IL-23 and IL-17A, but not IL-12 and IL-22, are required for optimal skin host defense against Candida albicans

Abstract

IL-23 and Th17 cells play important roles in host defense against systemic infections with extracellular bacteria and fungi, although their roles in immunity against localized skin infections are less well defined. Here, the contributions of IL-23 and Th17 cytokines in host defense against cutaneous Candida albicans infection were evaluated. Mice deficient in IL-23 or IL-17A demonstrated delayed healing and decreased IL-17A production after skin infection with C. albicans compared with wild-type mice or mice deficient in IL-12 or IL-22. Histologic examination revealed epidermal hyperplasia overlying infected dermis four days postinoculation in wild-type mice. In IL-23-deficient mice, fungal burden was greater in skin, neither IL-17A nor IL-22 mRNAs were expressed postinfection, and these mice demonstrated only minimal epidermal hyperplasia. Exogenous recombinant IL-17A injected at the site of skin infection promoted more rapid healing of candidiasis in both wild-type mice and mice deficient in IL-23 and IL-12. Taken together, these results demonstrate that IL-23 and IL-17A, but not IL-12 and IL-22, are required for optimal host defense against cutaneous candidiasis. In addition, recombinant IL-17A may serve as a potential therapy to enhance healing in individuals with chronic cutaneous candidiasis.

FIGURE 1

Optimal resolution of cutaneous C. albicans infection is dependent upon IL-23. A, Kaplan–Meier curves demonstrating the percentage of C. albicans-infected mice with disease, as defined by the presence of nodule, ulcer, erythema, or crust at the skin site of inoculation. This was assessed by an investigator (blinded to the genotype of the mice) three times weekly postinfection. In three separate experiments, at least 12 mice for each genetic background were evaluated. *p < 0.025 versus WT. B, Quantification of C. albicans fungal burden in skin 4 d after inoculation. Infected tissue was isolated, weighed, minced, and cultured on agar plates to determine CFU/gram skin tissue. **p < 0.025 versus WT.

FIGURE 2

Increased numbers of infiltrating cells in WT mouse skin compared with knockout mouse skin. A, H&E, CD3 (T cells), F4/80 (macrophages), and Gr-1 (neutrophils) staining of skin 4 d postinfection with C. albicans showing a large collection of fungal elements in the deep dermis and superficial fat. H&E and Gr-1 staining, original magnification ×10; scale bars, 0.2 mm. CD3 and F4/80 staining, original magnification ×40; scale bars, 0.025 mm. B, Quantification of CD3⁺ and F4/80⁺ cells and grading of Gr-1 staining in each of the four groups of mice shown in A. Data are expressed as mean ± SD. Bar graphs were analyzed with Kruskal–Wallis test and Scheffe's F test.*p < 0.05; **p < 0.01 versus WT.

FIGURE 3

C. albicans infection in skin induces epidermal hyperplasia that is dependent upon IL-23. A, H&E staining of mouse skin 4 d postinfection with C. albicans. Original magnification ×20; scale bars, 0.05 mm. B, Quantification of epidermal hyperplasia in each of the five groups of mice in A. Data are expressed as mean epidermal thickness (μm) ± SD. Data were analyzed using Kruskal–Wallis test and Scheffe's F test. *p < 0.001 versus WT.

FIGURE 4

Impaired production of IL-17A, IL-17F, and IL-22 in IL-23–deficient mice. IL-17A, IL-17F, IL-22, IL-21, IFN-γ, and TNF-α mRNA expression 48 h after C. albicans skin infection in WT and the various knockout mice as measured by qPCR. Each symbol indicates the amount of mRNA transcript as measured by arbitrary units (AU) of a single specimen, and horizontal bars denote the mean of scatterplots. Data were analyzed using Kruskal–Wallis test and Scheffe's F test. IL-17A: *p < 0.001 versus WT. IL-17F: *p < 0.001 versus WT. IL-22: *p < 0.001 versus WT. IFN-γ: *p < 0.01 for IL-23p19^−/− and IL-12p35^−/− versus WT; *p < 0.01 for IL-12/23p40^−/− versus WT. TNF-α: *p < 0.01 versus WT.

