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

Abstract

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.

Figure 1.

ISG15 expression in H-K ATCC-treated primary HCECs and conditioned media. Cultured HCECs were challenged with 100:1 H-K P. aeruginosa cells. Cells and conditioned media were collected at 4, 8, and 16 hours postchallenge and processed for ISG15 Western immunoblotting analysis. The results are representative of two independent experiments.

Figure 2.

Isg15 deficiency increased the severity of P. aeruginosa keratitis in B6 mice. WT or Isg15^−/− B6 mouse corneas were scarified and inoculated with 1.0 × 10⁴ CFU of P. aeruginosa. The infected corneas (n = 5) were photographed at 1 and 3 dpi (A). At 3 dpi, the corneas were clinically scored (B), excised, and subjected to bacterial counting (C) with the results presented as CFU P. aeruginosa per cornea and to MPO determination (units/cornea) (D). The results are representative of two independent experiments (n = 5 each), and P values were generated using 1-way ANOVA. **P < 0.01.

Figure 3.

Effects of Isg15 deficiency on the gene expression of B6 mouse corneas in response to P. aeruginosa infection. WT or Isg15−/− mouse corneas were inoculated with 1.0 × 10⁴ CFU of P. aeruginosa. The scrapped epithelia (A) or whole corneas (B, C) were subjected to RNA isolation and real-time PCR analysis at 6 hpi (A), 24 hpi (B), and 3 dpi (C). The results are presented as the increase (fold) over the value for WT naive corneas (set at 1) after normalization to the level of β-actin as the internal control. The results are representative of two independent experiments, each with three corneas. *P < 0.05, **P < 0.01 (1-way ANOVA).

Figure 4.

Effects of Ifnar deficiency on Isg15 and Cxcl10 expression in B6 mouse corneas in response to P. aeruginosa infection. WT or Ifnar⁻/⁻ mouse corneas were scarified and inoculated with 1.0 × 10⁴ CFU of P. aeruginosa at 0 hours. At 1 dpi, the infected corneas, along the corneas isolated from naive WT and Ifnar-deficient mice, were processed for qPCR analysis for the mRNA expressions of Isg15, Il1b, and Cxcl10. The results are presented as the increase (fold) over the value for wild-type naive corneas (set at 1) after normalization to the level of β-actin as the internal control. The results are representative of two independent experiments, each with three corneas. *P < 0.05, **P < 0.01 (1-way ANOVA).

Figure 5.

Ifnar deficiency increased the severity of P. aeruginosa keratitis in B6 mice. WT or Ifnar⁻/⁻ mouse corneas were subconjuctivally injected with ISG15 or BSA at –6 hours, scarified, and inoculated with 1.0 × 10⁴ CFU of P. aeruginosa at 0 hours. The infected corneas were photographed (A) at 1 and 3 dpi. At 3 dpi, the corneas were excised and subjected to CFU counting (CFU/corneas) (B) and MPO determination (units/cornea) (C) (n = 5). Another set of corneas was processed for ELISA determination of the levels of IL-1β (D) and IFN-γ (E) (n ≥ 3). The results are representative of two independent experiments. *P < 0.05, **P < 0.01.

Figure 6.

Recombinant ISG15 affects gene expression in PHCECs (Primary Human Corneal Epithelial Cells) and in vitro bactericidal activity of ISG15-treated corneas. Cultured primary HCECs were treated with human recombinant ISG15 (200 ng) and collected at 1 or 2 hours and subjected to RNA isolation and real-time PCR analysis (A). Naive B6 mouse corneas with subconjuctival injection or ISG15, with BSA as the control, for 6 hours, excised, minced, and homogenized in 100 µL PBS with a TissueLyser (Retch). A total of 200 ng recombinant ISG15 or BSA was added to the naive corneal homogenates. P. aeruginosa (100 CFU) in 400 µL PBS was incubated with corneal homogenates for 30 and 60 minutes at 37°C. At the end of incubation, the samples were subjected to bacterial plate-colony counting. The results are representative of two independent experiments, each with three samples (B). *P < 0.05, **P < 0.01 (1-way ANOVA).

Figure 7.

ISG15-induced protection against P. aeruginosa infection in B6 mouse corneas is LFA-1 dependent. B6 mouse corneas were subconjuctivally injected IFA1 inhibitor A286982 or A286982 plus ISG15 at –6 h, scarified, and inoculated with 1.0 × 10⁴ CFU of P. aeruginosa at 0 hours. The infected corneas were photographed (A) and clinically scored (B) at 1 dpi. The corneas were excised and subjected to CFU counting (CFU/corneas) (C) and MPO determination (units/cornea) (D) (n = 5). The results are representative of two independent experiments. *P < 0.05, **P < 0.01 (1-way ANOVA).