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

Inflammation can be infectious and/or sterile depending on the initiating event. The proinflammatory mediator High mobility group box protein 1 (HMGB1) is a nuclear protein released from cells during both sterile and infectious inflammation and once extracellular, initates and potentiates inflammation by inducing cytokine production and by recruiting inflammatory cells. Autoimmune diseases are characterised by chronic sterile inflammation leading to tissue destruction. HMGB1 has been implicated in the pathogenesis of several autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosis, multiple sclerosis and myositis. The involvement of HMGB1 in arthritis has been shown by overexpression of HMGB1 in RA synovial tissue and synovial fluid, by beneficial outcome of therapeutic HMGB1-blockade in several experimental arthritis models and by the induction of arthritis by intra-articular injection of recombinant HMGB1 into mice. In this thesis work I set out to investigate the potential role of HMGB1 in juvenile idiopathic arthritis (JIA), to further delineate mechanisms by which HMGB1 can contribute to arthritis pathogenesis and to study the means by which HMGB1 activity can be suppressed. I could report for the first time that HMGB1 levels were increased in synovial fluid as compared to plasma during JIA. HMGB1 levels in synovial fluid did not correlate to disease duration. In contrast, the recorded levels of IL-8 and S100 proteins were higher in synovial fluid during early phases of disease. This indicates a change in the inflammatory phenotype during the progression of JIA. High HMGB1 levels in synovial fluid correlated with early JIA onset, suggesting differences in immunopathogenesis between patient groups. I have also demonstrated that HMGB1 may form complexes with the exogenous TLR ligand LPS or the endogenous inflammatory mediators IL-1α and IL-1β, respectively. Compared to each mediator alone such complexes stimulated synovial fibroblasts from arthritis patients to enhanced production of cytokines and tissue degrading enzymes. This enhancement is mediated via the reciprocal receptor for each HMGB1-partner molecule. Since all the studied mediators are present in arthritic joint during inflammation, this is a potential mechanism through which HMGB1 enhances ongoing inflammation and destruction during rheumatic diseases. Finally, I have demonstrated that the proinflammatory activity of HMGB1 can be therapeutically targeted, either by inhibiting its active release by clinically approved anti-rheumatic drugs or by neutralization with a HMGB1-specific monoclonal antibody. Extracellular secretion of HMGB1 from LPS+IFN-γ stimulated human primary monocytes was inhibited by dexamethasone, chloroquine and gold sodium thiomalate in vitro as recorded using an ELISPOT assay. Therapeutic administration of an HMGB1-specific HMGB1 monoclonal antibody ameliorated arthritis in two separate experimental models. In conclusion, my thesis work has added to the growing evidence that HMGB1 is involved in the pathogenesis of arthritis, has revealed a potential mechanism for its proinflammatory function and has demonstrated a means by which HMGB1-mediated activities can be counteracted

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

collagen type II-induced arthriti

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

HMGB1 in inflammation : Secretion and function

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by progressive joint destruction. The inflammatory and joint destructive processes in RA are mediated by resident synovial cells and cells recruited from the blood stream and bone marrow. A variety of cytokines, chemokines and proteases contribute to the cartilage and bone destruction. High mobility group box chromosomal protein 1 (HMGB1) was discovered over three decades ago as a transcription-regulating protein. In addition to its nuclear role, HMGB1 expression was detected at the leading edges in motile cells and its active secretion from immune cells was demonstrated. An excessive HMGB1 expression has been recorded in arthritic joints and in synovial fluid from RA patients and HMGB1-blocking therapies have been demonstrated to attenuate the disease course in murine arthritis models, suggesting that HMGB1 is a key player in arthritis. The focus of this thesis work has been to further the understanding of HMGB1 as an inflammatory mediator and its role in arthritis. More specifically, I have studied the induction of HMGB1 secretion from a variety of inflammatory cells, how HMGB1 blockade affects the proinflammatory cytokine pattern in cell cultures and how HMGB1-targeting therapy affects the disease development in collagen-induced arthritis (CIA). Finally, I have also studied the inflammation-inducing capacity of HMGB1 alone and in complex with other proinflammatory molecules. In order to quantify HMGB1 secretion from different cell types an HMGB1-specific ELIspot method was developed. We could demonstrate that HMGB1 was secreted from macrophage/monocytic cells during inflammatory conditions and that the secretion could be inhibited by gold salts and oxaliplatin treatment as detected by ELIspot. We could demonstrate that oxaliplatin-treatment attenuated disease development in murine CIA. A rebound effect with severe and aggressive disease course was demonstrated after one week of treatment which correlated with an excessive extranuclear HMGB1 pattern in the affected joints, indicating an HMGB1-mediated joint inflammation. Furthermore, we have demonstrated in vitro that the proinflammatory activity of HMGB1 is dependent on complex formation between HMGB1 and other inflammation-promoting molecules, such as IL-1beta, LPS and CpG-DNA. Studies using synovial fibroblasts obtained from arthritis patients demonstrated enhanced induction of TNF, IL-6 and IL-8 production and an enchased production of matrix metalloproteinase-1 and -3 when stimulated with HMGB1 in complex with IL-1beta or LPS as compared to either substance alone. Thus, these results suggest that HMGB1 in complex with IL-1beta or LPS can mediate both inflammation and destruction in RA. In conclusion, the studies presented in this thesis strengthen the view of HMGB1 as an inflammation- and destruction-promoting molecule. I have demonstrated HMGB1 secretion from cell types present in the arthritic joint, defined two therapies in clinical use which have the potential to block HMGB1 secretion and verified the anti-rheumatic effect of one of these therapies in murine CIA. By in vitro studies, I have extended the knowledge of the proinflammatory features of HMGB1 and demonstrated both a proinflammatory and prodestructive effect of HMGB1 on synovial fibroblasts. Taken together, the studies in this thesis suggest that HMGB1 is one of the key mediators of arthritic inflammation and joint destruction

