Systemic sclerosis (SSc), or scleroderma, is a chronic connective tissue disease characterized by autoimmunity, fibrosis of the skin and internal organs, and vascular dysfunction 1 . While the pathogenic mechanisms of the disease are still largely elusive, a number of findings indicate that the immune response may play a key role 2 . First, antinuclear antibodies (ANA) are characteristically present and segregate with distinct clinical presentations. Second, genetic studies indicate that most of the gene polymorphisms associated with SSc involve genes coding for molecules controlling the immune response, shared with other auto-immune disorders like systemic lupus erythematosus (SLE) 3 . Third, histological examination of the skin of patients with SSc during the early edematous inflammatory phase of the disease demonstrates the presence of mononuclear cell infiltrates containing T cells with perivascular distribution preceding the development of fibrosis and overt vasculopathy 4 5 . Of interest, fibroblasts with increased expression of type I and III procollagen mRNA are frequently detected in areas adjacent to mononuclear cell infiltrates suggesting that inflammatory cells, and in particular T cells, are responsible for the altered functional fibroblast phenotype 6 .

Chemokine receptors sense the appropriate ligands in the extracellular environment and transduce the signal directing cell movement 7 . Importantly, in conjunction with adhesion molecules, they determine the combinatorial code used by immune cells to transmigrate across the endothelium and reach target tissues, both in homeostatic and inflamed conditions 8 . CCR4, and to a more limited degree CCR10, contribute to homing into the skin 9 10 11 . CCR6 has been shown to allow homing into the skin and other tissues including the lung, particularly under inflammatory conditions 11 12 13 . CD4+ T cells differentiate into a variety of effector subsets, which include T helper (Th)1, Th2, Th17, and the more recently identified Th22 cells 14 15 . Of interest, chemokine receptor distribution is characteristically restricted to discrete Th cell subsets 13 16 17 18 . Th1 cells mainly produce interferon gamma (IFN-γ) and are thought to preferentially express CXCR3 16 . Th2 cells produce interleukin (IL)-4 and when activated preferentially express CCR4 19 . Th17 cells produce IL-17A, IL-17F and IL-22 and mostly express CCR6 13 . Moreover, Th17 cells have been recently shown to express the lectin receptor CD161, previously known as a natural killer cell marker 20 21 . IL-22 is produced by many cell types, including Th17 cells and Th1, while Th22 cells characteristically produce IL-22 in the absence of IL-17A and IFN-γ. Of interest, Th22 are enriched in cells expressing CCR4, CCR6, and CCR10 17 18 .

Th2 cells have been shown to be overrepresented in SSc tissues and to be linked to active disease compared to Th1 cells, since IL-4 has direct pro-fibrotic properties 22 23 24 25 . While Th17 cells are thought to play an important role in the induction of autoimmune tissue injury 26 , little is known about their role in SSc. However, increased levels of IL-17 were detected in the sera and bronchoalveolar lavage fluid of SSc individuals 27 28 , and in a recent study Th17 cells were found to be increased, especially in patients with early diffuse SSc 29 . As far as we know, no studies have yet directly assessed the presence and the functional characteristics of Th22 cells in SSc.

The objective of the present study was to revisit the contribution of various CD4+ T cell subsets to the peripheral cell pool characterizing SSc with major focus on cells producing IL-22 and IL-17A. We studied the chemokine receptor usage for assessing their potential to transmigrate into SSc affected tissues, and verified whether Th cell characteristics distinguishing SSc from healthy individuals (HD) could associate with specific SSc clinical features. The results indicate an SSc-specific increase in the number of Th cells producing IL-22 and IL-17A, skewed to preferential homing into the lung and associated with interstitial lung disease (ILD).

In the present study, we demonstrate that Th22 and Th17 cells are specifically increased in the peripheral blood of individuals affected by SSc compared to HD. Moreover, the number of IL-17A and IL-22-producing T cells correlated with CCR6 expression in SSc and not in HD, consistent with enhanced skin- and lung-homing properties for these cells under inflammatory conditions. In addition, we identified a strong relationship between high numbers of IL-22 producing T-cells and SSc ILD.

The relatively low number of individuals included limits the power of our study. In addition, we did not have data on disease activity and quantitative evaluation of the skin involvement. However, our cohort was prospectively recruited and the patients included were not receiving disease-modifying drugs, cytokine blocking reagents or immunosuppressant agents at the time of sampling, which strengthen the reliability of our findings.

