Background/Purpose: Fibroblast-like synoviocyte (FLS) activation is a key component of rheumatoid arthritis (RA) inflamed synovium.  Their aggressive phenotype in RA contributes to initiation and perpetuation of destructive joint inflammation. Understanding changes in RA FLS metabolism is becoming a new focus of research in RA. We previously showed that the glutaminase gene, which encodes for the enzyme that generates glutamate from glutamine, is highly epigenetically regulated in RA FLS compared to osteoarthritis (OA) FLS. Here, we determined the importance of glutamine (gln) as an alternate carbon source to glucose for FLS and whether or not interference of this pathway can decrease the aggressive phenotype of FLS in RA.

Methods: Uptake of glutamine was determined by Glutamine/Glutamate-Glo kit by Promega. Dependence of glutamine by RA FLS was determined by functional assays, including invasion (matrigel assay), proliferation assays (EdU assay), and a migration assay (scratch assay), that were conducted using DMEM without glucose supplemented with dialyzed fetal bovine serum. We then used increasing amounts of glutamine before and after PDGF stimulation in the presence or absence of glucose. RA FLS were also incubated with CB-839 (300nM), a glutaminase inhibitor.

Results: Glutamine/glutamate luminescence test displayed significant glutamine uptake in FLS starved of glucose after PDGF stimulation (3938±10425 vs 1165±371.8, p< 0.0001). Functional assays revealed that 6mM glutamine (FLS cultured in a media without glucose) is sufficient as an alternate source of energy to 6mM glucose (FLS cultured in media without gln) in proliferation (6mM gln 0.3±0.07 vs 6mM glucose 0.37±0.21; ns) and invasion (6mM gln 21.47±1.34 vs 6mM glucose 20.91±1.3; ns). In addition, the use of glutamine allows for an increase of invasion (0mM gln 12.87±3.316 vs 6mM 18.83±3.236; p< 0.002), proliferation (0mM gln 8.752±4.7 vs. 6mM gln 40.55±14.07; p< 0.02), and migration (0mM gln 219.8±49.88 vs 6mM gln 186.4±45.90; p=0.0002) in the absence of glucose. Of interest, OA FLS do not migrate more on 6mM glutamine compared to 0mM glutamine (0mM gln 216.1±46.35 vs 6mM gln 219.4±58.64; ns). Experiments with CB-839 showed significant decrease in invasion (vehicle 23.11±3.161 vs CB-839 14.46±2.036 p< 0.0001) and proliferation (vehicle 0.2548±0.03589 vs CB-839 0.1278±0.02861 p< 0.0001) of RA FLS under glucose depletion with varying amounts of glutamine compared with vehicle control.

Conclusion: RA FLS are able to utilize glutamine as an alternative carbon source in the absence of glucose, which mimics the starvation conditions within the joint. Glutaminase could potentially be a good target to reduce the aggressive phenotype of FLS in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-glutamine-metabolism-in-rheumatoid-arthritis-fibroblast-like-synoviocyte-aggressive-phenotype/