Identification of early risk factors for anti-citrullinated-protein-antibody positive rheumatoid arthritis-a prospective cohort study.

OBJECTIVE: Individuals positive for anti-cyclic-peptide-antibodies (anti-CCP) and musculoskeletal complaints (MSK-C) are at risk for developing rheumatoid arthritis (RA). In this study we aimed to investigate factors involved in arthritis progression. METHODS: Anti-CCP2-positive individuals with MSK-C referred to a rheumatologist were recruited. Individuals lacked arthritis at clinical and ultrasound examination and were followed for ≥three years or until clinical arthritis diagnosis. Blood samples from inclusion were analyzed for; nine anti-citrullinated-protein-antibody (ACPA) reactivities (citrullinated α-1-enolase, fibrinogen, filaggrin, histone, vimentin and tenascin peptides); 92 inflammation-associated proteins; and HLA-shared epitope alleles. Cox regression was applied to the data to identify independent predictors in a model. RESULTS: 267 individuals were included with median follow up of 49 months (IQR: 22-60). 101 (38%) developed arthritis after median 14 months (IQR: 6-27). The analysis identified that presence of at least one ACPA reactivity (HR 8.0, 95% CI 2.9-22), ultrasound detected tenosynovitis (HR 3.4, 95% CI 2.0-6.0), IL6 levels (HR 1.5, 95% CI 1.2-1.8) and IL15-Rα levels (HR 0.6, 95% CI 0.4-0.9) are significant independent predictors for arthritis progression in a prediction model (Harrell's C 0.76 [SE 0.02], AUC 0.82 [95% CI 0.76-0.89], cross-validated AUC 0.70 [95% CI 0.56-0.85]). CONCLUSION: We propose a high-Risk-RA phase characterized by presence of ACPA reactivity, tenosynovitis, IL6, and IL15-Rα and suggest that these factors need to be further investigated for their biological effects and clinical values, to identify individuals at particular low risk and high risk for arthritis progression.

Citrullination Controls Dendritic Cell Transdifferentiation into Osteoclasts.

An increased repertoire of potential osteoclast (OC) precursors could accelerate the development of bone-erosive OCs and the consequent bone damage in rheumatoid arthritis (RA). Immature dendritic cells (DCs) can develop into OCs, however, the mechanisms underlying this differentiation switch are poorly understood. We investigated whether protein citrullination and RA-specific anti-citrullinated protein Abs (ACPAs) could regulate human blood-derived DC-OC transdifferentiation. We show that plasticity toward the OC lineage correlated with peptidyl arginine deiminase (PAD) activity and protein citrullination in DCs. Citrullinated actin and vimentin were present in DCs and DC-derived OCs, and both proteins were deposited on the cell surface, colocalizing with ACPAs binding to the cells. ACPAs enhanced OC differentiation from monocyte-derived or circulating CD1c+ DCs by increasing the release of IL-8. Blocking IL-8 binding or the PAD enzymes completely abolished the stimulatory effect of ACPAs, whereas PAD inhibition reduced steady-state OC development, as well, suggesting an essential role for protein citrullination in DC-OC transdifferentiation. Protein citrullination and ACPA binding to immature DCs might thus promote differentiation plasticity toward the OC lineage, which can facilitate bone erosion in ACPA-positive RA.

Human Lymph Node Stromal Cells Have the Machinery to Regulate Peripheral Tolerance during Health and Rheumatoid Arthritis.

BACKGROUND: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. METHOD: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon γ (IFNγ) stimulation (n = 15). RESULTS: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. CONCLUSION: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance.

Anticitrullinated protein antibodies facilitate migration of synovial tissue-derived fibroblasts.

OBJECTIVES: Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology. METHODS: Fibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting. RESULTS: Challenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis. CONCLUSION: We propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration.

JAK/STAT1 signaling promotes HMGB1 hyperacetylation and nuclear translocation.

Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release.

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss.

OBJECTIVES: Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. METHODS: Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. RESULTS: Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. CONCLUSIONS: We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Rheumatoid Arthritis-Specific Autoimmunity in the Lung Before and at the Onset of Disease.

