Managing Cardiovascular and Cancer Risk Associated with JAK Inhibitors.

Janus kinase inhibitors (JAKi) have enormous appeal as immune-modulating therapies across many chronic inflammatory diseases, but recently this promise has been overshadowed by questions regarding associated cardiovascular and cancer risk emerging from the ORAL Surveillance phase 3b/4 post-marketing requirement randomized controlled trial. In that study of patients with rheumatoid arthritis with existing cardiovascular risk, tofacitinib, the first JAKi registered for chronic inflammatory disease, failed to meet non-inferiority thresholds when compared with tumor necrosis factor inhibitors for both incident major adverse cardiovascular events and incident cancer. While this result was unexpected by many, subsequently published observational data have also supported this finding. Notably, however, such a risk has largely not yet been demonstrated in patients outside the specific clinical situation examined in the trial, even in the face of many studies examining this. Nevertheless, this signal has practically re-aligned approaches to both tofacitinib and other JAKi to varying extents, in other patient populations and contexts: within rheumatoid arthritis, but also in psoriatic arthritis, axial spondyloarthritis, inflammatory bowel disease, atopic dermatitis, and beyond. Application to individual patients can be more challenging but remains important to harness the substantive potential of JAKi to the maximum extent safely possible. This review not only explores the evolution of the regulatory response to the signal, its informing data, biological plausibility, and its impact on guidelines, but also the many factors that clinicians must consider in navigating cardiovascular and cancer risk for their patients considering JAKi as immune-modulating therapy.

Increased Galectin-9 Levels Correlate with Disease Activity in Patients with DMARD-Naïve Rheumatoid Arthritis and Modulate the Secretion of MCP-1 and IL-6 from Synovial Fibroblasts.

Background: Fibroblast-like synoviocytes (FLSs) are essential mediators in the expansive growth and invasiveness of rheumatoid synovitis, and patients with a fibroblastic-rich pauci-immune pathotype respond poorly to currently approved antirheumatic drugs. Galectin-9 (Gal-9) has been reported to directly modulate rheumatoid arthritis (RA) FLSs and to hold both pro- and anti-inflammatory properties. The objective of this study was to evaluate clinical and pathogenic aspects of Gal-9 in RA, combining national patient cohorts and cellular models. Methods: Soluble Gal-9 was measured in plasma from patients with newly diagnosed, treatment-naïve RA (n = 98). The disease activity score 28-joint count C-reactive protein (DAS28CRP) and total Sharp score were used to evaluate the disease course serially over a two-year period. Plasma and synovial fluid samples were examined for soluble Gal-9 in patients with established RA (n = 18). A protein array was established to identify Gal-9 binding partners in the extracellular matrix (ECM). Synovial fluid mononuclear cells (SFMCs), harvested from RA patients, were used to obtain synovial-fluid derived FLSs (SF-FLSs) (n = 7). FLSs from patients suffering from knee Osteoarthritis (OA) were collected from patients when undergoing joint replacement surgery (n = 5). Monocultures of SF-FLSs (n = 6) and autologous co-cultures of SF-FLSs and peripheral blood mononuclear cells (PBMCs) were cultured with and without a neutralizing anti-Gal-9 antibody (n = 7). The mono- and co-cultures were subsequently analyzed by flow cytometry, MTT assay, and ELISA. Results: Patients with early and established RA had persistently increased plasma levels of Gal-9 compared with healthy controls (HC). The plasma levels of Gal-9 were associated with disease activity and remained unaffected when adding a TNF-inhibitor to their standard treatment. Gal-9 levels were elevated in the synovial fluid of established RA patients with advanced disease, compared with corresponding plasma samples. Gal-9 adhered to fibronectin, laminin and thrombospondin, while not to interstitial collagens in the ECM protein array. In vitro, a neutralizing Gal-9 antibody decreased MCP-1 and IL-6 production from both RA FLSs and OA FLSs. In co-cultures of autologous RA FLSs and PBMCs, the neutralization of Gal-9 also decreased MCP-1 and IL-6 production, without affecting the proportion of inflammatory FLSs. Conclusions: In RA, pretreatment plasma Gal-9 levels in early RA were increased and correlated with clinical disease activity. Gal-9 levels remained increased despite a significant reduction in the disease activity score in patients with early RA. The in vitro neutralization of Gal-9 decreased both MCP-1 and IL-6 production in an inflammatory subset of RA FLSs. Collectively these findings indicate that the persistent overexpression of Gal-9 in RA may modulate synovial FLS activities and could be involved in the maintenance of subclinical disease activity in RA.

