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Staphyloccocus aureus is one of the major pathogens in orthopedic periprosthetic joint infection (PJI), a devastating complication of total joint arthroplasty that often results in chronic and persistent infections that are refractory to antibiotics and require surgical interventions. Biofilm formation has been extensively investigated as a reason for persistent infection. The cellular composition, activation status, cytokine profile, and role of the immune response during persistent S. aureus PJI are incompletely understood. In this study, we used histology, multiparametric flow cytometry, and gene expression analysis to characterize the immune response in a clinically relevant orthopedic PJI model. We tested the hypothesis that persistent S. aureus infection induces feedback mechanisms that suppress immune cell activation, thereby affecting the course of infection. Surprisingly, persistent infection was characterized by strikingly high cytokine gene expression indicative of robust activation of multiple components of innate and adaptive immunity, along with ongoing severe neutrophil-dominated inflammation, in infected joint and bone tissues. Activation and expansion of draining lymph nodes and a bone marrow stress granulopoiesis reaction were also maintained during late phase infection. In parallel, feedback mechanisms involving T-cell inhibitory receptors and exhaustion markers, suppressive cytokines, and regulatory T cells were activated and associated with decreased T-cell proliferation and tissue infiltration during the persistent phase of infection. These results identify the cellular and molecular components of the mouse immune response to persistent S. aureus PJI and indicate that neutrophil infiltration, inflammatory cytokine responses, and ongoing lymph node and bone marrow reactions are insufficient to clear infection and that immune effector mechanisms are suppressed by feedback inhibitory pathways. These immune-suppressive mechanisms are associated with diminished T-cell proliferation and tissue infiltration and can be targeted as part of adjuvant immunotherapeutic strategies in combination with debridement of biofilm, antibiotics, and other therapeutic modalities to promote eradication of infection. © 2021 American Society for Bone and Mineral Research (ASBMR).

The chemokine CXCL4 activates myeloid cells and contributes to the pathogenesis of inflammatory and fibrotic diseases. One mechanism of CXCL4 action is binding of nucleic acids to promote their internalization and activation of endosomal TLRs. However, the signaling pathways and receptors that mediate myeloid cell responses to CXCL4 alone are not well characterized. Here, we report that in primary human monocytes, CXCL4 activated NF-κB and a TBK1-JNK signaling axis that drive the expression of inflammatory, fibrotic and neutrophil chemokine genes, and also RIPK3-dependent necroptosis. Surprisingly, six distinct lines of evidence targeting TLR4 expression and function suggested a role for TLR4 in CXCL4 responses. However, in contrast to the canonical TLR4 agonist LPS, CXCL4 did not effectively activate an autocrine IFN response or IL-12 family genes, even after IFN-γ priming of monocytes. In accord with its role in inducing chemokine and inflammatory gene expression, CXCL4 regulated neutrophil and macrophage infiltration into skin wounds, and promoted wound healing in a mouse model. Our findings suggest that CXCL4 modulates the profile of TLR4 responses to induce a macrophage phenotype divergent from LPS-activated cells, provide new insights into mechanisms by which CXCL4 activates human monocytes, and implicate CXCL4 in regulation of skin wound healing.