OBJECTIVE: The lung is implicated as a site for breach of tolerance prior to onset of seropositive rheumatoid arthritis (RA). To substantiate this, we investigated lung-resident B cells in bronchoalveolar lavage (BAL) samples from untreated early RA patients and anti-citrullinated protein antibody (ACPA)-positive individuals at risk for developing RA. METHODS: Single B cells (n = 7,680) were phenotyped and isolated from BAL samples from individuals at risk of RA (n = 3) and at RA diagnosis (n = 9). The immunoglobulin variable region transcripts were sequenced and selected for expression as monoclonal antibodies (n = 141). Monoclonal ACPAs were tested for reactivity patterns and binding to neutrophils. RESULTS: Using our single-cell approach, we found significantly increased proportions of B lymphocytes in ACPA+ compared to ACPA- individuals. Memory and double-negative B cells were prominent in all subgroups. Upon antibody re-expression, 7 highly mutated citrulline-autoreactive clones originating from different memory B cell subsets were identified, both in individuals at risk of RA and early RA patients. Lung IgG variable gene transcripts from ACPA+ individuals carried frequent mutation-induced N-linked Fab glycosylation sites (P < 0.001), often in the framework 3 of the variable region. Two of the lung ACPAs bound to activated neutrophils, 1 from an individual at risk of RA and 1 from an early RA patient. CONCLUSION: T cell-driven B cell differentiation resulting in local class switching and somatic hypermutation are evident in lungs before as well as in early stages of ACPA+ RA. Our findings add to the notion of lung mucosa being a site for initiation of citrulline autoimmunity preceding seropositive RA.

Anti-Citrullinated Protein Antibody Reactivity towards Neutrophil-Derived Antigens: Clonal Diversity and Inter-Individual Variation.

BACKGROUND: Why the adaptive immune system turns against citrullinated antigens in rheumatoid arthritis (RA) and whether anti-citrullinated protein antibodies (ACPAs) contribute to pathogenesis are questions that have triggered intense research, but still are not fully answered. Neutrophils may be crucial in this context, both as sources of citrullinated antigens and also as targets of ACPAs. To better understand how ACPAs and neutrophils contribute to RA, we studied the reactivity of a broad spectrum of RA patient-derived ACPA clones to activated or resting neutrophils, and we also compared neutrophil binding using polyclonal ACPAs from different patients. METHODS: Neutrophils were activated by Ca2+ ionophore, PMA, nigericin, zymosan or IL-8, and ACPA binding was studied using flow cytometry and confocal microscopy. The roles of PAD2 and PAD4 were studied using PAD-deficient mice or the PAD4 inhibitor BMS-P5. RESULTS: ACPAs broadly targeted NET-like structures, but did not bind to intact cells or influence NETosis. We observed high clonal diversity in ACPA binding to neutrophil-derived antigens. PAD2 was dispensable, but most ACPA clones required PAD4 for neutrophil binding. Using ACPA preparations from different patients, we observed high patient-to-patient variability in targeting neutrophil-derived antigens and similarly in another cellular effect of ACPAs, the stimulation of osteoclast differentiation. CONCLUSIONS: Neutrophils can be important sources of citrullinated antigens under conditions that lead to PAD4 activation, NETosis and the extrusion of intracellular material. A substantial clonal diversity in targeting neutrophils and a high variability among individuals in neutrophil binding and osteoclast stimulation suggest that ACPAs may influence RA-related symptoms with high patient-to-patient variability.

An Image-based Dynamic High-throughput Analysis of Adherent Cell Migration.

In this protocol, we describe a method to monitor cell migration by live-cell imaging of adherent cells. Scratching assay is a common method to investigate cell migration or wound healing capacity. However, achieving homogenous scratching, finding the optimal time window for end-point analysis and performing an objective image analysis imply, even for practiced and adept experimenters, a high chance for variability and limited reproducibility. Therefore, our protocol implemented the assessment for cell mobility by using homogenous wound making, sequential imaging and automated image analysis. Cells were cultured in 96-well plates, and after attachment, homogeneous linear scratches were made using the IncuCyte ® WoundMaker. The treatments were added directly to wells and images were captured every 2 hours automatically. Thereafter, the images were processed by defining a scratching mask and a cell confluence mask using a software algorithm. Data analysis was performed using the IncuCyte ® Cell Migration Analysis Software. Thus, our protocol allows a time-lapse analysis of treatment effects on cell migration in a highly reliable, reproducible and re-analyzable manner.

