Targeting cellular metabolism in CD4-stimulated synovial fibroblasts reduces inflammation and joint degradation in rheumatoid arthritis
Andreea Petrasca , Monika Biniecka , Douglas J Veale , Ursula Fearon [2,3] and Jean M Fletcher [1,2]
 School of Biochemistry & Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland  School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland  Rheumatology, St. Vincent’s University Hospital, Dublin, Ireland
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by synovial tissue proliferation and degradation of articular cartilage. Activated synovial fibroblasts proliferate and express matrix-degrading proteases and pro-inflammatory cytokines, which contribute to cartilage and joint destruction. Moreover, synoviocyte activation correlates with infiltration of inflammatory lymphocytes and monocytes.
To characterise the functional relationship linking fibroblasts and T lymphocytes in this complex microenvironment, we established an in-vitro model to examine the outcomes of co-culturing activated human CD4 T cells with RA synovial fibroblast cells (SFC) and subsequently altered a range of cellular metabolic pathways to identify key molecular players in joint inflammation.
Anti-CD3/28-stimulated CD4 T cells from healthy human donors were co-cultured with SFC derived from arthroscopy biopsies of RA patients for 5 days. Supernatants were harvested and assayed for cytokine production by ELISA, while the cells were examined for proliferation, adhesion molecules, RANK ligand and glucose transporter, GLUT1, by flow cytometry. Furthermore, SFC were cultured with conditioned medium from stimulated healthy CD4 T cells and manipulated using metabolic manipulators AICAR and 2-DG and analysed by Seahorse assay and for invasion across a matrigel membrane by microscopy.
We found that CD4 T cells induced increased levels of adhesion molecules in SFC, independent of cell contact. Furthermore, CD4 T cells promoted pro-inflammatory cytokine secretion and invasiveness in these SFC. Interestingly, AICAR and 2-DG inhibited invasiveness while reducing the levels of adhesion molecules and IL-8. Seahorse flux analysis showed that T cells enhanced glycolysis, while concomitantly reducing oxidative phosphorylation in SFC, which was reversed by the addition of AICAR or 2-DG. Thus by targeting specific metabolic pathways, these inflammatory responses could be reversed.
Our results show that CD4 T cells work mutually with fibroblasts to create an inflammatory microenvironment which directly contributes to joint destruction through pro-inflammatory mediators, as well as a switch from oxidative phosphorylation to glycolysis. Therapeutic altering of these metabolic pathways using compounds that reduce inflammation, could have potential clinical implications for RA treatment.