Via analysis of two cohorts of established RA (one cross-sectional and one prior to and after exercise training), we identified a phenotype for an exercise training-induced anti-inflammatory response as being older, more inflamed, less aerobically fit, and with multiple alterations in skeletal muscle metabolic pathways. Based on our transcriptomic analyses of top highly associated genes and IPA canonical pathways, we have identified the apparent skeletal muscle cellular metabolic pathways that 1) are connected to RA inflammation and 2) are associated with exercise training modulation of systemic immune responses (Fig. 2). These skeletal muscle pathways are highlighted by major gene expression alterations in amino acid catabolism and the regulation of glycolysis and TCA cycle flux. Taken together, rewiring of protein homeostasis and oxidative metabolism in sedentary RA skeletal muscle may be connected to the perpetuation of systemic inflammation. This association of the RA muscle transcriptional profile with HIIT-mediated improvements in RA disease activity suggests that exercise modulation of inflammation occurs in concert with reprogramming of skeletal muscle metabolism.
Amino acid homeostasis: In RA, amino acids are critical to sustain chronic inflammation. In RA synovium, chronic inflammation upregulates amino acid transporters, fueling maladaptive synovial proliferation and bone remodeling pathways (27). In the circulation, activated RA immune cells have high energetic demands that are met with glutamine, the majority of which is produced by skeletal muscle (28–30). Further, inhibition of glutaminolysis is a promising therapeutic target for management of Th17- driven autoimmune diseases (31, 32). Our data demonstrate that muscle catabolism and interconversion of multiple amino acids—including glycine, lysine, glutamate/glutamine and the branched-chain amino acids leucine, isoleucine, and valine—are linked to both RA inflammation and exercise training-induced reduction of inflammation (Fig. 2).
Chronic immune activity and exercise training-related modulation appear strongly related to the RA muscle mitochondrial glycine cleavage pathway. Baseline upregulation of multiple key glycine cleavage system genes (GLDC, GCSH, LIAS, AMT) associated with HIIT-related improvements in disease activity, while GLDC (glycine dehydrogenase) was significantly down-regulated following HIIT. Glycine is a proteogenic amino acid and critical for multiple metabolic pathways in humans (33). Catabolism of glycine via the glycine cleavage pathway to N5,N10-methylenetetrahydrofolate is utilized for the biosynthesis of purine nucleotides (34, 35), key regulators of T lymphocyte proliferation and survival (36, 37). Thus, increased activity of the glycine cleavage system may be one way that impaired RA skeletal muscle metabolism contributes to and is impacted by chronic RA immune dysfunction.
Altered skeletal muscle metabolism of other amino acids also link to RA inflammation. Skeletal muscle branched-chain amino acid (BCAA) catabolism—via increased mitochondrial branched-chain α-ketoacid (BCKA) dehydrogenase (BCKDH) complex activity—was associated with both baseline RA disease activity and with exercise-mediated disease activity improvements. We theorize that increased RA muscle BCKDH activity might lead to glutamate/glutamine formation via multiple pathways. First, BCAA are metabolized to both BCKA and glutamate directly. Second, BCKA are converted to acetyl-CoA; subsequent α-ketoglutarate flux from the TCA cycle leads to further glutamate production via ornithine aminotransferase/OAT (25, 34). Glutamate is then readily converted to glutamine with glutamine production augmented by lysine degradation via increased muscle AACC activity. Glutamine released into circulation can then sustain activation of RA immune cells (30, 34, 38)(Fig. 2).
We additionally identified that HIIT-related improvements in disease activity were associated with baseline upregulation of muscle metabolism of dopamine to homocysteine via COMT/LRTOMT and glutamate conversion to proline via PYCR. Increased systemic homocysteine is linked to the human inflammatory response (39), though the direct pathway in which muscle proline production contributes to immune cell activation is less clear (40).
Interestingly, opposing protein homeostatic pathways involved in both protein synthesis and muscle atrophy associated with subsequent improvement in RA inflammatory disease activity following HIIT. These pathways were highlighted by greater gene expression of RPL15 (a ribosomal protein critical for muscle protein synthesis) (34) and the nNOS calcium signaling canonical pathway important for driving muscle atrophy (41). Taken together, these altered pathways suggest that RA skeletal muscle tissue is in a heightened state of protein turn-over; further, exercise training may work in part to regulate these protein catabolic and anabolic programs. Our findings of upregulated RA muscle protein turnover are in agreement with the prominent clinical phenotype of muscle loss due to “rheumatoid cachexia” or sarcopenic obesity (15). Therefore, the increased ribosomal activity in RA muscle may represent a failed compensatory response to maintain muscle mass in the face of constant protein breakdown and atrophy signaling triggered by chronic inflammation. One unanswered question is whether this state of dysregulated RA muscle amino acid/protein homeostasis is merely a byproduct of inflammation or if muscle protein breakdown truly contributes to direct fueling of chronic immune activity (30).