Immunomodulatory drugs regulate HMGB1 release from activated human monocytes.

Several HMGB1-specific antagonists have provided beneficial results in multiple models of inflammatory disease-preclinical trials including arthritis. Since no HMGB1-specific targeted therapy has yet reached the clinic, we have performed in vitro studies to investigate whether any of a selection of well-established antirheumatic drugs inhibit HMGB1 release as part of its mode of action. Freshly purified peripheral blood monocytes from healthy donors were stimulated in cultures with LPS and IFNγ to cause HMGB1 and TNF release detected in ELISPOT assays. Effects on the secretion were assessed in cultures supplemented with dexamethasone, cortisone, chloroquine, gold sodium thiomalate, methotrexate, colchicine, etanercept or anakinra. Pharmacologically relevant doses of dexamethasone, gold sodium thiomalate and chloroquine inhibited the extracellular release of HMGB1 in a dose-dependent mode. Immunostaining demonstrated that dexamethasone caused intracellular HMGB1 retention. No effects on HMGB1 secretion were observed in cultures with activated monocytes by any of the other studied agents. TNF production in LPS/IFNγ-activated monocytes was readily downregulated by dexamethasone and, to some extent, by chloroquine and etanercept. We conclude that dexamethasone, gold sodium thiomalate and chloroquine share a capacity to inhibit HMGB1 release from activated monocytes.

Extracellular miR-574-5p Induces Osteoclast Differentiation via TLR 7/8 in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and joint destruction. Cell-derived small extracellular vesicles (sEV) mediate cell-to-cell communication in the synovial microenvironment by carrying microRNAs (miRs), a class of small non-coding RNAs. Herein, we report that sEV from synovial fluid promote osteoclast differentiation which is attributed to high levels of extracellular miR-574-5p. Moreover, we demonstrate for the first time that enhanced osteoclast maturation is mediated by Toll-like receptor (TLR) 7/8 signaling which is activated by miR-574-5p binding. This is a novel mechanism by which sEV and miRs contribute to RA pathogenesis and indicate that pharmacological inhibition of extracellular miR-574-5p might offer new therapeutic strategies to protect osteoclast-mediated bone destruction in RA.

Development and formative evaluation of patient research partner involvement in a multi-disciplinary European translational research project.

PLAIN ENGLISH SUMMARY: Patient and public involvement (PPI) improves the quality of health research and ensures that research is relevant to patients' needs. Though PPI is increasingly evident in clinical and health services research, there are few examples in the research literature of effective PPI in translational and laboratory-based research. In this paper, we describe the development and evaluation of PPI in a multi-centre European project (EuroTEAM - Towards Early biomarkers in Arthritis Management) that included both translational and laboratory-based and psychosocial research. We found that although most PPI in EuroTEAM was centred around the psychosocial research, there were examples of PPI in the laboratory studies. As the project evolved, researchers became better at accommodating PPI and identifying PPI opportunities. It was generally agreed that PPI had a positive impact on the project overall, particularly on public engagement with the research. We concluded that the inclusion of both psychosocial and laboratory-based research in the same project facilitated PPI across all aspects of the research. In future projects, we would try to specify individual PPI activities in more detail at the project-planning stage, and better accommodate patient partners who are not native speakers of English. ABSTRACT: Background Patient and public involvement (PPI) enhances research quality and relevance and is central to contemporary health policy. The value of PPI has been recognised in rheumatology research, though there are limited examples of PPI in basic and translational science. The EU FP7 funded 'EuroTEAM' (Towards Early biomarkers in Arthritis Management) project was established to develop biomarker-based approaches to predict the future development of rheumatoid arthritis and incorporated psychosocial research to investigate the perceptions of 'at risk' individuals about predictive testing, and to develop informational resources about rheumatoid arthritis (RA) risk. Patient involvement was central to EuroTEAM from the inception of the project. The objective of this paper is to describe the development of PPI in EuroTEAM, formatively assess the impact of PPI from the perspectives of researchers and patient research partners (PRPs), reflect on successes and lessons learned, and formulate recommendations to guide future projects.Methods Two mixed-methods surveys (for PRPs and researchers) and a teleconference were undertaken to assess the impact of PPI on individual work packages and on EuroTEAM overall.Results There was consensus about the positive impact of PPI on the research and on the experiences of those involved. In particular, the positive impact of PPI on the personal development of researchers, and on effective public engagement with EuroTEAM research were highlighted. Researchers described adapting their practice in future projects to facilitate PPI. Spin-off projects and ongoing collaborations between PRPs and researchers reflected the value of PPI to participants. PPI was more frequently integrated in psychosocial research, though examples of PPI in laboratory/translational science were also described. PRPs asked for more opportunities to contribute meaningfully to basic scientific research and for more extensive feedback on their contributions.Conclusions The findings were used to formulate recommendations to guide effective involvement of patients in future similar projects, including identifying specific training requirements for PRPs and researchers, the identification of PRP focused tasks/deliverables at the project planning stage, and supporting access to involvement for all PRPs. Importantly, the distinctive multidisciplinary approach of EuroTEAM, incorporating both basic science and psychosocial research, facilitated patient involvement in the project overall.

