No evidence of increased anti-M3 muscarinic acetylcholine receptor autoantibodies in SSA-positive connective tissue disease patients.

Anti-muscarinic type 3 receptor autoantibodies (M3R) and anti-SSA antibodies are both related to salivary secretion. The presence of M3R antibodies in Sjögren's syndrome is previously demonstrated; nevertheless, the relationship between the anti-SSA antibodies and M3R fragment antibodies, namely the N terminal, first, second, and third extracellular loops, remains to be elucidated. In this study, we analyzed the antibodies against the M3R epitopes in healthy controls and anti-SSA antibody-positive connective tissue disease patients through ELISA method. Antibodies against the first, second, and third extracellular loop (M3R211-230 ) were not increased in anti-SSA positive patients compared to healthy controls. Indeed, antibodies against the N terminal (M3R1-33 ) were found to be high in healthy controls. High levels of M3R1-33 in healthy controls are a novel original finding; further research is needed for the clinical significance. There is no significant difference between SSA-positive patients and healthy controls in terms of autoantibodies against the remainder of the linear M3R fragments.

Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis.

BACKGROUND: Lymphocyte activation gene-3 (LAG-3) inhibits T cell activation and interferes with the immune response by binding to MHC-II. As antigen presentation is central in rheumatoid arthritis (RA) pathogenesis, we studied aspects of LAG-3 as a serological marker and mediator in the pathogenesis of RA. Since Galectin-3 (Gal-3) is described as an additional binding partner for LAG-3, we also aimed to study the functional importance of this interaction. METHODS: Plasma levels of soluble (s) LAG-3 were measured in early RA patients (eRA, n = 99) at baseline and after 12 months on a treat-to-target protocol, in self-reportedly healthy controls (HC, n = 32), and in paired plasma and synovial fluid (SF) from chronic RA patients (cRA, n = 38). Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were examined for LAG-3 expression by flow cytometry. The binding and functional outcomes of LAG-3 and Gal-3 interaction were assessed with surface plasmon resonance (SPR) and in cell cultures using rh-LAG3, an antagonistic LAG-3 antibody and a Gal-3 inhibitor. RESULTS: Baseline sLAG-3 in the plasma was increased in eRA compared to HC and remained significantly elevated throughout 12 months of treatment. A high level of sLAG-3 at baseline was associated with the presence of IgM-RF and anti-CCP as well as radiographic progression. In cRA, sLAG-3 was significantly increased in SF compared with plasma, and LAG-3 was primarily expressed by activated T cells in SFMCs compared to PBMCs. Adding recombinant human LAG-3 to RA cell cultures resulted in decreased cytokine secretion, whereas blocking LAG-3 with an antagonistic antibody resulted in increased cytokine secretion. By SPR, we found a dose-dependent binding between LAG-3 and Gal-3. However, inhibiting Gal-3 in cultures did not further change cytokine production. CONCLUSIONS: sLAG-3 in the plasma and synovial fluid is increased in both early and chronic RA patients, particularly in the inflamed joint. High levels of sLAG-3 are associated with autoantibody seropositivity and radiographic progression in eRA, and LAG-3 plays a biologically active role in cRA by decreasing inflammatory cytokine production. This functional outcome is not affected by Gal-3 interference. Our results suggest that LAG-3 is a faceted regulator of inflammation in early and chronic RA.

Validation of an indirect ELISA assay for assessment of autoantibodies against full-length TRIM21 and its individual domains.

Anti-SSA-autoantibodies are common in patients with rheumatologic disease, especially Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis. They consist of both autoantibodies towards Ro60 and Ro52, the latter also known as TRIM21. TRIM21 is an intracellular protein consisting of four domains; PRY/SPRY, Coiled-Coil, B-box and RING. The aim of this study was to establish an indirect ELISA detecting autoantibodies towards both the full-length TRIM21 protein and its four domains. We expressed the five constructs, created, and validated indirect ELISA protocols for each target using plasma from anti-SSA positive patients and healthy controls. Our findings were validated to the clinically used standards. We measured significantly higher levels of autoantibodies towards our full-length TRIM21, and the PRY/SPRY, Coiled-Coil and RING domains in patients compared to healthy controls. No significant difference in the level of autoantibodies were detected against the B-box domain. Our setups had a signal to noise ratio in the range of 30 to 184, and an OD between 2 and 3. Readings did not decline using NaCl of 500 mM as wash, affirming the high binding affinity of the autoantibodies measured. Our protocols allow us to further study the different autoantibodies of anti-SSA positive patients. This creates the possibility to stratify our patients into subgroups regarding autoantibody profile and specific pheno- or endotype.

