moreover, TSLP could induce Th2 development. THCE-derived TSLP induced activation of CD4+ T cells, CD8+ T cells and B cells and enhanced the production of Th2 cytokines (IL-4 and IL-13) and IgG. TSLP produced by A. fumigatus-infected THCEs could act as a key molecule in adaptive immune responses of FK via skewing Th2 differentiation and promoting humoral immunity.