Multi-omics profiling of collagen-induced arthritis mouse model reveals early metabolic dysregulation via SIRT1 axis.

Rheumatoid arthritis (RA) is characterized by joint infiltration of immune cells and synovial inflammation which leads to progressive disability. Current treatments improve the disease outcome, but the unmet medical need is still high. New discoveries over the last decade have revealed the major impact of cellular metabolism on immune cell functions. So far, a comprehensive understanding of metabolic changes during disease development, especially in the diseased microenvironment, is still limited. Therefore, we studied the longitudinal metabolic changes during the development of murine arthritis by integrating metabolomics and transcriptomics data. We identified an early change in macrophage pathways which was accompanied by oxidative stress, a drop in NAD+ level and induction of glucose transporters. We discovered inhibition of SIRT1, a NAD-dependent histone deacetylase and confirmed its dysregulation in human macrophages and synovial tissues of RA patients. Mining this database should enable the discovery of novel metabolic targets and therapy opportunities in RA.

TNF induces glycolytic shift in fibroblast like synoviocytes via GLUT1 and HIF1A.

TNF is a central cytokine in the pathogenesis of rheumatoid arthritis (RA). Elevated level of TNF causes local inflammation that affects immune cells and fibroblast-like synoviocytes (FLS). Nowadays, only 20-30% of patients experience remission after the standard of care therapy-antibodies against TNF. Interestingly, responders show reduced levels of GLUT1 and GAPDH, highlighting a potential link to cellular metabolism. The aim of the study was to investigate whether TNF directly affects the metabolic phenotype of FLS. Real-time respirometry displayed TNF-induced upregulation of glycolysis along with a modest increase of oxidative phosphorylation in FLS from healthy donors. In addition, TNF stimulation enhanced HIF1A and GLUT1 expression. The upregulation of HIF1A and GLUT1 reflects their enriched level in FLS from RA patients (RA-FLS). The inhibition of TAK1, HIF1a and hexokinase deciphered the importance of TNF/TAK1/HIF1A/glycolysis signaling axis. To prove that inhibition of glycolysis reduced the pathogenic phenotype, we showed that 2-deoxyglucose, a hexokinase inhibitor, partially decreased secretion of RA biomarkers. In summary, we identified a direct role of TNF on glycolytic reprogramming of FLS and confirmed the potency of immunometabolism for RA. Further studies are needed to evaluate the therapeutic impact especially regarding non-responder data.

Glucose transporter 1 in rheumatoid arthritis and autoimmunity.

Knowledge about metabolism of immune cells increased almost exponentially during the last two decades and thereby created the new area immunometabolism. Increased glucose uptake and glycolysis were identified as one of the major drivers in immune cells for rapid adaptation to changes in the microenvironment or external stimuli. These metabolic switches are crucial to generate macromolecules for immune cell proliferation and activation. Glucose transporter 1 (GLUT1), a ubiquitously expressed glucose transporter, is strongly upregulated after innate and adaptive immune cell activation. Deletion or inhibition of GLUT1 blocked T cell proliferation and effector function, antibody production from B cells and reduced inflammatory responses in macrophages. Increased glucose uptake and GLUT1 expression are not only observed in proinflammatory conditions, but also in murine models of autoimmunity as well as in human patients. Rheumatoid arthritis (RA), the most common autoimmune disease, is characterized by infiltration of immune cells, hyperproliferation of fibroblast-like synoviocytes, and destruction of cartilage and bone. These processes create a hypoxic microenvironment in the synovium. Moreover, synovial samples including fibroblast-like synoviocytes from RA patients showed increased lactate level and upregulate GLUT1. Similar upregulation of GLUT1 is observed in systemic lupus erythematosus and psoriasis patients as well as in murine autoimmune models. Inhibition of GLUT1 using either T cell specific knockouts or small molecule GLUT1/glycolysis inhibitors improved phenotypes of different murine autoimmune disease models like arthritis, lupus, and psoriasis. Thereby the therapeutic potential of immunometabolism and especially interference with glycolysis was proven. This article is categorized under: Biological Mechanisms > Metabolism Translational, Genomic, and Systems Medicine > Translational Medicine Physiology > Mammalian Physiology in Health and Disease.

