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Objectives: Bone destruction in rheumatoid arthritis is mediated by osteoclasts (OC), which are derived from precursor cells of the myeloid lineage. The role of the two monocyte subsets, classical monocytes (expressing CD115, Ly6C and CCR2) and non-classical monocytes (which are CD115 positive, but low in Ly6C and CCR2), in serving as precursors for OC in arthritis is still elusive., Methods: We investigated CCR2 -/- mice, which lack circulating classical monocytes, crossed into hTNFtg mice for the extent of joint damage. We analysed monocyte subsets in hTNFtg and K/BxN serum transfer arthritis by flow cytometry. We sorted monocyte subsets and analysed their potential to differentiate into OC and their transcriptional response in response to RANKL by RNA sequencing. With these data, we performed a gene ontology enrichment analysis and gene set enrichment analysis., Results: We show that in hTNFtg arthritis local bone erosion and OC generation are even enhanced in the absence of CCR2. We further show the numbers of non-classical monocytes in blood are elevated and are significantly correlated with histological signs of joint destruction. Sorted non-classical monocytes display an increased capacity to differentiate into OCs. This is associated with an increased expression of signal transduction components of RANK, most importantly TRAF6, leading to an increased responsiveness to RANKL., Conclusion: Therefore, non-classical monocytes are pivotal cells in arthritis tissue damage and a possible target for therapeutically intervention for the prevention of inflammatory joint damage., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Chronic inflammation of articular joints causing bone and cartilage destruction consequently leads to functional impairment or loss of mobility in affected joints from individuals affected by rheumatoid arthritis (RA). Even successful treatment with complete resolution of synovial inflammatory processes does not lead to full reversal of joint functionality, pointing to the crucial contribution of irreversibly damaged structural components, such as bone and cartilage, to restricted joint mobility. In this context, we investigated the impact of the distinct components, including synovial inflammation, bone erosion or cartilage damage, as well as the effect of blocking tumor necrosis factor (TNF) on functional impairment in human-TNF transgenic (hTNFtg) mice, a chronic inflammatory erosive animal model of RA. We determined CatWalk-assisted gait profiles as objective quantitative measurements of functional impairment. We first determined body-weight-independent gait parameters, including maximum intensity, print length, print width and print area in wild-type mice. We observed early changes in those gait parameters in hTNFtg mice at week 5 - the first clinical signs of arthritis. Moreover, we found further gait changes during chronic disease development, indicating progressive functional impairment in hTNFtg mice. By investigating the association of gait parameters with inflammation-mediated joint pathologies at different time points of the disease course, we found a relationship between gait parameters and the extent of cartilage damage and bone erosions, but not with the extent of synovitis in this chronic model. Next, we observed a significant improvement of functional impairment upon blocking TNF, even at progressed stages of disease. However, blocking TNF did not restore full functionality owing to remaining subclinical inflammation and structural microdamage. In conclusion, CatWalk gait analysis provides a useful tool for quantitative assessment of functional impairment in inflammatory destructive arthritis. Our findings indicate that cartilage damage and bone erosion, but not synovial inflammation, are the most important determinants for progressive functional impairment in this chronic erosive arthritis model., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Introduction: Autoreactive T cells are a central element in many systemic autoimmune diseases. The generation of these pathogenic T cells is instructed by antigen-presenting cells (APCs). However, signaling pathways in APCs that drive autoimmune diseases, such as rheumatoid arthritis, are not understood., Methods: We measured phenotypic maturation, cytokine production and induction of T cell proliferation of APCs derived from wt mice and mice with a myeloid-specific deletion of PTEN (myeloid PTEN(-/-)) in vitro and in vivo. We induced collagen-induced arthritis (CIA) and K/BxN serum transfer arthritis in wt and myeloid-specific PTEN(-/-) mice. We measured the cellular composition of lymph nodes by flow cytometry and cytokines in serum and after ex vivo stimulation of T cells., Results: We show that myeloid-specific PTEN(-/-) mice are almost protected from CIA. Myeloid-specific deletion of PTEN leads to a significant reduction of cytokine expression pivotal for the induction of systemic autoimmunity such as interleukin (IL)-23 and IL-6, leading to a significant reduction of a Th17 type of immune response characterized by reduced production of IL-17 and IL-22. In contrast, myeloid-specific PTEN deficiency did not affect K/BxN serum transfer arthritis, which is independent of the adaptive immune system and solely depends on innate effector functions., Conclusions: These data demonstrate that the presence of PTEN in myeloid cells is required for the development of CIA. Deletion of PTEN in myeloid cells inhibits the development of autoimmune arthritis by preventing the generation of a pathogenic Th17 type of immune response.

