ISG15 Acts as a Mediator of Innate Immune Response to Pseudomonas aeruginosa Infection in C57BL/6J Mouse Corneas

PURPOSE: IFN-stimulated gene (ISG) 15 is a type 1 IFN-induced protein and known to modify target proteins in a manner similar to ubiquitylation (protein conjugation by ISG15 is termed ISGylation). We sought to determine the role of ISG15 and its underlying mechanisms in corneal innate immune defense against Pseudomonas aeruginosa keratitis. METHODS: ISG15 expression in cultured human corneal epithelial cells (HCECs) and mouse corneas was determined by PCR and Western blot analysis. Gene knockout mice were used to define the role of ISG15 signaling in controlling the severity of P. aeruginosa keratitis, which was assessed with photographing, clinical scoring, bacterial counting, myeloperoxidase assay, and quantitative PCR determination of cytokine expression. Integrin LFA-1 inhibitor was used to assess its involvement of ISG15 signaling in P. aeruginosa–infected corneas. RESULTS: Heat-killed P. aeruginosa induced ISG15 expression in cultured HCECs and accumulation in the conditioned media. Isg15 deficiency accelerated keratitis progress, suppressed IFNγ and CXCL10, and promoted IL-1β while exhibiting no effects on IFNα expression. Moreover, exogenous ISG15 protected the corneas of wild-type mice from P. aeruginosa infection while markedly reducing the severity of P. aeruginosa keratitis in type 1 IFN-receptor knockout mice. Exogenous ISG15 increased bacteriostatic activity of B6 mouse corneal homogenates, and inhibition of LFA-1 exacerbated the severity of and abolished protective effects of ISG15 on P. aeruginosa keratitis. CONCLUSIONS: Type 1 INF-induced ISG15 regulates the innate immune response and greatly reduces the susceptibility of B6 mouse corneas to P. aeruginosa infection in an LFA-1-dependent manner.