Inflammatory Responses of Corneal Epithelial Cells to Pseudomonas aeruginosa Infection

Abstract

Purpose: We hypothesized that corneal epithelium plays a role in the innate immune response by sensing the presence of pathogens and providing signals that activate the corneal defense system. We sought to determine the mechanisms involved in the activation of the signaling pathways and subsequent production of proinflammatory cytokines in human corneal epithelial cells (HCECs) in response to Pseudomonas aeruginosa infection. Methods: Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary cultures of HCECs were exposed to P. aeruginosa (PA01 strain) with or without the presence of the NF-κ B inhibitor kamebakaurin, the p38 inhibitor SB203580, or the JNK inhibitor SP600125. Iκ B-α phosphorylation and degradation and p38 and JNK phosphorylation were assessed at different time points by Western blot analysis. Interleukin (IL)-6, IL-8, and TNF-α levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Results: Exposure of HUCL cells and primary HCECs to P. aeruginosa resulted in rapid activation of NF-κ B as indicated by an increase in IκB-α phosphorylation observed within 15 min and by IκB-α degradation, which peaked in 1 hr. Two stress-activated mitogen-activated protein kinases, p38 and JNK, were also activated as their phosphorylation was induced by P. aeruginosa infection. Concomitant with the activation of these Toll-like receptor–mediated signaling pathways, transcriptional expression and subsequent secretion of IL-6 and IL-8 in HUCL cells were also induced by P. aeruginosa. Presence of the NF-κ B inhibitor kamebakaurin in culture medium blocked P. aeruginosa–induced NF-κ B activation and inhibited IL-6, IL-8, and TNF-α expression and secretion. Inhibition of p38 or JNK also resulted in a decrease in bacteria-induced expression and secretion of these cytokines. Conclusions: P. aeruginosa triggers an innate immune response in HCECs, and NF-κ B and, to a lesser extent, the p38/JNK signal pathways are responsible for P. aeruginosa–induced proinflammatory cytokine production in these cells.

KEYWORDS:

• bacterial keratitis
• corneal epithelium
• proinflammatory cytokines
• Pseudomonas aeruginosa
• Toll-like receptors