Objective: Local bone destruction in rheumatic diseases, which often leads to disability and severely reduced quality of life, is almost exclusively mediated by osteoclasts. Therefore, it is important to understand pathways regulating the generation of osteoclasts. Here, we analysed the impact of the Phosphoinositide-3-Kinase (PI3K)/Phosphatase and tensin homolog (PTEN) axis on osteoclast generation and bone biology under basal and inflammatory conditions., Methods: We analysed osteoclastogenesis of wildtype (wt) and PTEN(-/-) cells in vitro and in vivo, pit resorption and qPCR of osteoclasts in vitro. Mice with a myeloid cell-specific deletion of PTEN and wt littermate mice were investigated by bone histomorphometry and clinical and histological assessment in the human tumour necrosis factor (TNF)-transgenic (hTNFtg) arthritis model., Results: We show that myeloid-specific PTEN(-/-) mice display increased osteoclastogenesis in vitro and in vivo compared to wt mice. Loss of PTEN did not affect the generation or survival of osteoclast precursor cells. However, PTEN deficiency greatly enhanced receptor activator of nuclear factor κ-B ligand (RANKL)-induced expression of the master transcription factor of osteoclastogenesis, nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), resulting in markedly increased terminal differentiation of osteoclasts in vitro. We also observed increased osteoclastogenesis under inflammatory conditions in the hTNFtg mouse model of arthritis, where hTNFtg/myeloid-specific PTEN(-/-) mice displayed enhanced local bone destruction as well as osteoclast formation in the inflamed joints. The extent of synovial inflammation, however, as well as recruitment of osteoclast precursor cells was not different between wt and myeloid-specific PTEN(-/-) mice., Conclusions: These data demonstrate that loss of PTEN and, therefore, sustained PI3-Kinase signalling in myeloid cells especially, elevates the osteoclastogenic potential of myeloid cells, leading to enhanced inflammatory local bone destruction. Therefore, although our study allows no direct translational conclusion since we used a conditional knockout approach, the therapeutic targeting of the PI3-Kinase pathway may be of benefit in preventing structural joint damage., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: MicroRNAs (miRNA) are a new class of regulatory elements. Altered expression of miRNA has been demonstrated in the inflamed joints of patients with rheumatoid arthritis (RA). The aim of this study was to examine the role of miRNA in the pathogenesis of autoimmune arthritis, using 2 murine models., Methods: Collagen-induced arthritis (CIA) and K/BxN serum-transfer arthritis were induced in wild-type (WT) and miR-155-deficient (miR-155(-/-) ) mice. The severity of arthritis was determined clinically and histologically. Anticollagen antibodies and cytokines were measured by enzyme-linked immunosorbent assay. The cellular composition of the draining lymph nodes after induction of CIA was measured by flow cytometry., Results: The miR-155(-/-) mice did not develop CIA. Deficiency in miR-155 prevented the generation of pathogenic autoreactive B and T cells, since anticollagen antibodies and the expression levels of antigen-specific T cells were strongly reduced in miR-155(-/-) mice. Moreover, Th17 polarization of miR-155(-/-) mouse T cells was impaired, as shown by a significant decrease in the levels of interleukin-17 (IL-17) and IL-22. In the K/BxN serum-transfer arthritis model, which only depends on innate effector mechanisms, miR-155(-/-) mice showed significantly reduced local bone destruction, attributed to reduced generation of osteoclasts, although the severity of joint inflammation was similar to that in WT mice., Conclusion: These results demonstrate that miR-155 is essentially involved in the adaptive and innate immune reactions leading to autoimmune arthritis, and therefore miR-155 might provide a novel target for the treatment of patients with RA., (Copyright © 2011 by the American College of Rheumatology.)

