The Transcriptional Regulation Of Flagellin-Induced Innate Protection Of The Cornea: Role Of Irf1 And Atf3

Pre-exposure of the cornea to TLR5 ligand flagellin induces profound mucosal innate protection against infections by reprogramming gene expression. This study explored the flagellin-induced modifications of transcription factor expression and function, specifically of IRF1 and ATF3 in corneal epithelial cells to elucidate the transcriptional mechanisms underlying the protective function of flagellin on the cornea. Initially we used Superarray to screen for transcription factors and identified Interferon Regulatory Factor (IRF) 1 and Activating Transcription Factor (ATF) 3 as the most drastically affected genes by flagellin pretreatment in P. aeruginosa challenged human corneal epithelial cells (CEC). However, flagellin pretreatment had opposite effects on IRF1 (inhibition) and ATF3 (enhancement) gene expression in response to P. aeruginosa, and other IRFs were not affected. To find the functional target gene of IRF1, we knocked-down IRF1 using siRNA and identified the pleiotropic chemokine CXCL10, but not IL12-p35 or iNOS, as a specific target. We then attempted to understand the role of IRF1 and CXCL10 in mucosal innate protection against infections in vivo. Flagellin augmented the P. aeruginosa-induced expression of CXCL10 in CECs in WT, but not in IRF1-/- mice at 6 hpi. IRF1 deficiency markedly increased the severity of P. aeruginosa keratitis and significantly attenuated flagellin-elicited protection compared to WT controls at 3 dpi. CXCL10 neutralization in the cornea of WT mice displayed similar pathogenesis to that of IRF1-/- mice. To understand the regulation of CXCL10 expression in vivo, we used neutralizing antibodies to target IFNγ and its producer NK cells. The neutralization of IFNγ receptor or NK cells prevented flagellin-augmented IRF1 and CXCL10 expression and increased the susceptibility to P. aeruginosa infection in mouse corneas. Together, our results indicate that IRF1 plays a role in the corneal innate immune response by regulating CXCL10 expression and that IFNγ-producing NK cells induces epithelial expression of IRF1, thus significantly contributing to the protection of the cornea from P. aeruginosa infection. Next we sought understand the functional role of ATF3 in mucosal innate protection against infections in vivo. Consistent with in vitro results, flagellin augmented the P. aeruginosa-induced epithelial expression of ATF3 in CECs in WT at 6 hpi. ATF3 deficiency markedly increased the severity of P. aeruginosa keratitis, indicated by significantly increased bacterial count, neutrophil infiltration and CXCL2 expression. In addition, ATF3-null mice displayed compromised flagellin-induced corneal protection compared to PBS-pretreated ATF3-/- littermates at 3 dpi. Together, our results indicate that ATF3 plays a role in the corneal innate immune response by regulating inflammation and bacterial clearance, and ATF3 is indispensible to maintain the protective properties of flagellin against bacterial keratitis. In conclusion, flagellin pretreatment of corneal epithelial cells reprograms the expression of epithelial IRF1 and ATF3, via different mechanisms, resulting in the enhancement of the innate immune response of the cornea. Both transcription factors play an critical role in protecting the cornea from bacterial invasion and regulating inflammation, and may serve as important markers for the development of therapeutic drugs to treat and/or prevent bacterial keratitis