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Zhitao Su, Lili Zhang, Fan Lu, Cintia S. De Paiva, Stephen C. Pflugfelder, De-Quan Li; Short Ragweed Pollen Induces Proallergic Cytokine TSLP via TLR4-dependent Signaling Pathways in Human and Murine Ocular Surface. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4012.
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Pollen is the most common allergen that triggers seasonal conjunctivitis, rhinitis and asthma, as well as exacerbates atopic dermatitis. However, the underlying mechanism by which pollen induces pro-allergic cytokine thymic stromal lymphopoietin (TSLP) via mucosal innate immunity is largely unknown. This study was to explore the direct effects and signaling pathway of short ragweed (SRW) pollen on TSLP expression and production by human and murine ocular surface epithelia.
Two models were used for this study, a culture model of primary human corneal epithelial cells (HCECs) and a topical challenge model on mouse ocular surface, exposed to aqueous extract of defatted SRW pollen (SRWe). Confluent HCECs were treated for 4-48 hours with SRWe (0.1-50 µg/ml) without or with pre-incubated TLR4 monoclonal antibody, its isotype mouse IgG2a Κ, or quinazoline. BALB/c, TLR4 deficient, MyD88 wild type and knockout mice at 8-10 weeks of age were topically challenged for 4-24 hours by SRWe at 150µg/5µl/eye without or with TLR4 antibody, or by PBS as controls. The corneal epithelial and conjunctival tissues were collected for evaluation by total RNA reverse transcription and real time PCR, immunohistochemical staining, ELISA or Western blotting.
TSLP mRNA was upregulated at 4 hours and its protein was increased at 24 hours in primary HCECs exposed to SRWe with a concentration-dependent manner. SRWe at 10µg/ml increased the TSLP protein to the levels comparable to that stimulated by 10ng/ml of TNF-α in HCECs. Interestingly, the SRWe-stimulated TSLP was significantly blocked by pre-incubation with TLR4 antibody or a NF-ΚB activation inhibitor quinazoline, but not the isotype. In vivo study, topical exposure to SRWe significantly stimulated TSLP mRNA by 2 fold in corneal and conjunctival epithelia, and its protein levels by 3.2 fold in corneal epithelia and 2.2 fold in conjunctiva in BALB/c mice when compared with untreated or PBS-treated controls. The SRWe-stimulated TSLP was significantly blocked at both mRNA and protein levels by a TLR4 antibody but not by its isotype. Furthermore, SRWe did not stimulate TSLP mRNA and protein levels by corneal and conjunctival epithelia in TLR4 deficient and MyD88 knockout mice.
These findings demonstrate for the first time that SRW pollen, acting as a functional TLR4 agonist, initiates TLR4-dependent innate signaling that induces TSLP production in human and murine ocular surface epithelia, a novel mechanism by which pollen triggers allergic inflammation. These findings shed light on the understanding of mucosal epithelial innate immunity, and may discover new therapeutic targets to cure allergic diseases.
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