ROCK2 interacts with p22phox to phosphorylate p47phox and to control NADPH oxidase activation in human monocytes.

Monocytes play a key role in innate immunity by eliminating pathogens, releasing high levels of cytokines, and differentiating into several cell types, including macrophages and dendritic cells. Similar to other phagocytes, monocytes produce superoxide anions through the NADPH oxidase complex, which is composed of two membrane proteins (p22phox and gp91phox/NOX2) and four cytosolic proteins (p47phox, p67phox, p40phox and Rac1). The pathways involved in NADPH oxidase activation in monocytes are less known than those in neutrophils. Here, we show that p22phox is associated with Rho-associated coiled-coil kinase 2 (ROCK2) in human monocytes but not neutrophils. This interaction occurs between the cytosolic region of p22phox (amino acids 132 to 195) and the coiled-coil region of ROCK2 (amino acids 400 to 967). Interestingly, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox in vitro but phosphorylates p47phox on Ser304, Ser315, Ser320 and Ser328. Furthermore, KD025, a selective inhibitor of ROCK2, inhibited reactive oxygen species (ROS) production and p47phox phosphorylation in monocytes. Specific inhibition of ROCK2 expression in THP1-monocytic cell line by siRNA inhibited ROS production. These data show that ROCK2 interacts with p22phox and phosphorylates p47phox, and suggest that p22phox could be a shuttle for ROCK2 to allow p47phox phosphorylation and NADPH oxidase activation in human monocytes.

Impaired p47phox phosphorylation in neutrophils from patients with p67phox-deficient chronic granulomatous disease.

Superoxide production by the phagocyte reduced NAD phosphate (NADPH) oxidase is essential for innate immunity as shown in chronic granulomatous disease (CGD), an immunodeficiency disease resulting from mutations in 1 of its genes. The NADPH oxidase is composed of 2 membrane proteins (gp91phox/NOX2 and p22phox) and 4 cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). The phosphorylation of p47phox is required for NADPH oxidase activation in cells. As p47phox and p67phox can form a tight complex in cells, we hypothesized that p67phox could regulate p47phox phosphorylation. To investigate this hypothesis, we used phospho-specific antibodies against 5 major p47phox-phosphorylated sites (Ser304, Ser315, Ser320, Ser328, and Ser345) and neutrophils from healthy donors and from p67phox-/- CGD patients. Results showed that formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate induced a time- and a concentration-dependent phosphorylation of p47phox on Ser304, Ser315, Ser320, and Ser328 in healthy human neutrophils. Interestingly, in neutrophils and Epstein-Barr virus-transformed B lymphocytes from p67phox-/- CGD patients, phosphorylation of p47phox on serine residues was dramatically reduced. In COSphox cells, the presence of p67phox led to increased phosphorylation of p47phox. In vitro studies showed that recombinant p47phox was phosphorylated on Ser304, Ser315, Ser320, and Ser328 by different PKC isoforms and the addition of recombinant p67phox alone or in combination with p40phox potentiated this process. Thus, p67phox and p40phox are required for optimal p47phox phosphorylation on Ser304, Ser315, Ser320, and Ser328 in intact cells. Therefore, p67phox and p40phox are novel regulators of p47phox-phosphorylation.

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.

Distinct stromal and immune cell interactions shape the pathogenesis of rheumatoid and psoriatic arthritis.