MAPK activated kinase 2 inhibition shifts the chemokine signature in arthritis synovial fluid mononuclear cells from CXCR3 to CXCR2.

BACKGROUND: The development of novel treatment strategies of immune-mediated inflammatory arthritis (IMIA) is still a clinical unmet need. The mitogen-activated protein kinase (MAPK) pathway is activated by environmental stressors, growth factors and inflammatory cytokines. However, the inhibition of central MAPK proteins has so far had undesirable side effects. The MAPK-activated protein kinase 2 (MK2) is a downstream mediator in the MAPK signaling pathway. OBJECTIVES: The objective of this study was to explore the effects of a small molecule inhibiting MK2 on synovial fluid mononuclear cells from patients with IMIA. METHODS: Synovial fluid mononuclear cells (SFMCs) were obtained from a study population consisting of patients with active rheumatoid arthritis (RA), peripheral spondyloarthritis (SpA) or psoriatic arthritis (PsA) with at least one swollen joint (for obtaining synovial fluid) (n = 11). SFMCs were cultured for 48 h with and without the MK2 inhibitor CC0786512 at 1000 nM, 333 nM and 111 nMand cell free supernatants were harvested and frozen before they were analyzed by the Olink proseek multiplex interferon panel. RESULTS: In SFMCs cultured for 48 h, the MK2 inhibitor decreased the production of chemokine (C-X-C motif) ligand 9 (CXCL9) (P < 0.001), CXCL10 (P < 0.01), hepatocyte growth factor (HGF) (P < 0.01), CXCL11 (P < 0.01), tumor necrosisfactor-like weak inducer of apoptosis (TWEAK) (P < 0.05), and interleukin 12B (IL-12B) (P < 0.05) and increased the production of CXCL5 (P < 0.0001), CXCL1 (P < 0.0001), CXCL6 (P < 0.001), transforming growthfactoralpha (TGFα) (P = 0.01), monocyte-chemotactic protein 3 (MCP-3) (P < 0.01), latency-associated peptide (LAP) TGFß (P < 0.05) dose-dependently. CONCLUSIONS: This study reveals the downstream effects of MK2 inhibition on the secretory profile of SFMCs. Specifically, C-X-C motif chemokine receptors 3 (CXCR3) chemokines were decreased and CXCR2 chemokines were increased. This shift in the chemokine milieu may be one of the mechanisms behind the anti-inflammatory effects of MK2 inhibitors.

Immunomodulatory and immunosuppressive therapies in cardiovascular disease and type 2 diabetes mellitus: A bedside-to-bench approach.

OBJECTIVE: To assess which immunosuppressive drugs have been investigated and proven efficacious in patients with cardiovascular disease (CVD) or type 2 diabetes (T2D) without preexisting immune mediated disorders to validate in vitro and animal model findings on low grade inflammation (bedside-to-bench). METHODS: Clinical trials on immunosuppressive drugs in CVD or T2D were found in PubMed. Studies on patients with preexisting immune mediated inflammatory disease were excluded. A total of 19 clinical trials testing canakinumab, anakinra, methotrexate, colchicine, hydroxychloroquine, etanercept and sulfasalazine were found. RESULTS: Canakinumab and colchicine significantly reduced the risk of CVD, whereas methotrexate did not. Sulfasalazine showed no effect on vascular function. Anakinra and hydroxychloroquine had a positive effect on glycemic control and ß-cell function in T2D. Etanercept had no effect in patients with T2D. CONCLUSION: The observed results indicate that immunosuppressive drugs specifically targeting IL-1ß hold promise for dampening CVD and T2D. These findings validate in vitro and animal models showing involvement of the IL-1-axis in the pathogenesis of CVD and T2D. The use of immunosuppressive drugs targeting the chronic inflammation in these diseases could be a possible future treatment strategy as an add-on to the existing pharmacological treatment of CVD and T2D. However, potential treatment effects, adverse events and cost-effectiveness should be carefully considered with importance for drug development.

The Programmed Death-1 Pathway Counter-Regulates Inflammation-Induced Osteoclast Activity in Clinical and Experimental Settings.

