Amino Acid Deprivation Impacts Rheumatoid Arthritis Fibroblast-Like Synoviocytes

With approval of the local ethics committee, synovial tissues from patients fulfilling the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria for RA (Aletaha et al., 2010 (link)) and patients suffering from OA were obtained as discarded specimens following synovectomy or joint replacement. Culture of FLSs was performed in DMEM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone) and with 1% penicillin and streptomycin (P/S) and nonessential amino acids (both Gibco) as previously described (Kiener et al., 2009 (link)). FLSs between passages 4 and 8 were used for all experiments. The following cytokines, inhibitors, and blocking antibody were used: rhTNF (R&D), JAK inhibitor I (Calbiochem), rapamycin (Calbiochem), MK2206 (Selleckchem), PP242 (Sigma), and Torin-1 (Tocris). All experiments were repeated with FLS cell lines from different donors.
To study the effects of amino acid deprivation, aa-free DMEM (Pan Biotech) was reconstituted with amino acids (Sigma) except the amino acid or amino acids to be omitted. The culture medium (DMEM ± aa) was changed 1 hr before TNF stimulation.