OBJECTIVES: Immune and stromal cell communication is central in the pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA), however, the nature of these interactions in the synovial pathology of the two pathotypes can differ. Identifying immune-stromal cell crosstalk at the site of inflammation in RA and PsA is challenging. This study creates the first global transcriptomic analysis of the RA and PsA inflamed joint and investigates immune-stromal cell interactions in the pathogenesis of synovial inflammation. METHODS: Single cell transcriptomic profiling of 178 000 synovial tissue cells from five patients with PsA and four patients with RA, importantly, without prior sorting of immune and stromal cells. This approach enabled the transcriptomic analysis of the intact synovial tissue and identification of immune and stromal cell interactions. State of the art data integration and annotation techniques identified and characterised 18 stromal and 14 immune cell clusters. RESULTS: Global transcriptomic analysis of synovial cell subsets identifies actively proliferating synovial T cells and indicates that due to differential λ and κ immunoglobulin light chain usage, synovial plasma cells are potentially not derived from the local memory B cell pool. Importantly, we report distinct fibroblast and endothelial cell transcriptomes indicating abundant subpopulations in RA and PsA characterised by differential transcription factor usage. Using receptor-ligand interactions and downstream target characterisation, we identify RA-specific synovial T cell-derived transforming growth factor (TGF)-ß and macrophage interleukin (IL)-1ß synergy in driving the transcriptional profile of FAPα+THY1+ invasive synovial fibroblasts, expanded in RA compared with PsA. In vitro characterisation of patient with RA synovial fibroblasts showed metabolic switch to glycolysis, increased adhesion intercellular adhesion molecules 1 expression and IL-6 secretion in response to combined TGF-ß and IL-1ß treatment. Disrupting specific immune and stromal cell interactions offers novel opportunities for targeted therapeutic intervention in RA and PsA.

The Kinesin Light Chain-Related Protein PAT1 Promotes Superoxide Anion Production in Human Phagocytes.

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function.

Priming of the neutrophil respiratory burst: role in host defense and inflammation.

Neutrophils are the major circulating white blood cells in humans. They play an essential role in host defense against pathogens. In healthy individuals, circulating neutrophils are in a dormant state with very low efficiency of capture and arrest on the quiescent endothelium. Upon infection and subsequent release of pro-inflammatory mediators, the vascular endothelium signals to circulating neutrophils to roll, adhere, and cross the endothelial barrier. Neutrophils migrate toward the infection site along a gradient of chemo-attractants, then recognize and engulf the pathogen. To kill this pathogen entrapped inside the vacuole, neutrophils produce and release high quantities of antibacterial peptides, proteases, and reactive oxygen species (ROS). The robust ROS production is also called 'the respiratory burst', and the NADPH oxidase or NOX2 is the enzyme responsible for the production of superoxide anion, leading to other ROS. In vitro, several soluble and particulate agonists induce neutrophil ROS production. This process can be enhanced by prior neutrophil treatment with 'priming' agents, which alone do not induce a respiratory burst. In this review, we will describe the priming process and discuss the beneficial role of controlled neutrophil priming in host defense and the detrimental effect of excessive neutrophil priming in inflammatory diseases.

Altered expression of microRNA-23a in psoriatic arthritis modulates synovial fibroblast pro-inflammatory mechanisms via phosphodiesterase 4B.

OBJECTIVES: To investigate the functional role of miR-23a in synovial fibroblasts (SFC) activation in psoriatic arthritis (PsA). METHODS: Differential expression of the miR-23a-27a-24-2 cluster was identified by real-time quantitative PCR in PsA synovial tissue and peripheral blood mononuclear cells (PBMC) compared to osteoarthritis (OA) and correlated with disease activity. For regulation experiments, PsA synovial fibroblasts (SFC) were cultured with Toll-like receptor (TLR) ligands and pro-inflammatory cytokines. PsA SFC were transfected with a miR-23a inhibitor to assess the functional effect on migration, invasion and expression of pro-inflammatory meditators. The direct interaction between miR-23a and predicted target mRNA, phosphodiesterase 4B (PDE4B), was examined by luciferase reporter gene assay, with the expression and regulation confirmed by RT-PCR and western blot. A PDE4 inhibitor was used to analyse the function of PDE4B signalling in both miR-23a and Poly(I:C)-induced PsA SFC activation. RESULTS: Synovial tissue expression of miR-23a was lower in PsA compared to OA and correlated inversely with disease activity and synovitis. TLR activation via Poly(I:C) and LPS, but not Pam3CSK4, significantly decreased miR-23a expression, with no significant effect observed in reponse to stimulation with pro-inflammatory cytokines. Decreased miR-23a expression enhanced PsA SFC migration, invasion and secretion of IL-6, IL-8, MCP-1, RANTES and VEGF. We identified PDE4B as a direct target of miR-23a and demonstrated enhanced mRNA and protein expression of PDE4B in anti-miR-23a transfected PsA SFC. Poly(I:C) and/or miR-23a-induced migration and enhanced cytokine expression was suppressed by the blockade of PDE4 signalling. CONCLUSIONS: In PsA, dysregulated miR-23a expression contributes to synovial inflammation through enhanced SFC activation, via PDE4B signalling, and identifies a novel anti-inflammatory mechanism of PDE4 blockade.