By applying a multi-parameter cytofluorimetric analysis we found an increased number of CD4+ T cells producing IL-17A and IL-22 in SSc. They were identified as bona fide Th17 and Th22 cells, since single IL-17A+ cells (IL-17A+IL-22-IFNγ-IL4-), single IL-22+ cells (IL-17A-IL-22+IFNγ-IL4-), in addition to double IL-17A+IL-22+ cells (IL-17A+IL-22+IFNγ-IL4-) were distinctly increased in SSc upon seven days of culture. Although unlikely, we cannot exclude that a small percentage of NKT cells or γ/δ T cells co-expressing CD4 could contribute to IL-17 and IL-22 production in our culture system.

Noteworthy, in SSc, and not in HD, the IL-22 and IL-17A T cell numbers strongly correlated with the expression of CCR6, particularly in the absence of CCR10, which may indicate that these cells are prone to be recruited into inflamed target tissues, specifically in SSc, including the lung. Furthermore, we show for the first time that CD4+ T cells expressing the lectin receptor CD161 are increased in SSc and positively correlate with the number of Th17 cells 20 .

Our study is the first to assess the presence and functional characteristics of Th22 cells in SSc. It is interesting to note that Th22 cells appear to play important roles in inflammatory skin disorders. For instance, the frequency of IL-22+ T cells in skin derived T-cell lines from psoriasis, atopic eczema and allergic contact dermatitis was significantly higher than in the peripheral blood 17 37 38 39 . Furthermore, supernatants of skin-derived Th22 clones from psoriatic lesions enhanced wound healing in an in vitro injury model, and transcriptome profiling of epithelial cells submitted to the influence of these clones revealed up-regulation of genes involved in tissue remodeling, angiogenesis and fibrosis 37 . In our cohort of SSc, we found that SSc individuals presenting with ILD had increased numbers of IL-22 producing cells. The literature is controversial on the possible mechanisms underlying this association. For instance, lung inflammation was ameliorated in IL-22-deficient mice receiving high doses of bleomycin compared to IL-22-sufficient mice 40 . On the other hand, protection mediated by IL-22 produced by gamma/delta T cells has been reported in a mouse model of lung fibrosis induced by hypersensitivity to Bacillus subtilis 41 . While it remains to be established whether the participation of Th22 cells in SSc pathogenic events is harmful rather than protective, their association with ILD suggests an important function of these cells.

Consistent with our findings, IL-17 was previously shown to be increased in the serum and the bronchoalveolar lavage fluid of SSc individuals 27 28 . In addition, an increase in Th17 cells in the peripheral blood of SSc was recently reported 29 . What could be the contribution of IL-17 to SSc pathogenesis remains at the moment speculative. IL-17A has been reported to induce IL-6 and IL-8 production and inter-cellular adhesion molecule 1 (ICAM-1) expression in human fibroblasts 42 . It should be noticed that IL-17A has been shown to participate in an IL-1-dependent manner to the development of bleomycin-induced mouse lung fibrosis 43 and IL-17 may directly stimulate collagen synthesis in rodent fibroblasts 44 45 . However, Kurasawa and colleagues could not demonstrate an increased synthesis of type I and III collagen mRNA in human dermal fibroblasts activated by IL-17 27 . The different responses of mouse and human fibroblasts to IL-17 may be explained by species-specific characteristics. Thus, our data are in line with the hypothesis that Th17 cells in SSc could be more related to inflammation, autoimmunity, and possibly the generation of autoantibodies 46 , as it is speculated for several autoimmune disorders non-characterized by fibrosis, including systemic lupus erythematosus and rheumatoid arthritis in which Th17 cells are increased 26 47 48 .

SSc individuals have an increased frequency of circulating IL-22 and IL-17A, producing cells with skin- and lung-homing potential. The association we found between increased numbers of IL-22-producing cells and ILD indicate that Th22 may act as biomarkers to identify individuals at risk and need to be assessed in larger prospective cohorts of patients.

ANA: antinuclear antibody; dSSc: diffuse systemic sclerosis; FCS: fetal calf serum; HD: healthy donor; HRCT: high-resolution computerized tomography; IL: interleukin; ILD: interstitial lung disease; IFN-γ: interferon gamma; lSSc: limited systemic sclerosis; mAb: monoclonal antibody; PAH: pulmonary arterial hypertension; PBMC: peripheral blood mononuclear cell; PMA: phorbol myristate acetate; SLE: systemic lupus erythematosus; SSc: systemic sclerosis; Th: T helper; TLC: total lung capacity.

The authors declare that they have no competing interests.

MET and NCB conceived the experiments, performed research, analyzed the data and drafted the manuscript. EM performed research. YA provided samples from SSc individuals and critically revised the manuscript. CC conceived research, analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.