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.

Serum miRNA Signature in Rheumatoid Arthritis and "At-Risk Individuals".

Background: MicroRNAs (miRNAs) are small non-coding RNAs which have been implicated as potential biomarkers or therapeutic targets in autoimmune diseases. This study examines circulatory miRNAs in RA patients and further investigates if a serum miRNA signature precedes clinical manifestations of disease in arthralgia or "at-risk individuals". Methods: Serum was collected from HC subjects (N = 20), RA patients (N = 50), and arthralgia subjects (N = 10), in addition to a subgroup of the RA patients post-methotrexate (MTX) (N = 18). The FirePlex miRNA Immunology-V2 panel was selected for multiplex analysis of 68 miRNAs in each sample. DNA intelligent analysis (DIANA)-mirPath and Ingenuity Pathway Analysis (IPA) software were used to predict pathways targeted by the dysregulated miRNAs. Results: 8 miRNA (miR-126-3p, let-7d-5p, miR-431-3p, miR-221-3p, miR-24-3p, miR-130a-3p, miR-339-5p, let-7i-5p) were significantly elevated in RA serum compared to HC (all p < 0.01) and 1 miRNA (miR-17-5p) was significantly lower in RA (p < 0.01). High specificity and sensitivity were determined by receiver operating characteristic (ROC) curve analysis. Both miR-339-5p and let-7i-5p were significantly reduced post-MTX (both p < 0.01). MiR-126-3p, let-7d-5p, miR-431-3p, miR-221-3p, miR-24-3p, miR-130a-3p were also significantly elevated in subjects "at risk" of developing RA (all p < 0.05) compared to HC. IPA analysis of this miRNA signature identified downstream targets including key transcription factors NF-κB, STAT-1, STAT-3, cytokines IL-1ß, TNF-α, and matrix-metalloproteases all importantly associated with RA pathogenesis. Conclusion: This study identified six miRNAs that are altered in both RA and "at-risk individuals," which potentially regulate key downstream pathways involved in regulating inflammation. These may have potential as predictive signature for disease onset and early progression.

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.

Trypanosoma brucei Secreted Aromatic Ketoacids Activate the Nrf2/HO-1 Pathway and Suppress Pro-inflammatory Responses in Primary Murine Glia and Macrophages.

African trypanosomes, such as Trypanosoma brucei (T. brucei), are protozoan parasites of the mammalian vasculature and central nervous system that are best known for causing fatal human sleeping sickness. As exclusively extracellular parasites, trypanosomes are subject to constant challenge from host immune defenses but they have developed very effective strategies to evade and modulate these responses to maintain an infection while simultaneously prolonging host survival. Here we investigate host parasite interactions, especially within the CNS context, which are not well-understood. We demonstrate that T. brucei strongly upregulates the stress response protein, Heme Oxygenase 1 (HO-1), in primary murine glia and macrophages in vitro. Furthermore, using a novel AHADHinT. brucei cell line, we demonstrate that specific aromatic ketoacids secreted by bloodstream forms of T. brucei are potent drivers of HO-1 expression and are capable of inhibiting pro-IL1ß induction in both glia and macrophages. Additionally, we found that these ketoacids significantly reduced IL-6 and TNFα production by glia, but not macrophages. Finally, we present data to support Nrf2 activation as the mechanism of action by which these ketoacids upregulate HO-1 expression and mediate their anti-inflammatory activity. This study therefore reports a novel immune evasion mechanism, whereby T. brucei secretes amino-acid derived metabolites for the purpose of suppressing both the host CNS and peripheral immune response, potentially via induction of the Nrf2/HO-1 pathway.

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.

Dysregulated miR-125a promotes angiogenesis through enhanced glycolysis.