FIGURE 5

Impaired protein production of IL-17A and IL-22 in IL-23–deficient mice. A, Anti–IL-17A Ab-stained or anti–IL-22 Ab-stained sections of skin 4 d postinfection with C. albicans. Original magnification ×40; scale bars, 0.025 mm. B, Quantification of IL-17A⁺ and IL-22⁺ cells in each of the four groups of mice shown in A. Bar graphs were analyzed using Kruskal–Wallis test and Scheffe's F test. IL-17A: *p < 0.05; **p < 0.01 versus WT. IL-22: *p * 0.05; **p < 0.001 versus WT. C, IL-17A, IL-22, and CD3, or IL-17A and Gr-1 staining of serial sections of WT mouse skin 4 d postinfection with C. albicans. Original magnification ×100; scale bars, 0.025 mm. Black arrows indicate cells positive for IL-17A, IL-22, and CD3 (top panels) and cells positive for IL-17A and Gr-1 (bottom panels). Green arrows indicate cells positive for IL-22 and CD3, but negative for IL-17A. Red arrows indicate cells positive for IL-17A and CD3, but negative for IL-22.

FIGURE 6

Optimal resolution of cutaneous C. albicans infection is dependent upon IL-17A but not IL-22. A, Kaplan–Meier curves demonstrating the percentage of C. albicans-infected mice with disease, as defined by the presence of nodule, ulcer, erythema, or crust at the skin site of inoculation. This was assessed by an investigator (blinded to the genotype of the mice) three times weekly postinfection. *p < 0.025 versus WT. B, Quantification of C. albicans fungal burden in skin 4 d after inoculation. Infected tissue was isolated, weighed, minced, and cultured on agar plates to determine CFU/gram skin tissue. *p < 0.001 versus WT.

FIGURE 7

Higher numbers of CD3⁺ T cells in WT mouse skin compared with that in IL-17A^−/− and IL-22^−/− mice. A, H&E, CD3, F4/80, and Gr-1 staining of skin 4 d postinfection with C. albicans showing a large collection of fungal elements in the deep dermis and superficial fat. H&E and Gr-1 staining, original magnification ×10; scale bars, 0.2 mm. CD3 and F4/80 staining, original magnification ×40; scale bars, 0.025 mm. B, Quantification of CD3⁺ and F4/80⁺ cells and grading of Gr-1 staining in each of the four groups of mice shown in A. Data are expressed as mean ± SD. Bar graphs were analyzed with Kruskal–Wallis test and Scheffe's F test. *p < 0.025; **p, 0.001 versus WT.

FIGURE 8

IL-22 expression is decreased in IL-17A^−/− mice, whereas IL-17A expression is normal in IL-22^−/− mice after cutaneous C. albicans infection. IL-17A, IL-17F, IL-22, IL-21, IFN-γ, and TNF-α mRNA expression at 48 h after C. albicans skin infection in WT and the various knockout mice as measured by qPCR. Each symbol indicates the amount of mRNA transcript (as measured in AU) of a single specimen, and horizontal bars denote the mean of scatterplots. Data were analyzed using Kruskal–Wallis test and Scheffe's F test. IL-17A and IL-22: *p < 0.001 versus WT. IL-17F and TNF-α: *p < 0.01 versus WT. IL-21 and IFN-γ: no differences versus WT.

FIGURE 9

Administration of exogenous recombinant IL-17A at the onset of infection promotes more rapid healing of cutaneous candidiasis in both WT and IL-12/23p40^−/− mice. A, The percentage of mice healed 12 d postinfection. B, The percentage of mice healed 19 d postinfection. Data shown were generated after completing three independent experiments using a total of seven mice in each group.