Background Over 1 million Americans undergo joint replacement each year, and approximately 1 in 75 will incur a periprosthetic joint infection. Effective treatment necessitates pathogen identification, yet standard-of-care cultures fail to detect organisms in 10% to 20% of cases and require invasive sampling. We hypothesized that cell-free DNA (cfDNA) fragments from microorganisms in a periprosthetic joint infection can be found in the bloodstream and utilized to accurately identify pathogens via next-generation sequencing. Methods In this prospective observational study performed at a musculoskeletal specialty hospital in the U.S., we enrolled 53 adults with validated hip or knee periprosthetic joint infections. Participants had peripheral blood drawn immediately prior to surgical treatment. Microbial cfDNA from plasma was sequenced and aligned to a genome database with >1,000 microbial species. Intraoperative tissue and synovial fluid cultures were performed per the standard of care. The primary outcome was accuracy in organism identification with use of blood cfDNA sequencing, as measured by agreement with tissue-culture results. Results Intraoperative and preoperative joint cultures identified an organism in 46 (87%) of 53 patients. Microbial cfDNA sequencing identified the joint pathogen in 35 cases, including 4 of 7 culture-negative cases (57%). Thus, as an adjunct to cultures, cfDNA sequencing increased pathogen detection from 87% to 94%. The median time to species identification for cases with genus-only culture results was 3 days less than standard-of-care methods. Circulating cfDNA sequencing in 14 cases detected additional microorganisms not grown in cultures. At postoperative encounters, cfDNA sequencing demonstrated no detection or reduced levels of the infectious pathogen. Conclusions Microbial cfDNA from pathogens causing local periprosthetic joint infections can be detected in peripheral blood. These circulating biomarkers can be sequenced from noninvasive venipuncture, providing a novel source for joint pathogen identification. Further development as an adjunct to tissue cultures holds promise to increase the number of cases with accurate pathogen identification and improve time-to-speciation. This test may also offer a novel method to monitor infection clearance during the treatment period. Level of evidence Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Lung-infiltrating macrophages create a marked inflammatory milieu in a subset of patients with COVID-19 by producing a cytokine storm, which correlates with increased lethality. However, these macrophages are largely not infected by SARS-CoV-2, so the mechanism underlying their activation in the lung is unclear. Type I interferons (IFN-I) contribute to protecting the host against SARS-CoV-2 but may also have some deleterious effect, and the source of IFN-I in the lungs of infected patients is not well defined. Plasmacytoid dendritic cells (pDCs), a key cell type involved in antiviral responses, can produce IFN-I in response to SARS-CoV-2. We observed the infiltration of pDCs in the lungs of SARS-CoV-2–infected patients, which correlated with strong IFN-I signaling in lung macrophages. In patients with severe COVID-19, lung macrophages expressed a robust inflammatory signature, which correlated with persistent IFN-I signaling at the single-cell level. Hence, we observed the uncoupling in the kinetics of the infiltration of pDCs in the lungs and the associated IFN-I signature, with the cytokine storm in macrophages. We observed that pDCs were the dominant IFN-α–producing cells in response to the virus in the blood, whereas macrophages produced IFN-α only when in physical contact with infected epithelial cells. We also showed that IFN-α produced by pDCs, after the sensing of SARS-CoV-2 by TLR7, mediated changes in macrophages at both transcriptional and epigenetic levels, which favored their hyperactivation by environmental stimuli. Together, these data indicate that the priming of macrophages can result from the response by pDCs to SARS-CoV-2, leading to macrophage activation in patients with severe COVID-19.

The importance of a local tissue immune response in healing injured tissues such as skin and lung is well established. Little is known about whether sterile wounds elicit lymph node (LN) responses and inflammatory responses after injury of musculoskeletal tissues that are mechanically loaded during the repair response. We investigated LN and tissue immune responses in a tibial implant model of joint replacement surgery where wounded tissue is subjected to movement and mechanical loading postoperatively. Draining inguinal and iliac LNs expanded postoperatively, including increases in regulatory T cells and activation of a subset of T cells. Thus, tissue injury was actively sensed in secondary lymphoid organs, with the potential to activate adaptive immunity. Joint tissues exhibited three temporally distinct immune response components, including a novel interferon (IFN) response with activation of signal transducer and activator of transcription (STAT) and interferon regulatory factor (IRF) pathways. Fibrovascular tissue formation was not associated with a macrophage type 2 (M2) reparative immune response, but instead with delayed induction of interleukin-1 family (IL-1β, IL-33, IL-36), IL-17, and prostaglandin pathway genes concomitant with transforming growth factor (TGF)-β and growth factor signaling, fibroblast activation, and tissue formation. Tissue remodeling was associated with activity of the HOX antisense intergenic RNA (HOTAIR) pathway. These results provide insights into immune responses and regulation of tissue healing after knee arthroplasty that potentially can be used to develop therapeutic strategies to improve healing, prevent arthrofibrosis, and improve surgical outcomes. © 2021 American Society for Bone and Mineral Research (ASBMR).

