CD200+ fibroblasts form a pro-resolving mesenchymal network in arthritis.

Fibroblasts are important regulators of inflammation, but whether fibroblasts change phenotype during resolution of inflammation is not clear. Here we use positron emission tomography to detect fibroblast activation protein (FAP) as a means to visualize fibroblast activation in vivo during inflammation in humans. While tracer accumulation is high in active arthritis, it decreases after tumor necrosis factor and interleukin-17A inhibition. Biopsy-based single-cell RNA-sequencing analyses in experimental arthritis show that FAP signal reduction reflects a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalization) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalization). Spatial transcriptomics of human joints indicates that pro-resolving niches of CD200+DKK3+ fibroblasts cluster with type 2 innate lymphoid cells, whereas MMP3+/IL6+ fibroblasts colocalize with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilized the type 2 innate lymphoid cell phenotype and induced resolution of arthritis via CD200-CD200R1 signaling. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

Canagliflozin impairs T cell effector function via metabolic suppression in autoimmunity.

Augmented T cell function leading to host damage in autoimmunity is supported by metabolic dysregulation, making targeting immunometabolism an attractive therapeutic avenue. Canagliflozin, a type 2 diabetes drug, is a sodium glucose co-transporter 2 (SGLT2) inhibitor with known off-target effects on glutamate dehydrogenase and complex I. However, the effects of SGLT2 inhibitors on human T cell function have not been extensively explored. Here, we show that canagliflozin-treated T cells are compromised in their ability to activate, proliferate, and initiate effector functions. Canagliflozin inhibits T cell receptor signaling, impacting on ERK and mTORC1 activity, concomitantly associated with reduced c-Myc. Compromised c-Myc levels were encapsulated by a failure to engage translational machinery resulting in impaired metabolic protein and solute carrier production among others. Importantly, canagliflozin-treated T cells derived from patients with autoimmune disorders impaired their effector function. Taken together, our work highlights a potential therapeutic avenue for repurposing canagliflozin as an intervention for T cell-mediated autoimmunity.

The mammalian target of rapamycin contributes to synovial fibroblast pathogenicity in rheumatoid arthritis.

Objectives: The mammalian target of Rapamycin (mTOR) is a metabolic master regulator of both innate and adaptive immunity; however, its exact role in stromal cell biology is unknown. In this study we explored the role of the mTOR pathway on Rheumatoid Arthritis synovial fibroblast (RASF) metabolism and activation and determined if crosstalk with the Hippo-YAP pathway mediates their effects. Methods: Primary RA synovial fibroblasts (RASF) were cultured with TNFα alone or in combination with the mTOR inhibitor Rapamycin or YAP inhibitor Verteporfin. Chemokine production, matrix metalloproteinase (MMP) production, and adhesion marker expression were quantified by real-time PCR, ELISA, and/or Flow Cytometry. Invasion assays were performed using Transwell invasion chambers, while wound repair assays were used to assess RASF migration. Cellular bioenergetics was assessed using the Seahorse XFe96 Analyzer. Key metabolic genes (GLUT-1, HK2, G6PD) were measured using real-time PCR. Reanalysis of RNA-Seq analysis was performed on RA (n = 151) and healthy control (HC) (n = 28) synovial tissue biopsies to detect differential gene and pathway expression. The expression of YAP was measured by Western Blot. Results: Transcriptomic analysis of healthy donor and RA synovial tissue revealed dysregulated expression of several key components of the mTOR pathway in RA. Moreover, the expression of phospho-ribosomal protein S6 (pS6), the major downstream target of mTOR is specifically increased in RA synovial fibroblasts compared to healthy tissue. In the presence of TNFα, RASF display heightened phosphorylation of S6 and are responsive to mTOR inhibition via Rapamycin. Rapamycin effectively alters RASF cellular bioenergetics by inhibiting glycolysis and the expression of rate limiting glycolytic enzymes. Furthermore, we demonstrate a key role for mTOR signaling in uniquely mediating RASF migratory and invasive mechanisms, which are significantly abrogated in the presence of Rapamycin. Finally, we report a significant upregulation in several genes involved in the Hippo-YAP pathway in RA synovial tissue, which are predicted to converge with the mTOR pathway. We demonstrate crosstalk between the mTOR and YAP pathways in mediating RASF invasive mechanism whereby Rapamycin significantly abrogates YAP expression and YAP inhibition significantly inhibits RASF invasiveness. Conclusion: mTOR drives pathogenic mechanisms in RASF an effect which is in part mediated via crosstalk with the Hippo-YAP pathway.

