Post-transcriptional regulation of proinflammatory cytokines in arthritis using gene delivery of AU-rich element binding factors

Background

Monocytes and macrophages are abundant in rheumatoid synovial tissue and play a major role in the pathogenesis of rheumatoid arthritis (RA) by secreting proinflammatory cytokines such as tumour necrosis factor alpha (TNF-α). An innovative approach is based on the regulation of mRNA stability and degradation, which constitute a critical step in the control of gene expression rather than neutralization of the downstream protein. Stability and degradation of TNF-α mRNA are regulated by cis-acting sequences as AU-rich elements (AREs). Tristetraprolin (TTP), a class of Cys-Cys-Cys-His (CCCH) zinc finger proteins, was identified as the critical TNF-α ARE-binding protein. Importantly, TTP knockout mice develop inflammatory arthritis, dermatitis and myeloid hyperplasia, prevented by anti-TNF-α antibodies. A recent study suggested that a low TTP/TNF-α RNA expression ratio could indicate failure of RA patients to produce adequate amounts of TTP in response to increased TNF-α production. Our aim is to investigate the therapeutic potential of gene expression of ARE-binding elements for anti-TNF-α therapies.

Methods

We used the THP1 human monocyte cell line known for a high lipopolysaccharide (LPS)-induced TNF-α secretion. An expression vector containing TTP gene driven by the CMV promoter was constructed. TTP expression was evaluated by western blot following transfection by electroporation (320 mV). The secretion of TNF-α by THP1 was assessed by ELISA before and after TTP transfection (1 μg/10⁶ cells), following LPS stimulation (50 ng/ml).

Results

A significant decrease in TNF-α expression was observed in the TTP-transfected THP1 cells, compared with a GFP mock control. This suppression increased over time and last at least 48 hours after LPS stimulation (23% at 3 hours and 37% at 24 hours).

Conclusion

These preliminary results support an important role of TTP in regulating TNF-α in monocytes and might be the new target for gene delivery in an anti-TNF-α strategy in RA. In vivo evaluation of this strategy in experimental models of RA will be tested.