Metal implants are commonly used in orthopedic surgery. The mechanical stability and longevity of implants depend on adequate bone deposition along the implant surface. The cellular and molecular mechanisms underlying peri-implant bone formation (ie, osseointegration) are incompletely understood. Herein, our goal was to determine the specific bone marrow stromal cell populations that contribute to bone formation around metal implants. To do this, we utilized a mouse tibial implant model that is clinically representative of human joint replacement procedures. Using a lineage-tracing approach, we found that both Acta2.creERT2 and Tmem100.creERT2 lineage cells are involved in peri-implant bone formation, and Pdgfra- and Ly6a/Sca1-expressing stromal cells (PαS cells) are highly enriched in both lineages. Single-cell RNA-seq analysis indicated that PαS cells are quiescent in uninjured bone tissue; however, they express markers of proliferation and osteogenic differentiation shortly after implantation surgery. Our findings indicate that PαS cells are mobilized to repair bone tissue and participate in implant osseointegration after surgery. Biologic therapies targeting PαS cells might improve osseointegration in patients undergoing orthopedic procedures. © 2021 American Society for Bone and Mineral Research (ASBMR).

Osteoclastogenesis requires activation of RANK signaling as well as costimulatory signals from immunoreceptor tyrosine-based activation motif-containing (ITAM-containing) receptors/adaptors, predominantly tyrosine kinase-binding proteins DAP12 and FcRγ, in osteoclast precursors. It is not well understood how costimulatory signals are regulated and integrated with RANK signaling. Here, we found that osteopetrotic bone phenotypes in mice lacking DAP12 or DAP12 and FcRγ are mediated by the transcription factor RBP-J, as deletion of Rbpj in these mice substantially rescued the defects of bone remodeling. Using a TNF-α-induced model of inflammatory bone resorption, we determined that RBP-J deficiency enables TNF-α to induce osteoclast formation and bone resorption in DAP12-deficient animals. Thus, RBP-J imposes a requirement for ITAM-mediated costimulation of RANKL or TNF-α-induced osteoclastogenesis. Mechanistically, RBP-J suppressed induction of key osteoclastogenic factors NFATc1, BLIMP1, and c-FOS by inhibiting ITAM-mediated expression and function of PLCγ2 and activation of downstream calcium-CaMKK/PYK2 signaling. Moreover, RBP-J suppressed Plcg2 expression and downstream calcium oscillations indirectly by a TGF-β/PLCγ2/calcium axis. Together, our findings indicate that RBP-J suppresses ITAM-mediated costimulation, thereby limiting crosstalk between ITAM and RANK/TNFR signaling and allowing fine tuning of osteoclastogenesis during bone homeostasis and under inflammatory conditions. Furthermore, these data suggest that environmental cues that regulate RBP-J expression/function potentially modulate the requirement for costimulatory signaling for osteoclast differentiation and bone remodeling.

Hypoxia occurs in tumors, infections, and sites of inflammation, such as in the affected joints of patients with rheumatoid arthritis (RA). It alleviates inflammatory responses and increases bone resorption in inflammatory arthritis by enhancing osteoclastogenesis. The mechanism by which the hypoxia response is linked to osteoclastogenesis and inflammatory bone resorption is unclear. This study was undertaken to evaluate whether the protein lysine-specific demethylase 1 (LSD1) metabolically integrates inflammatory osteoclastogenesis and bone resorption in a state of inflammatory arthritis.LSD1-specific inhibitors and gene silencing with small interfering RNAs were used to inhibit the expression of LSD1 in human osteoclast precursor cells derived from CD14-positive monocytes, with subsequent assessment by RNA-sequencing analysis. In experimental mouse models of arthritis, inflammatory osteolysis, or osteoporosis, features of accelerated bone loss and inflammatory osteolysis were analyzed. Furthermore, in blood samples from patients with RA, cis-acting expression quantitative trait loci (cis-eQTL) were analyzed for association with the expression of hypoxia-inducible factor 1α (HIF-1α), and associations between HIF-1α allelic variants and extent of bone erosion were evaluated.In human osteoclast precursor cells, RANKL induced the expression of LSD1 in a mechanistic target of rapamycin-dependent manner. Expression of LSD1 was higher in synovium from RA patients than in synovium from osteoarthritis patients. Inhibition of LSD1 in human osteoclast precursors suppressed osteoclast differentiation. Results of transcriptome analysis identified several LSD1-mediated hypoxia and cell-cycle pathways as key genetic pathways involved in human osteoclastogenesis. Furthermore, HIF-1α protein, which is rapidly degraded by the proteasome in a normoxic environment, was found to be expressed in RANKL-stimulated osteoclast precursor cells. Induction of LSD1 by RANKL stabilized the expression of HIF-1α protein, thereby promoting glycolysis, in conjunction with up-regulation of the transcription factor E2F1. Analyses of cis-eQTL revealed that higher HIF-1α expression was associated with increased bone erosion in patients with RA. Inhibition of LSD1 decreased pathologic bone resorption in mice, both in models of accelerated osteoporosis and models of arthritis and inflammatory osteolysis.LSD1 metabolically regulates osteoclastogenesis in an energy-demanding inflammatory environment. These findings provide potential new therapeutic strategies targeting osteoclasts in the management of inflammatory arthritis, including in patients with RA.

