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.

Association of synovial tissue polyfunctional T-cells with DAPSA in psoriatic arthritis.

OBJECTIVE: This study examines polyfunctional T-cells in psoriatic arthritis (PsA) synovial tissue and their associations with clinical disease and implications for therapy. METHODS: PsA synovial tissue was enzymatically/mechanically digested to generate synovial tissue single cell suspensions. Frequencies of polyfunctional CD4, CD8, T-helper 1 (Th1), Th17 and exTh17 cells, using CD161 as a marker of Th17 plasticity, were determined by flow cytometry in matched PsA synovial tissue and peripheral blood. Synovial T-cell polyfunctionality was assessed in relation to Disease Activity in PSoriatic Arthritis (DAPSA) and in synovial cell suspensions cultured with a current mode of treatment, phosphodiesterase 4 (PDE4) inhibitor. RESULTS: PsA synovial tissue infiltrating CD4+ T-cells expressed higher levels of interleukin (IL)-17A, interferon gamma (IFN-γ), GM-CSF and CD161, with parallel enrichment of Th1, Th17 and exTh17 T-helper subsets (all p<0.05). Interestingly, a significant proportion of synovial T-cell subsets were triple-positive for GM-CSF, tumour necrosis factor (-TNF), -IL-17 or IFN-γ compared with matched blood (all p<0.05). Importantly, frequencies of polyfunctional T-cells correlated with DAPSA: Th1-GM-CSF+/TNF+/IFN-γ+ (r=0.7, p<0.01), Th17-GM-CSF+/TNF+/IL-17+ (r=0.6, p<0.057) and exTh17-GM-CSF+/TNF+/IFN-γ+ (r=0.7, p=0.0096), with no associations observed for single cytokine-producing T-cells. Following ex vivo culture of PsA synovial tissue cell suspensions, polyfunctional GM-CSF+TNFα+IL-17A+ or/IFN-γ+-producing T-cells (p<0.05), but not single cytokine-producing T-cells, were inhibited with a PDE4 inhibitor. CONCLUSION: These data demonstrate enrichment of polyfunctional T-cells in PsA synovial tissue which were strongly associated with DAPSA and ex vivo therapeutic response.

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.

Interleukin 12 and interleukin 23 play key pathogenic roles in inflammatory and proliferative pathways in giant cell arteritis.

OBJECTIVES: The pathogenesis of giant cell arteritis (GCA) remains unclear. TH1 and TH17 pathways are implicated, but the proximal initiators and effector cytokines are unknown. Our aim was to assess the role of interleukin 12 (IL-12) and interleukin 23 (IL-23) in GCA pathogenesis. METHODS: IL-12 and IL-23 expression were quantified by immunohistochemistry in temporal artery biopsies (TABs). Temporal artery (TA) explant, peripheral blood mononuclear cell (PBMC) and myofibroblast outgrowth culture models were established. PBMCs and TA explants were cultured for 24 hours in the presence or absence of IL-12 (50 ng/mL) or IL-23 (10 ng/mL). Gene expression in TA was quantified by real-time PCR and cytokine secretion by ELISA. Myofibroblast outgrowths were quantified following 28-day culture. RESULTS: Immunohistochemistry demonstrated increased expression of interleukin 12p35 (IL-12p35) and interleukin 23p19 (IL-23p19) in biopsy-positive TAs, localised to inflammatory cells. IL-12p35 TA expression was significantly increased in those with cranial ischaemic complications (p=0.026) and large vessel vasculitis (p=0.006). IL-23p19 TA expression was increased in those with two or more relapses (p=0.007). In PBMC cultures, exogenous IL-12 significantly increased interleukin 6 (IL-6) (p=0.009), interleukin 22 (IL-22) (p=0.003) and interferon γ (IFN-γ) (p=0.0001) and decreased interleukin 8 (IL-8) (p=0.0006) secretion, while exogenous IL-23 significantly increased IL-6 (p=0.029), IL-22 (p=0.001), interleukin 17A (IL-17A) (p=0.0003) and interleukin 17F (IL-17F) (p=0.012) secretion. In ex vivo TA explants, IL-23 significantly increased gene expression of IL-8 (p=0.0001) and CCL-20 (p=0.027) and protein expression of IL-6 (p=0.002) and IL-8 (p=0.004). IL-12 (p=0.0005) and IL-23 (p<0.0001) stimulation increased the quantity of myofibroblast outgrowths from TABs. CONCLUSION: IL-12 and IL-23 play central and distinct roles in stimulating inflammatory and proliferative pathways relevant to GCA pathogenesis.

Serum MicroRNA Signature as a Diagnostic and Therapeutic Marker in Patients with Psoriatic Arthritis.

OBJECTIVE: MicroRNA (miRNA) are small endogenous regulatory RNA molecules that have emerged as potential therapeutic targets and biomarkers in autoimmunity. Here, we investigated serum miRNA levels in patients with psoriatic arthritis (PsA) and further assessed a serum miRNA signature in therapeutic responder versus nonresponder PsA patients. METHODS: Serum samples were collected from healthy controls (HC; n = 20) and PsA patients (n = 31), and clinical demographics were obtained. To examine circulatory miRNA in serum from HC and PsA patients, a focused immunology miRNA panel was analyzed utilizing a miRNA Fireplex assay (FirePlex Bioworks Inc.). MiRNA expression was further assessed in responders versus nonresponders according to the European League Against Rheumatism response criteria. RESULTS: Six miRNA (miR-221-3p, miR-130a-3p, miR-146a-5p, miR-151-5p, miR-26a-5p, and miR-21-5p) were significantly higher in PsA compared to HC (all P < 0.05), with high specificity and sensitivity determined by receiver-operating characteristic curve analysis. Analysis of responder versus nonresponders demonstrated higher baseline levels of miR-221-3p, miR-130a-3p, miR-146a-5p, miR-151-5p, and miR-26a-5p were associated with therapeutic response. CONCLUSION: This study identified a 6-serum microRNA signature that could be attractive candidates as noninvasive markers for PsA and may help to elucidate the disease pathogenesis.

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).

Enriched Cd141+ DCs in the joint are transcriptionally distinct, activated, and contribute to joint pathogenesis.

CD141+ DC are implicated in antiviral and antitumor immunity. However, mechanistic studies in autoimmune disease are limited. This is the first study to our knowledge examining CD141+ DC in autoimmune disease, specifically inflammatory arthritis (IA). We identified significant enrichment of CD141+ DC in the inflamed synovial joint, which were transcriptionally distinct from IA and healthy control (HC) blood CD141+ DC and significantly more activated, and they exhibited increased responsiveness to TLR3. Synovial CD141+ DC represent a bone fide CD141+ DC population that is distinct from CD1c+ DC. Synovial CD141+ DC induced higher levels of CD4+ and CD8+ T cell activation compared with their peripheral blood counterparts, as made evident by expression of IFN-γ, TNF-α, and granulocyte-macrophage CSF (GMCSF). Autologous synovial CD141+ DC cocultures also induce higher levels of these cytokines, further highlighting their contribution to synovial inflammation. Synovial CD141+ DC-T cell interactions had the ability to further activate synovial fibroblasts, inducing adhesive and invasive pathogenic mechanisms. Furthermore, we identify a mechanism in which synovial CD141+ DC are activated, via ligation of the hypoxia-inducible immune-amplification receptor TREM-1, which increased synovial CD141+ DC activation, migratory capacity, and proinflammatory cytokines. Thus, synovial CD141+ DC display unique mechanistic and transcriptomic signatures, which are distinguishable from blood CD141+ DC and can contribute to synovial joint inflammation.