Fibroblast activation protein and disease severity, progression, and survival in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive fibrosis in the lungs. Activated fibroblasts play a central role in fibrogenesis and express fibroblast activation protein α. A truncated, soluble form (sFAP) can be measured in blood and is a potential novel biomarker of disease activity. The aim was to study the association between sFAP and clinical, radiological, and histopathological measures of disease severity, progression, and survival in a prospective, multicentre, real-world cohort of patients with IPF. Patients with IPF were recruited from the tertiary interstitial lung disease centres in Denmark and followed for up to 3 years. Baseline serum levels of sFAP were measured by ELISA in patients with IPF and compared to healthy controls. Pulmonary function tests, 6-minute walk test and quality of life measures were performed at baseline and during follow-up. The study included 149 patients with IPF. Median sFAP in IPF was 49.6 ng/mL (IQR: 43.1-61.6 ng/mL) and in healthy controls 73.8 ng/mL (IQR: 62.1-92.0 ng/mL). Continuous sFAP was not associated with disease severity, progression or survival (p > 0.05). After dichotomization of sFAP below or above mean sFAP + 2 SD for healthy controls, higher levels of sFAP were associated with lower FVC % predicted during follow-up (p < 0.01). Higher than normal serum levels of sFAP were associated with longitudinal changes in FVC % predicted, but sFAP did not show clear associations with other baseline or longitudinal parameters. As such, sFAP has limited use as a biomarker of disease progression or survival in patients with IPF.

Synovial fibroblasts in juvenile idiopathic arthritis: A scoping review.

Synovial fibroblasts and their role in juvenile idiopathic arthritis have received limited attention compared to other immune mediated disease such as rheumatoid arthritis. Furthermore, no review exists regarding synovial fibroblasts, their interaction with immune cells and their potential involvement in juvenile idiopathic arthritis pathogenesis. This scoping review set out to identify and compile the current knowledge of all peer-reviewed studies on synovial fibroblasts from patients with juvenile idiopathic arthritis. The aim was to map the current knowledge and to produce a tool to assist future studies. The entire MEDLINE, EMBASE and Web of Science databases were used to identify all published studies in English regarding synovial fibroblasts from patients with juvenile idiopathic arthritis. We identified 18 eligible studies out of a total of 1778 screened entries. The majority of studies identified synovial fibroblast subsets or functional characteristics that may be involved in disease pathogenesis. We identified mechanisms of cell-cell interaction with leukocytes, pro-inflammatory signaling and unfavorable connective tissue homeostasis that may contribute to cartilage damage or bony overgrowth. All included studies identified mechanisms potentially linking synovial fibroblasts to specific disease traits in juvenile idiopathic arthritis. Most findings were similar to mechanisms also described in synovial fibroblast from adults with arthritis. However, the limited number of studies found identifies an unmet need for additional studies on synovial fibroblasts and their potential role in juvenile idiopathic arthritis.

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.

Phenotypic and functional characterization of synovial fluid-derived fibroblast-like synoviocytes in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLS) play an important pathological role in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). These cells have primarily been characterized in the RA synovial membrane. Here we aim to phenotypically and functionally characterize cultured synovial fluid-derived FLS (sfRA-FLS). Paired peripheral blood mononuclear cells (PBMC) and sfRA-FLS from patients with RA were obtained and monocultures of sfRA-FLS and autologous co-cultures of sfRA-FLS and PBMC were established. The in situ activated sfRA-FLS were CD34-, CD45-, Podoplanin+, Thymocyte differentiation antigen-1+. SfRA-FLS expressed uniform levels of NFкB-related pathway proteins and secreted several pro-inflammatory cytokines dominated by IL-6 and MCP-1. In a co-culture model with autologous PBMC, the ICAM-1 and HLA-DR expression on sfRA-FLS and secretion of IL-1ß, IL-6, and MCP-1 increased. In vivo, human sfRA-FLS were cartilage invasive both at ipsilateral and contralateral implantation site. We conclude that, sfRA-FLS closely resemble the pathological sublining layer FLS subset in terms of surface protein expression, cytokine production and leukocyte cross-talk potential. Further, sfRA-FLS are comparable to tissue-derived FLS in their capabilities to invade cartilage at implantation sites but also spread tissue destruction to a distant site. Collectively, sfRA-FLS can serve as a an easy-to-obtain source of pathological sublining FLS 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.

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 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.

Inverse Correlation at the Hip Between Areal Bone Mineral Density Measured by Dual-Energy X-ray Absorptiometry and Cortical Volumetric Bone Mineral Density Measured by Quantitative Computed Tomography.

Quantitative computed tomography (QCT) is considered to measure true volumetric bone mineral density (vBMD; mg/cm3) and enables differentiation between cortical and trabecular bone. We aimed to determine the value of QCT by correlating areal BMD (aBMD) by dual-energy X-ray absorptiometry (DXA) with vBMD when using a fixed threshold to delineate cortical from trabecular bone. In a cross-sectional study, 98 postmenopausal women had their hip scanned by DXA and by QCT. At the total hip and the trabecular bone compartment, aBMD correlated significantly with vBMD (r=0.74 and r=0.63; p<0.01, respectively). A significant inverse correlation was found between aBMD and cortical vBMD (r=-0.57; p<0.01). Total hip volume by QCT did not change with aBMD. However, increased aBMD was associated with a decreased trabecular bone volume (r=-0.36; p<0.01) and an increased cortical volume (r=0.69; p<0.01). Changing the threshold used to delineate cortical from trabecular bone from default 350 mg/cm3 to either 300 or 400 mg/cm3 did not affect integral vBMD (p=89) but had marked effects on estimated vBMD at the cortical (p<0.001) and trabecular compartments (p<0.001). Furthermore, increasing the threshold decreased cortical thickness (p<0.001), whereas the strength parameter in terms of buckling ratio increased (p<0.001). Our results show good agreement between aBMD and integral vBMD. However, using a fixed threshold to differentiate cortical from trabecular bone causes an apparent increase in cortical volume with a decrease in cortical density as aBMD increases. This may be caused by the classification of a larger part of the transition zone as cortical bone with increased aBMD.