Objective: The programmed death-1 (PD-1) pathway is essential for maintaining self-tolerance and plays an important role in autoimmunity, including rheumatoid arthritis (RA). Here, we investigated how membrane-bound and soluble (s)PD-1 influence bone homeostasis during chronic inflammation, exemplified in RA. Methods: Bone mineral density and bone microstructure were examined in PD-1 and PD-L1 knockout (KO) mice and compared with wild-type (WT) mice. Receptor activator of nuclear factor kappa-B ligand (RANKL) was measured in serum, and the expression examined on activated bone marrow cells. Osteoclast formation was examined in cells from murine spleen and bone marrow and from human synovial fluid cells. sPD-1 was measured in chronic and early (e)RA patients and correlated to markers of disease activity and radiographic scores. Results: PD-1 and PD-L1 KO mice showed signs of osteoporosis. This was supported by a significantly reduced trabecular bone volume fraction and deteriorated microstructure, as well as increased osteoclast formation and an increased RANKL/OPG ratio. The recombinant form of sPD-1 decreased osteoclast formation in vitro, but was closely associated with disease activity markers in eRA patients. Sustained elevated sPD-1 levels indicated ongoing inflammation and were associated with increased radiographic progression. Conclusion: The PD-1 pathway is closely associated with bone homeostasis, and lacking members of this pathway causes a deteriorated bone structure. The immunological balance in the microenvironment determines how the PD-1 pathway regulates osteoclast formation. In eRA patients, sPD-1 may serve as a biomarker, reflecting residual but clinically silent disease activity and radiographic progression.

Nitro-fatty acids decrease type I interferons and monocyte chemoattractant protein 1 in ex vivo models of inflammatory arthritis.

BACKGROUND: Inflammatory arthritis including rheumatoid arthritis (RA) and spondyloarthritis (SpA) is characterized by inflammation and destruction of the joints. Approximately one third of patients do not respond to first-line treatments. Nitro-fatty acids are bioactive lipids with anti-inflammatory properties and tissue-protective functions. The nitro-fatty acid 10-NO2-oleic acid (10-NO2-OA) is being tested in clinical trials for patients with fibrotic and inflammatory conditions. Here, we tested whether 10-NO2-OA could inhibit immune reactions involved in the inflammatory and joint destructive processes in inflammatory arthritis. METHODS: Synovial fluid and blood samples were obtained from 14 patients with active RA or SpA. The in vitro models consisted of synovial fluid mononuclear cells (SFMCs) cultured for 48 h, SFMCs cultured for 21 days, and fibroblast-like synovial cells (FLSs) co-cultured with peripheral blood mononuclear cells (PBMCs) for 48 h. Cells were treated with or without 10-NO2-OA or the tumor necrosis factor alpha (TNFα) inhibitor etanercept. Supernatants were analyzed for type I interferon, monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase 3 (MMP3) and tartrate resistant acid phosphatase (TRAP). RESULTS: In SFMCs cultured for 48 h, 10-NO2-OA dose-dependently decreased the secretion of bioactive type I interferons and MCP-1 but not MMP3 (P = 0.032, P = 0.0001, and P = 0.58, respectively). Both MCP-1 and MMP3 were decreased by etanercept (P = 0.0031 and P = 0.026, respectively). In SFMCs cultured for 21 days, 10-NO2-OA significantly decreased the production of MCP-1 but not TRAP (P = 0.027 and P = 0.1523, respectively). Etanercept decreased the production of TRAP but not MCP-1 (P < 0.001 and P = 0.84, respectively). In co-cultures of FLSs and PBMCs, 10-NO2-OA decreased the production of MCP-1 (P < 0.0001). This decrease in MCP-1 production was not seen with etanercept treatment (P = 0.47). CONCLUSION: 10-NO2-OA decreased the release of MCP-1 in three models of inflammatory arthritis. Of particular interest, 10-NO2-OA inhibited type I interferon, and 10-NO2-OA was more effective in reducing MCP-1 production in cultures dominated by FLSs compared with etanercept. Our results encourage clinical investigations of 10-NO2-OA in patients with inflammatory arthritis.

Plasma ACE2 predicts outcome of COVID-19 in hospitalized patients.

