A9.6 Identification of New Potential Therapeutic Targets for the Treatment of Rheumatoid Arthritis: ENTPD1 (CD39) and 5NTE1 (CD73)

Background and Objectives

Adenosine and ATP are known to have important immunomodulatory properties. Extracellular ATP has multiple roles in inflammation and can act as a damage-associated molecular pattern (DAMP) that can activate the immune system. Conversely, adenosine is primarily anti-inflammatory and can inhibit the production of pro-inflammatory molecules by immune cells. The modulation of ATP and adenosine levels are an essential part of the induction and resolution of an inflammatory response. ENTPD1 (CD39) is a membrane-bound ectonucleoside triphosphate diphosphohydrolase enzyme that converts ATP and ADP to AMP 5NTE1 (CD73) is a 5′ ecto-nucleotidase that dephosphorylates AMP to form adenosine. We investigated the role of genes in the adenosine pathway in patients with rheumatoid arthritis (RA) and determined whether expression of CD39 and CD73 would have an effect in an in vitro inflammation model.

Materials and Methods

Gene expression analysis using 43k cDNA microarrays (Stanford Functional Genomics Facility) was performed on total RNA extracted from RA synovial tissues obtained by arthroscopy. Adenosine pathway gene expression was compared between high-inflammation versus low-inflammation tissue type synovial biopsies. ATPase levels were measured in synovial fluid (SF) from RA (n = 10) or osteoarthritis (OA) (n = 6) patients. Adeno-associated viral (AAV) vectors expressing CD39 or CD73 were generated and used to transduce HEK 293 cells or RA fibroblast-like synoviocytes (FLS) and these transduced cells were co-cultured with LPS-activated human monocytes (THP-1) in the presence of ATP. Pro-inflammatory cytokine/chemokine (IL-6, CCL2) production was measured by ELISA.

Results

Genes involved in the ATP:adenosine pathway, including CD73, were differentially expressed in high-inflammation synovial tissues, consistent with the hypothesis that there is skewing of the ATP:adenosine balance during inflammation. The half-life of ATP was significantly increased in SF from RA patients compared with OA (t_1/2 = 8.0 versus 4.5 min., p = <0.05), indicating that there was a significant decrease in ATPase activity in RA SF HEK 293 cells and RA FLS cell lines transduced with CD39-and/or CD73-expressing AAV5 vectors demonstrated high CD39 and CD73 activity. THP-1 cells stimulated with LPS showed lower levels (>80% reduction p = <0.05) of IL-6 and CCL2 secretion when co-cultured with CD39 and CD73 expressing HEK293 cells or FLS cells in the presence of ATP.

Conclusions

Together, these data suggest that synovial inflammation in RA is characterised by skewing of the ATP:adenosine balance. This could be reversed by overexpression of CD39 or CD73. Thus, these data show that the ATP:adenosine pathway may be a novel therapeutic target for the treatment of RA.