Bona fide dendritic cells are pivotal precursors for osteoclasts.

OBJECTIVES: Osteoclasts (OCs) are myeloid-derived multinucleated cells uniquely able to degrade bone. However, the exact nature of their myeloid precursors is not yet defined. METHODS: CD11c-diphtheria toxin receptor (CD11cDTR) transgenic mice were treated with diphtheria toxin (DT) or phosphate buffered saline (PBS) during serum transfer arthritis (STA) and human tumour necrosis factor transgenic (hTNFtg) arthritis and scored clinically and histologically. We measured cytokines in synovitis by quantitative polymerase chain reaction (qPCR). We performed ovariectomy in CD11cDTR mice treated with PBS or DT. We analysed CD11cDTR, CD11c-Cre/CX3CR1-STOP-DTR and Zbtb46-DTR-treated mice with DT using histomorphometry and OC of CD11c and Zbtb46 fate reporter mice by fluorescent imaging. We sorted murine and human OC precursors and stimulated them with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) to generate OCs. RESULTS: Targeting CD11c+ cells in vivo in models of inflammatory arthritis (STA and hTNFtg) ameliorates arthritis by reducing inflammatory bone destruction and OC generation. Targeting CD11c-expressing cells in unchallenged mice removes all OCs in their long bones. OCs do not seem to be derived from CD11c+ cells expressing CX3CR1+, but from Zbtb46+conventional dendritic cells (cDCs) as all OCs in Zbtb46-Tomato fate reporter mice are Tomato+. In line, administration of DT in Zbtb46-DTR mice depletes all OCs in long bones. Finally, human CD1c-expressing cDCs readily differentiated into bone resorbing OCs. CONCLUSION: Taken together, we identify DCs as important OC precursors in bone homeostasis and inflammation, which might open new avenues for therapeutic interventions in OC-mediated diseases.

Amino Acids Fueling Fibroblast-Like Synoviocyte Activation and Arthritis By Regulating Chemokine Expression and Leukocyte Migration.

OBJECTIVE: We analyzed the impact of amino acid (AA) availability on the inflammatory response in arthritis. METHODS: We stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) with tumor necrosis factor (TNF) in the presence or absence of proteinogenic AAs and measured their response by QuantSeq 3' messenger RNA sequencing, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. Signal transduction events were determined by Western blot. We performed K/BxN serum transfer arthritis in mice receiving a normal and a low-protein diet and analyzed arthritis clinically and histologically. RESULTS: Deprivation of AAs decreased the expression of a specific subset of genes, including the chemokines CXCL10, CCL2, and CCL5 in TNF-stimulated FLSs. Mechanistically, the presence of AAs was required for the TNF-induced activation of an interferon regulatory factor 1 (IRF1)-STAT1 signaling circuit that drives the expression of chemotactic factors. The expression of IRF1 and the IRF1-dependent gene set in FLSs was highly correlated with the presence of inflammatory cells in human RA, emphasizing the important role of this AA-dependent pathway in inflammatory cell recruitment to the synovial tissue. Finally, we show that mice receiving a low-protein diet expressed less IRF1 in the inflamed synovium and consequently developed reduced clinical and histologic signs of arthritis. CONCLUSION: AA deprivation reduces the severity of arthritis by suppressing the expression of IRF1-STAT1-driven chemokines, which are crucial for leukocyte recruitment to the arthritic joint. Overall, our study provides novel insights into critical determinants of inflammatory arthritis and may pave the way for dietary intervention trials in RA.

microRNA-146a controls age-related bone loss.

Bone loss is one of the consequences of aging, leading to diseases such as osteoporosis and increased susceptibility to fragility fractures and therefore considerable morbidity and mortality in humans. Here, we identify microRNA-146a (miR-146a) as an essential epigenetic switch controlling bone loss with age. Mice deficient in miR-146a show regular development of their skeleton. However, while WT mice start to lose bone with age, animals deficient in miR-146a continue to accrue bone throughout their life span. Increased bone mass is due to increased generation and activity of osteoblasts in miR-146a-deficient mice as a result of sustained activation of bone anabolic Wnt signaling during aging. Deregulation of the miR-146a target genes Wnt1 and Wnt5a parallels bone accrual and osteoblast generation, which is accompanied by reduced development of bone marrow adiposity. Furthermore, miR-146a-deficient mice are protected from ovariectomy-induced bone loss. In humans, the levels of miR-146a are increased in patients suffering fragility fractures in comparison with those who do not. These data identify miR-146a as a crucial epigenetic temporal regulator which essentially controls bone homeostasis during aging by regulating bone anabolic Wnt signaling. Therefore, miR-146a might be a powerful therapeutic target to prevent age-related bone dysfunctions such as the development of bone marrow adiposity and osteoporosis.

