High IL-17E and low IL-17C dermal expression identifies a fibrosis-specific motif common to morphea and systemic sclerosis

Abstract

Background: High interleukin (IL)-17A levels are characteristically found in the skin of systemic sclerosis (SSc) individuals. Our aim was to investigate whether the dermal expression of IL-17A and related IL-17 family members (i.e. IL-17C, IL-17E and IL-17F) could distinguish fibrotic from healthy skin and could show similarities in SSc and morphea, two disorders with presumed distinct pathogenesis, but characterized by skin fibrosis.

Methods: Biopsies were obtained from the involved skin of 14 SSc, 5 morphea and 8 healthy donors (HD) undergoing plastic surgery. Immunohistochemistry/immunofluorescence techniques were coupled to a semi-automated imaging quantification approach to determine the presence of the IL-17 family members in the skin. The in vitro effects induced by the IL-17 family members on fibroblasts from normal and SSc individuals were assessed by ELISA and RIA.

Results: Positive cells for each of the IL-17 isoforms investigated were present in the dermis of all the individuals tested, though with variable frequencies. SSc individuals had increased frequency of IL-17A+ (p = 0.0237) and decreased frequency of IL-17F+ (p = 0.0127) and IL-17C+ cells (p = 0.0008) when compared to HD. Similarly, morphea individuals had less frequent IL-17C+ cells (p = 0.0186) in their skin but showed similar number of IL-17A+ and IL-17F+ cells when compared to HD. Finally, IL-17E+ cells were more numerous in morphea (p = 0.0109) and tended to be more frequent in SSc than in HD. Fibroblast production of IL-6, MMP-1 and MCP-1 was enhanced in a dose-dependent manner in the presence of IL-17E and IL-17F, but not in the presence of IL-17C. None of the cytokine tested had significant effect on type I collagen production. Of interest, in SSc the frequency of both IL-17A and IL-17F positive cells increased with disease duration.

Conclusions: The frequency of IL-17A and IL-17F distinguish SSc to morphea individuals while dermal expression of IL-17C (low) and IL-17E (high) identifies a fibrosis-specific motif. The specific IL-17C/IL-17E cytokine combination may thus play a role in the development of fibrosis.

Figure 1. Frequency of IL-17A+ and IL-17F + cells in SSc and morphea.

A. Immunohistochemical analysis of IL-17A and IL-17F in healthy (HD), lesional SSc and morphea skin. Arrows indicate IL-17A (upper panels) or IL-17F (lower panels) positive cells. Results are representative of 14 SSc, 5 Morphea and 8 HD individuals. Original magnification 20X, scale bar 100 µm. Insets, 2X. B. Frequency of IL-17A+ and IL-17F+ cells expressed as percentage of total cells. Each symbol represents a distinct individual and the line depicts the mean. Empty and full dots refer to limited and diffuse SSc, respectively. Significance was assessed by Mann-Whitney test.

Figure 2. Presence of CD3+IL-17+ T cells and tryptase+IL17+ mast cells in morphea.

A. Indirect immunofluorescence analysis was used to assess the expression of IL-17A (green), in combination with CD3 (red, left panel) or Tryptase (red, right panel) and DAPI staining of nuclei (blue) in the dermis of a representative of 5 morphea biopsies. Original magnification 40X. B. Box-plots show the quantification of CD3+IL-17A+ and Tryptase+IL-17A+ cells expressed as percentage of total IL-17A+ cells in 4 HD, 4 SSc and 4 morphea skin section. The box represents values between 25^th and 75^th percentile with a line at the median (50^th percentile). The whiskers extend above and below the box to show the highest and the lowest values.

Figure 3. High frequency of IL-17E+ and low frequency of IL-17C + cells identifies a fibrosis-specific motif common to SSc and morphea.

A. Immunohistochemical analysis for IL-17C and IL-17E in healthy (HD), lesional SSc and morphea skin. Arrows indicate IL-17E (upper panels) or IL-17C (lower panels) positive staining. Results shown are representative of 14 SSc, 5 Morphea and 8 HD individuals. Original magnification 20X, scale bar 100 µm. Insets, 2X. B. Frequency of IL-17E+ and IL-17C+ cells expressed as percentage of total cells. Each symbol represents a distinct individual and the line depicts the mean. Empty and full dots refer to limited and diffuse SSc, respectively. Significance was assessed by Mann-Whitney test.

Figure 4. Co-expression of IL-17A with IL-17C or IL-17E.

Indirect immunofluorescence analysis was used to assess the expression of IL-17A (green), in combination with IL-17C (A) or IL-17E (B) or IL-17F (C) (red), and DAPI staining for nuclei (blue) in the dermis of one biopsy of 3 assessed (1 HD, 1 SSc and 1 morphea). Original magnification 40X.

Figure 5. IL-17A+ and IL-17F+ cell frequency positively correlates with disease duration.

IL-17A+ and IL-17F+ cells were identified by immunohistochemistry. Each symbol represents a single individual. Significance was assessed by Pearson's correlation test. mo  =  months.

Figure 6. IL-17F and IL-17E, but not IL-17C, enhance MCP-1 and MMP-1 production by normal and SSc fibroblasts.

Dermal fibroblasts were cultured in triplicate in the presence of increasing amounts (3, 30, 300, 600 ng/ml) of IL-17C, IL-17E or IL-17F for 48 h. Box-plot show the levels of MCP-1, IL-6, IL-8, MMP-1 and type I collagen assessed in fibroblast culture supernatants from 4 HD and 4 SSc. The box represents values between 25^th and 75^th percentile with a line at the median (50^th percentile). The whiskers extend above and below the box to show the highest and the lowest values. IL-17A (30 ng/ml), TNF (A, 1 ng/ml) or TGF-β (B, 10 ng/ml) were used as controls. Significance versus nil condition was assessed by one-sample t-test.