BACKGROUND: Although neoangiogenesis is a hallmark of chronic inflammatory diseases such as inflammatory arthritis and many cancers, therapeutic agents targeting the vasculature remain elusive. Here we identified miR-125a as an important regulator of angiogenesis. METHODS: MiRNA levels were quantified in Psoriatic Arthritis (PsA) synovial-tissue by RT-PCR and compared to macroscopic synovial vascularity. HMVEC were transfected with anti-miR-125a and angiogenic mechanisms quantified using tube formation assays, transwell invasion chambers, wound repair, RT-PCR and western blot. Real-time analysis of EC metabolism was assessed using the XF-24 Extracellular-Flux Analyzer. Synovial expression of metabolic markers was assessed by immunohistochemistry and immunofluorescent staining. MiR-125a CRISPR/Cas9-based knock-out zebrafish were generated and vascular development assessed. Finally, glycolytic blockade using 3PO, which inhibits Phosphofructokinase-fructose-2,6-bisphophatase 3 (PFKFB3), was assessed in miR-125a-/- ECs and zebrafish embryos. FINDINGS: MiR-125a is significantly decreased in PsA synovium and inversely associated with macroscopic vascularity. In-vivo, CRISPR/cas9 miR-125a-/- zebrafish displayed a hyper-branching phenotype. In-vitro, miR-125a inhibition promoted EC tube formation, branching, migration and invasion, effects paralleled by a shift in their metabolic profile towards glycolysis. This metabolic shift was also observed in the PsA synovial vasculature where increased expression of glucose transporter 1 (GLUT1), PFKFB3 and Pyruvate kinase muscle isozyme M2 (PKM2) were demonstrated. Finally, blockade of PFKFB3 significantly inhibited anti-miR-125a-induced angiogenic mechanisms in-vitro, paralleled by normalisation of vascular development of CRISPR/cas9 miR-125a-/- zebrafish embryos. INTEPRETATION: Our results provide evidence that miR-125a deficiency enhances angiogenic processes through metabolic reprogramming of endothelial cells. FUND: Irish Research Council, Arthritis Ireland, EU Seventh Framework Programme (612218/3D-NET).

S100A8 acts as an autocrine priming signal for heme-induced human Mϕ pro-inflammatory responses in hemolytic inflammation.

Intravascular hemolysis, in addition to reducing red cell counts, incurs extensive vascular inflammation and oxidative stress. One product of hemolysis, heme, is a potent danger associated molecular pattern (DAMP), activating leukocytes and inducing cytokine expression and processing, among other pro-inflammatory effects. We explored pathways by which heme-induced inflammation may be amplified under sterile conditions. Incubation of human Mϕs, differentiated from CD14+ cells, with heme induced time- and concentration-dependent gene and protein expression of S100A8, a myeloid cell-derived alarmin. Human Mϕ stimulation with recombinant S100A8, in turn, induced robust pro-IL-1ß expression that was dependent upon NF-κB activation, gene transcription, and partially dependent upon TLR4-mediated signaling. Moreover, heme itself stimulated significant Mϕ pro-IL-1ß gene and protein expression via an S100A8-mediated mechanism and greatly amplified S100A8-driven NLRP3 inflammasome-mediated IL-1ß secretion. In vivo, induction of acute intravascular hemolysis in mice induced a rapid elevation of plasma S100A8 that could be abolished by hemopexin, a heme scavenger. Finally, plasma S100A8 levels were found to be significantly elevated in patients with the inherited hemolytic anemia, sickle cell anemia, when compared with levels in healthy individuals. In conclusion, we demonstrate that hemolytic processes are associated with S100A8 generation and that some of the inflammatory effects of heme may be amplified by autocrine S100A8 production. Findings suggest a mechanism by which hemolytic inflammation could be propagated via leukocyte priming by endogenous proteins, even in sterile inflammatory environments such as those that occur in the hemolytic diseases. S100A8 may represent a therapeutic target for reducing inflammation in hemolytic disorders.

Orthopaedic implant materials drive M1 macrophage polarization in a spleen tyrosine kinase- and mitogen-activated protein kinase-dependent manner.