Environmental arginine controls multinuclear giant cell metabolism and formation.

Multinucleated giant cells (MGCs) are implicated in many diseases including schistosomiasis, sarcoidosis and arthritis. MGC generation is energy intensive to enforce membrane fusion and cytoplasmic expansion. Using receptor activator of nuclear factor kappa-Β ligand (RANKL) induced osteoclastogenesis to model MGC formation, here we report RANKL cellular programming requires extracellular arginine. Systemic arginine restriction improves outcome in multiple murine arthritis models and its removal induces preosteoclast metabolic quiescence, associated with impaired tricarboxylic acid (TCA) cycle function and metabolite induction. Effects of arginine deprivation on osteoclastogenesis are independent of mTORC1 activity or global transcriptional and translational inhibition. Arginine scarcity also dampens generation of IL-4 induced MGCs. Strikingly, in extracellular arginine absence, both cell types display flexibility as their formation can be restored with select arginine precursors. These data establish how environmental amino acids control the metabolic fate of polykaryons and suggest metabolic ways to manipulate MGC-associated pathologies and bone remodelling.

Histone deacetylase 1 (HDAC1): A key player of T cell-mediated arthritis.

Rheumatoid Arthritis (RA) represents a chronic T cell-mediated inflammatory autoimmune disease. Studies have shown that epigenetic mechanisms contribute to the pathogenesis of RA. Histone deacetylases (HDACs) represent one important group of epigenetic regulators. However, the role of individual HDAC members for the pathogenesis of arthritis is still unknown. In this study we demonstrate that mice with a T cell-specific deletion of HDAC1 (HDAC1-cKO) are resistant to the development of Collagen-induced arthritis (CIA), whereas the antibody response to collagen type II was undisturbed, indicating an unaltered T cell-mediated B cell activation. The inflammatory cytokines IL-17 and IL-6 were significantly decreased in sera of HDAC1-cKO mice. IL-6 treated HDAC1-deficient CD4+ T cells showed an impaired upregulation of CCR6. Selective inhibition of class I HDACs with the HDAC inhibitor MS-275 under Th17-skewing conditions inhibited the upregulation of chemokine receptor 6 (CCR6) in mouse and human CD4+ T cells. Accordingly, analysis of human RNA-sequencing (RNA-seq) data and histological analysis of synovial tissue samples from human RA patients revealed the existence of CD4+CCR6+ cells with enhanced HDAC1 expression. Our data indicate a key role for HDAC1 for the pathogenesis of CIA and suggest that HDAC1 and other class I HDACs might be promising targets of selective HDAC inhibitors (HDACi) for the treatment of RA.

Innate immunity as the trigger of systemic autoimmune diseases.

The innate immune system consists of a variety of elements controlling and participating in virtually all aspects of inflammation and immunity. It is crucial for host defense, but on the other hand its improper activation is also thought to be responsible for the generation of autoimmunity and therefore diseases such as autoimmune arthritides like rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS) or inflammatory bowel disease. The innate immune system stands both at the beginning as well as the end of autoimmunity. On one hand, it regulates the activation of the adaptive immune system and the breach of self-tolerance, as antigen presenting cells (APCs), especially dendritic cells, are essential for the activation of naïve antigen specific T cells, a crucial step in the development of autoimmunity. Various factors controlling the function of dendritic cells have been identified that directly regulate lymphocyte homeostasis and in some instances the generation of organ specific autoimmunity. Moreover, microbial cues have been identified that are prerequisites for the generation of several specific autoimmune diseases. On the other hand, the innate immune system is also responsible for mediating the resulting organ damage underlying the clinical symptoms of a given autoimmune disease via production of proinflammatory cytokines that amplify local inflammation and further activate other immune or parenchymal cells in the vicinity, the generation of matrix degrading and proteolytic enzymes or reactive oxygen species directly causing tissue damage. In the last decades, molecular characterization of cell types and their subsets as well as both positive and negative regulators of immunity has led to the generation of various scenarios of how autoimmunity develops, which eventually might lead to the development of targeted interventions for autoimmune diseases. In this review, we try to summarize the elements that are contributing to the initiation and perpetuation of autoimmune responses.

MicroRNA-155 Controls T Helper Cell Activation During Viral Infection.