Rheumatoid arthritis macrophages are primed for inflammation and display bioenergetic and functional alterations.

OBJECTIVES: Myeloid cells with a monocyte/macrophage phenotype are present in large numbers in the RA joint, significantly contributing to disease; however, distinct macrophage functions have yet to be elucidated. This study investigates the metabolic activity of infiltrating polarized macrophages and their impact on pro-inflammatory responses in RA. METHODS: CD14+ monocytes from RA and healthy control (HC) bloods were isolated and examined ex vivo or following differentiation into 'M1/M2' macrophages. Inflammatory responses and metabolic analysis ± specific inhibitors were quantified by RT-PCR, western blot, Seahorse XFe technology, phagocytosis assays and transmission electron microscopy along with RNA-sequencing (RNA-seq) transcriptomic analysis. RESULTS: Circulating RA monocytes are hyper-inflammatory upon stimulation, with significantly higher expression of key cytokines compared with HC (P < 0.05) a phenotype which is maintained upon differentiation into mature ex vivo polarized macrophages. This induction in pro-inflammatory mechanisms is paralleled by cellular bioenergetic changes. RA macrophages are highly metabolic, with a robust boost in both oxidative phosphorylation and glycolysis in RA along with altered mitochondrial morphology compared with HC. RNA-seq analysis revealed divergent transcriptional variance between pro- and anti-inflammatory RA macrophages, revealing a role for STAT3 and NAMPT in driving macrophage activation states. STAT3 and NAMPT inhibition results in significant decrease in pro-inflammatory gene expression observed in RA macrophages. Interestingly, NAMPT inhibition specifically restores macrophage phagocytic function and results in reciprocal STAT3 inhibition, linking these two signalling pathways. CONCLUSION: This study demonstrates a unique inflammatory and metabolic phenotype of RA monocyte-derived macrophages and identifies a key role for NAMPT and STAT3 signalling in regulating this phenotype.

Cellular metabolic adaptations in rheumatoid arthritis and their therapeutic implications.

Activation of endothelium and immune cells is fundamental to the initiation of autoimmune diseases such as rheumatoid arthritis (RA), and it results in trans-endothelial cell migration and synovial fibroblast proliferation, leading to joint destruction. In RA, the synovial microvasculature is highly dysregulated, resulting in inefficient oxygen perfusion to the synovium, which, along with the high metabolic demands of activated immune and stromal cells, leads to a profoundly hypoxic microenvironment. In inflamed joints, infiltrating immune cells and synovial resident cells have great requirements for energy and nutrients, and they adapt their metabolic profiles to generate sufficient energy to support their highly activated inflammatory states. This shift in metabolic capacity of synovial cells enables them to produce the essential building blocks to support their proliferation, activation and invasiveness. Furthermore, it results in the accumulation of metabolic intermediates and alteration of redox-sensitive pathways, affecting signalling pathways that further potentiate the inflammatory response. Importantly, the inflamed synovium is a multicellular tissue, with cells differing in their metabolic requirements depending on complex cell-cell interactions, nutrient supply, metabolic intermediates and transcriptional regulation. Therefore, understanding the complex interplay between metabolic and inflammatory pathways in synovial cells in RA will provide insight into the underlying mechanisms of disease pathogenesis.

Metabolites as drivers and targets in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by neovascularization, immune cell infiltration, and synovial hyperplasia, which leads to degradation of articular cartilage and bone, and subsequent functional disability. Dysregulated angiogenesis, synovial hypoxia, and immune cell infiltration result in a 'bioenergetic crisis' in the inflamed joint which further exacerbates synovial invasiveness. Several studies have examined this vicious cycle between metabolism, immunity, and inflammation and the role metabolites play in these interactions. To add to this complexity, the inflamed synovium is a multicellular tissue with many cellular subsets having different metabolic requirements. Metabolites can shape the inflammatory phenotype of immune cell subsets during disease and act as central signalling hubs. In the RA joint, the increased energy demand of stromal and immune cells leads to the accumulation of metabolites such as lactate, citrate, and succinate as well as adipocytokines which can regulate downstream signalling pathways. Transcription factors such as HIF1ɑ and mTOR can act as metabolic sensors to activate synovial cells and drive pro-inflammatory effector function, thus perpetuating chronic inflammation further. These metabolic intermediates may be potential therapeutic targets and so understanding the complex interplay between metabolites and synovial cells in RA may allow for identification of novel therapeutic strategies but also may provide significant insight into the underlying mechanisms of disease pathogenesis.

