IL-22 promotes allergic airway inflammation in epicutaneously sensitized mice

Abstract

Background: Serum IL-22 levels are increased in patients with atopic dermatitis, which commonly precedes asthma in the atopic march. Epicutaneous sensitization in mice results in T_H2-dominated skin inflammation that mimics atopic dermatitis and sensitizes the airways for antigen challenge-induced allergic inflammation characterized by the presence of both eosinophils and neutrophils. Epicutaneous sensitization results in increased serum levels of IL-22.

Objective: We sought to determine the role of IL-22 in antigen-driven airway allergic inflammation after inhalation challenge in epicutaneously sensitized mice.

Methods: Wild-type (WT) and Il22^-/- mice were sensitized epicutaneously or immunized intraperitoneally with ovalbumin (OVA) and challenged intranasally with antigen. OVA T-cell receptor-specific T cells were T_H22 polarized in vitro. Airway inflammation, mRNA levels in the lungs, and airway hyperresponsiveness (AHR) were examined.

Results: Epicutaneous sensitization preferentially elicited an IL-22 response compared with intraperitoneal immunization. Intranasal challenge of mice epicutaneously sensitized with OVA elicited in the lungs Il22 mRNA expression, IL-22 production, and accumulation of CD3⁺CD4⁺IL-22⁺ T cells that coexpressed IL-17A and TNF-α. Epicutaneously sensitized Il22^-/- mice exhibited diminished eosinophil and neutrophil airway infiltration and decreased AHR after intranasal OVA challenge. Production of IL-13, IL-17A, and TNF-α was normal, but IFN-γ production was increased in lung cells from airway-challenged and epicutaneously sensitized Il22^-/- mice. Intranasal instillation of IFN-γ-neutralizing antibody partially reversed the defect in eosinophil recruitment. WT recipients of T_H22-polarized WT, but not IL-22-deficient, T-cell receptor OVA-specific T cells, which secrete both IL-17A and TNF-α, had neutrophil-dominated airway inflammation and AHR on intranasal OVA challenge. Intranasal instillation of IL-22 with TNF-α, but not IL-17A, elicited neutrophil-dominated airway inflammation and AHR in WT mice, suggesting that loss of IL-22 synergy with TNF-α contributed to defective recruitment of neutrophils into the airways of Il22^-/- mice. TNF-α, but not IL-22, blockade at the time of antigen inhalation challenge inhibited airway inflammation in epicutaneously sensitized mice.

Conclusion: Epicutaneous sensitization promotes generation of antigen-specific IL-22-producing T cells that promote airway inflammation and AHR after antigen challenge, suggesting that IL-22 plays an important role in the atopic march.

Keywords: IL-22; asthma; atopic dermatitis; neutrophils.

Figure 1. EC sensitization elicits a systemic IL-22 response and an antigen-specific IL-22 response in the lungs

A-B. IL-22 secretion by OVA stimulated splenocytes (A) and IL-22 serum levels (B). C,D. Il22 mRNA expression in the lungs (C), and IL-22 secretion by OVA stimulated lung cells (D). E. Representative FACS analysis and quantitation of intracelluar expression of IL-22⁺ cells among CD3⁺CD4⁺ T cells and of IL-17A⁺ and TNFα⁺ cells among CD3⁺CD4⁺IL-22⁺ cells in the lung. Mice were EC sensitized with OVA or saline in A and B, followed by i.n. challenged with OVA in C-F. Bars represent mean±SEM (n=5–10 per group). *p<0.05.

Figure 2. EC sensitization preferentially promotes an IL-22 response

A-D. serum IL-22 levels (A), IL-22 secretion by OVA stimulated splenocytes (B), Il22 and Tnfa mRNA expression in the lungs (C) and IL-22 and TNFα secretion by OVA stimulated lung cells of mice EC or IP sensitized with OVA and i.n. challenged with OVA. Bars represent mean±SEM (n=3–5 per group). *p<0.05, **p<0.005 and ***p<0.001.

Figure 3. IL-22 is important for the development of airway inflammation and AHR following intranasal antigen challenge of EC sensitized mice

A-D. Total and differential cell counts in BALF (A), H&E stained lung sections (B), and lung resistance in response to increasing doses of methacholine (C) and cytokine secretion by OVA stimulated lung cells (D) in WT and Il22^−/− mice EC sensitized with OVA or saline and i.n. challenged with OVA. Bars represent mean±SEM (n=4–7 per group). *p<0.05, **p<0.05, ***p<0.001.

Figure 4. Increased IFNγ production underlies the decreased airway eosinophilia in inhalation challenged Il22^−/− mice EC sensitized with OVA

A, B. Ifng, Tbx21, Cxcr3 and Ccl5 mRNA levels in the lungs (A), Representative FACS analysis and quantitation of intracellular expression of IFNγ⁺ cells among CD3⁺Lin⁻C90⁺ cells, CD3⁺CD4⁺ T and CD3⁺CD8⁺ T cells in the lungs (B) of WT and Il22^−/− mice EC sensitized and intranasally challenged with OVA. C, D. Effect of i.n. administration of anti-IFNγ antibody, or isotype control, on the recruitments of eosinophils (C) and neutrophils (D) to the antigen challenged airways of Il22^−/− mice EC sensitized with OVA. Bars represent mean±SEM (n=5–7 per group). *p<0.05.

Figure 5. Th22 antigen-specific CD4⁺ T cells drive neutrophil-dominated airway inflammation and AHR in response to intranasal antigen challenge

A. Cytokine secretion by vitro polarized Th22 cells from in WT and Il22^−/− mice. B-F. Total and differential cell counts in BALF (B), frequency of neutrophils in lungs (C), H&E stained lung sections (D), chemokine mRNA levels in lungs (F), and lung resistance in response to increasing doses of methacholine (G) in WT recipients of Th22 polarized OVA-specific CD4⁺ T cells from DO11.10 and DO11.10/Il22^−/− mice following n. challenge with OVA. Mice that received no T cells were used as controls. Bars represent mean±SEM (n=4–6 per group). *p<0.05, **p<0.05, ***p<0.001.

Figure 6. IL-22 synergizes with TNFα to promote neutrophil airway inflammation

A-D. Total and differential counts in BALF (A), frequency of neutrophils in lungs (B), H&E stained lung sections (C), chemokine mRNA levels in the lungs (D) and lung resistance in response to increasing doses of methacholine (E) in WT mice treated intranasally with saline, rIL-22, rTNFα or rIL-22+rTNFα. Bars represent mean±SEM (n=4–6 per group). *p<0.05, **p<0.05, ***p<0.001

Figure 7. TNFα, but not IL-22 blockade during the challenge phase inhibits airway inflammation

A-D. Total and differential counts in BALF (A), Numbers of neutrophils in lungs (B), H&E staining of lung sections (C), chemokine mRNA levels in the lungs (D) in WT mice EC sensitized with OVA then subjected to i.n. instillation of OVA together with neutralizing anti-IL-22 IgG antibody, neutralizing anti-TNFα IgG antibody or IgG isotype controls. Bars represent mean±SEM (n=4–6 per group). *p<0.05, **p<0.05, ***p<0.001.