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.

Galectin-3 interacts with PD-1 and counteracts the PD-1 pathway-driven regulation of T cell and osteoclast activity in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and bone erosions. The glycosylated programmed death-1 (PD-1) receptor plays an important role in regulating immune responses and maintaining tolerance. In this study, we focus on two features observed in RA: impaired PD-1 signalling and Galectin-3 (Gal-3) upregulation. We hypothesize that Gal-3 binds PD-1 and PD-1 ligands, potentially contributing to impaired PD-1 signalling. PD-1 and Gal-3 levels in RA synovial fluid (SF) and plasma were evaluated by ELISA. PD-1 and Gal-3 interaction was examined by Surface Plasmon Resonance and ELISA. PD-1, PD-L1 and Gal-3 expression on mononuclear cells from SF and peripheral blood as well as fibroblast-like synoviocytes were examined by flow cytometry. Effects of Gal-3 and PD-L1 on osteoclast formation was evaluated by tartrate-resistant acid phosphatase assay. We show that Gal-3 binds PD-1 and PD-L1. Results demonstrated high expression of PD-1 and Gal-3 on mononuclear cells, especially from SF. Gal-3 inhibited PD-1 signalling when PD-L1 was present. Furthermore, a role of Gal-3 in osteoclast formation was observed in vitro, both directly but also through PD-1:PD-L1 inhibition. Effects of Gal-3 on the PD-1 signalling axis are proposed to be inhibitory, meaning high Gal-3 levels in the complex synovial microenvironment are not desirable in RA. Preventing Gal-3's inhibitory role on PD-1 signalling could, therefore, be a therapeutic target in RA by affecting inflammatory T cell responses and osteoclasts.

T-cell immunoglobulin and mucin domain 3 is upregulated in rheumatoid arthritis, but insufficient in controlling inflammation.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease, that involves both pro- and anti-inflammatory mechanisms. The purpose of the present study is to investigate T-cell immunoglobulin and mucin domain 3 (Tim-3) in RA. METHODS: Plasma levels of soluble (s) Tim-3 in early RA (n=98), were followed, to evaluate association with treatment and disease activity, acquired from a prospective collected biobank (clinicaltrials.gov (NCT00660647)). We also investigate the influence of Tim-3 on spontaneous cytokine production in synovial fluid mononuclear cells (SFMC) from RA patients after addition of neutralizing anti-Tim-3's antibodies, either alone or in combination with neutralizing anti-Programmed Cell death protein 1 (PD-1) antibodies. RESULTS: Long-time stimulated CD4 T-cells expressed high levels of Tim-3, but tended to decrease their PD-1 expression. Tim-3 expression was exclusively seen co-expressed with PD-1 by CD3, CD4, CD45RO positive cells in the inflamed RA joint. Addition of neutralizing Tim-3 antibodies increased the secretion of IFNγ and MCP-1, in SFMC cultures from RA. Whereas neutralizing anti-PD-1 antibodies showed a broader impact on cytokine production. Finally, we observed that soluble Tim-3 is increased in plasma and is associated with disease activity in early RA. CONCLUSION: Taken together, our findings indicate disease-suppressive functions of Tim-3 in RA.

Galectin-3 Decreases 4-1BBL Bioactivity by Crosslinking Soluble and Membrane Expressed 4-1BB.

4-1BB is a T cell costimulatory receptor and a member of the tumor necrosis factor receptor superfamily. Here, we show that Galectin-3 (Gal-3) decreases the cellular response to its ligand (4-1BBL). Gal-3 binds to both soluble 4-1BB (s4-1BB) and membrane-bound 4-1BB (mem4-1BB), without blocking co-binding of 4-1BBL. In plasma, we detected complexes composed of 4-1BB and Gal-3 larger than 100 nm in size; these complexes were reduced in synovial fluid from rheumatoid arthritis. Both activated 4-1BB+ T cells and 4-1BB-transfected HEK293 cells depleted these complexes from plasma, followed by increased expression of 4-1BB and Gal-3 on the cell surface. The increase was accompanied by a 4-fold decrease in TNFα production by the 4-1BBhighGal-3+ T cells, after exposure to 4-1BB/Gal-3 complexes. In RA patients, complexes containing 4-1BB/Gal-3 were dramatically reduced in both plasma and SF compared with healthy plasma. These results support that Gal-3 binds to 4-1BB without blocking the co-binding of 4-1BBL. Instead, Gal-3 leads to formation of large soluble 4-1BB/Gal-3 complexes that attach to mem4-1BB on the cell surfaces, resulting in suppression of 4-1BBL's bioactivity.

