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A1.20 Epigenetic modificationsand TLR8 signalling contribute to systemic sclerosis pathogenesis by ros and profibrotic genes induction
  1. M Ciechomska1,2,
  2. P Zarecki1,
  3. D Piatek1,
  4. M Merdas1,
  5. J Swierkot3,
  6. E Morgiel3,
  7. JM van Laar4,
  8. W Maslinski2,
  9. K Bogunia-Kubik1
  1. 1Institute of Immunology and Experimental Therapy, Wroclaw, Poland
  2. 2National Institute of Geriatrics Rheumatology and Rehabilitation, Warsaw, Poland
  3. 3Wroclaw Medical University, Wroclaw, Poland
  4. 4University Medical Centre, Utrecht, The Netherlands

Abstract

Background and objectives Systemic sclerosis (SSc) is a chronic multisystem autoimmune disease characterised by skin and  internal organs fibrosis and immune abnormalities. Recent evidence suggests that activated circulating monocytes from SSc patients play a role in SSc pathogenesis due to enhanced expression of tissue inhibitor of metalloproteinases 1 (TIMP-1), IL-8 and reactive oxygen species (ROS) induction, which contribute to fibrosis progression and chronic inflammation. The exact factors driving TIMP-1, IL-8 and ROS secretion are still unknown.

The aim of this study was to investigate the expression pattern of profibrotic IL-8, TIMP-1, AP1 transcription factor-Fra2 and ROS induction in peripheral blood monocytes following DZNep (histone methyltransferase inhibitor) and TLR8 agonist (ssRNA) stimulation.

Materials and methods The expression of Fra2, IL-8 and TIMP-1 and anti-oxidant superoxide dismutase 1 (SOD1) was measured by qRT-PCR in stimulated and unstimulated HC (n = 14) and SSc (n = 17) monocytes. Generation of ROS was determined using luciferase based assay. Fra2 DNA-binding activity was measured in AP-1 transcription assay in monocytic U937 cell line following epigenetic and TLR8 modifications. The level of anti-fibrotic miRNA-5196, which is predicted to bind and inhibit 3’UTR of Fra-2 gene, was also determined in HC and SSc monocytes.

Results Combination of DZNep+TLR8 enhanced Fra2 (2-fold, p = 0.02), TIMP-1 (2-fold) and IL-8 (7.87-fold, p < 0.001) expression in SSc monocytes. Fra2 DNA-binding activity was 1.5-fold increased upon stimulation. Secreted level of TIMP-1 was 1.46-fold higher in SSc monocytes compared to unstimulated cells. Generated ROS was 2.21-fold (p = 0.0395) higher following DZNep+TLR8 stimulation in monocytic U937 cells. In contrast, miRNA-5196 expression was 2.13-fold decreased in SSc monocytes upon DZNep+TLR8 stimulation. Also the level of SOD1 was decreased in HC and SSc monocytes following stimulation, 2.16-fold (p = 0.025) and 1.56-fold, respectively.

Conclusions These data suggest that DZNep and TLR8 agonist are able to enhance pro-fibrotic TIMP-1, IL-8 and oxidative stress generation. As opposed by the decrease of anti-fibrotic miRNA-5196 and anti-oxidant SOD1 expression in SSc monocytes, which might be used as a potential modulators of fibrogenesis in SSc.

Supported by Homing Plus grant/2013–8/4 from Foundation for Polish Science and UMO-2015/16/S/NZ6/00041 from National Science Centre, Poland.

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