Pivotal advance: inhibition of HMGB1 nuclear translocation as a mechanism for the anti-rheumatic effects of gold sodium thiomalate.

Gold compounds such as gold sodium thiomalate (GST) can reduce the symptoms of rheumatoid arthritis (RA), although their mechanism of action is not well defined. As the proinflammatory mediator high mobility group box chromosomal protein 1 (HMGB1) may play a role in the pathogenesis of RA, we have performed in vitro studies to investigate whether GST inhibits HMGB1 release as the basis of its mode of action. Murine RAW 264.7 or human THP-1 macrophage cells were stimulated in culture with agents causing extracellular HMGB1 release, including LPS, IFN-gamma, polyinosinic:polycytidylic acid, IFN-beta, or NO in the presence of GST, ranging from 0 microM to 250 microM. Secretion and intracellular location of HMGB1 were assessed by Western blotting, HMGB1-specific ELISPOT assay, and immunofluorescent staining. In parallel, TNF and IFN-beta levels were analyzed by ELISPOT and/or ELISA. Supernatant NO production was analyzed by the Griess method. At pharmacologically relevant doses, GST inhibited the extracellular release of HMGB1 from activated macrophages and caused the nuclear retention of this protein; in contrast, no effects were observed on the secretion or production of TNF. Release of the key endogenous mediators of HMGB1 translocation, IFN-beta and NO, was inhibited by GST. This inhibition required gold, as sodium thiomalate did not affect the responses measured. Furthermore, gold chloride also inhibited release of HMGB1. Together, these results suggest a new mechanism for the anti-rheumatic effects of gold salts in RA and the potential of drugs, which interfere with intracellular HMGB1 transport mechanisms, as novel agents to treat RA.

HMGB1-secreting capacity of multiple cell lineages revealed by a novel HMGB1 ELISPOT assay.

High mobility group box protein 1 (HMGB1) exerts different biological functions dependent on its cellular localization. Nuclear HMGB1 maintains chromatin architecture and is required for undisturbed transcription activity, and extracellularly released HMGB1 mediates inflammation and tissue regeneration. A present paucity of readily accessible methods to quantify released HMGB1 represents a problem concerning the exploration of HMGB1 biology. We have now developed a HMGB1-specific ELISPOT assay enabling enumeration of individual HMGB1-releasing cells. The method also allows automated, semiquantitative assessment of released HMGB1 by evaluating areas of single HMGB1 spots. Actively secreted HMGB1 as well as cells passively releasing the protein following necrotic cell death are visualized distinctly using this ELISPOT assay. Kinetics of HMGB1 secretion after different stimuli was studied using cell lines of various lineages. IFN-gamma already induced substantial HMGB1 secretion from the monocytic cell line RAW 264.7 within 24 h and even more so after 48 h. LPS only stimulated a modest HMGB1 release within 24 h, but this increased considerably by 48 h. TNF-induced HMGB1 release was unexpectedly low. Mast cells, which share the secretory, lysosomal pathway with macrophages/monocytes, did not secrete HMGB1 in response to any studied mode of activation. Most transformed cells overexpress HMGB1, but the ELISPOT assay revealed that all transformed cell lines will not actively secrete the protein. We believe the ELISPOT method provides a novel tool to study pathways promoting or inhibiting HMGB1 secretion.

