Background/Purpose: Altered generation of adenosine from extracellular nucleotides by ectonucleotidases may orchestrate chronic injury responses and promote fibrosis via the adenosine A2A receptor (ADORA2A). We have explored perturbed adenosine signaling pathways and potential links with fibroblast activation in early stage diffuse scleroderma (SSc).

Methods: Dermal fibroblasts were cultured from diffuse SSc (n=15) and healthy controls (n=5). RNA-Seq explored expression of enzymes involved in degradation of adenosine (adenosine deaminase [ADA] and adenosine kinase [AK]), ectonucleotidases(CD39, CD73) and adenosine receptors.Production of key profibrotic mediators (collagen α1[Col1], alpha smooth muscle actin [αSMA] and connective tissue growth factor [CTGF]) was analysed by western blot of cell lysates in response to adenosine receptor agonist 5’-N-ethylcarboxamidoadenosine (NECA), A2A adenosine agonist (CGS-21680), A2A adenosine antagonist (ZM241385) and polyethylene glycol conjugated recombinant bovine ADA[PEG-rADA] (EZN 2279- elapegademase). Cell migration was assessed at 48h using scratch wound assay and contraction of fibroblast populated 3D collagen gel lattices at 24h.

Results: ADA transcript was increased (1.85-fold, p=0.01, n=12) in SSc fibroblasts (SScF) compared to normal fibroblasts (NF, n=5). In contrast, no significant difference in AK expression (0.79-fold, p=0.19) was observed. ADORA2A was significantly lower in SScF compared to NF (0.33 fold, p=0.04) and CD73 expression reduced in SScF compared to NF (0.61-fold, p=0.004). SScF produced higher levels of Col1 than NF (11.54±2.66 vs 22.07±1.57, p=0.02, n=3). Whilst there was no significant increase in Col1 in response to EZN2279 (9.24±1.18, p=0.2) or ZM241385 (8.95±1.55, p=0.17) in NF, there was with CGS-21680 (1.86 fold, p=0.04). In contrast, there was a smaller induction in Col1 in SScF with CGS-21680 (1.34 fold, p=0.02). The increase in Col1 in SScF was attenuated with EZN2279 and ZM241385 to similar extent (0.56 fold and 0.55 fold respectively, p< 0.05). Analogous response was observed for both αSMA and CTGF. Migratory capacity was enhanced with CGS-21680 in NF (62.83±4.80 vs 45.73±4.86 p< 0.05, n=3) and in SScF (31.17±3.62 vs 25.06±2.01, p< 0.05, n=3). In SScF, this was abrogated with EZN2279 and ZM241385 (56.29±6.26 and 52.32±4.33 respectively, p< 0.05). Gel contraction was induced by CGS-21680 in SScF (120.56±21.19 vs 78.77±6.91, p< 0.05,n=3) compared to NF (196.01±20.87 vs 133.82±19.46, p=0.06,n=3). In SScF, induction of contractile activity was further attenuated with EZN2279 and ZM241385 (189.83±16.44 and 181.02±13.18 respectively, p< 0.05). When stimulated with NECA, increased IL-6 was detected in media of fibroblasts (NF, n=3) (3.09±1.04 vs 14.18±1.56, p< 0.05) and this was attenuated with both EZN2279 and ZM241385 (3.88±0.99 and 3.03±1.10 respectively, p< 0.05).

Conclusion: These data suggest adenosinergic signalling is profibrotic and altered in SSc and that an ADORA2A-IL6 dependent loop may contribute to fibrosis. This may have therapeutic relevance for SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dysregulated-il-6-dependent-dermal-adenosine-signaling-via-adenosine-a2a-receptor-may-drive-fibrosis-in-systemic-sclerosis/