MicroRNA (miR) 155 has been implicated in the regulation of innate and adaptive immunity as well as autoimmune processes. Importantly, it has been shown to regulate several antiviral responses, but its contribution to the immune response against cytopathic viruses such as vesicular stomatitis virus (VSV) infections is not known. Using transgenic/recombinant VSV expressing ovalbumin, we show that miR-155 is crucially involved in regulating the T helper cell response against this virus. Our experiments indicate that miR-155 in CD4+ T cells controls their activation, proliferation, and cytokine production in vitro and in vivo upon immunization with OVA as well as during VSV viral infection. Using intravital multiphoton microscopy we analyzed the interaction of antigen presenting cells (APCs) and T cells after OVA immunization and found impaired complex formation when using miR-155 deficient CD4+ T cells compared to wildtype CD4+ T cells ex vivo. In contrast, miR-155 was dispensable for the maturation of myeloid APCs and for their T cell stimulatory capacity. Our data provide the first evidence that miR-155 is required for efficient CD4+ T cell activation during anti-viral defense by allowing robust APC-T cell interaction required for activation and cytokine production of virus specific T cells.

Non-classical monocytes as mediators of tissue destruction in arthritis.

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.

A T cell-specific deletion of HDAC1 protects against experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a human neurodegenerative disease characterized by the invasion of autoreactive T cells from the periphery into the CNS. Application of pan-histone deacetylase inhibitors (HDACi) ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model for MS, suggesting that HDACi might be a potential therapeutic strategy for MS. However, the function of individual HDAC members in the pathogenesis of EAE is not known. In this study we report that mice with a T cell-specific deletion of HDAC1 (using the Cd4-Cre deleter strain; HDAC1-cKO) were completely resistant to EAE despite the ability of HDAC1cKO CD4+ T cells to differentiate into Th17 cells. RNA sequencing revealed STAT1 as a prominent upstream regulator of differentially expressed genes in activated HDAC1-cKO CD4+ T cells and this was accompanied by a strong increase in phosphorylated STAT1 (pSTAT1). This suggests that HDAC1 controls STAT1 activity in activated CD4+ T cells. Increased pSTAT1 levels correlated with a reduced expression of the chemokine receptors Ccr4 and Ccr6, which are important for the migration of T cells into the CNS. Finally, EAE susceptibility was restored in WT:HDAC1-cKO mixed BM chimeric mice, indicating a cell-autonomous defect. Our data demonstrate a novel pathophysiological role for HDAC1 in EAE and provide evidence that selective inhibition of HDAC1 might be a promising strategy for the treatment of MS.

Therapeutic Modulation of Plasmacytoid Dendritic Cells in Experimental Arthritis.

OBJECTIVE: The role of plasmacytoid dendritic cells (PDCs) and type I interferons (IFNs) in rheumatoid arthritis (RA) remains a subject of controversy. This study was undertaken to explore the contribution of PDCs and type I IFNs to RA pathogenesis using various animal models of PDC depletion and to monitor the effect of localized PDC recruitment and activation on joint inflammation and bone damage. METHODS: Mice with K/BxN serum-induced arthritis, collagen-induced arthritis, and human tumor necrosis factor transgene insertion were studied. Symptoms were evaluated by visual scoring, quantification of paw swelling, determination of cytokine levels by enzyme-linked immunosorbent assay, and histologic analysis. Imiquimod-dependent therapeutic effects were monitored by transcriptome analysis (using quantitative reverse transcriptase-polymerase chain reaction) and flow cytometric analysis of the periarticular tissue. RESULTS: PDC-deficient mice showed exacerbation of inflammatory and arthritis symptoms after arthritogenic serum transfer. In contrast, enhancing PDC recruitment and activation to arthritic joints by topical application of the Toll-like receptor 7 (TLR-7) agonist imiquimod significantly ameliorated arthritis in various mouse models. Imiquimod induced an IFN signature and led to reduced infiltration of inflammatory cells. CONCLUSION: The therapeutic effects of imiquimod on joint inflammation and bone destruction are dependent on TLR-7 sensing by PDCs and type I IFN signaling. Our findings indicate that local recruitment and activation of PDCs represents an attractive therapeutic opportunity for RA patients.

MicroRNA-146a governs fibroblast activation and joint pathology in arthritis.

Synovial fibroblasts are key cells orchestrating the inflammatory response in arthritis. Here we demonstrate that loss of miR-146a, a key epigenetic regulator of the innate immune response, leads to increased joint destruction in a TNF-driven model of arthritis by specifically regulating the behavior of synovial fibroblasts. Absence of miR-146a in synovial fibroblasts display a highly deregulated gene expression pattern and enhanced proliferation in vitro and in vivo. Deficiency of miR-146a induces deregulation of tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) in synovial fibroblasts, leading to increased proliferation. In addition, loss of miR-146a shifts the metabolic state of fibroblasts towards glycolysis and augments the ability of synovial fibroblasts to support the generation of osteoclasts by controlling the balance of osteoclastogenic regulatory factors receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). Bone marrow transplantation experiments confirmed the importance of miR-146a in the radioresistant mesenchymal compartment for the control of arthritis severity, in particular for inflammatory joint destruction. This study therefore identifies microRNA-146a as an important local epigenetic regulator of the inflammatory response in arthritis. It is a central element of an anti-inflammatory feedback loop in resident synovial fibroblasts, who are orchestrating the inflammatory response in chronic arthritis. MiR-146a restricts their activation, thereby preventing excessive tissue damage during arthritis.