AIMS: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) enabling entrance of the virus into cells and causing the infection termed coronavirus disease of 2019 (COVID-19). Here, we investigate associations between plasma ACE2 and outcome of COVID-19. METHODS AND RESULTS: This analysis used data from a large longitudinal study of 306 COVID-19 positive patients and 78 COVID-19 negative patients (MGH Emergency Department COVID-19 Cohort). Comprehensive clinical data were collected on this cohort, including 28-day outcomes. The samples were run on the Olink® Explore 1536 platform which includes measurement of the ACE2 protein. High admission plasma ACE2 in COVID-19 patients was associated with increased maximal illness severity within 28 days with OR = 1.8, 95%-CI: 1.4-2.3 (P < 0.0001). Plasma ACE2 was significantly higher in COVID-19 patients with hypertension compared with patients without hypertension (P = 0.0045). Circulating ACE2 was also significantly higher in COVID-19 patients with pre-existing heart conditions and kidney disease compared with patients without these pre-existing conditions (P = 0.0363 and P = 0.0303, respectively). CONCLUSION: This study suggests that measuring plasma ACE2 is potentially valuable in predicting COVID-19 outcomes. Further, ACE2 could be a link between COVID-19 illness severity and its established risk factors hypertension, pre-existing heart disease and pre-existing kidney disease.

Protein array-based companion diagnostics in precision medicine.

INTRODUCTION: The development of companion diagnostics (CDx) will increase efficacy and cost-benefit markedly, compared to the currently prevailing trial-and-error approach for treatment. Recent improvements in high-throughput protein technology have resulted in large amounts of predictive biomarkers that are potentially useful components of future CDx assays. Current high multiplex protein arrays are suitable for discovery-based approaches, while low-density and more simple arrays are suitable for use in point-of-care facilities. AREA COVERED: This review discusses the technical platforms available for protein array focused CDx, explains the technical details of the platforms and provide examples of clinical use, ranging from multiplex arrays to low-density clinically applicable arrays. We thereafter highlight recent predictive biomarkers within different disease areas, such as oncology and autoimmune diseases. Lastly, we discuss some of the challenges connected to the implementation of CDx assays as point-of-care tests. EXPERT OPINION: Recent advances in the field of protein arrays have enabled high-density arrays permitting large biomarker discovery studies, which are beneficial for future CDx assays. The density of protein arrays range from a single protein to proteome-wide arrays, allowing the discovery of protein signatures that may correlate with drug response. Protein arrays will undoubtedly play a key role in future CDx assays.

Programmed death ligand 2 - A link between inflammation and bone loss in rheumatoid arthritis.

OBJECTIVE: Active rheumatoid arthritis (RA) is accompanied by increased appendicular and axial bone loss, closely associated to the degree of inflammation. The programmed death-1 (PD-1) pathway is important for maintaining peripheral tolerance, and its ligand PD-L2 has recently been associated with bone morphogenetic protein activity. Here, we report that PD-L2 plays a central role in RA osteoimmunology. METHODS: Femoral bone mineral density (BMD) and trabecular bone microstructure were evaluated by micro-CT in wild type (WT) and PD-L2-/- mice. Osteoclasts were generated from RA synovial fluid mononuclear cells and peripheral blood monocytes. The effects of recombinant PD-L2, was evaluated by tartrate-resistant acid phosphatase (TRAP) activity and the development of bone erosions in the presence of anti-citrullinated protein antibodies (ACPA). Plasma soluble (s)PD-L2 levels were measured in patients with early (e)RA (n â€‹= â€‹103) treated with methotrexate alone or in combination with the TNF inhibitor Adalimumab. RESULTS: PD-L2-/- mice had a decreased BMD and deteriorated trabecular bone microstructure that was not related to the RANKL/OPG pathway. PD-L2 decreased TRAP activity in osteoclasts and decreased ACPA-induced erosions. In the RA synovial membrane PD-L2 was highly expressed especially in the lining layer and plasma sPD-L2 levels were increased in eRA patients and decreased with treatment. One-year sPD-L2 correlated inversely with erosive progression two years after treatment initiation with methotrexate and placebo. CONCLUSION: PD-L2 regulates bone homeostasis in RA. Our findings provide new insight into the relationship between the immune system and bone homeostasis, and suggest a potential therapeutic target for limiting inflammatory bone loss in RA.

Responses to Cytokine Inhibitors Associated with Cellular Composition in Models of Immune-Mediated Inflammatory Arthritis.

