Ulrike Harre

Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin

Autoimmunity is complicated by bone loss. In human rheumatoid arthritis (RA), the most severe inflammatory joint disease, autoantibodies against citrullinated proteins are among the strongest risk factors for bone destruction. We therefore hypothesized that these autoantibodies directly influence bone metabolism. Here, we found a strong and specific association between autoantibodies against citrullinated proteins and serum markers for osteoclast-mediated bone resorption in RA patients. Moreover, human osteoclasts expressed enzymes eliciting protein citrullination, and specific N-terminal citrullination of vimentin was induced during osteoclast differentiation. Affinity-purified human autoantibodies against mutated citrullinated vimentin (MCV) not only bound to osteoclast surfaces, but also led to robust induction of osteoclastogenesis and bone-resorptive activity. Adoptive transfer of purified human MCV autoantibodies into mice induced osteopenia and increased osteoclastogenesis. This effect was based on the inducible release of TNF-α from osteoclast precursors and the subsequent increase of osteoclast precursor cell numbers with enhanced expression of activation and growth factor receptors. Our data thus suggest that autoantibody formation in response to citrullinated vimentin directly induces bone loss, providing a link between the adaptive immune system and bone.

Bone loss before the clinical onset of rheumatoid arthritis in subjects with anticitrullinated protein antibodies

Objective Anticitrullinated protein antibodies (ACPA) are a major risk factor for bone loss in rheumatoid arthritis (RA). We have recently shown that ACPA directly induce bone loss by stimulating osteoclast differentiation. As ACPA precede the clinical onset of RA by years, we hypothesised that ACPA positive healthy individuals may already show skeletal changes. Methods We performed a comparative micro-CT analysis of the bone microstructure in the metacarpophalangeal joints of ACPA positive and ACPA negative healthy individuals without clinical signs of arthritis. Results ACPA positive (n=15) and negative (n=15) healthy individuals were not different in age (48.2±4.1 vs 51.4±3.8 years, p=0.57) or gender (eight women and two men in both groups). Bone mineral density was significantly reduced in ACPA positive individuals (mean±SEM 280±11 mg/cm3) compared with controls (327±6). Bone loss was based on cortical bone changes, with significant (p=0.044) reduction in cortical thickness in the ACPA positive group (mean±SEM 0.22±0.03 mm) compared with controls (0.32±0.03 mm). Areas of cortical porosity were significantly (p=0.0005) more widespread in ACPA positive (mean±SEM 7.4±1.4%) than in ACPA negative individuals (1.0±0.3%). Discussion Structural bone damage starts before the clinical onset of arthritis in subjects with ACPA. These findings revise the concept that bone damage is an exclusive consequence of synovitis in patients with RA.

Glycosylation of immunoglobulin G determines osteoclast differentiation and bone loss

Immunglobulin G (IgG) sialylation represents a key checkpoint that determines the engagement of pro- or anti-inflammatory Fcγ receptors (FcγR) and the direction of the immune response. Whether IgG sialylation influences osteoclast differentiation and subsequently bone architecture has not been determined yet, but may represent an important link between immune activation and bone loss. Here we demonstrate that desialylated, but not sialylated, immune complexes enhance osteoclastogenesis in vitro and in vivo. Furthermore, we find that the Fc sialylation state of random IgG and specific IgG autoantibodies determines bone architecture in patients with rheumatoid arthritis. In accordance with these findings, mice treated with the sialic acid precursor N-acetylmannosamine (ManNAc), which results in increased IgG sialylation, are less susceptible to inflammatory bone loss. Taken together, our findings provide a novel mechanism by which immune responses influence the human skeleton and an innovative treatment approach to inhibit immune-mediated bone loss.

Regulation of autoantibody activity by the IL-23-TH17 axis determines the onset of autoimmune disease

The checkpoints and mechanisms that contribute to autoantibody-driven disease are as yet incompletely understood. Here we identified the axis of interleukin 23 (IL-23) and the TH17 subset of helper T cells as a decisive factor that controlled the intrinsic inflammatory activity of autoantibodies and triggered the clinical onset of autoimmune arthritis. By instructing B cells in an IL-22- and IL-21-dependent manner, TH17 cells regulated the expression of β-galactoside α2,6-sialyltransferase 1 in newly differentiating antibody-producing cells and determined the glycosylation profile and activity of immunoglobulin G (IgG) produced by the plasma cells that subsequently emerged. Asymptomatic humans with rheumatoid arthritis (RA)-specific autoantibodies showed identical changes in the activity and glycosylation of autoreactive IgG antibodies before shifting to the inflammatory phase of RA; thus, our results identify an IL-23–TH17 cell–dependent pathway that controls autoantibody activity and unmasks a preexisting breach in immunotolerance.

Moonlighting osteoclasts as undertakers of apoptotic cells.