Phosphatase and tensin homolog (PTEN) in antigen-presenting cells controls Th17-mediated autoimmune arthritis.

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.

Loss of Phosphatase and Tensin Homolog in APCs Impedes Th17-Mediated Autoimmune Encephalomyelitis.

The PI3K signaling cascade in APCs has been recognized as an essential pathway to initiate, maintain, and resolve immune responses. In this study, we demonstrate that a cell type-specific loss of the PI3K antagonist phosphatase and tensin homolog (PTEN) in myeloid cells renders APCs toward a regulatory phenotype. APCs deficient for PTEN exhibit reduced activation of p38 MAPK and reduced expression of T cell-polarizing cytokines. Furthermore, PTEN deficiency leads to upregulation of markers for alternative activation, such as Arginase 1, with concomitant downregulation of inducible NO synthase in APCs in vitro and in vivo. As a result, T cell polarization was dysfunctional in PTEN(-/-) APCs, in particular affecting the Th17 cell subset. Intriguingly, mice with cell type-specific deletions of PTEN-targeting APCs were protected from experimental autoimmune encephalomyelitis, which was accompanied by a pronounced reduction of IL-17- and IL-22-producing autoreactive T cells and reduced CNS influx of classically activated monocytes/macrophages. These observations support the notion that activation of the PI3K signaling cascade promotes regulatory APC properties and suppresses pathogenic T cell polarization, thereby reducing the clinical symptoms and pathology of experimental autoimmune encephalomyelitis.

Loss of phosphatase and tensin homolog (PTEN) in myeloid cells controls inflammatory bone destruction by regulating the osteoclastogenic potential of myeloid cells.

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.

Macrophage PTEN regulates expression and secretion of arginase I modulating innate and adaptive immune responses.

The activation of innate immune cells triggers numerous intracellular signaling pathways, which require tight control to mount an adequate immune response. The PI3K signaling pathway is intricately involved in innate immunity, and its activation dampens the expression and release of proinflammatory cytokines in myeloid cells. These signaling processes are strictly regulated by the PI3K antagonist, the lipid phosphatase, PTEN, a known tumor suppressor. Importantly, PTEN is responsible for the elevated production of cytokines such as IL-6 in response to TLR agonists, and deletion of PTEN results in diminished inflammatory responses. However, the mechanisms by which PI3K negatively regulates TLR signaling are only partially resolved. We observed that Arginase I expression and secretion were markedly induced by PTEN deletion, suggesting PTEN(-/-) macrophages were alternatively activated. This was mediated by increased expression and activation of the transcription factors C/EBPß and STAT3. Genetic and pharmacologic experimental approaches in vitro, as well as in vivo autoimmunity models, provide convincing evidence that PI3K/PTEN-regulated extracellular Arginase I acts as a paracrine regulator of inflammation and immunity.

The PI3 kinase/mTOR blocker NVP-BEZ235 overrides resistance against irreversible ErbB inhibitors in breast cancer cells.

Resistance against first and second generation (irreversible) ErbB inhibitors is an unsolved problem in clinical oncology. The purpose of this study was to examine the effects of the irreversible ErbB inhibitors pelitinib and canertinib on growth of breast and ovarian cancer cells. Although in vitro growth-inhibitory effects of both drugs exceeded by far the effects of all reversible ErbB blockers tested (lapatinib, erlotinib, and gefitinib), complete growth inhibition was usually not reached. To define the mechanism of resistance, we examined downstream signaling pathways in drug-exposed cells by Western blot analysis. Although ErbB phosphorylation was reduced by pelitinib and canertinib, activation of the AKT/mTOR pathway remained essentially unaltered in drug-resistant cells. Correspondingly, transfection of tumor cells with constitutively activated AKT was found to promote resistance against all ErbB inhibitors tested, whereas dominant negative AKT reinstalled sensitivity in drug-resistant cells. In a next step, we applied PI3K/AKT/mTOR blockers including the dual PI3K/mTOR kinase inhibitor NVP-BEZ235. These agents were found to cooperate with pelitinib and canertinib in producing in vitro growth inhibition in cancer cells resistant against ErbB-targeting drugs. In conclusion, our data show that ErbB drug-refractory activation of the PI3K/AKT/mTOR pathway plays a crucial role in resistance against classical and second-generation irreversible ErbB inhibitors, and NVP-BEZ235 can override this form of resistance against pelitinib and canertinib.