Abstract
Purpose: :
The transcription factor interferon regulatory factor 1 (IRF1) is known to upregulate antibacterial and proinflammatory enzymes/cytokines such as inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (Cox2) and IL-12. This study was conducted to elucidate the expression of IRF1 induced by flagellin (TLR5 ligand) and P. aeruginosa, and its role in corneal epithelial inflammatory response in human corneal epithelial cells (HCECs) and in B6 mouse corneas.
Methods: :
Superarray was used to screen for transcription factors differentially expressed in flagellin- or P. aeruginosa-treated HCECs. Superarray results were confirmed using RT-PCR and Western blot techniques. Pharmacological inhibitors were used to block NF-ΚB and STAT1 in flagellin or bacterium challenged cells. siRNA approach to was used to assess the role of IRF1in the expression of iNOS, Cox-2, and IL-12. In vivo expression of IRF1 was assessed with Western blotting of corneas infected with different PA strains.
Results: :
The Superarray screen identified, and RT-PCR and Western blotting confirmed IRF1 as one of the transcription factors greatly upregulated in bacterial challenged cells. Interestingly, flagellin pretreatment that dampens bacterium-induced cytokine expression also attenuated IRF1 upregulation. STAT1 and NF-ΚB was involved in bacterium-induced IRF1 expression. The expression pattern of several IRF1 target genes in flagellin and bacterial-treated cells were similar to that of IRF1 protein levels. Knockdown of IRF1 resulted in reduced target gene expression. In vivo, P. aeruginosa infection resulted in the induction of IRF1 and its levels appeared to be correlated reversely to the severity of keratitis.
Conclusions: :
IRF1 is a critical transcription factor required for P. aeruginosa-induced expression of certain proinflammatory and defense genes, such as iNOS, Cox2, and IL-12 and its lack of expression may contribute to the dampening of inflammation in flagellin-pretreated HCECs.
Keywords: cornea: basic science • inflammation • pseudomonas