IL-22 promotes fibroblast-mediated wound repair in the skin

Abstract

Skin wound repair requires complex and highly coordinated interactions between keratinocytes, fibroblasts, and immune cells to restore the epidermal barrier and tissue architecture after acute injury. The cytokine IL-22 mediates unidirectional signaling from immune cells to epithelial cells during injury of peripheral tissues such as the liver and colon, where IL-22 causes epithelial cells to produce antibacterial proteins, express mucins, and enhance epithelial regeneration. In this study, we used IL-22(-/-) mice to investigate the in vivo role for IL-22 in acute skin wounding. We found that IL-22(-/-) mice displayed major defects in the skin's dermal compartment after full-thickness wounding. We also found that IL-22 signaling is active in fibroblasts, using in vitro assays with primary fibroblasts, and that IL-22 directs extracellular matrix (ECM) gene expression and myofibroblast differentiation both in vitro and in vivo. These data define roles of IL-22 beyond epithelial cross talk, and suggest that IL-22 has a previously unidentified role in skin repair by mediating interactions between immune cells and fibroblasts.

Figure 1. IL-22 mRNA Is Expressed During Acute Skin Wounding

CD45 (A), IL-22 (B) and IL-22Rα (C) mRNA expression via Real Time PCR in unwounded skin or wounds of mice after indicated timepoints. n= 3 wounds from 3 mice for each timepoint. All data are ± SEM. Asterisks indicate significance.

Figure 2. IL-22^−/− mice exhibit defects in the dermis during wound healing

A) Images of WT and IL-22^−/− backskin 3 days after wounding with 2mm punch biopsies. Quantification of the external area of WT and IL-22^−/− small wounds at 3 and 5 days post-wounding. n>18 wounds for each genotype (6 wounds from 6 mice for each of 3 independent experiments). Ruler notches=1mm. B) Hematoxylin and eosin stained sections of WT and IL-22^−/− backskins wounded with 8mm punch biopsies (large wounds). Epidermis (e) is indicated by dotted line and wound bed (WB) is outlined by solid line. Quantification of wound bed area (i.e. dermal area) of small wounds (2mm) 3 days post wounding and large wounds (8mm) 5 and 8 days post wounding in WT and IL-22^−/− mice are shown. For small wounds, n = 18 (6 wounds from 6 mice for each of 3 independent experiments). For large wounds, n= 12 wounds from 6 mice. Bars = 400μm. All data are ± SEM. Asterisks indicate significance.

Figure 3. IL-22^−/− mice exhibit defects in extracellular matrix production

A) Skin sections of WT and IL-22^−/− mice 3, 7 and 28 days post wounding with 2mm punch biopsies were stained with Movat Pentachrome (collagen, yellow; mucin, blue) and Sirius Red (Collagen Type III, red). Bars = 200 μm. Arrows indicate collagen, arrowhead indicates disorganized collagen. B) Real Time PCR analysis reveals decreased Fibronectin, Collagen IaI and Collagen IIIaI mRNA levels in 2mm IL-22^−/− skin wounds. n= 4 mice for each genotype and timepoint. C) Quantification of % ER-TR7+ cells in the total dermal cell population derived from the dermis of WT and IL-22^−/− mice wounded with 2mm punch biopsies. n=4 mice for each genotype. D) Skin sections of IL-22^−/− skin 5 days post-wounding that were injected with PBS or recombinant IL-22 on day 3 and stained with trichrome histological stain (collagen, blue). Bars = 200 μm. Quantification of the area of collagen in the wound beds of IL-22^−/− mice injected with PBS or 25ng recombinant IL-22. n= 6 mice. Asterisks indicate significance. All data are mean +/− SEM.

Figure 4. IL-22 can stimulate STAT3 activation and ECM production in dermal fibroblasts

A) Real time PCR reveals that isolated dermal fibroblasts (FB) and keratinocytes (KC) express IL-22Rα mRNA. n=3 mice. Data are mean +/− SEM. B) Skin sections of were immunostained with antibodies against IL-22Rα and ER-TR7. Arrows indicate ER-TR7+ and IL-22Rα^Ecells. Bar = 100 μm. epi, epidermis. C) Representative images of primary mouse fibroblasts immunostained with antibodies against phospho-STAT3 (STAT3-P) after treated with IL-22 or oncostatin M (OSM) for 20 minutes. D) Western blot analysis confirms the increased activation of STAT3-P in primary dermal fibroblasts treated with IL-22 or Oncostatin M. E) Real time PCR analysis of mRNA in WT or IL-22^−/− primary mouse fibroblasts reveals enhanced expression of ECM genes after IL-22 stimulation for 48 hours. F) Western blot analysis shows that IL-22 increases the expression of fibronectin protein in WT fibroblasts. β-actin is included as a loading control.

Figure 5. IL-22 induces myofibroblast differentiation

A) Skin sections of WT and IL-22^−/− mice 3 days after wounding with 2mm punch biopsies were immunostained with antibodies against α-SMA. Quantification of α-SMA staining, n = 6 mice for each genotype. B) Real time PCR reveals reduced mRNA expression of α-SMA in 2mm IL-22^−/− wounds 3 days post-wounding. n= 4 mice for each genotype and each timepoint. C) Real time PCR analysis of WT or IL-22^−/− primary dermal fibroblasts cultured with vehicle or recombinant IL-22 for 48 hours confirms that α-SMA mRNA expression increases with IL-22 stimulation. n = 3 independent experiments. D) α-SMA protein expression increases in primary fibroblasts after stimulation with IL-22 for 48 hours as indicated by representative image of western analysis. Quantification of the ratio of α-SMA/β-actin expression is shown. n = 3 independent experiments. All data are mean +/− SEM.