Research On The Role And Regulation Of Toll-like Receptor-mediated Mechanism Of TSLP In The Allergic Conjunctivitis

Objective:We sought to explore the role of Toll-like receptor (TLR) mediated induction of TSLP and its signaling pathways in patients with allergic conjunctivitis.Methods:Three models were used for this study. Based on this, this study was divided into three parts:1. A well characterized murine model of allergic conjunctivitis induced by SRW pollen: Wild type BALB/c mice, TLR4-deficient (Tlr4-d) mice, myeloid differentiation primary response gene88(MyD88) knockout (MyD88-/-) mice, MyD88+/+mice were used in this part. Mice were immunized with SRW pollen by means of footpad injection on day0. Allergic conjunctivitis was induced by means of topical application of SRW pollen into each eye once a day from days10to15.(Quantitative real-time PCR) qPCR, Immunohistochemical and immunofluorescent staining were carried out to evaluate the expression of TSLP, its downstream singaling pathways and Th2associated cytokines in the ocular mucosa and draining cervical lymph nodes.2. A topical challenge model by extract of SRW (SRWe) on the murine ocular surface:Wild type BALB/c mice, TLR4-deficient (Tlr4-d) mice, myeloid differentiation primary response gene88(MyD88) knockout (MyD88-/-) mice, MyD88+/+mice were used in this part. SRWe was topically instilled per eye or PBS as a control for4to24hours. qPCR and ELISA were carried out to evaluate the expression of TSLP in the ocular mucosa.3. A culture model of primary human corneal epithelium exposed to SRWe:Human corneal epithelial cultures were treated with different concentration of SRWe. SRWe was applied without or with preincubated mouse anti-human TLR4antibody; its isotype, murine IgG2a k; or quinazoline, a nuclear factor kB (NF-kB) activation inhibitor. qPCR and ELISA were carried out to evaluate the expression of TSLP.Results:1. Murine model of allergic conjunctivitis induced by SRW pollen:①Evaluated by qPCR, the mRNA expression of TSLP, TSLP signaling pathways (TSLPR, OX40L, OX40, CD11C and CD4) and Th2cytokones (IL-4, IL-5and IL-13) were significantly upregulated in the ocular mucosa and draining cervical lymph nodes from SRW mice, compared with the PBS control group. The expression of Thl cytokine did not change in SRW mice. A corresponding response was detected by immunohistochemical and immunofluorescent staining. ②Compared with wild-type BALB/c mice, the stimulated TSLP/OX40L/OX40signaling, and TH2-dominant inflammatory response by ocular mucosa, especially conjunctival tissues, were dramatically reduced or eliminated in BALB/c-based Tlr4-d mice.③The expression of TSLP and its signaling molecules, as well as the TH2cytokines were significantly stimulated in the cornea, conjunctiva, and CLNs from SRW-challenged wild-type MyD88+/+mice at both the mRNA and protein levels. Those responses were dramatically reduced or eliminated in SRW-challenged MyD88-/-mice, as evaluated by means of RT-qPCR and immunostaining.2. A topical challenge model by extract of SRW (SRWe) on the murine ocular surface:①SRWe significantly stimulated TSLP expression in corneal epithelia and conjunctiva in BALB/c mice when compared with PBS-treated control animals. The SRWe-stimulated TSLP was significantly blocked at both the mRNA and protein levels by a rat antimouse TLR4antibody but not by its isotype, rat IgG2a.②Compared with wild-type BALB/c mice, the SRWe topical challenge did not increase TSLP expression in corneal and conjunctival epithelia of TLR4-d mice. SRWe promoted TSLP production by ocular epithelia at both the mRNA and protein levels only in MyD88+/+mice, but not in MyD88-/-mice.3. A culture model of primary human corneal epithelium exposed to SRWe:TSLP induction at the mRNA and protein levels was concentration-dependently stimulated by SRWe in primary HCECs. Interestingly, the SRWe stimulated TSLP was significantly blocked by preincubation of cells with neutralizing mAb against human TLR4or NF-K B activation inhibitor.Conclusion:1. TSLP and its downstream pathways play an important role in allergic conjunctivitis.2. TSLP signaling in TH2-dominant inflammation is stimulated by SRW pollen through a TLR4/Myd88-dependent innate immune response.3. When blocking TLR4or Myd88, the TSLP induction and Th2response were reduced in allergic conjunctivitis. These novel findings shed light on the understanding of innate mucosal epithelial immunity involved in allergic inflammation and might create new therapeutic targets to cure allergic disease.