NOX5 and p22phox are 2 novel regulators of human monocytic differentiation into dendritic cells.

Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells and are key cells of the immune system, acquiring different phenotypes in accordance with their localization during the immune response. A subset of inflammatory DCs is derived from circulating monocytes (Mo) and has a key role in inflammation and infection. The pathways controlling Mo-DC differentiation are not fully understood. Our objective was to investigate the possible role of nicotinamide adenine dinucleotide phosphate reduced form oxidases (NOXs) in Mo-DC differentiation. In this study, we revealed that Mo-DC differentiation was inhibited by NOX inhibitors and reactive oxygen species scavengers. We show that the Mo-DC differentiation was dependent on p22phox, and not on gp91phox/NOX2, as shown by the reduced Mo-DC differentiation observed in chronic granulomatous disease patients lacking p22phox. Moreover, we revealed that NOX5 expression was strongly increased during Mo-DC differentiation, but not during Mo-macrophage differentiation. NOX5 was expressed in circulating myeloid DC, and at a lower level in plasmacytoid DC. Interestingly, NOX5 was localized at the outer membrane of the mitochondria and interacted with p22phox in Mo-DC. Selective inhibitors and small interfering RNAs for NOX5 indicated that NOX5 controlled Mo-DC differentiation by regulating the JAK/STAT/MAPK and NFκB pathways. These data demonstrate that the NOX5-p22phox complex drives Mo-DC differentiation, and thus could be critical for immunity and inflammation.

Syzygium aromaticum aqueous extract inhibits human neutrophils myeloperoxidase and protects mice from LPS-induced lung inflammation.

CONTEXT: Syzygium aromaticum (L.) Merr. & Perry (Myrtaceae), commonly known as clove, originally found in the Muluku Islands in East Indonesia, is widely used as a spice and has numerous medicinal properties. OBJECTIVE: This study investigated the antioxidant potential of S. aromaticum aqueous extract (SAAE) in vitro and its protective effects on lipopolysaccharide (LPS)-induced lung inflammation in mice. MATERIAL AND METHODS: Neutrophils were isolated from healthy donors and reactive oxygen species (ROS) generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by cytochrome c reduction assay. H2O2 was detected by DCFH fluorescence assay. Myeloperoxidase (MPO) activity was mesured by tetramethyl benzidine oxidation method. To study the anti-inflammatory activity of SAAE, lung inflammation was induced in mice (BALB/c) by intra-tracheal instillation of lypopolysaccharide (5 µg/mouse), and SAAE (200 mg/kg body weight) was injected intraperitoneally prior to LPS administration. Bronchoalveolar lavage and lung tissue were collected to assess inflammatory cells count and total protein content. Metalloproteinases activity was detected by zymography technique. RESULTS: SAAE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl-methionyl-leucyl-phenylalanine- or phorbol myristate acetate-stimulated neutrophils, with an inhibitory effect starting at a concentration as low as 0.5 µg/mL. Moreover, SAAE reduced significantly MPO activity and it exhibits a dose-dependent action (IC50 = 0.5 µg/mL). In vivo results showed that SAAE decreased markedly neutrophil count (From 61% to 15%) and proteins leakage into bronchoalveolar lavage fluid. Gelatin zymography assay showed that S. aromaticum inhibited MMP-2 (15%) and MMP-9 (18%) activity in lung homogenates. DISCUSSION AND CONCLUSION: Our results suggest that the anti-inflammatory activity of SAAE, in vivo, is due to the inhibition of ROS production and metalloproteinases activity via its action on MPO. According to these findings, SAAE could be a potential source of new compounds with anti-inflammatory activity.