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.

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.

Juvenile idiopathic arthritis: lymphocyte activation gene-3 is a central immune receptor in children with oligoarticular subtypes.

BACKGROUND: We investigated the role of inhibitory receptors (IRs) and especially lymphocyte activation gene-3 (LAG-3) in the pathogenesis of oligoarticular juvenile idiopathic arthritis (o-JIA). METHODS: Paired samples of synovial fluid (SF) and plasma and peripheral blood (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from o-JIA patients along with their clinical data (n = 24). Plasma from healthy controls (n = 14) and paired SF and plasma samples from five non-arthritic juvenile orthopedic patients (n = 5) served as controls. Spontaneously differentiated fibroblast-like synoviocytes (FLSs) from SFMCs were co-cultured with autologous PBMCs/SFMCs and used as ex vivo disease model. Soluble levels and cellular expressions of IRs together with their functional properties in the ex vivo model were analyzed. RESULTS: In patients with o-JIA, soluble levels of LAG-3 and expression of LAG-3 and T cell immunoglobulin mucin03 (TIM-3) on CD3+CD4+CD45RO+ T cells were increased, especially in SF. Major histocompatibility complex (MHC) class II expression was induced on FLSs when these were co-cultured with autologous PBMCs/SFMCs, together with an increased monocyte chemoattractant protein-1 (MCP-1) production. In PBMC and FLS + PBMC co-cultures, neutralizing antibodies to IRs were added. Only anti-LAG-3 antibodies significantly increased MCP-1 secretion. The addition of agonistic LAG-3 antibody resulted in decreased effector cytokine secretion. CONCLUSIONS: This is the first report comparing the effects of different IRs in o-JIA and suggests that LAG-3 might contribute to the pathogenesis of this disease. IMPACT: This is the first study addressing the role of different co-IRs in o-JIA. We showed that LAG-3 and TIM-3 seem more important in juvenile arthritis in contrast to adult rheumatoid arthritis, where cytotoxic T-lymphocyte-associated antigen-4 and programmed cell death-1 were reported to be more important. We designed an ex vivo disease model for o-JIA, examined the effects of co-IRs in this model, and demonstrated that they might contribute to the pathogenesis of the disease. LAG-3 might play a role in o-JIA pathogenesis and might be a potential therapeutic option for o-JIA patients.

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.

The OX40 Axis is Associated with Both Systemic and Local Involvement in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic, or chronically relapsing, inflammatory skin disease associated with asthma and allergic rhinitis, and is dominated by Th2 cells. The co-stimulatory T-cell receptor OX40 and its ligand, OX40L, play a central role in the pathogenesis of AD, as their interactions are crucial for the generation of TH2 memory cells. Using enzyme-linked immunoassay (ELISA) and flow cytometry on blood samples from patients with AD and healthy volunteers, this study shows that the serum level of soluble (s) OX40 is decreased in patients with AD, and the expression of OX40 by activated skin-homing CD4+ T cells is increased. This study further shows, using immunofluorescence on skin biopsies, that OX40+ and OX40L+ cells are co-located within the dermis, indicating local activity of OX40/OX40L. Serum levels of sOX40 were associated with atopic diseases and, together, these results support that the OX40 system is important for chronic inflammation in AD skin.

Parenteral vaccination protects against transcervical infection with Chlamydia trachomatis and generate tissue-resident T cells post-challenge.