Sphingosine kinase 2 is a negative regulator of inflammatory macrophage activation.

Sphingosine kinases (SPHK) generate the sphingolipid sphingosine-1-phosphate, which, among other functions, is a potent regulator of inflammation. While SPHK1 produces S1P to promote inflammatory signaling, the role of SPHK2 is unclear due to divergent findings in studies utilizing gene depletion versus inhibition of catalytic activity. We sought to clarify how SPHK2 affects inflammatory signaling in human macrophages, which are main regulators of inflammation. SPHK2 expression and activity were rapidly decreased within 6 h upon stimulating primary human macrophages with lipopolysaccharide (LPS), but was upregulated after 24 h. At 24 h following LPS stimulation, targeting SPHK2 with the inhibitor ABC294640, a specific siRNA or by using Sphk2-/- mouse peritoneal macrophages increased inflammatory cytokine production. Downregulation of SPHK2 in primary human macrophages within 6 h of LPS treatment was blocked by inhibiting autophagy. SPHK2 overexpression or inhibiting autophagy 6 h after human macrophage activation with LPS suppressed inflammatory cytokine release. Mechanistically, SPHK2 suppressed LPS-triggered NF-κB activation independent of its catalytic activity and prevented increased mitochondrial ROS formation downstream of LPS. In conclusion, SPHK2 is an anti-inflammatory protein in human macrophages that is inversely coupled to inflammatory cytokine production. This needs consideration when targeting SPHK2 with specific inhibitors.

A Novel Function for 15-Lipoxygenases in Cholesterol Homeostasis and CCL17 Production in Human Macrophages.

Arachidonate 15-lipoxygenase (ALOX15) and arachidonate 15-lipoxygenase, type B (ALOX15B) catalyze the dioxygenation of polyunsaturated fatty acids and are upregulated in human alternatively activated macrophages (AAMs) induced by Th2 cytokine interleukin-4 (IL-4) and/or interleukin-13. Known primarily for roles in bioactive lipid mediator synthesis, 15-lipoxygenases (15-LOXs) have been implicated in various macrophage functions including efferocytosis and ferroptosis. Using a combination of inhibitors and siRNAs to suppress 15-LOX isoforms, we studied the role of 15-LOXs in cellular cholesterol homeostasis and immune function in naïve and AAMs. Silencing or inhibiting the 15-LOX isoforms impaired sterol regulatory element binding protein (SREBP)-2 signaling by inhibiting SREBP-2 processing into mature transcription factor and reduced SREBP-2 binding to sterol regulatory elements and subsequent target gene expression. Silencing ALOX15B reduced cellular cholesterol and the cholesterol intermediates desmosterol, lanosterol, 24,25-dihydrolanosterol, and lathosterol as well as oxysterols in IL-4-stimulated macrophages. In addition, attenuating both 15-LOX isoforms did not generally affect IL-4 gene expression but rather uniquely impacted IL-4-induced CCL17 production in an SREBP-2-dependent manner resulting in reduced T cell migration to macrophage conditioned media. In conclusion, we identified a novel role for ALOX15B, and to a lesser extent ALOX15, in cholesterol homeostasis and CCL17 production in human macrophages.

Mitochondrial fragmentation in human macrophages attenuates palmitate-induced inflammatory responses.

