Background/Purpose: Interleukin-35 (IL-35) is the most recent addition to the IL-12 family, which also comprises IL-12, IL-23 and IL-27. IL-35 consists of two chains, p35/IL-12a and EBI3/IL-27b, and exerts immunomodulatory activities in experimental and human autoimmune inflammatory conditions. Its potential role has been proposed in the pathogenesis of rheumatoid arthritis, sarcoidosis and systemic lupus erythematosus. However, its role in systemic sclerosis (SSc) has not been studied to date. The aim of this study was to assess IL-35 expression in the skin, dermal fibroblasts and circulation of SSc patients and characterize its potential association with SSc-related features.

Methods: Expression of IL-35 in the skin and dermal fibroblasts was quantified by qPCR, immunohistochemistry and immunofluorescence. Serum levels of IL-35 were analyzed by high sensitivity ELISA (Life Sciences, Hubei, PRC), and routine laboratory parameters such as CRP (by turbidimetry), ANA (by immunofluorescence) and autoantibodies of the ENA complex (by immunoblot) were measured in 40 SSc patients who met the EULAR/ACR 2013 criteria for SSc. Serum IL-35 was also determined in 40 healthy volunteers matched by age and sex.

Results: IL-35 chains EBI3 and p35 were overexpressed in the fibrotic skin of patients with SSc compared to that of healthy controls on both mRNA (p<0.05, p<0.05) and protein level (p<0.001, p<0.001). In fibrotic SSc skin, in contrast to non-fibrotic healthy skin, prominent staining for both IL-35 subunits was detected in dermal fibroblasts, myofibroblasts and perivascular inflammatory cells. Overexpression of IL-35 persisted in cultured dermal fibroblasts from SSc patients which maintained an increased mRNA (p<0.05, p<0.05) and protein level of both IL-35 subunits and released increased levels of IL-35 into supernatants (p<0.05) compared to healthy dermal fibroblasts. Stimulation with the main pro-fibrotic cytokine, TGF-β, increased the mRNA (p<0.05, p<0.05) and protein levels of both IL-35 chains, and enhanced the release of IL-35 into the supernatants (p<0.05), as well. Incubation with recombinant IL-35 induced an activated phenotype in resting fibroblasts (increased the expression of α-smooth muscle actin and stress fibers) and enhanced the release of collagen in a dose-dependent manner. IL-35 serum levels were increased in patients with SSc compared to healthy controls (median(IQR): 83.9 (45.1–146.1) vs. 36.2 (17.2–49.4) pg/ml, p<0.0001). Furthermore, serum IL-35 negatively correlated with disease duration (p<0.01, r=-0.434), and was increased in patients with early SSc pattern on capillaroscopy assessment compared to those with active and late SSc patterns (p<0.05, p<0.05). No correlations of serum IL-35 with other SSc-related features were observed.

Conclusion:

The present study demonstrates an overexpression of IL-35 in the skin, dermal fibroblasts and serum of SSc patients. TGF-β induces IL-35, which in turn activates resting fibroblasts and enhances the release of collagen thereby contributing to aberrant TGF-β signaling in SSc. Elevated serum IL-35 is associated with early, inflammatory stages of SSc.

Acknowledgement: Supported by IGA12440-4, MHCR023728.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/interleukin-35-is-upregulated-in-systemic-sclerosis-and-its-serum-levels-are-increased-in-early-disease/