The optimal protective immunity against Chlamydia trachomatis (C.t.) is still not fully resolved. One of the unresolved issues concerns the importance of resident immunity, since a recent study showed that optimal protection against a transcervical (TC) infection required genital tissue-resident memory T cells. An important question in the Chlamydia field is therefore if a parenteral vaccine strategy, inducing only circulating immunity primed at a nonmucosal site, should be pursued by Chlamydia vaccine developers. To address this question we studied the protective efficacy of a parenteral Chlamydia vaccine, formulated in the Th1/Th17 T cell-inducing adjuvant CAF01. We found that a parenteral vaccination induced significant protection against a TC infection and against development of chronic pathology. Protection correlated with rapid recruitment of Th1/Th17 T cells to the genital tract (GT), which efficiently prevented infection-driven generation of low quality Th1 or Th17 T cells, and instead maintained a pool of high quality multifunctional Th1/Th17 T cells in the GT throughout the infection. After clearance of the infection, a pool of these cells settled in the GT as tissue-resident Th1 and Th17 cells expressing CD69 but not CD103, CD49d, or CCR7, where they responded rapidly to a reinfection. These results show that a nonmucosal parenteral strategy inducing Th1 and Th17 T cells mediates protection against both infection with C.t. as well as development of chronic pathology, and lead to post-challenge protective tissue-resident memory immunity in the genital tract.

Anti-tumor necrosis factor treatment increases both the Th17 and Th22 T helper subsets in spondyloarthritis.

The aim was to examine anti-tumor necrosis factor α (anti-TNFα) therapy influence changes on Th17 and Th22 cells in patients with spondyloarthritis (SpA), and its correlation with changes in clinical and magnetic resonance imaging (MRI) activity and chronicity scores. The Th17 and Th22 cells were assessed at baseline, after 12 and 52 weeks of anti-TNFα therapy by flow cytometry (ClinicalTrials.gov NCT4682724). The percentages of both Th17 and Th22 cells were increased by 70% at baseline compared with healthy controls (both p < 0.01). During treatment, these two subsets increased further to be 170% (Th17) and 123% (Th22) above levels in healthy controls (both p < 0.01). The same subsets decrease their expression of IL-23R significantly during the observation period (p < 0.05). High levels of Th17 and Th22 cells at baseline were associated with the degree of chronic changes in the sacroiliac joints on MRI and a good clinical response to anti-TNFα treatment after one year. Plasma levels were not associated with clinical changes. Th17 cells, and Th22 subsets, increased during one year of anti-TNF-α therapy in SpA, regardless of their clinical improvement. This supports that both the Th17 and Th22 subsets could be involved in the progression in SpA.

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.

Extracellular Vesicles Transfer the Receptor Programmed Death-1 in Rheumatoid Arthritis.

INTRODUCTION: Extracellular vesicles (EVs) have been recognized as route of communication in the microenvironment. They transfer proteins and microRNAs (miRNAs) between cells, and possess immunoregulatory properties. However, their role in immune-mediated diseases remains to be elucidated. We hypothesized a role for EVs in the rheumatoid arthritis (RA) joint, potentially involving the development of T cell exhaustion and transfer of the co-inhibitory receptor programmed death 1 (PD-1). METHODS: Synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from RA patients were investigated for PD-1 and other markers of T cell inhibition. EVs were isolated from RA plasma and synovial fluid. In addition, healthy control (HC) and RA PBMCs and SFMCs were cultured to produce EVs. These were isolated and investigated by immunogold electron microscopy (EM) and also co-cultured with lymphocytes and PD-1 negative cells to investigate their functions. Finally, the miRNA expression profiles were assessed in EVs isolated from RA and HC cell cultures. RESULTS: Cells from the RA joint expressed several T cell co-inhibitory receptors, including PD-1, TIM-3, and Tigit. ELISA demonstrated the presence of PD-1 in EVs from RA plasma and synovial fluid. Immunogold EM visualized PD-1 expression by EVs. Co-culturing lymphocytes and the PD-1 negative cell line, U937 with EVs resulted in an induction of PD-1 on these cells. Moreover, EVs from RA PBMCs increased proliferation in lymphocytes when co-cultured with these. All EVs contained miRNAs associated with PD-1 and other markers of T cell inhibition and the content was significantly lower in EVs from RA PBMCs than HC PBMCs. Stimulation of the cells increased the miRNA expression. However, EVs isolated from stimulated RA SFMCs did not change their miRNA expression profile to the same extend. CONCLUSION: EVs carrying both the PD-1 receptor and miRNAs associated with T cell inhibition were present in RA cell cultures. Upon stimulation, these miRNAs failed to be upregulated in EVs from RA SFMCs. This was in line with increased expression of T cell co-inhibitory markers on SFMCs. In conclusion, we suggest EVs to play a significant role in the RA microenvironment, potentially favoring the progression of T cell exhaustion.

Soluble CD206 plasma levels in rheumatoid arthritis reflect decrease in disease activity.