Regulation of endosomal Toll-like receptor (TLR) responses by the chemokine CXCL4 is implicated in inflammatory and fibrotic diseases, with CXCL4 proposed to potentiate TLR responses by binding to nucleic acid TLR ligands and facilitating their endosomal delivery. Here we report that in human monocytes/macrophages, CXCL4 initiates signaling cascades and downstream epigenomic reprogramming that change the profile of the TLR8 response by selectively amplifying inflammatory gene transcription and interleukin (IL)−1β production, while partially attenuating the interferon response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activates TBK1 and IKKε, repurposes these kinases towards an inflammatory response via coupling with IRF5, and activates the NLRP3 inflammasome. CXCL4 signaling, in a cooperative and synergistic manner with TLR8, induces chromatin remodeling and activates de novo enhancers associated with inflammatory genes. Our findings thus identify new regulatory mechanisms of TLR responses relevant for cytokine storm, and suggest targeting the TBK1-IKKε-IRF5 axis may be beneficial in inflammatory diseases.

The number of total joint replacements (TJRs) in the United States is increasing annually. Cementless implants are intended to improve upon traditional cemented implants by allowing bone growth directly on the surface to improve implant longevity. One major complication of TJR is implant loosening, which is related to deficient osseointegration in cementless TJRs. Although poor osseointegration in aged patients is typically attributed to decreased basal bone mass, little is known about the molecular pathways that compromise the growth of bone onto porous titanium implants. To identify the pathways important for osseointegration that are compromised by aging, we developed an approach for transcriptomic profiling of peri‐implant tissue in young and aged mice using our murine model of osseointegration. Based on previous findings of changes of bone quality associated with aging, we hypothesized that aged mice have impaired activation of bone anabolic pathways at the bone‐implant interface. We found that pathways most significantly downregulated in aged mice relative to young mice are related to angiogenic, Notch, and Wnt signaling. Downregulation of these pathways is associated with markedly increased expression of inflammatory and immune genes at the bone‐implant interface in aged mice. These results identify osseointegration pathways affected by aging and suggest that an increased inflammatory response in aged mice may compromise peri‐implant bone healing. Targeting the Notch and Wnt pathways, promoting angiogenesis, or modulating the immune response at the peri‐implant site may enhance osseointegration and improve the outcome of joint replacement in older patients. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Chemokines control the migratory patterns and positioning of immune cells to organize immune responses to pathogens. However, many chemokines have been associated with systemic autoimmune diseases that have chronic IFN signatures. We report that a series of chemokines, including CXCL4, CXCL10, CXCL12, and CCL5, can superinduce type I IFN (IFN-I) by TLR9-activated plasmacytoid DCs (pDCs), independently of their respective known chemokine receptors. Mechanistically, we show that chemokines such as CXCL4 mediate transcriptional and epigenetic changes in pDCs, mostly targeted to the IFN-I pathways. We describe that chemokines physically interact with DNA to form nanoparticles that promote clathrin-mediated cellular uptake and delivery of DNA in the early endosomes of pDCs. Using two separate mouse models of skin inflammation, we observed the presence of CXCL4 associated with DNA in vivo. These data reveal a noncanonical role for chemokines to serve as nucleic acid delivery vectors to modulate TLR signaling, with implications for the chronic presence of IFN-I by pDCs in autoimmune diseases.