Rheumatoid arthritis CD14+ monocytes display metabolic and inflammatory dysfunction, a phenotype that precedes clinical manifestation of disease.

INTRODUCTION: This study investigates the metabolic activity of circulating monocytes and their impact on pro-inflammatory responses in RA and explores whether this phenotype is already primed for inflammation before clinical manifestations of disease. METHODS: Blood was collected and CD14+ monocytes isolated from healthy control donors (HC), individuals at-risk (IAR) and RA patients. Monocyte frequency in blood and synovial tissue was assessed by flow cytometry. Inflammatory responses and metabolic analysis ± specific inhibitors were quantified by RT-PCR, Western blot, migration assays, Seahorse-XFe-technology, mitotracker assays and transmission electron microscopy. Transcriptomic analysis was performed on HC, IAR and RA synovial tissue. RESULTS: CD14+ monocytes from RA patients are hyper-inflammatory following stimulation, with significantly higher expression of cytokines/chemokines than those from HC. LPS-induced RA monocyte migratory capacity is consistent with increased monocyte frequency in RA synovial tissue. RA CD14+ monocytes show enhanced mitochondrial respiration, biogenesis and alterations in mitochondrial morphology. Furthermore, RA monocytes display increased levels of key glycolytic enzymes HIF1α, HK2 and PFKFB3 and demonstrate a reliance on glucose consumption, blockade of which abrogates pro-inflammatory mediator responses. Blockade of STAT3 activation inhibits this forced glycolytic flux resulting in metabolic reprogramming and resolution of inflammation. Interestingly, this highly activated monocytic phenotype is evident in IAR of developing disease, in addition to an enhanced monocyte gene signature observed in synovial tissue from IAR. CONCLUSION: RA CD14+ monocytes are metabolically re-programmed for sustained induction of pro-inflammatory responses, with STAT3 identified as a molecular regulator of metabolic dysfunction. This phenotype precedes clinical disease onset and may represent a potential pathway for therapeutic targeting early in disease.

STAT3 Mediates the Differential Effects of Oncostatin M and TNFα on RA Synovial Fibroblast and Endothelial Cell Function.

Objectives: Oncostatin M (OSM), a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family, has been implicated in the pathogenesis of autoimmune diseases. Here we investigate the mechanisms by which its synergistic interactions with TNFα regulate the cellular bioenergetics and invasive function of synovial cells from patients with Rheumatoid Arthritis. Methods: Primary RA synovial fibroblasts (RAFLS) and human umbilical vein endothelial cells (HUVEC) were cultured with OSM alone or in combination with TNFα. Pro-inflammatory cytokines, angiogenic growth factors and adhesion molecules were quantified by real-time PCR and ELISA. Invasion, angiogenesis and cellular adhesion were quantified by Transwell invasion chambers, Matrigel tube formation assays, and adhesion binding assays. Cellular bioenergetics was assessed using the Seahorse XFe96 Analyser. Key metabolic genes (GLUT-1, HK2, PFKFB3, HIF1α, LDHA, PKM2) and transcription factor STAT3 were measured using real-time PCR and western blot. Results: OSM differentially regulates pro-inflammatory mediators in RAFLS and HUVEC, with IL-6, MCP-1, ICAM-1, and VEGF all significantly induced, in contrast to the observed inhibition of IL-8 and GROα, with opposing effects observed for VCAM-1 depending on cell type. Functionally, OSM significantly induced angiogenic network formation, adhesion, and invasive mechanisms. This was accompanied by a change in the cellular bioenergetic profile of the cells, where OSM significantly increased the ECAR/OCR ratio in favor of glycolysis, paralleled by induction of the glucose transporter GLUT-1 and key glycolytic enzymes (HK2, PFKFB3, HIF1α). OSM synergizes with TNFα to differentially regulate pro-inflammatory mechanisms in RAFLS and HUVEC. Interestingly, OSM differentially synergizes with TNFα to regulate metabolic reprogramming, where induction of glycolytic activity with concomitant attenuation of mitochondrial respiration and ATP activity was demonstrated in RAFLS but not in HUVEC. Finally, we identified a mechanism, whereby the combination of OSM with TNFα induces transcriptional activity of STAT3 only in RAFLS, with no effect observed in HUVEC. Conclusion: STAT3 mediates the differential effects of OSM and TNFα on RAFLS and EC function. Targeting OSM or downstream signaling pathways may lead to new potential therapeutic or adjuvant strategies, particularly for those patients who have sub-optimal responses to TNFi.