IL-13 induces skin fibrosis in atopic dermatitis by thymic stromal lymphopoietin

Abstract

Skin fibrotic remodeling is a major feature in human atopic dermatitis (AD). Inflammation and tissue fibrosis are common consequences of Th2 responses. Elevated IL-13 and thymic stromal lymphopoietin (TSLP) have been found in the AD skin lesions. Fibrocytes can be recruited to inflamed tissues to promote wound healing and fibrosis. Dermal transgenic expression of IL-13 causes an AD-like phenotype with fibrosis and increased TSLP. However, the role of TSLP in fibrotic remodeling is unknown. In this study, we investigated the role of TSLP and fibrocytes in the generation of IL-13-induced skin fibrosis. In AD lesion, cessation of IL-13 transgene expression resulted in reduced skin inflammation but with no effect on further progression of fibrosis. This was accompanied by markedly increased CD34(+)/procollagen 1(+) fibrocytes. Furthermore, fibrocytes express TSLP receptor (TSLPR), and TSLP directly promotes PBMC-derived fibrocytes to produce collagen. Neutralization of TSLP or genetic deletion of TSLPR in IL-13 transgenic mice resulted in a significant reduction in fibrocytes and in skin fibrosis. Furthermore, reduction of fibrosis by depletion of TSLP was independent of IL-13. Interestingly, the number of fibrocytes was highly increased in the skin samples of AD patients. These data indicate that the progression of skin fibrosis in IL-13-induced AD occurs via TSLP/TSLPR-dependent but IL-13-independent novel mechanisms by promoting fibrocyte functions.

Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1

Progression of skin fibrosis in AD despite the attenuation of skin inflammation. After withdrawal of Dox in the drinking water (the transgene was turned on) for 8–10 wk, IL-13 Tg(+) mice began to develop AD (clinical score A, Clinical score of AD: amelioration of the AD after the IL-13 transgene was turned off in the Tg(+) On-Off mice (solid line) and On-On group (dotted line). B, Quantification of skin inflammatory cells (HPF) in the skin stained by H&E. C, Quantification of the skin fibrosis (fibrosis score): progression of skin fibrosis in AD in the Tg(+) On-On mice (dotted line) and in the Tg(+) On-Off mice (solid line). D, Quantification of collagen contents (by Sircol assay; Biocolor) in the skin. E, No significant difference in the skin collagen deposition between the two groups of mice by Masson’s Trichrome staining (original magnification ×10). Shown are representatives of six individual samples for each group. For A–D, n = 6 for each group. *p < 0.05, **p < 0.005.

FIGURE 2

Increased CD34+/ProCol 1+ fibrocytes and mediators critical in the biology of fibrocytes in the AD skin of Tg(+) mice. A, CD34+/ProCol 1+ fibrocytes (white arrows) in the skin of Tg(+) mice were evaluated by confocal microscopy. Scale bar, 10 μm. B, Quantification of fibrocytes in the skin by fluorescent IHC. Double-positive cells were counted in five HPF per section, five sections per mouse (n = 4 for each group, Mean ± SEM. C, Percentage of CD45+/collagen-1+ fibrocytes in the skin tissue by flow cytometry. D, Quantitative comparison by real-time PCR of mRNA encoding CXCL12, CXCR4, PDGF, and ET-1. The results were normalized to GAPDH (reference gene) and expressed in arbitrary units relative to the lowest detectable expression. The data were representative of two or three independent experiments. *p < 0.05.

FIGURE 3

Specific mAb-mediated neutralization of TSLP resulted in amelioration of IL-13–induced skin fibrosis and inflammation. Six-week-old Tg(+) mice received the first dose of anti-TSLP mAb or control IgG2a at the time of the transgene being activated. Anti-TSLP was given i.p. at 10 mg/kg weekly for four doses and biweekly for three doses. The mice were examined for scores of dermatitis and skin fibrosis (n = 4 for anti-TSLP group and n = 5 for IgG2a group). A, Using Masson’s Trichrome staining, increased collagen deposition in the dermal and s.c. areas of the skin from IgG2a-treated Tg(+) mice compared with markedly reduced collagen deposition in anti-TSLP–treated Tg(+) mice. Original magnification ×10. B, Quantitative evaluation of skin fibrosis score. C, Quantification of collagen content in the skin using the Sircol collagen assay (Biocolor). D, The levels of total and active forms of TGF-β1 assessed by ELISA. Histological evaluation of skin samples from Tg(+) mice given IgG2a and Tg(+) mice given anti-TSLP (E; H&E, original magnification ×10) and quantification of skin inflammatory cells per HPF (F). For B–D, results are shown as mean ± SEM. *p < 0.05, **p < 0.001.