Stabilized Cyclic Peptides as Scavengers of Autoantibodies: Neutralization of Anticitrullinated Protein/Peptide Antibodies in Rheumatoid Arthritis.

The occurrence of autoantibodies is a hallmark of rheumatoid arthritis, specifically those autoantibodies targeting proteins containing the arginine-derived amino acid citrulline. There is strong evidence showing that the occurrence of anticitrullinated protein/peptide antibodies (ACPA) are involved in disease progression, and ACPA was recently shown to induce pain in animals. Here, we explore a novel concept useful for research, diagnostics, and possibly therapy of autoimmune diseases, namely, to directly target and neutralize autoantibodies using peptide binders. A high-affinity peptide-based scavenger of ACPA was developed by grafting a citrullinated epitope derived from human fibrinogen into a naturally occurring stable peptide scaffold. The best scavenger comprises the truncated epitope α-fibrinogen, [Cit573]fib(566-580), grafted into the scaffold sunflower trypsin inhibitor-1, SFTI-1. The final peptide demonstrates low nanomolar apparent affinity and superior stability.

Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by synovial joint inflammation and by development of pathogenic humoral and cellular autoimmunity to citrullinated proteins. Neutrophil extracellular traps (NETs) are a source of citrullinated autoantigens and activate RA synovial fibroblasts (FLS), cells crucial in joint damage. We investigated the molecular mechanisms by which NETs promote proinflammatory phenotypes in FLS, and whether these interactions generate pathogenic anti-citrulline adaptive immune responses. NETs containing citrullinated peptides are internalized by FLS through a RAGE-TLR9 pathway promoting FLS inflammatory phenotype and their upregulation of MHC class II. Once internalized, arthritogenic NET-peptides are loaded into FLS MHC class II and presented to Ag-specific T cells. HLADRB1*0401 transgenic mice immunized with mouse FLS loaded with NETs develop antibodies specific to citrullinated forms of relevant RA autoantigens implicated in RA pathogenesis as well as cartilage damage. These results implicate FLS as mediators in RA pathogenesis, through the internalization and presentation of NET citrullinated peptides to the adaptive immune system leading to pathogenic autoimmunity and cartilage damage.

High mobility group box protein 1 in complex with lipopolysaccharide or IL-1 promotes an increased inflammatory phenotype in synovial fibroblasts.

INTRODUCTION: In addition to its direct proinflammatory activity, extracellular high mobility group box protein 1 (HMGB1) can strongly enhance the cytokine response evoked by other proinflammatory molecules, such as lipopolysaccharide (LPS), CpG-DNA and IL-1ß, through the formation of complexes. Extracellular HMGB1 is abundant in arthritic joint tissue where it is suggested to promote inflammation as intra-articular injections of HMGB1 induce synovitis in mice and HMGB1 neutralizing therapy suppresses development of experimental arthritis. The aim of this study was to determine whether HMGB1 in complex with LPS, interleukin (IL)-1α or IL-1ß has enhancing effects on the production of proinflammatory mediators by rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). Furthermore, we examined the toll-like receptor (TLR) 4 and IL-1RI requirement for the cytokine-enhancing effects of the investigated HMGB1-ligand complexes. METHODS: Synovial fibroblasts obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were stimulated with HMGB1 alone or in complex with LPS, IL-1α or IL-1ß. Tumour necrosis factor (TNF) production was determined by enzyme-linked immunospot assay (ELISPOT) assessment. Levels of IL-10, IL-1-ß, IL-6 and IL-8 were measured using Cytokine Bead Array and matrix metalloproteinase (MMP) 3 production was determined by ELISA. RESULTS: Stimulation with HMGB1 in complex with LPS, IL-1α or IL-1ß enhanced production of TNF, IL-6 and IL-8. HMGB1 in complex with IL-1ß increased MMP production from both RASF and OASF. The cytokine production was inhibited by specific receptor blockade using detoxified LPS or IL-1 receptor antagonist, indicating that the synergistic effects were mediated through the partner ligand-reciprocal receptors TLR4 and IL-1RI, respectively. CONCLUSIONS: HMGB1 in complex with LPS, IL-1α or IL-1ß boosted proinflammatory cytokine- and MMP production in synovial fibroblasts from RA and OA patients. A mechanism for the pathogenic role of HMGB1 in arthritis could thus be through enhancement of inflammatory and destructive mechanisms induced by other proinflammatory mediators present in the arthritic joint.

