IL-36α Enhances Host Defense against Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas

Abstract

The IL-36 cytokines are known to play various roles in mediating the immune response to infection in a tissue- and pathogen-dependent manner. The present study seeks to investigate the role of IL-36R signaling in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. IL-36α^-/-, IL-36γ^-/-, and IL-36R^-/- mice had significantly more severe keratitis than wild-type mice. At six hours postinfection, IL-36α pretreatment augmented P. aeruginosa-induced expression of IL-1Ra, IL-36γ, LCN2, and S100A8/A9. At one day postinfection, exogenous IL-36α suppressed, whereas IL-36α deficiency promoted, the expression of IL-1β. At three days postinfection, exogenous IL-36α suppressed Th1 but promoted Th2 immune response. IL-36α stimulated the infiltration of IL-22-expressing immune cells, and IL-22 neutralization resulted in more severe keratitis. IL-36α alone stimulated dendritic cell infiltration in B6 mouse corneas. Taken together, our study suggests that IL-36R signaling plays a protective role in the pathogenesis of P. aeruginosa keratitis by promoting the innate immune defense, Th2, and/or Th22/IL-22 immune responses. Exogenous IL-36α might be a potential therapy for improving the outcome of P. aeruginosa keratitis.

Figure 1.. IL-36 signaling pathway protects B6 mouse corneas from PA infection.

WT, IL-36α^−/−, IL-36γ^−/−, and IL-36R^−/− mouse corneas were gently scratched with a needle to create three 1mm epithelium incisions and inoculated with 1.0×10⁴ CFU PA. (A & B) Eyes were photographed (original magnification × 10) and clinically scored from 1 to 3 dpi. The 1–12 scale clinical scores were graded by an independent blinded observer. All mice were euthanized at 3 dpi. The corneas were excised and subjected to (C) bacterial plate counting presented in log scale and (D) Myeloperoxidase (MPO) determination. The data in (B-D) were presented as median of clinical score (median ± interquartile range), average of CFU or MPO units per cornea (mean ± SD) in dot plots. P values were analyzed with one-way ANOVA, followed by a Bonferroni test. N=5, *P < 0.05, ** P < 0.01, ***P<0.001. The results were representative of three independent experiments.

Figure 2.. PA-infection increases IL-36α expression in B6 mouse cornea.

Mouse corneas were gently scratched with a needle to create three 1mm epithelium incisions and inoculated with 1.0×10⁴ CFU PA at 0h. (A) Whole corneas were collected at 3hi, 6hpi, 9hpi 18hpi for q-PCR. P values were analyzed with one-way ANOVA. N=5, *P < 0.05, ** P < 0.01, ***P<0.001. (B) Mouse corneal epithelial cells were collected at 6 hpi for Western-blot analysis of IL-36α. β-actin serves as loading control. (C) Cornea samples representing 5 time points were also collected for Western-blot analysis of IL-36α with β-actin as loading control. Three samples of each time point were used in qPCR and two samples in Western blot. (D) Corneas at 1 dpi were excised and processed for immunohistochemistry analysis. The 6 μm cryostat sections were stained with anti-IL-36α (red), and DAPI (blue) for nuclei. E, epithelium; S, stroma. (E) Flow cytometric analyses of IL-36α, CD45, Ly6G, F4/80 positive immune cells in 1dpi infected corneas. 6 corneas were pooled for each sample. IL36α, CD45+Ly6G+, CD45+F4/80+ cells are shown in the flow cytometric plots. The results were representative of three independent experiments.

Figure 3.. IL-36α plays a protective role in PA keratitis in B6 mice.

At 4h before PA inoculation, WT mice were subconjunctivally injected with recombinant mouse (rm) IL-36α (100ng/5μl), or 0.1% BSA as control. IL-36α^−/− mice were subconjunctivally injected with 0.1% BSA. At 0h, the corneas were gently scratched with a needle to create three 1mm epithelium incisions and inoculated with 1.0×10⁴ CFU PA. (A & E) Mouse corneas were monitored and photographed at 1, 3 dpi, (B & F) and clinical scores were assigned as described in Figure 1. The corneas were excised and subjected to (C & G) bacterial plate counting, the results were presented at log scale; and (D & H) MPO unit determination. The data in (B-D, F-H) were presented as a median of clinical score (median ± interquartile range), average of CFU or MPO units per cornea (mean ± SD). P values were generated by one-way ANOVA, followed by a Bonferroni test. N=5, *P < 0.05, ** P < 0.01, ***P<0.001. The results were representative of three independent experiments.

