Interleukin-9 over-expression and T helper 9 polarization in systemic sclerosis patients

Abstract

T helper 9 (Th9) cells and interleukin (IL)-9 are involved in the pathogenesis of several autoimmune diseases. The exact role of IL-9 and Th9 cells in patients with systemic sclerosis (SSc) have not yet been studied adequately. IL-9, IL-9R, transcription factor PU.1 (PU.1), IL-4, thymic stromal lymphopoietin (TSLP) and transforming growth factor (TGF)-β expression were assessed in skin and kidney biopsies of SSc patients and healthy controls (HC) by immunohistochemistry (IHC). The cellular source of IL-9 was also analysed by confocal microscopy analysis. Peripheral IL-9-producing cells were also studied by flow cytometry. The functional relevance of IL-9 increased expression in SSc was also investigated. Our results demonstrated a strong expression of IL-9, IL-9R, IL-4, TSLP and TGF-β in skin tissues of patients with both limited and diffuse SSc. IL-9 expression was observed mainly in the context of skin infiltrating mononuclear cells and keratinizing squamous epithelium. IL-9 over-expression was also observed in renal biopsies of patients with SSc. IL-9 producing cells in the skin were identified as Th9 cells. Similarly, Th9 cells were expanded and were the major source of IL-9 among SSc peripheral blood mononuclear cells (PBMC), their percentage being correlated directly with the modified Rodnan skin score. Infiltrating mononuclear cells, mast cells and neutrophils expressed IL-9R. In in-vitro studies stimulation with rIL-9 significantly induced NET (neutrophil extracellular traps) release by dying cells (NETosis) in neutrophils, expansion of mast cells and increase of anti-systemic scleroderma 70 (Scl70) production by B cells. Our findings suggest that Th9 cells and IL-9 could be implicated in the pathogenesis of SSc.

Keywords: IL-9; ILC2; Th9; systemic sclerosis.

Figure 1

Interleukin (IL)‐9 and IL‐9R expression in the skin of systemic sclerosis (SSc) and HD. IL‐9 expression in the skin of SSc patients (a–c) and controls (d). Single staining for IL‐9 (e), IL‐17 (f) and transcription factor PU.1 (PU.1) (g). (h) Merged triple staining for IL‐9, IL‐17 and PU.1 in the skin of SSc patients. IL‐9R expression in the skin of SSc patients (i,k) and controls (j). Quantification of IL‐9 and IL‐9R‐producing cells from SSc patients and controls (l,m). Original magnification ×400 (i), ×250 (a,b,d,j,k), ×630 (c). Data are expressed as mean ± standard error of the mean (s.e.m.); *P < 0·05. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Interleukin (IL)‐4, transforming growth factor (TGF)‐β, thymic stromal lymphopoietin (TSLP), IL‐25, IL‐17RB and tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) expression in systemic sclerosis (SSc) and healthy donors (HD). IL‐4, TGF‐β, TSLP, IL‐25, IL‐17RB and TRAF6 expression in the skin of SSc patients (b,e,h,m,p,s) and controls (a,d,g,l,o,r). Quantification of IL‐4‐, TGF‐β–, TSLP‐, IL‐25‐, IL‐17RB‐ and TRAF6‐producing cells from SSc patients and controls (c,f,i,n,q,t). Original magnification ×250. Data are expressed as mean ± standard error of the mean (s.e.m.); *P < 0·05. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Percentage of T helper type 9 (Th9), Th17 and Th2 in systemic sclerosis (SSc) and healthy donors (HD). Representative dot‐plot analysis and gating strategy in an SSc patient (a). Percentage of Th2 (b), Th9 (c) and Th17 (d) cells in SSc and HD. Mean percentage of Th9 cells in patients with diffuse or limited disease (e). Direct correlation of Th9 percentage in SSc and pulmonary hypertension (f) and Rodnan skin score (g). Data are expressed as mean ± standard error of the mean (s.e.m.). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Innate lymphoid cells (ILC) type 2 are expanded in systemic sclerosis (SSc) patients. Representative dot‐plot analysis and gating strategy in a SSc patient (a). Mean percentage of ILC2 cells among peripheral blood mononuclear cells (PBMC) of patients and HD (b). Single staining for chemoattractant receptor Th2 (CRTH2) (c), GATA binding protein 3 (GATA3) (d) and interleukin (IL)‐4 (e). (f) Merged triple staining for CRTH2, GATA3 and IL‐4. ILC2 are directly correlated with Th9 (g). Data are expressed as mean ± standard error of the mean (s.e.m.); *P < 0·05. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Effect of recombinant interleukin (IL)‐9 on neutrophils, mast cells and B lymphocytes. Immunofluorescences for neutrophil extracellular traps release by dying cells (Netosis): single staining for myeloperoxidase (MPO) in untreated and interleukin (IL)‐9 treated neutrophils (a). Expansion of CD117‐positive mast cells after in‐vitro culture with recombinant IL‐9 (b). Anti‐systemic scleroderma 70 (Scl70) production after in‐vitro stimulation of B lymphocytes with recombinant IL‐9 (c). [Colour figure can be viewed at wileyonlinelibrary.com]