Anvillea garcinii extract inhibits the oxidative burst of primary human neutrophils.

BACKGROUND: Anvillea garcinii Coss. & Durieu (Anv) plant is used as a traditional North African medicine against several diseases associated with inflammation. At inflammatory sites, reactive oxygen species (ROS) produced in excess by activated phagocyte NADPH oxidase (NOX2) can accentuate inflammatory responses. Thus, we investigated if Anv-water soluble polysaccharides could modulate primary human neutrophil oxidative burst in vitro. METHODS: Human neutrophils were isolated from fresh whole blood and O2.- generation was measured by cytochrome c reduction assays. Western blots were used to analyse the translocation of PKC, p47phox (a key component of NOX2 activity) to neutrophil plasma membrane. Also, myeloperoxidase (MPO) release in the extracellular medium was studied by western blots. Flow cytometric analysis was used to detect CD11b membrane expression. RESULTS: Water soluble polysaccharides from Anv dose-dependently inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLF)- and phorbol myristate acetate (PMA)-induced O2.- generation by human neutrophils. Moreover, Anv-polysaccharides strongly inhibited PMA-induced PKCß and p47phox translocation to membranes and p47phox phosphorylation on Ser328, a main PKC target. In contrast, polysaccharides extract from Zygophyllum gaetulum plant, which is also used as a traditional North African medicine against inflammatory diseases, was ineffective on this PKCß-p47phox pathway. Further, Anv inhibited important neutrophil degranulation markers corresponding to myeloperoxidase (MPO) release and CD11b membrane expression. CONCLUSION: The process of down-regulating NADPH oxidase by polysaccharides extracts from Anv provides new insights into the mechanism of Anv's anti-inflammatory actions.

IL-22 suppresses IFN-γ-mediated lung inflammation in asthmatic patients.

BACKGROUND: IL-22 controls tissue homeostasis by both proinflammatory and anti-inflammatory effects. However, the anti-inflammatory mechanisms of IL-22 remain poorly investigated. OBJECTIVE: We sought to investigate the anti-inflammatory role for IL-22 in human asthma. METHODS: T-cell lines derived from lung biopsy specimens of asthmatic patients were characterized by means of flow cytometry. Human bronchial epithelial cells from healthy and asthmatic subjects were stimulated with IL-22, IFN-γ, or the combination of both cytokines. Effects of cytokine stimulation were investigated by using whole-genome analysis, ELISA, and flow cytometry. The functional consequence of cytokine stimulation was evaluated in an in vitro wound repair model and T cell-mediated cytotoxicity experiments. In vivo cytokine expression was measured by using immunohistochemistry and Luminex assays in bronchoalveolar lavage fluid of healthy and asthmatic patients. RESULTS: The current study identifies a tissue-restricted antagonistic interplay of IL-22 and the proinflammatory cytokine IFN-γ. On the one hand, IFN-γ antagonized IL-22-mediated induction of the antimicrobial peptide S100A7 and epithelial cell migration in bronchial epithelial cells. On the other hand, IL-22 decreased epithelial susceptibility to T cell-mediated cytotoxicity by inhibiting the IFN-γ-induced expression of MHC-I, MHC-II, and CD54/intercellular adhesion molecule 1 molecules. Likewise, IL-22 inhibited IFN-γ-induced secretion of the proinflammatory chemokines CCL5/RANTES and CXCL10/interferon-inducible protein 10 in vitro. Consistently, the IL-22 expression in bronchoalveolar lavage fluid of asthmatic patients inversely correlated with the expression of CCL5/RANTES and CXCL10/interferon-inducible protein 10 in vivo. CONCLUSIONS: IL-22 might control the extent of IFN-γ-mediated lung inflammation and therefore play a tissue-restricted regulatory role.

Histamine contributes to increased RANKL to osteoprotegerin ratio through altered nuclear receptor 4A activity in human chondrocytes.

