Role of IL-36γ/IL-36R Signaling in Corneal Innate Defense Against Candida albicans Keratitis

Abstract

Purpose: Interleukin (IL)-36 cytokines have been shown to play either beneficial or detrimental roles in the infection of mucosal tissues in a pathogen-dependent manner, but their involvement in fungal keratitis remains elusive. We herein investigated their expression and function in mediating corneal innate immunity against Candida albicans infection.

Methods: Gene expression in mouse corneas with or without C. albicans infection was determined by regular RT- and real-time (q)-PCR, Western blot analysis, ELISA or proteome profile assay. The severity of C. albicans keratitis was assessed using clinical scoring, bacterial counting, and myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration. IL36R knockout mice and IL-33-specific siRNA were used to assess the involvement IL-33 signaling in C. albicans-infected corneas. B6 CD11c-DTR mice and clodronate liposomes were used to define the involvement of dendritic cells (DCs) and macrophages in IL-36R signaling and C. albicans keratitis, respectively.

Results: IL-36γ were up-regulated in C57BL6 mouse corneas in response to C. albicans infection. IL-36 receptor-deficient mice display increased severity of keratitis, with a higher fungal load, MPO, and IL-1β levels, and lower soluble sIL-1Ra and calprotectin levels. Exogenous IL-36γ prevented fungal keratitis pathogenesis with lower fungal load and MPO activity, higher expression of sIL-1Ra and calprotectin, and lower expression of IL-1β, at mRNA or protein levels. Protein array analysis revealed that the expression of IL-33 and REG3G were related to IL-36/IL36R signaling, and siRNA downregulation of IL-33 increased the severity of C. albicans keratitis. Depletion of dendritic cells or macrophages resulted in severe C. albicans keratitis and yet exhibited minimal effects on exogenous IL-36γ-induced protection against C. albicans infection in B6 mouse corneas.

Conclusions: IL-36/IL36R signaling plays a protective role in fungal keratitis by promoting AMP expression and by suppressing fungal infection-induced expression of proinflammatory cytokines in a dendritic cell- and macrophage-independent manner.

Figure 1.

The expression of IL-36 cytokines and IL-36R in response CA infection in B6 mouse corneas. Mouse corneas were gently scratched with a needle, 1-mm epithelium incisions, and inoculated with 1.0 × 10⁵ CFU CA. Whole corneas were collected at indicated times and subjected for RNA or protein extraction. (A) RT-PCR assessment of IL-36α and IL-36γ expression with uninfected naïve corneas as the controls and β-actin as internal loading controls. Two samples were used for each time point. For the sham controls, mouse corneas were scratched but not inoculated with CA (A’). Corneas were later collected at indicated time, followed by RT-PCR assay. (B) The qPCR analysis of the expression of IL-36 cytokines in CA-infected corneas at 6 hpi. Results are normalized with β-actin and presented as fold increase (mean ± SD) relative to the control, naïve corneas, set as a value of 1. Stars on top of columns are P value results: *P < 0.05, ***P < 0.001 determined by Student′s t-test, N = 3. Western blotting assessing the expression of IL-36γ (C) and IL-36R (D) at indicated times with naïve corneas as the control. The band intensity of IL-36γ (C’) and IL-36R (D’) were quantitated by densitometric analysis and the results were presented as the ratio of IL-36γ or IL-36R versus β-actin, which served as protein loading controls. P values were generated using unpaired Student's t-test o of naïve versus 6, 9, 18, or 24 hpi, **P < 0.01, **P < 0.01, n = 3.

Figure 2.

IL-36R deficiency increases the severity of CA keratitis. The centers of WT and IL-36R^−/− mouse corneas were gently scratched and inoculated with 1.0 × 10⁵ cfu of CA. (A) Eyes were photographed and clinically scored at 1 and 3 dpi. The clinical scores were analyzed with nonparametric Mann-Whitney U test (N = 5) and presented as inserts (low right corner). ***P < 0.001, n = 6. (B, C) Six WT and IL-36^−/− were euthanized at 1 dpi and the corneas were excised and subjected to fungal plate counting, the results were presented as cfu CA per cornea (B) or to MPO determination, units/cornea (C). The data in B and C were presented as dot plots with the average of CFU or MPO units per cornea and error bars (mean ± SD). P values were generated using unpaired Student's t test. (***P < 0.001), N = 6.

Figure 3.

IL-36R deficiency affects the expression of calprotectin, IL-1β, and sIL-1Ra in B6 mouse corneas in response to CA infection. WT and IL36R^−/− mouse corneas were inoculated with CA as in Figure 2. (A) Corneal epithelial cells were collected at 6 hpi and subjected to qPCR analysis for the expression of S100A8, A9, IL-1β, and soluble IL-1Ra. The results of qPCR are presented as fold increase compared to the naïve, WT corneas, set as value 1 after normalization with the level of β-actin as the internal control. NS, no statistical significance. P values were generated using unpaired Student's t-test, **P < 0.01, ***P < 0.001, n = 3. (B) Whole corneas were dissected and processed for ELISA analysis of calprotectin and IL-1β at 1 dpi. The results of ELISA are presented as pg/µg total protein. P values were generated using unpaired Student's t-test, **P < 0.01, ***P < 0.001, N = 3. UD, undetectable. Data are presented as mean ± SD and representative of two independent experiments.

Figure 4.