OBJECTIVE: Immune-mediated inflammatory arthritis (IMIA) is a heterogeneous group of diseases including rheumatoid arthritis (RA), psoriatic arthritis (PsA), and spondyloarthritis (SpA). Disease-modifying antirheumatic drugs (DMARDs) target very different cellular components of the disease processes. Characterization of the pathobiological subtypes of IMIA could provide more specific treatment approaches for each disease. For example, RA has been proposed to consist of at least three synovial pathotypes (lymphoid, myeloid, and fibroid), and only a subgroup of RA patients have erosive disease. The objective of this study was to evaluate the effects of various DMARDs on different synovial cell subsets using human ex vivo models of IMIA. METHODS: Synovial fluid and blood samples were obtained from a study population consisting of patients with RA, PsA, or peripheral SpA with at least one swollen joint (n = 18). The DMARDs used in this study were methotrexate, adalimumab, etanercept, tocilizumab, anakinra, ustekinumab, secukinumab, tofacitinib, and baricitinib. Paired synovial fluid mononuclear cells (SFMCs), peripheral blood mononuclear cells (PBMCs), and fibroblast-like synovial cells (FLSs) were used in three different previously optimized ex vivo models. RESULTS: In SFMCs cultured for 48 hours, all DMARDs except anakinra decreased the production of monocyte chemoattractant protein (MCP)-1. In SFMCs cultured for 21 days, only the two tumor necrosis factor alpha (TNFα) inhibitors adalimumab and etanercept decreased the secretion of tartrate-resistant acid phosphatase (P < 0.01, P < 0.001). In the FLS and PBMC 48-hour co-cultures, only tocilizumab (P < 0.001) and the two Janus kinase inhibitors tofacitinib and baricitinib (both P < 0.05) decreased the production of MCP-1 by around 50%. CONCLUSION: TNFα inhibition was effective in preventing inflammatory osteoclastogenesis, whereas tocilizumab, tofacitinib, and baricitinib had superior efficacy in cultures dominated by FLSs. Taken together, this study reveals that responses to cytokine inhibitors associate with cellular composition in models of IMIA. In particular, this study provides new evidence on the differential effect of DMARDs on leukocytes compared with stromal cells.

Fibroblast-like synovial cell production of extra domain A fibronectin associates with inflammation in osteoarthritis.

BACKGROUND: The pathophysiology of osteoarthritis (OA) involves wear and tear, and a state of low-grade inflammation. Tissue repair responses include transforming growth factor beta (TGFß)-induced myofibroblast production of extracellular matrix. Fibronectins are an essential part of the extracellular matrix, and injection of fibronectin fragments into rabbit joints is a previously established animal model of OA. Fibronectin containing the ED-A domain is currently being used as drug delivery target in the development of anti-inflammatory drugs (e.g. Dekavil). METHODS: In this study, samples of synovial membrane were obtained from patients with knee OA undergoing joint replacement surgery. Immunostaining for ED-A fibronectin and the myofibroblast marker alpha smooth muscle actin (αSMA) was performed on fibroblast-like synovial cells (FLS) and synovial membranes. RAW 264.7 macrophages were incubated with recombinant ED-A fibronectin. RESULTS: The staining of ED-A fibronectin in OA FLS was increased by TGFß but not by TNFα, lipopolysaccharide, or IL-6 (n = 3). ED-A fibronectin co-stained with the myofibroblast marker αSMA in both the OA FLS (n = 3) and in the OA synovial membranes (n = 8). ED-A fibronectin staining was associated with both number of lining layer cells (rho = 0.85 and p = 0.011) and sublining cells (rho = 0.88 and p = 0.007) in the OA synovium (n = 8), and co-distributed with TNFα (n = 5). Recombinant ED-A fibronectin increased the production of TNFα by RAW 264.7 macrophages (n = 3). CONCLUSIONS: The disease process in OA shares features with the chronic wound healing response. Our findings support utilizing ED-A fibronectin for drug delivery or therapeutic targeting to reduce pro-inflammatory responses in OA.

IL-12/IL-23p40 identified as a downstream target of apremilast in ex vivo models of arthritis.

BACKGROUND: Apremilast (Otezla®) is a phosphodiesterase 4 (PDE4) inhibitor approved for the treatment of psoriasis and psoriatic arthritis (PsA), but the reason why apremilast shows clinical effect is not fully understood. The objective of this study was to study the downstream effects of apremilast on cells of inflamed joints in immune-mediated inflammatory arthritis. METHODS: Synovial fluid was obtained from patients with active rheumatoid arthritis (RA), PsA or peripheral spondyloarthritis (SpA; n = 18). The in vitro models consisted of synovial fluid mononuclear cells (SFMCs) or fibroblast-like synovial cells (FLSs) cultured for 48 h, SFMCs cultured for 21 days, an osteoclast pit formation assay, and a mineralization assay. RESULTS: In SFMCs cultured for 48 h, apremilast decreased the production of interleukin (IL)-12/IL-23p40 (the shared subunit of IL-12 and IL-23), colony-stimulating factor 1, CD6, and CD40 and increased the production of C-X-C motif chemokine 5 dose-dependently. Apremilast had a very different response signature compared with the tumor necrosis factor alpha inhibitor adalimumab with a substantially greater inhibition of IL-12/IL-23p40. In SFMCs cultured for 21 days, apremilast increased the secretion of IL-10. In FLS cultures, apremilast decreased matrix metalloproteinase-3 production. Apremilast decreased osteoclastogenesis but did not affect mineralization by human osteoblasts. CONCLUSION: This study reveals the downstream effects of apremilast in ex vivo models of arthritis with a strong inhibition of IL-12/IL-23p40 by SFMCs. Our findings could explain some of the efficacy of apremilast seen in IL-12/IL-23-driven immune-mediated inflammatory diseases such as psoriasis and PsA.