Rheumatoid arthritis (RA) is characterized by chronic joint inflammation and infiltration by activated macrophages. TNFα is a central mediator in this process. The mannose receptor, CD206, is a scavenger receptor expressed by M2A-macrophages and dendritic cells. It is involved in collagen internalization and degradation. The soluble form has been suggested as a biomarker of M2A-macrophage activation. The aim of this study was to investigate sCD206 plasma levels in early RA patients initiating anti-TNFα treatment. Plasma levels of sCD206 were measured by ELISA in samples from 155 early RA patients with an average symptom duration of 3 months. Patients were randomized to 12 months' methotrexate and placebo (PLA) or methotrexate and adalimumab (ADA) treatment, followed by open-label treatment with disease-modifying anti-rheumatic drugs (DMARD) and if needed, ADA. Disease activity was assessed at baseline and after 3, 6, 12 and 24 months. Baseline plasma level of sCD206 in treatment naïve RA patients was 0.33 mg/L (CI: 0.33-0.38 mg/L) corresponding to the upper part of the reference interval for healthy controls (0.10-0.43 mg/L). In the PLA group, sCD206 levels decreased after 3 months, but did not differ from baseline after 6 months. In the ADA group, however, levels remained lower than baseline throughout the treatment period. In conclusion, initially, plasma sCD206 in early RA patients decreased in accordance with disease activity and initiation of DMARD treatment. Treatment with anti-TNFα preserved this decrease throughout the study period.

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.

Expression of soluble CD83 in plasma from early-stage rheumatoid arthritis patients is not modified by anti-TNF-α therapy.

Rheumatoid arthritis (RA) is an autoimmune disease which may lead to severe disabilities due to structural joint damage and extraarticular manifestations The dendritic cell marker CD83 belongs to the immunoglobulin superfamily and has previously been associated with autoimmune diseases. In RA the levels of soluble CD83 (sCD83) are elevated in synovial fluid, however little is known about CD83 expression and regulation in RA. Therefore, we studied how CD83 is expressed in RA and further evaluated the effect of anti-TNF-α therapy hereon. Early RA patients were randomized to conventional disease modifying anti-rheumatic drugs with or without additional anti-TNF-α therapy. Rheumatoid arthritis patients had increased levels of sCD83 in plasma compared with healthy volunteers. The increase in sCD83 plasma levels were unaffected by anti-TNF-α therapy. In chronic RA patients the levels of sCD83 were higher in synovial fluid than in plasma, and only a limited amount of membrane bound CD83 expression was detected on the surface of cells from peripheral blood and synovial fluid. Finally, confocal microscopy of RA synovial membranes revealed that CD83 was mainly localized intracellularly in a group of cells with diverse morphology including both antigen-presenting cells and non-antigen-presenting cells. Our findings demonstrate that early-stage RA patients have elevated levels of sCD83 in plasma and that anti-TNF-α treatment has no effect on the sCD83 plasma level. This suggest that in RA patients sCD83 regulation is beyond control of TNF-α.

The "Alarmins" HMBG1 and IL-33 Downregulate Structural Skin Barrier Proteins and Impair Epidermal Growth.

The epidermal-derived "alarmins" high-mobility group box 1 (HMGB1) protein and interleukin-33 (IL-33) are upregulated in patients with atopic dermatitis. How-ever, their capacity as pro-inflammatory cytokines and their derived effects on skin barrier regulation are poorly elucidated. We investigated the impact of HMGB1 and IL-33 on gene transcription, protein expression and epidermal differentiation across 3 distinct keratinocyte in vitro models. Primary keratinocytes from healthy donors were used in submerged monolayer cultures, 3D human epidermis equivalents and 3D human skin equivalents. All keratinocyte models underwent 96-h stimulation with HMGB1 (100 µM) or IL-33 (100 ng/ml) using IL-4 (50 ng/ml) as positive control of regulation and vehicle as negative control. We found that HMGB1 and IL-33 downregulated transcription of several genes from members of the epidermal differentiation complex, including filaggrin. Furthermore, HMGB1 downregulated the expression of the encoded proteins in the upper epidermis. Finally, IL-33 and HMGB1 ultimately led to impaired epidermal growth and maturation. In conclusion, HMGB1 and IL-33 could play a significant role in the atopic dermatitis pathophysiology due to negative regulation of structural proteins, stratum corneum formation and epidermal growth.