Total joint replacements (TJR) are costly procedures required to relieve pain and restore function in patients suffering from end-stage arthritis. Despite great progress in the development and durability of TJRs, the generation of prosthesis-associated wear particles over time leads to an inflammatory cascade which culminates in periprosthetic osteolysis. Studies suggest that wear particles drive the polarization/differentiation of immature macrophages towards a pro-inflammatory M1 phenotype rather than an anti-inflammatory M2 phenotype associated with normal bone and wound healing. This, in turn, contributes to the initiation of peri-implant inflammation. As a result, modulating M1 macrophage cytokine production has been recognised as a viable therapeutic option. The aim of this study was to examine the impact of hydroxyapatite (HA) and poly(methyl methacrylate) (PMMA) particles on human macrophage polarization by comparing their effect on M1/M2-associated gene expression using real-time PCR. Furthermore, using immunoblotting to assess kinase activation, we sought to identify the intracellular signalling molecules activated by PMMA/HA particles and to determine whether pharmacological blockade of these molecules impacts on macrophage phenotype and cytokine production as measured by ELISA. We report that wear particles preferentially polarize macrophages towards an M1 phenotype, an effect that is dependent on activation of the membrane proximal kinase, Syk and members of the mitogen-activated protein kinase (MAPK) family of signalling molecules. Pre-treatment of macrophages with Syk inhibitors (R788/piceatannol) or MAPK inhibitors (SB203580 and PD98059), not only prevents M1 polarization, but also attenuates production of key pro-inflammatory mediators that have been specifically implicated in periprosthetic osteolysis and osteoclast differentiation. STATEMENT OF SIGNIFICANCE: It is now well established that wear-debris particles from implanted materials drive deleterious inflammatory responses which can eventually lead to implant loosening. In this study, we provide further insight into the specific cellular pathways activated by wear particles in primary human immune cells. We demonstrate that PMMA bone cement and hydroxyapatite, a commonly used biomaterial, drive the polarization of macrophages towards an inflammatory phenotype and identify the specific signalling molecules that are activated in this process. Pre-treatment of macrophages with pharmacological inhibitors of these molecules in turn prevents macrophage polarization and dampens inflammatory cytokine production. Hence these signalling molecules represent potential therapeutic targets to treat or possibly prevent particulate induced osteolysis.

Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells.

BACKGROUND: Osteoarthritis (OA) is a chronic debilitating joint disorder of particularly high prevalence in the elderly population. Intra-articular basic calcium phosphate (BCP) crystals are present in the majority of OA joints and are associated with severe degeneration. They are known to activate macrophages, synovial fibroblasts, and articular chondrocytes, resulting in increased cell proliferation and the production of pro-inflammatory cytokines and matrix metalloproteases (MMPs). This suggests a pathogenic role in OA by causing extracellular matrix degradation and subchondral bone remodelling. There are currently no disease-modifying drugs available for crystal-associated OA; hence, the aim of this study was to explore the inflammatory pathways activated by BCP crystals in order to identify potential therapeutic targets to limit crystal-induced inflammation. METHODS: Primary human macrophages and dendritic cells were stimulated with BCP crystals, and activation of spleen tyrosine kinase (Syk), phosphoinositide-3 kinase (PI3K), and mitogen-activated protein kinases (MAPKs) was detected by immunoblotting. Lipopolysaccharide (LPS)-primed macrophages were pre-treated with inhibitors of Syk, PI3K, and MAPKs prior to BCP stimulation, and cytokine production was quantified by enzyme-linked immunosorbent assay (ELISA). Aa an alternative, cells were treated with synovial fluid derived from osteoarthritic knees in the presence or absence of BCP crystals, and gene induction was assessed by real-time polymerase chain reaction (PCR). RESULTS: We demonstrate that exposure of primary human macrophages and dendritic cells to BCP crystals leads to activation of the membrane-proximal tyrosine kinases Syk and PI3K. Furthermore, we show that production of the pro-inflammatory cytokines interleukin (IL)-1α and IL-1ß and phosphorylation of downstream MEK and ERK MAPKs is suppressed following treatment with inhibitors of Syk or PI3K. Finally, we demonstrate that treatment of macrophages with BCP crystals induces the production of the damage-associated molecule S100A8 and MMP1 in a Syk-dependent manner and that synovial fluid from OA patients together with BCP crystals exacerbates these effects. CONCLUSIONS: We identify Syk and PI3K as key signalling molecules activated by BCP crystals prior to inflammatory cytokine and DAMP expression and therefore propose that Syk and PI3K represent potential targets for the treatment of BCP-related pathologies.