The novel inflammatory cytokine high mobility group box protein 1 (HMGB1) is expressed by human term placenta

High mobility group box protein 1 (HMGB1) was previously considered a strict nuclear protein, but lately data are accumulating on its extranuclear functions. In addition to its potent proinflammatory capacities, HMGB1 has a prominent role in a number of processes of specific interest for the placenta. Our overall aim was to investigate the expression of HMGB1 in human term placenta and elucidate a potential difference in HMGB1 expression comparing vaginal deliveries with elective Caesarean sections. In addition, placentas from normal pregnancies were compared with placentas from pregnancies complicated by pre-eclampsia. Twenty-five placentas, 12 from normal term pregnancies and 13 from pregnancies complicated by pre-eclampsia were analysed with immunohistochemistry for HMGB1 and its putative receptors; receptor for advanced glycation end-products (RAGE), Toll-like receptor 2 (TLR2) and TLR4. We present the novel finding that in addition to a strong nuclear HMGB1 expression in almost all cells in investigated placentas, an individual variation of cytoplasmic HMGB1 expression was detected in the syncytiotrophoblast covering the peripheral chorionic villi, by cells in the decidua and in amnion. Production of HMGB1 was confirmed by in situ hybridization. Although labour can be described as a controlled inflammatory-like process no differences in HMGB1 expression could be observed comparing active labour and elective Caesarean sections. However, a tendency towards a higher expression of cytoplasmic HMGB1 in the decidua from women with pre-eclampsia was demonstrated. The abundant expression of the receptors RAGE, TLR2 and TLR4 implicates a local capability to respond to HMGB1, although the precise role in the placenta remains to be elucidated

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

Animals treated with oxaliplatin demonstrated reduced cell infiltration, cartilage and bone destruction as well as less cytosolic and extracellular HMGB1

Oxaliplatin treated mice (= 8 with a mean clinical score of 1.0) and mice given vehicle alone (= 8 with a mean clinical score of 4.8) were killed on day 36 PI and intra-articular effects of oxaliplatin were evaluated by immunohistochemistry. Cell infiltration , cartilage and bone destruction were all significantly lower in oxaliplatin treated mice. Representative micrographs illustrating HMGB1 staining (in brown) in synovial tissue where an abundant cytosolic and extracellular HMGB1 staining was evident in mice treated with control vehicle as compared to the low extranuclear HMGB1 expression in oxaliplatin treated mice . Sequential sections stained with Safranin O demonstrating reduced proteoglycan content in articular cartilage in the control than in oxaliplatin treated animals . Signs of pronounced articular destructions are more evident in control treated animals, where destained cartilage layers reflected loss of matrix proteoglycans (see arrow). No cartilage destruction was detected in oxaliplatin treated mice indicated by a homogenous cartilage staining. CI, cell infiltration, JC, joint cavity, B, bone, C, cartilage. The boxes represent 25th to 75th percentiles and the lines inside the boxes stand for the median. The lines outside the boxes reflect 10th and 90th percentiles and circles indicate outliers. p = 0.05. Animals: control = 8, oxaliplatin = 8. Paws: control = 32, oxaliplatin = 32.<p><b>Copyright information:</b></p><p>Taken from "Oxaliplatin retains HMGB1 intranuclearly and ameliorates collagen type II-induced arthritis"</p><p>http://arthritis-research.com/content/10/1/R1</p><p>Arthritis Research & Therapy 2008;10(1):R1-R1.</p><p>Published online 7 Jan 2008</p><p>PMCID:PMC2374449.</p><p></p