Glycolysis regulation: In addition to amino acids, dysregulation of RA skeletal muscle glycolysis and lactate production appears to support chronic immune signaling. We previously showed that, as compared to healthy matched controls, RA skeletal muscle is characterized by increased glycolysis as evidenced by transcriptional downregulation of oxidative metabolism components and accumulation of pyruvate (16). Here, in a separate RA cohort, additional pro-glycolytic programs were linked to exercise-mediated improvements in RA disease activity. These transcriptomic features include increased pre-training breakdown of glycogen to glucose, decreased conversion of pyruvate to acetyl CoA, and increased interconversion between pyruvate and lactate (34)(Fig. 2). It is unclear from our data alone if an increased reliance on muscle glycolysis and subsequent lactate production contributes to RA immune cytokine signaling. Of note, release of lactate from tumor and inflamed tissue—including RA synovium—stimulates pro-inflammatory IL-17 secretion (42–44). Further, our data show that increased RA muscle hypoxia-inducible factor 1 alpha (HIF1A) expression is also associated with exercise-related improvements in inflammation; increased lactate can trigger increased HIF1A (45), where HIF1A can then stimulate immune cell IL-1β and IL-17 production (46).
TCA cycle flux and oxidative metabolism: Increased RA muscle glycolysis appears to be connected to remodeling of oxidative metabolic pathways. Oxidative phosphorylation requires reducing agents (i.e., NADH and FADH2), typically generated through acetyl CoA and TCA cycle metabolism. Our findings suggest that RA muscle amino acid sources contribute more and glucose and fatty acids less than expected to acetyl CoA generation (Fig. 2). This apparent reduction in beta oxidation is congruent with findings of increased lipid storage (i.e., intramuscular adiposity) in RA muscle. Intramuscular adiposity is linked to disability and an early aging phenotype in RA (47, 48). Taken together, this suggests that exercise training is potentially important for modulating both RA fat metabolism and functional impairments.
Remodeling of RA muscle oxidative metabolism was also evidenced by downregulation of multiple TCA cycle enzymes (i.e., OGDH, SUCLA2, MDH1B). We theorize that alterations in multiple TCA cycle nodes would result in inefficient reducing agent production to support ATP generation via OXPHOS (34). Despite increased electron transport chain complex I activity (i.e., increased NDUFV3 expression), reliance on reducing agents produced from pathways other than the TCA cycle could result in greater RA muscle reactive oxygen species (ROS) formation relative to ATP generation (49). Altered ROS and impaired redox balance could then contribute to perpetuating chronic systemic inflammation (50).
Immune pathways: In addition to metabolic alterations, we hypothesized that upregulated RA skeletal muscle immune/inflammatory cytokine pathways would be associated with disease activity and improvements in disease activity following exercise training, however, our data did not consistently support this theory. In the cross-sectional RA cohort #1, IL1RL2 and TAB1 were the only skeletal muscle genes specifically involved in immune function or inflammation to correlate highly with RA disease activity (Supplementary Table 1). Skeletal muscle IL-15 production was the top canonical pathway associated with disease activity (12/128 genes; p = 0.12; data not shown). Interestingly, muscle IL-15 has important roles in regulating fat mass and systemic metabolism, as well as modulating lymphocyte development and acute inflammation (11). In the HIIT RA cohort #2, only muscle expression of immune-related genes FCRL6, TNFRSF19, CMTM4, and NKG7 were highly correlated with improvements in RA disease activity following HIIT. In established RA, these data suggest that systemic inflammation impacts skeletal muscle metabolism to a greater extent than the direct effects of localized inflammation within skeletal muscle tissue.
Limitations: Though we have identified multiple skeletal muscle pathways that are connected to RA inflammation and can potentially be modulated by exercise training, our findings should be reviewed in the context of a few key limitations. Primarily, the bulk of our analyses relied on correlations based on stored human tissue samples and thus true causative pathways or intervenable targets could not be interrogated. Further, in the HIIT RA cohort #2, we analyzed correlations between baseline/pre-HIIT factors—as opposed to changes in these factors—with changes in RA disease activity following exercise training. While this decision to focus on baseline factors may limit our understanding of co-occurring exercise training-induced muscle and immune modifications, our analyses better show how immune overactivity influences RA muscle at baseline and the potential for exercise training to rectify those interactions. Finally, our HIIT cohort did not include a control RA group (i.e., not undergoing exercise training) which somewhat limits interpretation of exercise training-specific effects on RA muscle and immune function. Thus, further detailed study of cross-talk between RA skeletal muscle and immune cells is necessary to dissect the intricacies of these pathways so they can eventually be exploited to improve patient care.