Figure 4.. IL-36α ameliorates PA-induced inflammation in the B6 mouse corneas.

WT mouse corneas were treated with rmIL-36a or 0.1% BSA and inoculated with PA as in figure 3. Naïve corneas were used as negative control. The corneas were collected at 1dpi (A) The corneas were processed for immunohistochemistry analysis. 6μm cryostat sections were stained with NIMP-R14 antibody for neutrophils or F4/80 antibody for macrophages. The images of neutrophils (green), macrophages(green) were merged with DAPI (blue nuclei) staining. E, epithelium; S, stroma. Three independent experiments were performed; 1 representative image for each condition is presented. (B) The corneas were processed for flow cytometry analysis and stained with CD45, Ly6G, F4/80 antibodies. Six corneas were pooled for each sample. (C) Three samples of each groups were used for neutrophil and macrophage cell percentage statistical analysis (mean ± SD). P values were generated by one-way ANOVA, *P < 0.05, ** P < 0.01, ***P<0.001. Three independent experiments were performed; 1 representative image for each condition is presented. E, epithelium; S, stroma.

Figure 5.. IL-36α alters gene expression in B6 mouse corneas in response to PA-infection.

WT, IL-36α^−/− mouse corneas were treated with rmIL-36α, or 0.1% BSA and inoculated with PA as in Figure 3. (A) Corneal epithelial cells were collected at 6 hpi and (B) whole cornea samples were collected at 1dpi (immune cell infiltration occurs after 6 hpi) and analyzed by real-time PCR. The results were presented as a relative increase (fold) to those of naive corneas, which were set as a value of 1. Corneal epithelial cells were collected at 6 hpi for (C) ELISA analysis of S100A8/9 dimmer, (D) Western-blot analysis of LCN2. Data were representative of three independent experiments with three corneas per group (mean ± SD). *P<0.05, **P<0.01, ***P<0.001 (One-way ANOVA).

Figure 6.. IL-36 signaling regulates adaptive immune gene expression in B6 mouse corneas in response to PA-infection.

rmIL-36α, or 0.1% BSA treated WT mice, IL-36R^−/− mice were scratched and inoculated with PA. (A) At 3 dpi, whole cornea samples were collected and analyzed by real-time PCR. The results were presented as a relative increase (fold) to those of naive corneas, which were set as a value of 1. Three corneas per group (mean ± SD). (B) Six mouse corneas and two cervical lymph nodes were pooled for each sample for flow cytometric analysis. The samples were stained with CD45, CD4, T-bet, GATA3 antibody. Th1 and Th2 cell percentage of mouse lymph nodes were shown in FACS plots. (C)Th1 and Th2 cell percentage out of the total CD4+T cell population, as well as total cell number were analyzed. Data were representative of three independent experiments with three samples per group. *P<0.05, **P<0.01, ***P<0.001 (One-way ANOVA). LN, lymph node.

Figure 7.. IL-22 as a downstream cytokine of IL-36α improves the outcome of PA keratitis.

WT mice were subconjunctivally injected with rmIL-36α, or 0.1% BSA as control given 4h prior to PA inoculation. At 1 dpi, corneas were excised and processed for immunohistochemistry analysis. The 6μm cryostat sections were stained with (A) anti-IL22 (green) and DAPI (blue) for nuclei, arrowheads: IL-22 positive cells, or with anti-IL22 (red) and F4/80 (green) antibodies (B), arrowheads: IL-22 and F4/80 positive cells. (C-E) Infected corneas were photographed and scored at 1 and 3 dpi and collected at 3dpi for CFU and MPO determination. (C) The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. Data in (D) and (E) were presented as an average of CFU or MPO units per cornea (mean ± SD). N=5, *P<0.05, **P<0.01, ***P<0.001 (paired t-test). Data are representative of three independent experiments. E, epithelium; S, stroma; L, limbus.

Figure 8.. IL-36α regulates dendritic cell infiltration in B6 mouse corneas.

rmIL-36α or 0.1%BSA was subconjunctivally injected into WT mouse corneas. (A) At 1 day post injection, Mouse corneas were collected for whole mount staining with CD11c (green) antibody. (B) The number of CD11c positive cells was quantified and analyzed. Data were representative of three independent experiments with three corneas per group. ***P<0.001 (paired t-test).