OBJECTIVE: To elucidate histamine receptor-mediated signaling pathways, transcriptional events, and target gene expression in human cartilage. METHODS: Histamine modulation of cartilage destruction was assessed by Safranin O staining and proteoglycan release. H(1) , H(2) , H(3) , and H(4) histamine receptor-dependent regulation of transcription factors (nuclear receptor 4A1 [NR4A1], NR4A2, and NR4A3), RANKL, and osteoprotegerin (OPG) messenger RNA (mRNA) levels were measured in primary and SW-1353 chondrocyte cells using quantitative polymerase chain reaction and selective histamine receptor antagonists. Soluble RANKL and OPG protein levels were determined using enzyme-linked immunosorbent assays. NR4A protein levels and transactivity were evaluated by Western blot analysis, immunocytochemistry, and luciferase reporter assays. Stable depletion of NR4A1-3 was achieved by lentiviral transduction of NR4A short hairpin RNA. RESULTS: Primary human chondrocyte cells expressed differential steady-state levels of H(1) -H(4) histamine receptor mRNA. In combination with tumor necrosis factor α, histamine significantly promoted cartilage proteoglycan depletion and release. Histamine modulated the expression of NR4A1-3 orphan receptors in primary and immortalized human chondrocyte cells in a time- and concentration-dependent manner. Histamine selectively signaled through H(1) and H(2) histamine receptors in chondrocytes to modulate RANKL and NR4A2 expression. The temporal effects of histamine on NR4A2 gene transcription were reduced in cells pretreated with inhibitors directed against protein kinase A, MAPK, and NF-κB signaling pathways. Histamine modulated the expression of RANKL with modest effects on OPG levels, leading to increased RANKL:OPG mRNA and protein ratios. Stable knockdown of NR4A1-3 expression resulted in reduced endogenous OPG levels and the loss of histamine-dependent regulation of RANKL expression. CONCLUSION: Our findings indicate that histamine, via H(1) and H(2) histamine receptors, contributes to joint disease by enhancing the ratio of RANKL to OPG expression through altered NR4A activity in human chondrocyte cells.

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.

Inside the Joint of Inflammatory Arthritis Patients: Handling and Processing of Synovial Tissue Biopsies for High Throughput Analysis.

Inflammatory arthritis is a chronic systemic autoimmune disease of unknown etiology, which affects the joints. If untreated, these diseases can have a detrimental effect on the patient's quality of life, leading to disabilities, and therefore, exhibit a significant socioeconomic impact and burden. While studies of immune cell populations in arthritis patient's peripheral blood have been informative regarding potential immune cell dysfunction and possible patient stratification, there are considerable limitations in identifying the early events that lead to synovial inflammation. The joint, as the site of inflammation and the local microenvironment, exhibit unique characteristics that contribute to disease pathogenesis. Understanding the contribution of immune and stromal cell interactions within the inflamed joint has been met with several technical challenges. Additionally, the limited availability of synovial tissue biopsies is a key incentive for the utilization of high-throughput techniques in order to maximize information gain. This review aims to provide an overview of key methods and novel techniques that are used in the handling, processing and analysis of synovial tissue biopsies and the potential synergy between these techniques. Herein, we describe the utilization of high dimensionality flow cytometric analysis, single cell RNA sequencing, ex vivo functional assays and non-intrusive metabolic characterization of synovial cells on a single cell level based on fluorescent lifetime imaging microscopy. Additionally, we recommend important points of consideration regarding the effect of different storage and handling techniques on downstream analysis of synovial tissue samples. The introduction of new powerful techniques in the study of synovial tissue inflammation, brings new challenges but importantly, significant opportunities. Implementation of novel approaches will accelerate our path toward understanding of the mechanisms involved in the pathogenesis of inflammatory arthritis and lead to the identification of new avenues of therapeutic intervention.

CD209/CD14+ Dendritic Cells Characterization in Rheumatoid and Psoriatic Arthritis Patients: Activation, Synovial Infiltration, and Therapeutic Targeting.