The IL-20 Cytokine Family in Rheumatoid Arthritis and Spondyloarthritis.

This review describes the IL-20 family of cytokines in rheumatoid arthritis (RA) and spondyloartrhitits (SpA) including psoriatic arthritis. The IL-20 receptor (R) cytokines IL-19, IL-20, and IL-24 are produced in both the peripheral blood and the synovial joint and are induced by Toll-like receptor ligands and autoantibody-associated immune complexes in monocytes. IL-19 seems to have anti-inflammatory functions in arthritis. In contrast, IL-20 and IL-24 increase the production of proinflammatory molecules such as monocyte chemoattractant protein 1 and are associated with bone degradation and radiographic progression. IL-22 is also associated with progression of bone erosions. This suggests that the IL-22RA1 subunit shared by IL-20, IL-22, and IL-24 is important for bone homeostasis. In line with this, the IL-22RA1 has been found on preosteoclasts in early RA. IL-26 is produced in high amounts by myofibroblasts and IL-26 stimulation of monocytes is an important inducer of Th17 cells in RA. This indicates a role for IL-26 as an important factor in the interactions between resident synovial cells and infiltrating leukocytes. Clinical trials that investigate inhibitors of IL-20 (fletikumab) and IL-22 (fezakinumab) in psoriasis and RA have been terminated. Instead, it seems that the strategy for modulating the IL-20 cytokine family should take the overlap in cellular sources and effector mechanisms into account. The redundancy encourages inhibition of more than one cytokine or one of the shared receptors. All IL-20 family members utilize the Janus kinase signaling pathway and are therefore potentially inhibited by drugs targeting these enzymes. Effects and adverse effects in ongoing clinical trials with inhibitors of IL-22 and the IL-22RA1 subunit and recombinant IL-22 fusion proteins will possibly provide important information about the IL-20 subfamily of cytokines in the future.

The antifibrotic drug pirfenidone inhibits spondyloarthritis fibroblast-like synoviocytes and osteoblasts in vitro.

BACKGROUND: The pathogenesis of spondyloarthritis (SpA) involves both inflammation and new bone formation in the spine. In line with this, the disease has been characterized as both inflammatory and fibrotic. The current treatment dampens inflammation while new bone formation can progress. Therefore, there is an unmet therapeutic need for the treatment of new bone formation in SpA. Fibrosis is mediated by myofibroblasts and new bone formation is the result of increased osteoblast mineralization and decreased osteoclast resorption. Here, we evaluate the potential effect of the newly approved anti-fibrotic agent pirfenidone (PFD) on fibrosis and new bone formation in cell culture models of SpA. METHODS: Fibroblast-like synoviocytes (FLSs) were isolated from SpA patients (n = 6) while the osteoblast cell line Saos-2 was purchased. The cells were cultured with PFD at 0.25 0.5, or 1.0 mg/ml. The proliferation of FLSs was analyzed with light microscopy and flow cytometry. The differentiation and activation of FLSs was assessed with flow cytometry, a membrane-based antibody array and enzyme-linked immunosorbant assays. The mineralization capacity of osteoblasts was studied with an assay measuring deposition of hydroxyapatite. RESULTS: PFD reduced the Ki67 expression 7.1-fold in untreated FLSs (p = 0.001) and 11.0-fold in FLSs stimulated with transforming growth factor beta (TGFß), tumor necrosis factor alpha (TNFα), and interferon gamma (IFNγ) (p = 0.022). There were no statistically significant changes in membrane expression of alpha smooth muscle actin (αSMA), intercellular adhesion molecule 1 (ICAM-1), or human leukocyte antigen DR (HLA-DR). In supernatants from FLSs stimulated with TGFß, TNFα, and IFNγ, PFD decreased the secretion of 3 of 12 proteins more than 2-fold in the membrane-based antibody array. The changes in secretion of monocyte chemoattractant protein 1 (MCP-1) and chitinase-3-like protein 1 (CHI3L1, YKL-40) were validated with ELISA. PFD decreased the secretion of both Dickkopf-related protein 1 (DKK1) (p = 0.006) and osteoprotegerin (OPG) (p = 0.02) by SpA FLSs stimulated with TGFß, TNFα, and IFNγ. Finally, PFD inhibited the deposition of hydroxyapatite by osteoblasts in a dose-dependent manner (p = 0.0001). CONCLUSIONS: PFD inhibited SpA FLS proliferation and function and osteoblast mineralization in vitro. This encourages studies of the in vivo effect of PFD in SpA.