Cholesterol crystals activate Syk and PI3 kinase in human macrophages and dendritic cells.

BACKGROUND AND AIMS: Cholesterol crystals are a key component of atherosclerotic lesions where they promote pro-inflammatory cytokine production and plaque destabilization. Antagonists of inflammatory mediators and agents that dissolve or prevent the formation of cholesterol crystals are being explored as potential therapeutics for atherothrombosis. We sought to identify signalling molecules activated following exposure of immune cells to cholesterol crystals with the view to identifying novel therapeutic targets. METHODS: Human macrophages and dendritic cells (DC) were exposed to cholesterol crystals and activation of signalling molecules was assessed by immunoblotting. The role of Syk and PI3K in crystal-induced interleukin (IL)-1 production was determined by ELISA using specific kinase inhibitors. Real-time PCR was employed to examine the role of Syk/PI3K in cholesterol crystal-induced expression of S100 proteins and MMPs. RESULTS: Exposure of human macrophages and DC to cholesterol crystals induced robust activation of Syk and PI3K within 2-5 min. Pharmacological inhibition of Syk/PI3K reduced crystal-induced IL-1α/ß production by approximately 80%. Activation of the downstream MAP kinases, MEK and ERK, was suppressed following inhibition of Syk and PI3K. Finally, inhibition of both Syk and PI3K significantly reduced cholesterol crystal-induced S100A8 and MMP1 gene expression by >70% while inhibition of PI3K also reduced S100A12 expression. CONCLUSION: Cholesterol crystals activate specific cell signalling pathways which drive the production of inflammatory cytokines and degradative enzymes known to contribute to disease initiation and progression. These molecular events are dependent on activation of Syk and PI3K, hence, they represent potential therapeutic targets for the treatment of cholesterol crystal-related pathologies.

Investigating inflammasome activation under conditions of cellular stress and injury.

Inflammasomes are large multiprotein complexes that assemble in response to cellular stress and infection. NOD-like receptor-related proteins (NLRPs) are essential components of these complexes and are activated by exogenous and endogenous danger signals such as crystalline substances, extracellular ATP, and pore-forming toxins. In general, inflammasome activation is accompanied by perturbations in cellular homeostasis. For example, most inflammasome activators will trigger cation efflux, reactive oxygen species (ROS) generation and caspase-1-dependent cell death, commonly referred to as pyroptosis. In this chapter, we describe protocols to examine inflammasome activation and accompanying events in vitro.

Osteoarthritis-associated basic calcium phosphate crystals induce pro-inflammatory cytokines and damage-associated molecules via activation of Syk and PI3 kinase.

The pro-inflammatory cytokines, TNFα, IL-1 and IL-18, amplify cartilage destruction associated with osteoarthritis (OA). Current data suggest that basic calcium phosphate (BCP) crystals are potent drivers of inflammatory mediator and matrix metalloprotease expression in the OA joint. It has previously been demonstrated that synovial macrophages play a role in initiating and driving BCP-induced inflammation. However, the molecular mechanisms by which BCP crystals exert their effects remain unclear. Here we demonstrate that exposure of macrophages to BCP crystals leads to activation of Syk and PI3 kinase. Furthermore, we show that production of pro-inflammatory cytokines and phosphorylation of the downstream kinase, ERK, are suppressed following treatment with Syk and PI3 kinase inhibitors. Finally, we demonstrate that treatment of macrophages with BCP crystals induces the production of the damage-associated molecule, S100A8, in a Syk dependent manner. We therefore identify Syk and PI3 kinase as potential novel targets for the treatment of BCP-related pathologies.