Dendritic cells (DC) have a key role in the initiation and progression of inflammatory arthritis (IA). In this study, we identified a DC population that derive from monocytes, characterized as CD209/CD14+ DC, expressing classical DC markers (HLADR, CD11c) and the Mo-DC marker (CD209), while also retaining the monocytic marker CD14. This CD209/CD14+ DC population is present in the circulation of Healthy Control (HC), with increased frequency in Rheumatoid Arthritis (RA) and Psoriatic arthritic (PsA) patients. We demonstrate, for the first time, that circulatory IA CD209/CD14+ DC express more cytokines (IL1ß/IL6/IL12/TNFα) and display a unique chemokine receptor expression and co-expression profiles compared to HC. We demonstrated that CD209/CD14+ DC are enriched in the inflamed joint where they display a unique inflammatory and maturation phenotype, with increased CD40 and CD80 and co-expression of specific chemokine receptors, displaying unique patterns between PsA and RA. We developed a new protocol of magnetic isolation and expansion for CD209+ DC from blood and identified transcriptional differences involved in endocytosis/antigen presentation between RA and PsA CD209+ DC. In addition, we observed that culture of healthy CD209+ DC with IA synovial fluid (SF), but not Osteoarthritis (OA) SF, was sufficient to induce the development of CD209/CD14+ DC, leading to a poly-mature DC phenotype. In addition, differential effects were observed in terms of chemokine receptor and chemokine expression, with healthy CD209+ DC displaying increased expression/co-expression of CCR6, CCR7, CXCR3, CXCR4 and CXCR5 when cultured with RA SF, while an increase in the chemokines CCR3, CXCL10 and CXCL11 was observed when cultured with PsA SF. This effect may be mediated in part by the observed differential increase in chemokines expressed in RA vs PsA SF. Finally, we observed that the JAK/STAT pathway, but not the NF-κB pathway (driven by TNFα), regulated CD209/CD14+ DC function in terms of activation, inflammatory state, and migratory capacity. In conclusion, we identified a novel CD209/CD14+ DC population, which is active in the circulation of RA and PsA, an effect potentiated once they enter the joint. Furthermore, we demonstrated that JAK/STAT inhibition can be used as a therapeutic strategy to decrease the inflammatory state of the pathogenic CD209/CD14+ DC.

Functionally Mature CD1c+ Dendritic Cells Preferentially Accumulate in the Inflammatory Arthritis Synovium.

Objective: To examine the role of synovial CD1c+DCs in patients with Inflammatory Arthritis (IA) with a specific focus on the transcriptional and maturation signatures that govern their function. Methods: RNA sequencing was performed on healthy control (HC) peripheral blood (PB), IA PB, and IA synovial fluid (SF) CD1c+DCs. Multiparametric flow-cytometry and SPICE analysis were used to examine site [SF and Synovial Tissue (ST) CD1c+DCs] and disease specific characteristics of CD1c+DCs, while functional assays such as antigen processing, activation, and MMP production were also performed. Results: Increased frequency of CD1c+DCs (p<0.01) with a concomitant increase in CD80, CCR7 (p<0.01), and CXCR3 (p<0.05) expression was identified in IA PB compared to HC PB. Enrichment of CD1c+DCs was identified in IA synovial tissue (ST) (p<0.01) and IA SF (p<0.0001) compared to IA PB, while RNAseq revealed distinct transcriptional variation between PB and SF CD1c+DCs. Flow cytometry revealed increased expression of CD83, CD80, PD-L1, and BTLA (all p<0.05) in IA SF CD1c+DCs compared to PB, while SPICE identified synovial cells with unique co-expression patterns, expressing multiple DC maturation markers simultaneously. Functionally, synovial CD1c+DCs are hyper-responsive to TLR7/8 ligation (p<0.05), have decreased antigen processing capacity (p=0.07), and display dysregulated production of MMPs. Finally, examination of both synovial CD1c+DCs and synovial CD141+DCs revealed distinct maturation and transcriptomic profiles. Conclusion: Synovial CD1c+DCs accumulate in the inflamed IA synovium in a variety of distinct poly-maturational states, distinguishing them transcriptionally and functionally from CD1c+DCs in the periphery and synovial CD141+DCs.