Synovial cell production of IL-26 induces bone mineralization in spondyloarthritis.

Spondyloarthritis (SpA) is characterized by inflammation and new bone formation and can be treated by inhibition of the proinflammatory cytokines TNF-α and IL-17A. IL-26 is considered a proinflammatory cytokine, predominantly related to Th17 cells. In the present study, we investigate IL-26 expression in SpA patients, and examine the in vitro production of IL-26 by synovial cells and the effects of IL-26 on human osteoblasts. IL-26 was measured by ELISA in plasma and synovial fluid (SF) of 15 SpA patients and in plasma samples from 12 healthy controls. Facet joints from axial SpA patients were stained for IL-26 and analyzed by fluorescence microscopy. Synovial fluid mononuclear cells, C-C motif chemokine receptor 6 memory Th17 cells, and fibroblast-like synoviocytes (FLSs) were isolated, and supernatants were analyzed for IL-26 content by ELISA. FLSs were further stained for IL-26 production and the myofibroblast marker α-smooth-muscle-actin (αSMA) and analyzed by flow cytometry. Human osteoblasts were cultured in the presence of IL-26, and the degree of mineralization was quantified. We found that IL-26 levels in SF were increased compared with plasma (P < 0.0001). Moreover, IL-26 expression was found in facet joints of axial SpA patients within the bone marrow. IL-26 secretion was primarily found in αSMA+ myofibroblasts. In contrast, Th17 cells did not produce detectable amounts of IL-26. Human osteoblasts treated with IL-26 showed increased mineralization compared with untreated osteoblasts (P = 0.02). In conclusion, IL-26 seems to be produced by myofibroblasts in the inflamed synovium and could be a possible facilitator of bone mineralization in SpA. KEY MESSAGES: IL-26 levels are higher in synovial fluid compared to plasma in spondyloarthritis. IL-26 was identified in axial facet joints of spondyloarthritis patients. Myofibroblasts from the spondyloarthritis synovium produce large amounts of IL-26. IL-26 induces bone mineralization in human osteoblasts.

Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages.

Nonspecific binding of monoclonal antibodies (mAbs) to Fc-receptors on leukocytes is an important cause of background fluorescence in flow cytometry, and failing to block such nonspecific binding can lead to erroneous results. A major part of previous studies on blocking reagents for flow cytometry have been done in mice, and published results are not completely in agreement. In humans, Fc-receptors are found on most leukocytes, with highest abundance on monocytes/macrophages. Therefore, in the present study our aim was to thoroughly investigate the efficiency of different commonly used blocking reagents regarding inhibition of nonspecific binding of mouse mAbs to human peripheral blood mononuclear cells (MNCs) and monocyte-derived macrophages (MDMs). Monocytes and MDMs showed strong nonspecific binding of IgG1 and IgG2a isotypes, but not IgG2b. In contrast, B-cells, T-cells, and NK-cells did not substantially bind any of the mouse isotype control antibodies evaluated (IgG1, IgG2a, and IgG2b). Importantly, we show that binding of IgG1 and IgG2a to monocytes and MDMs can be eliminated by blocking, either with a commercial Fc-blocking reagent, with mouse or human serum, or with mouse or human IgG in high concentration. Previously, isotype controls have been widely used in flow cytometry assays. However, we show that such controls may be highly unreliable, and we believe they should not be used as gating controls, or to determine background signal. Based on these results, as well as considerations of price and applicability, our recommendation is not to use isotypes as gating controls in flow cytometry, but instead to use 100 µg/mL of purified human IgG as blocking reagent for elimination of nonspecific binding of mouse mAbs to human MNCs and MDMs. © 2016 International Society for Advancement of Cytometry.

