Toll–Like Receptor 2–Mediated Expression of Human ß–defensin–2 in Corneal Epithelial Cells

Abstract

Abstract: : Purpose: We previously showed that corneal epithelial cells possess the ability to recognize Gram–positive bacteria and to initiate the innate immune responses by the expression and/or release of proinflammatory cytokines and human ß–defensin–2 (hBD–2).This study sought to elucidate the underlying mechanisms regulating hBD–2 expression and its role in innate defense in corneal epithelial cells (HCECs) in response to S. aureus challenge. Methods: HUCL, a telomerase–immortalized HCEC line, and primary culture of HCECs were challenged with live or heat–killed S. aureus, its exo–products, or cell wall components, lipopeptide (Pam3Cys), and lipoteichoic acid (LTA). The expression of TLRs and ß–defensin were determined using RT–PCR and secretion of IL–6, IL–8, TNF–α, and hBD–2 was assessed using ELISA and slot–blot, respectively. TLR2 neutralizing antibody was used to block S. aureus–induced hBD2 expression. The NF–ΚB inhibitors isohelenin and karmebakaurin, p38 MAPkinase inhibitor SB203580 and JNK inhibitor SP600125 were used to elucidate the role of these intracellular signaling pathways in hBD–2 expression. Results: HCECs constitutively expressed TLR1, 2, 6 and 9 mRNA. The expression of TLR2 at both mRNA and protein levels was up–regulated in HCECs challenged with S.aureus–exoproducts or lipopeptide. S.aureus–exoproducts and lipopeptide also induced upregulation of hBD–2 mRNA and protein in a time–dependent manner in both HUCL and primary HCECs, whereas the levels of hBD–1and hBD–3 mRNA remained unchanged. Furthermore, both live and heat–killed S. aureus induced hBD–2 expression in HCECs. Blocking TLR2 with antibodies resulted in a decrease in the expression of IL–6, IL–8, TNF–α, and hBD–2 secretion. Conditioned media derived from HCECs challenged with lipopeptide or S.aureus–exoproducts exhibited bactericidal activity against S. aureus, P. aureginosa and E.coli. The NF–ΚB inhibitors completely blocked lipopeptide–induced expression of hBD–2, while both p38 and JNK inhibitors partially blocked, 50% and 60%, respectively. Conclusions: S.aureus exoproducts and lipopeptide induce proinflammatory cytokine and hBD–2 expression in HCECs in a TLR2 dependent manner; pathogen–challenged and TLR–activated HCECs possess antimicrobial activity. Thus, HCECs may play an important role in innate defense against bacterial infection in the cornea.

Keywords: cornea: epithelium • inflammation • Staphylococcus