Targeted Metabolic Profiling of the Tg197 Mouse Model Reveals Itaconic Acid as a Marker of Rheumatoid Arthritis

Abstract

Rheumatoid arthritis is a progressive, highly debilitating disease where early diagnosis, enabling rapid clinical intervention, would provide obvious benefits to patients, healthcare systems, and society. Novel biomarkers that enable noninvasive early diagnosis of the onset and progression of the disease provide one route to achieving this goal. Here a metabolic profiling method has been applied to investigate disease development in the Tg197 arthritis mouse model. Hind limb extract profiling demonstrated clear differences in metabolic phenotypes between control (wild type) and Tg197 transgenic mice and highlighted raised concentrations of itaconic acid as a potential marker of the disease. These changes in itaconic acid concentrations were moderated or indeed reversed when the Tg197 mice were treated with the anti-hTNF biologic infliximab (10 mg/kg twice weekly for 6 weeks). Further in vitro studies on synovial fibroblasts obtained from healthy wild-type, arthritic Tg197, and infliximab-treated Tg197 transgenic mice confirmed the association of itaconic acid with rheumatoid arthritis and disease-moderating drug effects. Preliminary indications of the potential value of itaconic acid as a translational biomarker were obtained when studies on K4IM human fibroblasts treated with hTNF showed an increase in the concentrations of this metabolite.

Metabolic Checkpoints in Rheumatoid Arthritis

“…The role of itaconate in RA is still debated. Despite evidence suggests an anti-inflammatory role (Mills et al, 2018) other studies have shown that reduced levels of itaconate correlate with a decreased pro-inflammatory (M1) signature in human macrophages isolated from healthy control subjects (Papathanassiu et al, 2017) and with a reduced arthritis severity in vivo (Michopoulos et al, 2016;Papathanassiu et al, 2017). It would be valuable to investigate how these observations in murine models translate into the human disease setting (i.e., OA vs. RA macrophages).…”

Altered metabolic pathways regulate synovial inflammation in rheumatoid arthritis

“…Lactic acid enhances secretion of IL-6 and IL-23 from monocytes and macrophages [47]. Itaconate, a metabolic inhibitor of succinate dehydrogenase which has been shown to regulate succinate levels and secretion of inflammatory cytokines in activated macrophages [48], is also increased in the RA joint, and is associated with disease activity and response to therapy in animal models of arthritis [27]. Itaconate, a metabolic inhibitor of succinate dehydrogenase which has been shown to regulate succinate levels and secretion of inflammatory cytokines in activated macrophages [48], is also increased in the RA joint, and is associated with disease activity and response to therapy in animal models of arthritis [27].…”

“…Evidence for this metabolic switch in RA has been demonstrated by several studies showing an increase in mitochondrial dysfunction in the RA synovium [20], along with increased expression of glucose transporters (GLUTs) [21] and glycolytic enzymes, including hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate isomerase (GPI) and LDH [7,8,[21][22][23][24][25]. Marked accumulation of metabolic intermediates including lactate, glutamine, succinate [26] and itaconate [27] have been demonstrated in the RA joint which, in turn, can activate synovial cells Fig. 1.…”

“…Accumulation of succinate in macrophages promotes HIF-1α activation which, in turn, induces IL-1β production [39] and in animal models of RA, mice lacking the succinate receptor GPR91 show reduced macrophage activation and secretion of IL-1β [33]. Itaconate, a metabolic inhibitor of succinate dehydrogenase which has been shown to regulate succinate levels and secretion of inflammatory cytokines in activated macrophages [48], is also increased in the RA joint, and is associated with disease activity and response to therapy in animal models of arthritis [27]. RA macrophages express high amounts of the glycolytic enzyme α-enolase, which through autoantibody recognition induces secretion of proinflammatory cytokines [49].…”

Cultures of a human synovial cell line to evaluate platelet-rich plasma and hyaluronic acid effects

“…K4IM cells were seeded at a density of 12,500 cells/cm 2 in 12‐well culture plates in 1‐ml 1:1 Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 (DMEM/F12; HyClone Lab, Thermo Scientific, Logan, Utah, USA) supplemented with 1% penicillin/streptomycin and 10% FBS. The day after the medium was replaced with 1 ml of fresh serum‐free culture medium and K4IM cells were maintained for 24 hr with 10 ng/ml TNF‐α (Bio Basic Inc, Markham Ontario, Canada) as pro‐inflammatory stimulus (Michopoulos et al, ; Mrosewski et al, ). Unstimulated cells were used as control.…”