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A3.1 Epigenetic and TLR8-dependent regulation of profibrotic genes expression in monocytes in systemic sclerosis and its implication on fibroblasts trans-differentiation
  1. M Ciechomska1,2,
  2. S O’Reilly1,
  3. M Merdas2,
  4. P Zarecki2,
  5. J Swierkot3,
  6. E Morgiel3,
  7. K Bogunia-Kubik2,
  8. JM van Laar1,4
  1. 1 Newcastle University, Newcastle Upon Tyne, UK
  2. 2 Institute of Immunology and Experimental Therapy, Wroclaw, Poland
  3. 3 Wroclaw Medical University, Wroclaw, Poland
  4. 4 University Medical Centre, Utrecht, The Netherlands

Abstract

Background and objectives To investigate whether epigenetic changes induced by 3’deazaneplanocin - DZNep and TLR signalling pathway can modulate monocytes to produce tissue-inhibitor of metalloproteinase-1 (TIMP-1) via Fra2 (Fos-related antigen 2) activity, a novel downstream mediator promoting fibrogenesis.

Methods AP-1 and TIMP-1 expression following TLR8 treatment was measured by qRT-PCR in monocytes from Systemic sclerosis (SSc) patients (n = 13) and healthy controls (HC) (n = 13). TIMP-1 promoter activity was measured in U397 monocytic cell line using luciferase reporter assay. The effect of DZNep treatment on inhibition of tri-methylation of lysine 27 on histone 3 was analysed by Western Blot. Expression of TIMP-1 and Fra2 was determined in response to DZNep. The functional effect of TLR8 and DZNep-treated HC monocytes was studied on dermal fibroblasts’ trans-differentiation.

Results Increased Fra2 and TIMP-1 expression was correlated in SSc monocytes (p = 0.021), but not in HC monocytes upon TLR8 stimulation. In contrast, the expression of anti-fibrotic Fra1 was significantly (p = 0.037) reduced in SSc monocytes compared to HC. Inhibiting AP-1 activity reduced TIMP-1 production in TLR8 stimulated HC and SSc monocytes. Also, TLR8 stimulation induced significant (p = 0.015) TIMP-1 promoter activity in monocytic U937 cells. Combination of DZNep plus TLR8 enhanced Fra2 (4.1 times) and TIMP-1 (4.7 times) expression in HC monocytes. However, the reverse effect on Fra2 and TIMP-1 expression was observed in SSc monocytes following stimulation. Finally, DZNep plus TLR8-treated HC monocytes induced strong production of collagen and a-SMA in dermal fibroblasts reflecting their trans-differentiation, which is a key event in the pathogenesis of SSc.

Conclusions These data demonstrate that histone modification induces by DZNep has an opposite effect of Fra2-mediated TIMP-1 production on HC versus SSc monocytes. Therefore, DZNep cloud be used as a selective regulator of downstream mediators, which orchestrates SSc development.

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