Targeting JAK-STAT Signalling Alters PsA Synovial Fibroblast Pro-Inflammatory and Metabolic Function.

Objectives: Psoriatic arthritis (PsA) is a chronic inflammatory disease associated with psoriasis. Janus Kinase inhibitors (JAKi) have emerged as an encouraging class of drugs for the treatment of PsA. Here, we compare the effect of four JAKi on primary PsA synovial fibroblasts (PsAFLS) activation, metabolic function, and invasive and migratory capacity. Methods: Primary PsAFLS were isolated and cultured with JAKi (Peficitinib, Filgotinib, Baricitinib and Upadacitinib) in the presence of Oncostatin M (OSM). pSTAT3 expression in response to OSM was quantified by Western Blot analysis. Pro-inflammatory cytokines/chemokines were quantified by ELISA and cell migration by wound-repair scratch assays. Invasive capacity was examined using Matrigel™ invasion chambers and MMP multiplex MSD assays. PsAFLS bioenergetics was assessed using the Seahorse XFe Extracellular Flux Analyzer, which simultaneously quantifies two energetic pathways- glycolysis (ECAR) and oxidative phosphorylation (OCR). In parallel, inflammatory, invasive, and migratory genes were quantified by RT-PCR. Results: OSM induces pSTAT3 expression in PsAFLS. OSM-induced secretion of MCP-1 and IL-6 was inhibited by all JAKi with Peficitinib, Baricitinib and Upadacitinib showing the greatest effect. In contrast, JAKi had no significant impact on IL-8 expression in response to OSM. PsAFLS cell invasion, migratory capacity and MMP1, 3, and 9 were suppressed following JAKi treatment, with Peficitinib showing the greatest effect. These functional effects were accompanied by a change in the cellular bioenergetic profile of PsAFLS, where JAKi significantly decreased glycolysis and the ECAR/OCR, resulting in a shift to a more quiescent phenotype, with Peficitinib demonstrating the most pronounced effect. Conclusion: This study demonstrates that JAK/STAT signalling mediates the complex interplay between inflammation and cellular metabolism in PsA pathogenesis. This inhibition shows effective suppression of inflammatory mechanisms that drive pathogenic functions of PsAFLS, further supporting the role of JAKi as a therapeutic target for the treatment of PsA.

Knowledge of disease, diagnosis, adherence and impact of research in an Irish cohort of patients with inflammatory arthritis.

Background: Patient engagement with clinicians results in shared decision making and increased adherence to medication. However, in order for strong patient: clinician partnerships to be achieved, communication barriers need to be identified. Therefore, the aim of this study was to examine the level of understanding of inflammatory arthritis patients and the need for strong patient-partnership in research. Methods: An online anonymous survey was distributed to patients living with inflammatory arthritis which addressed questions about diagnosis, routine tests, medications and how they work, medication adherence, disease flare, heredity, pregnancy, and patient involvement in research. Results: There were 1,873 respondents, 1416 of which had inflammatory arthritis (IA)- rheumatoid arthritis (RA) (65.8%) and psoriatic arthritis (PsA) (34.2%). They were predominantly female (RA 86%, PsA 85 %), aged 55±13 and 50±12 years. Less than 35% of patients had an understanding of diagnostic tests, what was measured and the implication for disease, with 75.5% also concerned about heredity. There was a high level of understanding of how specific medications treat inflammatory arthritis (72.9%). Adherence was also very high (>87%), with the main reasons for stopping medication without the advice of their clinician,  'feeling better' and 'side effects' however  a significant proportion of patients (69.9%) reported a disease-flare following cessation of medication. Patients (31%) were also concerned that inflammatory arthritis reduced their chances of getting pregnant, with only 8% believing arthritis medications were safe to take during pregnancy. Finally, only 9% of patients had ever been asked to participate in a research study. Conclusions: This study demonstrates a need for the development of stronger patient-partnerships with clinicians and researchers in relation to patient education and engagement with research, to create a platform where patients can have meaningful input and involvement in future research studies.

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.