IL-10 suppresses IL-17-mediated dermal inflammation and reduces the systemic burden of Vaccinia virus in a mouse model of eczema vaccinatum

Abstract

Individuals with atopic dermatitis (AD) are susceptible to a severe, potentially fatal, systemic infection and inflammatory response following exposure to Vaccinia virus (VV). IL-10 acts both as an inducer of Th2 responses and as a regulator of T cell activation. It has been shown to limit skin inflammation elicited by contact sensitizers. AD exacerbations have been associated with decreased IL-10 function. We used IL-10(-/-) mice to test the role of the cytokine in VV immunity. They exhibited larger primary lesions and increased cutaneous neutrophil infiltration compared to wild-type (WT) counterparts. This was associated with enhanced production of IL-17A, IL-17F and CXCL2. Paradoxically, despite intact adaptive immune responses, tissue viral burdens were increased in IL-10(-/-) mice. These findings suggest that IL-10 is important in limiting skin inflammation induced by VV and that abnormal IL-17-driven neutrophil recruitment at the primary infection site in the skin results in increased systemic viral dissemination.

Keywords: Atopic dermatitis; Eczema vaccinatum; Vaccine.

Figure 1. IL-10 limits primary lesion size after VV inoculation and regulates neutrophil infiltration and transcription of IL-17 and CXCL2

IL-10^−/− and WT mice were VV-infected via skin scarification with 1×10⁷ PFU. Primary lesions and cytokine production were analyzed 7 days later. A) Primary lesions (indicated by red ovals) on representative WT and IL-10^−/− mice B) Neutrophils/HPF (mean ± SEM) enumerated by light microscopic examination of H&E-stained sections 4–5 mice/group C) IL-17A, IL-17F and CXCL2 mRNA levels (mean ± SEM) determined by quantitative PCR and normalized to the WT infected group, n=3 for IL-10^−/−, uninfected mice; n=8–21 for other groups. ** p < 0.005, ***p < 0.0005 by unpaired t test, two-tailed.

Figure 2. Systemic adaptive immune responses to VV are intact in IL-10^−/− mice

IL-10^−/− and WT mice were VV-infected via skin scarification with 1×10⁷ PFU. Single-cell suspensions of splenocytes were prepared 7 days later and fluorescently stained to identify the number of cells A) CD8⁺ and VV-pentamer⁺ and B) CD8⁺ and IFNγ⁺ (3–6 mice per group). C) Splenocytes were cultured at 10⁶ cells/ml, and release of IFNγ, IL-6, IL-17, and IL-4 into the cell medium was measured by ELISA after 72 hours in culture (3–9 mice per uninfected group, 5–19 mice per infected group, mean ± SEM). * p < 0.05, ***p < 0.0005, by unpaired t test, two-tailed.

Figure 3. Lesion severity is increased in IL-10^−/− mice in an IL-17-dependent manner

IL-10^−/− and WT mice were VV-infected via skin scarification with 10⁷ PFU. Anti-IL-17 antibody or isotype control was administered retroorbitally, 3 doses of 100 μg per mouse, on days −1, 0, and 2 relative to infection. IL-17 was injected intradermally, one dose of 2.5 μg per mouse, immediately prior to infection. Primary lesions were evaluated 7 days after infection. A) Primary lesions of representative mice treated with anti-IL-17. B) Primary lesions of representative mice receiving intradermal IL-17. C) Lesion areas (5–22 mice per group, mean ± SEM). Administration of anti-IL-17 antibody decreased lesion sizes in IL-10^−/− mice. * p < 0.05, ** p < 0.005, ****p < 0.0001, by unpaired t test, two-tailed.

Figure 4. IL-10^−/− mice exhibit increased IFN-γ and decreased IL-4 expression in the skin following infection

IL-10^−/− and WT mice were VV-infected via skin scarification with 10⁷ PFU. Anti-IL-17 antibody or isotype control was administered retroorbitally, 3 doses of 100 μg per mouse, on days −1, 0, and 2 relative to infection. IL-17 was injected intradermally, one dose of 2.5 μg per mouse, immediately prior to infection. Transcripts encoding IFN-γ and IL-4 in skin RNA were evaluated by quanititative PCR and normalized to the WT infected group, mean ± SEM, (n=3 for IL-10^−/−, uninfected mice; n=5–22 for all other groups). * p < 0.05, by unpaired t test, two-tailed.

Figure 5. IL-10 deficiency results in and IL-17-dependent increase in viral burdens in VV-infected mice

WT and IL-10^−/− mice were VV-infected via skin scarification with 10⁷ PFU. Anti-IL-17 antibody or isotype control was administered retroorbitally, 3 doses of 100 μg per mouse, on days −1, 0, and 2 relative to infection. Sections of kidney (K), ovary (O), and lung (L) were collected 7 days after the infection and frozen immediately. DNA was isolated from the organs and viral genomes were quantified by real-time PCR using primers specific for vaccinia ribonucleotide reductase (Vvl4L) (13–19 mice per group, mean ± SEM). Results were normalized to the total amount of DNA. ** p < 0.005, ***p < 0.0005, by two-way ANOVA.