IL-36γ exerts protective effects on CA keratitis. WT B6 mice were subconjunctivally injected with 5 µL of IL-36γ (100 ng/µL) or BSA 4 hours (−4hpi) before CA inoculation. CA inoculation was carried out as in Figure 2. (A) Representative micrographs of the control (BSA) and IL-36γ pretreated, CA-infected corneas at 1 and 3 dpi. (B) Clinical scores of CA infected corneas pretreated with BSA (the control) and IL-36γ were presented as mean ± SD. **P < 0.01, ***P < 0.001, determined by nonparametric Mann-Whitney U test, n = 5. (C) Fungal burden determined by plate counting and (D) MPO enzymatic activity of CA infected, BSA (the control) and IL-36γ pretreated corneas at 1 dpi. The data are presented as total number of CA or MPO units per cornea and are representative of two independent experiments. The indicated P values were generated using unpaired Student's t-test, **P < 0.01, ***P < 0.001, N = 5.

Figure 5.

IL-36γ differentially regulates the expression of IL-1 cytokines and calprotectin in CA infected B6 mouse corneas. WT B6 mice were subconjunctivally injected with IL-36γ (100 ng/µL) or BSA 4 hours (−4 hpi) before CA inoculation as described for Figure 4. (A) The expression of IL-1β, IL-1Ra, S1000A8, and A9 in corneal epithelial cells collected at 6 hpi. The mRNA levels were assessed using qPCR analysis. The results are presented as mean of fold increase compared to naïve, WT corneas, set as 1 after normalization to the level of β-actin as the internal control. (B) Whole corneas were dissected and processed at 1 dpi for ELISA analysis of calprotectin and IL-1β. The results of ELISA are presented as pg of calprotectin and IL-1β in 1 µg of total protein. UD, undetectable. The P values were generated using unpaired Student's t test. BSA- and IL-36γ-treated corneas were compared to each other as uninfected or infected at 6 hpi, respectively. **P < 0.01, ***P < 0.001. Data are representative of three independent experiments with three corneas per group (mean ± SD).

Figure 6.

Proteome profiler analysis reveals IL-33 and REG3G as IL-36/IL-36R related genes. B6 mice were divided into four groups: (a) WT naïve, (b) WT CA-infected, (c) WT pretreated with rmIL-36γ and infected with CA, and (d) IL-36R^−/− infected with CA. (A) Micrographs of CA-infected corneas a–d photographed at 18 hpi. (B) Proteome Profiler Mouse XL Cytokine Array analysis of CA-infected corneas collected at 18 hpi. The corneas were homogenized and used for assessing cytokines with altered levels in group a–d, illustrated as dots for Reg3g and IL-33 (two dots per sample). The dot intensities for Reg3g and IL-33 were quantitated by densitometric analysis. (C) Western blotting of Reg3g and IL-33 in group a–d with actin staining as the internal loading control. (D) The band intensity of Western blot was quantitated by densitometric analysis and normalized with β-actin as internal control. C and D are representative of two independent experiments with two corneas per group (mean ± SD); **P < 0.01, ***P < 0.001 by one-way ANOVA).

Figure 7.

Downregulation of IL-33 increases severity of CA keratitis. WT B6 mice were subconjunctivally injected with IL-33 specific or the control, nonspecific siRNA (50 pmol in 5 µL RNase free water) at −1 dpi and at −6 hpi. Corneas were scratched and inoculated with 1.0 × 10⁵ cfu of CA at 0 hpi. (A) Micrographs of nonspecific siRNA (the control) and IL-33 siRNA-treated corneas photographed at indicated times, two corneas each presented. (B) Clinical scores of for the severity of keratitis were analyzed using nonparametric Mann-Whitney U test (**P < 0.01) at 1, 3, and 5 dpi. At 5 dpi, corneas were excised and subjected to MPO determination. This was analyzed using unpaired Student's t-test and presented as MPO units per cornea (C). The corneas were also subjected to qPCR analysis for the expression of IL-36α and IFN-γ and analyzed with one-way ANOVA (D), NL, naïve cornea, N = 5 each, *P < 0.05, **P < 0.01.

Figure 8.

DC depletion increases the severity of but exhibits no effects on IL-36γ-induced protection against CA keratitis. B6-DTR, as well as wildtype B6 mice were subconjunctivally injected with either 50 ng of DT in 5 µL of PBS at −1 dpi, followed by injection of IL-36γ at −4 hpi. Corneas were scratched and inoculated with 1.0 × 10⁵ cfu of CA at 0 hpi. (A) Micrographs of DT treated WT B6 (the control) and B6-DTR corneas with or without exogenous IL-36γ at 1 dpi; clinical scores were presented in the representative corneas. The diseased corneas were excised and subjected to fungal plate counting (B) and MPO determination (C) and analyzed using unpaired Student's t-test, n = 5 each, ***P < 0.001.

Figure 9.

Macrophage depletion increases the severity of but exhibits minimal effects on IL-36γ–induced protection against CA keratitis. B6 mice were subcutaneously injected with 1 µg of neutral clodronate liposomes or control liposomes at −1 dpi, followed by injection of IL-36γ at −4 hpi. Corneas were scratched and inoculated with 1.0 × 10⁵ cfu of CA at 0 hpi. (A) Micrographs of liposome treated B6 corneas at 1 dpi with or without exogenous IL-36γ; clinical scores were presented in the representative corneas. The diseased corneas were excised and subjected to fungal plate counting (B) and MPO determination (C) and analyzed using unpaired Student's t-test, n = 5 each, ***P < 0.001.