Rapid clearance of apoptotic cells, frequently referred to as efferocytosis, is crucial for the maintenance of tissue homeostasis and the prevention of autoimmunity. The common model of apoptotic cell clearance involves a system of released “Find me” and exposed “Eat me” signals on apoptotic cells, detected and recognized by matching receptors on macrophages or dendritic cells (DC), referred to as the phagocytic synapse. Osteoclasts share the monocyte lineage with these professional mononuclear phagocytes, thus raising the question if, in addition to bone resorption, osteoclasts can act as scavengers for apoptotic cells. Our qPCR data clearly show that osteoclasts express most of the genes required for dying cell clearance at mRNA levels similar to or even higher than those observed in M1-macrophages, M2-macrophages or DC. Our microscopical analyses reveal that osteoclasts in fact can bind and/or engulf apoptotic cells in an essentially serum-independent fashion. Together with our data on the abundance of the respective mRNAs, these results identify the vitronectin receptor (integrin ανβ3)/milk fat globule-EGF factor 8 protein (MFG-E8) axis, the scavenger receptors (CD36, CD68 and class A macrophage scavenger receptor (SR-A)), the complement/complement receptor axis, the Mer/Tyro3/Protein S axis, and the phosphatidylserine (PS) receptor brain-specific angiogenesis inhibitor 1 (BAI1) as the most promising candidates to be involved in osteoclast-mediated efferocytosis.

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines. Methods To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays. Results Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays. Conclusion Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.

ACPA and Bone Loss in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by bone loss. Degree of inflammation and autoantibody positivity have both been identified as important initiators of skeletal damage in RA. Whereas it is well appreciated that inflammation initiates bone loss via the action of cytokines, the effect of autoantibodies in initiating bone destruction has long been underestimated. It has, nonetheless, been known for years that antibodies against citrullinated proteins (ACPA) and rheumatoid factor are associated with a more destructive disease course. It was recently shown that ACPA bind osteoclast precursor cells and directly promote their differentiation into bone-resorbing osteoclasts. Other results have shown that in ACPA-positive individuals bone loss starts even before the onset of clinical disease; this is indicative of the independent effect of these antibodies in initiating skeletal damage. The observation that the mere presence of ACPA is associated with pathological changes suggests that these antibodies have functional properties and initiate the onset of disease long before patients consult the rheumatologist because of symptomatic joint disease. These findings also indicate that “RA” is a syndrome rather than a single disease, suggesting that autoantibody-positive patients are both genetically and clinically distinct phenotypes.

Autoantibody-Mediated Bone Loss

In rheumatoid arthritis (RA), the presence of autoantibodies such as the rheumatoid factor and antibodies against citrullinated proteins is highly correlated with the severity of disease and bone loss. For many years, the involvement of autoantibodies in bone resorption has merely been attributed to enhanced tissue infiltration and the production of inflammatory cytokines that promote osteoclastogenesis. However, recent research provides evidence for a direct activation of osteoclasts and their precursors by autoantibodies, which is independent of inflammation. The depletion of B-cells with rituximab that substantially reduces autoantibody levels seems to be as effective as the well-established treatment with tumor necrosis factor-antagonists in RA patients that do not respond to methotrexate, highlighting the significance of autoantibodies for RA and bone loss.

Cellular and molecular pathways of structural damage in rheumatoid arthritis

Structural damage of cartilage and bone tissue is a hallmark of rheumatoid arthritis (RA). The resulting joint destruction constitutes one of the major disease consequences for patients and creates a significant burden for the society. The main cells executing bone and cartilage degradation are osteoclasts and fibroblast-like synoviocytes, respectively. The function of both cell types is heavily influenced by the immune system. In the last decades, research has identified several mediators of structural damage, ranging from infiltrating immune cells and inflammatory cytokines to autoantibodies. These factors result in an inflammatory milieu in the affected joints which leads to an increased development and function of osteoclasts and the transformation of fibroblast-like synoviocytes towards a highly migratory and destructive phenotype. In addition, repair mechanisms mediated by osteoblasts and chondrocytes are strongly impaired by the presence of pro-inflammatory cytokines. This article will review the current knowledge on the mechanisms of joint inflammation and the destruction of bone and cartilage.

Nintedanib inhibits macrophage activation and ameliorates vascular and fibrotic manifestations in the Fra2 mouse model of systemic sclerosis

Background Nintedanib is an inhibitor targeting platelet-derived growth factor receptor, fibroblast growth factor receptor and vascular endothelial growth factor receptor tyrosine kinases that has recently been approved for the treatment of idiopathic pulmonary fibrosis. The aim of this study was to analyse the effects of nintedanib in the fos-related antigen-2 (Fra2) mouse model of systemic sclerosis (SSc). Methods The effects of nintedanib on pulmonary arterial hypertension with proliferation of pulmonary vascular smooth muscle cells (PVSMCs) and luminal occlusion, on microvascular disease with apoptosis of microvascular endothelial cells (MVECs) and on fibroblast activation with myofibroblast differentiation and accumulation of extracellular matrix were analysed. We also studied the effects of nintedanib on the levels of key mediators involved in the pathogenesis of SSc and on macrophage polarisation. Results Nintedanib inhibited proliferation of PVSMCs and prevented thickening of the vessel walls and luminal occlusion of pulmonary arteries. Treatment with nintedanib also inhibited apoptosis of MVECs and blunted the capillary rarefaction in Fra2-transgenic mice. These effects were associated with a normalisation of the serum levels of vascular endothelial growth factor in Fra2 mice on treatment with nintedanib. Nintedanib also effectively blocked myofibroblast differentiation and reduced pulmonary, dermal and myocardial fibrosis in Fra2-transgenic mice. The antifibrotic effects of nintedanib were associated with impaired M2 polarisation of monocytes and reduced numbers of M2 macrophages. Conclusion Nintedanib targets core features of SSc in Fra2-transgenic mice and ameliorates histological features of pulmonary arterial hypertension, destructive microangiopathy and pulmonary and dermal fibrosis. These data might have direct implications for the ongoing phase III clinical trial with nintedanib in SSc-associated interstitial lung disease.