FIGURE 4

Neutralization of TSLP in Tg(+) mice reduced accumulation of skin fibrocytes. A, Accumulation of skin fibrocytes in Tg(+) mice treated with anti-TSLP or IgG2a isotype control by confocal microscopy (white arrows: double-positive cells). Scale bars, 10 μm. B, Quantitative evaluation of fibrocytes in the skin by fluorescent IHC with double labeling of CD34 and ProCol 1 Abs. Double-positive cells were quantified (five HPF per section). The data are expressed as mean ± SEM (n = 6 for each group). C, Quantitative real-time PCR analysis of mRNA encoding CXCL12, CXCR4, PDGF, and ET-1. The results were normalized to GAPDH (reference gene) and expressed in arbitrary units relative to the lowest detectable expression. The data were representative of two or three independent experiments. D, The levels of IL-13 in the skin by ELISA in the anti-TSLP or IgG2a isotype control groups (n = 4 to 5 each group). *p < 0.05.

FIGURE 5

Role of TSLPR in IL-13–induced skin fibrosis in AD in IL-13 Tg(+) mice with WT(+/+) and null(−/−) TSLPR loci. Reduced collagen deposition by Masson’s Trichrome staining (A) and reduced skin inflammatory response by H&E staining (B) were seen in the skin of Tg(+)/TSLPR −/−mice compared with that in the skin of Tg(+)/TSLPR +/+ mice. A and B are representative of five individual samples from each group. Original magnification ×10. Total collagen content of the skin by Sircol assay (C) and the levels of total and active forms of TGF-β1 in the skin by ELISA (D). E, IHC with fluorescent anti-CD34/anti-ProCol 1 was used to assess double-positive fibrocytes in the skin. F, Real-time RT-PCR was used to evaluate the levels of mRNA encoding CXCR12, CXCR4, PDGF and ET-1 in the skin samples from Tg(−) and IL-13 Tg(+) mice with WT(+/+) and null(−/−) TSLPR loci (n = 7 for each group). G, The level of IL-13 in the skin by ELISA. The values in C–G are the mean ± SEM of three separate evaluations of at least eight animals for each group. *p < 0.05, **p < 0.005.

FIGURE 6

TSLPR expression and TSLP effects on skin fibrocytes. A, Expression of TSLPR on fibrocytes in the AD skin samples of K5-tTA–IL-13 mice. Skin samples from Tg(−) and Tg(+) mice were stained for TSLPR, CD34, and ProCol 1 by immunofluorescence using specific Abs and examined by confocal microscopy. White arrows indicate triple-positive cells. Slides are representatives of five different samples from each group. Scale bars, 10 μm. B, CD45 and Col 1 double-positive fibrocytes purified from the skin of Tg(+) mice were stained and analyzed for TSLPR. C, Morphology of fibrocytes purified from PBMC of WT mice that were cultured in the presence of TSLP (50 ng/ml) or IL-13 (10 ng/ml) for 3 d (original magnification ×100), and using Sircol assay (D), soluble collagen content from supernatants of cultured fibrocytes stimulated with TSLP (50 ng/ml) and IL-13 (10 ng/ml) for 10 d were measured. Data are representative of three independent experiments. n = 5 per group. *p < 0.05, **p < 0.01.

FIGURE 7

TSLP blockade induced attenuation of skin fibrogenesis in AD was independent of IL-13. Tg(+) mice, when the IL-13 level in the skin reached the level comparable to that in Tg(−) skin, after inactivation of the transgene IL-13, were randomly assigned to receive neutralizing TSLP mAb [Tg(+) On-Off-αTSLP group] and control Ab IgG2a [Tg(+)-On-Off-IgG group]. The mice received the treatments i.p. every 5 d for seven injections. A, Kinetics of the levels of IL-13 and TSLP in the skin samples of Tg(+) and Tg(−) mice. Collagen deposition in the dermal and subdermal areas by Masson’s Trichrome staining (blue color) was evaluated, and the kinetics of quantification of the collagen deposition are shown (B, C). Quantification (HPF) of CD34+/ProCol 1+ fibrocytes by IHC in the skin (D). The skin inflammatory response by H&E staining and numbers of inflammatory cells (E, F). Original magnification (C, E) ×10. **p < 0.01.

FIGURE 8

Increased collagen and fibrocytes in human AD skin. In skin biopsy specimens from normal subjects and patients with AD, Gomori’s Trichrome staining (original magnification ×10) was used to evaluate collagen deposition (A) and IHC (B) with fluorescent anti-CD34/anti-ProCol 1 was used to assess expression of double-positive fibrocytes (white arrows) in the skin. Shown are representatives of three individual samples from each group. Scale bars, 10 μm.