Objective: To evaluate the role of interleukin 6 (IL-6) in the pathogenesis of bilateral erosive sacroiliitis in human tumour necrosis factor transgenic (hTNFtg) mice, an animal model of ankylosing spondylitis (AS)., Methods: Histological sections of the sacroiliac joints from hTNFtg and IL-6(-/-)hTNFtg mice were evaluated, and wild type and IL-6(-/-)mice served as controls. mRNA levels of inflammation and tissue degradation related genes isolated from sacroiliac joints were also evaluated by quantitative PCR., Results: Severe, erosive bilateral sacroiliitis in 14-week-old hTNFtg animals was accompanied by an upregulation of mRNAs related to tissue inflammation such as matrix metalloproteinase 3 (MMP3), MMP9 and MMP13 or osteoclast activation such as cathepsin K and tartrate-resistant acid phosphatase. In addition, IL-6 was increased in the sera and in the sacroiliac joints of hTNFtg animals. However, high expression of these marker genes in sacroiliac joints from IL-6(-/-)hTNFtg mice was also found. Moreover, absence of IL-6 in these animals did not alter bilateral, erosive sacroiliitis when compared to hTNFtg littermates., Conclusions: IL-6 is not a crucial regulator in an animal model of TNF-mediated bilateral, erosive sacroiliitis. This finding questions the potential of IL-6 blockade as a new treatment in patients with AS.

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.

Tumour necrosis factor (TNF) signalling molecules are considered as promising therapeutic targets of antirheumatic therapy. Among them, mitogen-activated protein kinases are thought to be of central importance. Herein, we investigate the role in vivo of TNF-alpha signalling through c-Jun N-terminal kinase (JNK)1 in destructive arthritis. Human TNF transgenic (hTNFtg) mice, which develop inflammatory arthritis, were intercrossed with JNK1-deficient (JNK1-/-) mice. Animals (n = 35) of all four genotypes (wild-type, JNK1-/-, hTNFtg, JNK1-/-hTNFtg) were assessed for clinical and histological signs of arthritis. Clinical features of arthritis (swelling and decreased grip strength) developed equally in hTNFtg and JNK1-/-hTNFtg mice. Histological analyses revealed no differences in the quantity of synovial inflammation and bone erosions or in the cellular composition of the synovial infiltrate. Bone destruction and osteoclast formation were observed to a similar degree in hTNFtg and JNK1-/-hTNFtg animals. Moreover, cartilage damage, as indicated by proteoglycan loss in the articular cartilage, was comparable in the two strains. Intact phosphorylation of JNK and c-Jun as well as expression of JNK2 in the synovial tissue of JNK1-/-hTNFtg mice suggests that signalling through JNK2 may compensate for the deficiency in JNK1. Thus, JNK1 activation does not seem to be essential for TNF-mediated arthritis.

Local bone erosion and systemic bone loss are hallmarks of rheumatoid arthritis and cause progressive disability. Tumor necrosis factor (TNF) is a key mediator of arthritis and acts catabolically on bone by stimulating bone resorption and inhibiting bone formation. We hypothesized that the concerted action of anti-TNF, which reduces inflammation and parathyroid hormone (PTH), which stimulates bone formation, or osteoprotegerin (OPG), which blocks bone resorption and could lead to repair of local bone erosions and reversal of systemic bone loss. To test this, human TNF-transgenic mice with established erosive arthritis and systemic bone loss were treated with PTH, OPG, and anti-TNF, alone or in combination. Local bone erosions almost fully regressed, on combined treatment with anti-TNF and PTH and/or OPG, suggesting repair of inflammatory skeletal lesions. In contrast, OPG and anti-TNF alone led to arrest of bone erosions but did not achieve repair. Treatment with PTH alone had no influence on the progression of bone erosions. Local bone erosions all showed signs of new bone formation such as the presence of osteoblasts, osteoid formation, and mineralization. Furthermore, systemic bone loss was completely reversed on combined treatment and this effect was mediated by osteoblast stimulation and osteoclast blockade. In summary, we conclude that local joint destruction and systemic inflammatory bone loss because of TNF can regress and that repair requires a combined approach by reducing inflammation, blocking bone resorption, or stimulating bone formation.