Macrophages in adipose tissue contribute to inflammation and the development of insulin resistance in obesity. Exposure of macrophages to saturated fatty acids alters cell metabolism and activates pro-inflammatory signaling. How fatty acids influence macrophage mitochondrial dynamics is unclear. We investigated the mechanism of palmitate-induced mitochondrial fragmentation and its impact on inflammatory responses in primary human macrophages. Fatty acids, such as palmitate, caused mitochondrial fragmentation in human macrophages. Increased mitochondrial fragmentation was also observed in peritoneal macrophages from hyperlipidemic apolipoprotein E knockout mice. Fatty acid-induced mitochondrial fragmentation was independent of the fatty acid chain saturation and required dynamin-related protein 1 (DRP1). Mechanistically, mitochondrial fragmentation was regulated by incorporation of palmitate into mitochondrial phospholipids and their precursors. Palmitate-induced endoplasmic reticulum stress and loss of mitochondrial membrane potential did not contribute to mitochondrial fragmentation. Macrophages treated with palmitate maintained intact mitochondrial respiration and ATP levels. Pharmacological or genetic inhibition of DRP1 enhanced palmitate-induced mitochondrial ROS production, c-Jun phosphorylation, and inflammatory cytokine expression. Our results indicate that mitochondrial fragmentation is a protective mechanism attenuating inflammatory responses induced by palmitate in human macrophages.

Hypoxia Potentiates Palmitate-induced Pro-inflammatory Activation of Primary Human Macrophages.

Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1ß. Although palmitate-induced endoplasmic reticulum stress and nuclear factor κB pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1ß gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1ß and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation.

Analysis of multipotent mesenchymal stromal cells used for acute graft-versus-host disease prophylaxis.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) are used for prophylaxis of acute graft-versus-host disease (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT). Not all samples of MSC are efficient for aGvHD prevention. The suitability of MSCs for aGvHD prophylaxis was studied. METHODS: MSCs were derived from the bone marrow (BM) of HCT donor and cultivated for no more than three passages. The characteristics of donor BM samples including colony-forming unit fibroblast (CFU-F) concentration, growth parameters of MSCs, and the relative expression levels (REL) of different genes were analyzed. MSCs were injected intravenously precisely at the moment of blood cell reconstitution. RESULTS: MSCs infusion induced a significant threefold decrease in aGvHD development and improved overall survival compared with the standard prophylaxis group. In ineffective MSC samples (9.4%), a significant decrease in total cell production and the REL of CSF1, FGFR1, and PDGFRB was observed. In all studied BM samples, the cumulative MSC production and CFU-F concentrations decreased with age. The expression levels of FGFR2, PPARG, and VEGF differed by age. CONCLUSIONS: A universal single indicator for the prediction of MSC eligibility for aGvHD prophylaxis was not identified. A multiparameter mathematical model for selecting MSC samples effective for the prevention of aGvHD was proposed.

Interleukin-1 beta enhances human multipotent mesenchymal stromal cell proliferative potential and their ability to maintain hematopoietic precursor cells.

Multipotent mesenchymal stromal cells (MMSCs) have been demonstrated to produce mature stromal cells and maintain hematopoietic progenitor cells (HPC). It was previously demonstrated that interleukin-1 beta (IL-1 beta) stimulates the growth of the stromal microenvironment in vivo. The aim of this study was to investigate the effect of IL-1 beta treatment of human MMSCs on their proliferative potential, gene expression, immunomodulating properties, and their ability to support HPCs in vitro. Human bone marrow-derived MMSCs were cultivated in standard conditions or with IL-1 beta. The cumulative cell production was assessed for five passages. After withdrawal of IL-1 beta, MMSC clonal efficiency was investigated, and the maintenance of HPCs on top of MMSCs layers was estimated using cobblestone area forming cell (CAFC) and long-term culture initiating cell (LTC-IC) assays. The effect of untreated MMSCs or MMSCs pretreated with IL-1 beta on lymphocyte proliferation was studied by CFSE staining. The relative expression level of various genes by MMSCs was analyzed using RT-qPCR. The administration of IL-1 beta elevated MMSCs clonal efficiency and total cell production but did not affect lymphocyte proliferation. MMSCs pretreatment with IL-1 beta enhanced their ability to maintain HPCs, as detected by CAFC assay, and it altered the expression levels of genes participating in HPC regulation by stromal cells, e.g., adhesion molecules (ICAM1) and growth factors (SDF1). This study revealed the ability of IL-1 beta to stimulate MMSCs proliferation and enhance their potential to maintain HPCs. MMSCs are considered a stromal niche component in vitro. The combined in vitro and previous in vivo data suggest that IL-1 beta is a systemic regulator of the stromal microenvironment.