Decreased plasma levels of soluble CD18 link leukocyte infiltration with disease activity in spondyloarthritis.

INTRODUCTION: Spondyloarthritis (SpA) comprises a group of diseases often associated with HLA-B27 and characterized by inflammation of the entheses and joints of the axial skeleton. The inflammatory process in SpA is presumably driven by innate immune cells but is still poorly understood. Thus, new tools for monitoring and treating inflammation are needed. The family of CD18 integrins is pivotal in guiding leukocytes to sites of inflammation, and CD18 hypomorphic mice develop a disease resembling SpA. Previously, we demonstrated that altered soluble CD18 (sCD18) complexes in the blood and synovial fluid of patients with arthritis have anti-inflammatory functions. Here, we study the mechanisms for these alterations and their association with SpA disease activity. METHODS: Plasma levels of sCD18 in a study population with 84 patients with SpA and matched healthy controls were analyzed with a time-resolved immunoflourometric assay (TRIFMA). Binding of sCD18 to endothelial cells and fibroblast-like synoviocytes (FLSs) was studied with confocal microscopy. Shedding of CD18 from peripheral blood mononuclear cells (PBMCs) was studied with flow cytometry and TRIFMA. RESULTS: Plasma levels of sCD18 were decreased in patients with SpA compared with healthy volunteers (P <0.001), and the lowest levels were in the HLA-B27-positive subgroup (P <0.05). In a multiple regression model, the sCD18 levels exhibited an inverse correlation with the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) (P <0.05), the level of morning stiffness (P <0.05), the Bath Ankylosing Spondilitis Metrology Index (P <0.05), the physician global assessment score (P <0.01), and the sacroiliac magnetic resonance imaging activity score (P <0.05). The mechanisms for these changes could be simulated in vitro. First, sCD18 in plasma adhered to inflammation-induced intercellular adhesion molecule 1 (ICAM-1) on endothelial cells and FLS, indicating increased consumption. Second, CD18 shedding from SpA PBMCs correlated inversely with the BASDAI (P <0.05), suggesting insufficient generation. CD18 was shed primarily from intermediate CD14⁺⁺ CD16⁺ monocytes, supporting the view that alterations in innate immunity can regulate the inflammatory processes in SpA. CONCLUSIONS: Taken together, the failure of patients with SpA to maintain adequate sCD18 levels may reflect insufficient CD18 shedding from monocytes to counterbalance the capture of sCD18 complexes to inflammation-induced ICAM-1. This could increase the availability of ICAM-1 molecules on the endothelium and in the synovium, facilitating leukocyte migration to the entheses and joints and aggregating disease activity.

A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients.

Rheumatoid arthritis (RA) and spondyloarthritis (SpA) are chronic diseases characterized by activation of the immune system and production of antibodies. Thus, rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies in plasma samples from arthritis patients can interfere with immunoassays such as sandwich enzyme-linked immunosorbent assay (ELISA) systems often used in arthritis research. However, standard methodologies on how to test for false results caused by these antibodies are lacking. The objective of this study was to design a simple set of steps to validate a sandwich ELISA before using it for measuring analytes in plasma from arthritis patients. An interleukin-24 (IL-24) sandwich ELISA system was prepared with a monoclonal mouse capture antibody and a polyclonal goat detection antibody and tested for interference by rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies. Plasma samples from 23 patients with RA and SpA were used. No differences were found between plasma samples measured in wells coated with anti-IL-24 specific antibody and in wells coated with isotype control antibody (false positive results), and recombinant human IL-24 was not recovered in spiked samples (false negative results). This interference was removed after preincubating the plasma samples from patients with arthritis with goat or bovine IgG, suggesting that anti-animal IgG antibodies found in the plasma of the arthritis patients caused the false results. Additional testing showed that the signal-to-noise ratio could be increased by titration of the capture and detection antibodies and by using the ELAST amplification system. Finally, the calculated concentration of IL-24 was increased in ethylenediaminetetraacetic acid (EDTA) plasma compared to heparin plasma and serum and decreased with repetitive freeze/thaw cycles of the samples illustrating how sample handling could additionally contribute to the variations reported by different laboratories in measurement of the same analyte. This study proposes a simple set of validation steps to evaluate and optimize a sandwich ELISA before using it for measuring analytes in plasma from arthritis patients. Anti-animal IgG antibodies are also present in healthy individuals, suggesting that validation of ELISA systems for measuring non-arthritis samples could also be improved by this simple set of validation steps.