Metabolomics and metabolic function analysis of the secretome of articular cartilage and isolated chondrocytes in response to pro-inflammatory cytokines

Abstract

Purpose: Chondrocytes rely primarily on glycolysis to meet their energy requirements, but possess the metabolic flexibility to support cell survival and matrix synthesis during periods of nutrient stress, by enhancing glycolysis with mitochondrial respiration. Accessing this 'spare respiratory capacity' requires optimal mitochondrial function, but since impaired mitochondrial function is implicated in osteoarthritis (OA), this mechanism may be deficient or attenuated in joint disease. Metabolic adaptation is evident in early-stage OA, however cartilage from late-stage disease does not seem to have this flexibility. A deeper understanding of these complex metabolic pathways may identify new metabolic markers of disease stage, and support therapeutic strategies for treating OA. Metabolomics has the potential to identify underlying metabolic changes, reveal pathological pathways, novel biomarkers and therapeutic targets. The aim of this study was to identify metabolic processes involved in early stage disease by analysis of metabolites and metabolic function in two inflammatory models of cartilage degradation. Methods: Macroscopically normal articular cartilage was obtained from equine and bovine metacarpophalangeal joints. Six-millimetre diameter equine cartilage explants (n¼6) and isolated primary equine chondrocytes (n¼4), seeded at high density (105,000/cm 2), were cultured for 7 days in serum-free low or high glucose Glutamax DMEM (Gibco), respectively, with or without 10ng/ml equine interleukin-1b (IL-1b) and 10ng/ml equine tumour necrosis factor-a (TNF-a). Spent media (secretome) was collected and subjected to metabolomic analysis. Secretome metabolite levels were measured using the AbsoluteIDQ® p180 targeted metabolomics kit (Biocrates), and a Waters Xevo TQ-S mass spectrometer coupled to an Acquity UPLC system. Multivariate analysis was performed by principal component analysis and orthogonalized partial least squares discriminant analysis using SIMCA-P v12.0 software (Umetrics). Metabolic function of primary equine (n¼9) and bovine chondrocytes (n¼3) was determined using Seahorse XFp and XFe24 analyzers (Agilent). Cells were seeded at high density, treated with species-specific 10ng/ml IL-1b and/or 10ng/ml TNF-a for 18 h, and metabolically challenged with the Mito Stress Test during analysis. Metabolite levels, and oxygen consumption rates, were normalised to total cell protein, and differences between means determined by one-way analysis of variance with Tukey's multiple comparison post-tests. Results: Secretome metabolites which decreased with pro-inflammatory cytokine treatment were proline, ornithine and alpha-aminoadipic acid (p<0.0001). Citrulline increased with cytokine treatment (p<0.0001) and glutamate, present in DMEM, was elevated with cytokine treatment (p<0.0001). Metabolomic analysis of the chondrocyte secretome showed that glutamine decreased (p<0.02) with cytokine treatment whereas citrulline was elevated (p<0.003). Metabolic analysis showed that combined cytokine treatment reduced basal re...

Ex Vivo Equine Cartilage Explant Osteoarthritis Model: A Metabolomics and Proteomics Study

“…Previously, an in vivo study of equine OA identified physiological levels of TNF-α and IL-1β within SF to be 40–80 pg/mL . However, our study, along with previous studies in the field, has used significantly higher cytokine concentrations to experimentally model OA. − ,, This approach was used within this study due to the short half-lives of these two cytokines. , Supplementation at a concentration closer to the physiological levels would have ultimately resulted in the experiment being largely conducted with cytokine levels significantly below that experienced during OA, thus producing results that may have been of insufficient benefit. Therefore, the approach used within this study to model OA should be taken into consideration when interpreting the results.…”

“…Furthermore, elevations in glutamate were identified within the culture media at 3−5 days, consistent with a previous study, which may be resultant of the catabolism of collagenous proline through proline oxidase. 27,95 Reduced levels of collagen type VI α 2 chain and collagen type X α 1 chain were identified at 3−5 and 6−8 days following cytokine treatment. This may reflect a reduction in collagen synthesis, which has previously been identified within other collagen types following TNF-α/IL-1β stimulation.…”

“…19 However, our study, along with previous studies in the field, has used significantly higher cytokine concentrations to experimentally model OA. [20][21][22][23][24][25]27,28 This approach was used within this study due to the short half-lives of these two cytokines. 111,112 Supplementation at a concentration closer to the physiological levels would have ultimately resulted in the experiment being largely conducted with cytokine levels significantly below that experienced during OA, thus producing results that may have been of insufficient benefit.…”

“…Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are both proinflammatory cytokines, which are central in OA pathogenesis . TNF-α and IL-1β are secreted by mononuclear cells, synoviocytes, and articular cartilage and upregulate the gene expression of MMPs, ADAMTS-4, and ADAMTS-5, leading to significant ECM degradation. − Elevations in TNF-α and IL-1β are regularly identified within the OA synovial fluid (SF), including that of horses. − TNF-α and IL-1β have therefore become established experimental treatments for modeling OA pathology within in vitro and ex vivo studies, having been used both independently and as a combined treatment. − …”