The alarmin HMGB1 acts in synergy with endogenous and exogenous danger signals to promote inflammation.

The nuclear protein HMGB1 has previously been demonstrated to act as an alarmin and to promote inflammation upon extracellular release, yet its mode of action is still not well defined. Access to highly purified HMGB1 preparations from prokaryotic and eukaryotic sources enabled studies of activation of human PBMC or synovial fibroblast cultures in response to HMGB1 alone or after binding to cofactors. HMGB1 on its own could not induce detectable IL-6 production. However, strong enhancing effects on induction of proinflammatory cytokine production occurred when the protein associated with each of the separate proinflammatory molecules, rhIL-1beta, the TLR4 ligand LPS, the TLR9 ligand CpG-ODN, or the TLR1-TLR2 ligand Pam3CSK4. The bioactivities were recorded in cocultures with preformed HMGB1 complexes but not after sequential or simultaneous addition of HMGB1 and the individual ligands. Individual A-box and B-box domains of HMGB1 had the ability to bind LPS and enhance IL-6 production. Heat denaturation of HMGB1 eliminated this enhancement. Cocultures with HMGB1 and other proinflammatory molecules such as TNF, RANKL, or IL-18 did not induce enhancement. HMGB1 thus acts broadly with many but not all immunostimulatory molecules to amplify their activity in a synergistic manner.

Oxaliplatin retains HMGB1 intranuclearly and ameliorates collagen type II-induced arthritis.

INTRODUCTION: High mobility group box chromosomal protein 1 (HMGB1) is a nuclear protein that acts as a pro-inflammatory mediator following extracellular release. The protein is aberrantly expressed extracellularly in the settings of clinical and experimental synovitis. Therapy based on HMGB1 antagonists has shown encouraging results in experimental arthritis and warrants further scientific exploration using independent methods. In the present study we asked whether nuclear sequestration of HMGB1 preventing HMGB1 release would be beneficial for synovitis treatment. METHODS: Oxaliplatin-based therapy was evaluated in collagen type II-induced arthritis in DBA/1 mice by clinical scoring and immunostaining of articular tissue. Oxaliplatin is an antineoplastic platinum-based compound that generates DNA adducts which tightly bind HMGB1. Secretion and intracellular location of HMGB1 were assessed by a novel HMGB1-specific ELISPOT assay and immunofluorescent staining. RESULTS: Intraperitoneal injections of oxaliplatin in early collagen type II-induced arthritis trapped HMGB1 with a distinct biphasic response pattern. Oxaliplatin therapy showed beneficial results for approximately 1 week. Microscopic evaluation of synovitis during this period showed strong nuclear HMGB1 staining in the oxaliplatin treated animals with much lower quantities of extracellular HMGB1 when compared to control treated animals. Furthermore, cellular infiltration, as well as cartilage and bone damage, were all reduced in the oxaliplatin treated group. A dramatic and as yet unexplained clinical relapse occurred later in the oxaliplatin exposed animals, which coincided with a massive synovial tissue expression of extracellular HMGB1 in all treated animals. This rebound-like reaction was also accompanied by a significantly increased incidence of arthritis in the oxaliplatin treated group. These results indicate a distinct temporal and spatial relationship between the clinical course of disease and the cellular localization of HMGB1. Beneficial effects were noted when extracellular HMGB1 expression was low, while severe inflammation coincided with substantial extracellular synovial HMGB1 expression. CONCLUSION: Therapeutic compounds like oxaliplatin and gold salts share a capacity to inhibit nuclear HMGB1 release and to ameliorate the course of synovial inflammation. These observations support the hypothesis that HMGB1 plays an important functional role in the pathogenesis of arthritis and may represent a novel target molecule for therapy.