Histopathology is widely used to analyze clinical biopsy specimens and tissues from pre-clinical models of a variety of musculoskeletal conditions. Histological assessment relies on scoring systems that require expertise, time, and resources, which can lead to an analysis bottleneck. Recent advancements in digital imaging and image processing provide an opportunity to automate histological analyses by implementing advanced statistical models such as machine learning and deep learning, which would greatly benefit the musculoskeletal field. This review provides a high-level overview of machine learning applications, a general pipeline of tissue collection to model selection, and highlights the development of image analysis methods, including some machine learning applications, to solve musculoskeletal problems. We discuss the optimization steps for tissue processing, sectioning, staining, and imaging that are critical for the successful generalizability of an automated image analysis model. We also commenting on the considerations that should be taken into account during model selection and the considerable advances in the field of computer vision outside of histopathology, which can be leveraged for image analysis. Finally, we provide a historic perspective of the previously used histopathological image analysis applications for musculoskeletal diseases, and we contrast it with the advantages of implementing state-of-the-art computational pathology approaches. While some deep learning approaches have been used, there is a significant opportunity to expand the use of such approaches to solve musculoskeletal problems.

CXCL4 regulates responses of immune cells to endosomal TLRs and has been implicated in the pathogenesis of inflammatory and fibrotic diseases. However, mechanisms by which CXCL4 modulates TLR responses, and its functions in monocytes/macrophages, are still unclear. Here we report that CXCL4 changes the profile of the TLR8 response in human monocytes by selectively and dramatically amplifying inflammatory gene transcription and IL-1b production while partially attenuating the IFN response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activated TBK1/IKKe and repurposed these kinases towards an inflammatory response via coupling with IRF5, and by activating the NLRP3 inflammasome without the need for a second signal. CXCL4 strongly induced chromatin remodeling in a cooperative and synergistic manner with TLR8 signaling, inducing de novo enhancers associated with inflammatory genes. These findings identify signaling and epigenomic mechanisms that underly synergistic activation of inflammatory genes by CXCL4 and TLR8, provide a new paradigm for modulation of TLR responses that is relevant for cytokine storm, and suggest targeting the TBK1/IKKe-IRF5 axis may be beneficial in inflammatory diseases.

Aims Aseptic loosening is a leading cause of uncemented arthroplasty failure, often accompanied by fibrotic tissue at the bone-implant interface. A biological target, neutrophil extracellular traps (NETs), was investigated as a crucial connection between the innate immune system’s response to injury, fibrotic tissue development, and proper bone healing. Prevalence of NETs in peri-implant fibrotic tissue from aseptic loosening patients was assessed. A murine model of osseointegration failure was used to test the hypothesis that inhibition (through Pad4-/- mice that display defects in peptidyl arginine deiminase 4 (PAD4), an essential protein required for NETs) or resolution (via DNase 1 treatment, an enzyme that degrades the cytotoxic DNA matrix) of NETs can prevent osseointegration failure and formation of peri-implant fibrotic tissue. Methods Patient peri-implant fibrotic tissue was analyzed for NETs biomarkers. To enhance osseointegration in loose implant conditions, an innate immune system pathway (NETs) was either inhibited ( Pad4-/- mice) or resolved with a pharmacological agent (DNase 1) in a murine model of osseointegration failure. Results NETs biomarkers were identified in peri-implant fibrotic tissue collected from aseptic loosening patients and at the bone-implant interface in a murine model of osseointegration failure. Inhibition ( Pad4-/- ) or resolution (DNase 1) of NETs improved osseointegration and reduced fibrotic tissue despite loose implant conditions in mice. Conclusion This study identifies a biological target (NETs) for potential noninvasive treatments of aseptic loosening by discovering a novel connection between the innate immune system and post-injury bone remodelling caused by implant loosening. By inhibiting or resolving NETs in an osseointegration failure murine model, fibrotic tissue encapsulation around an implant is reduced and osseointegration is enhanced, despite loose implant conditions. Cite this article: Bone Joint J 2021;103-B(7 Supple B):135–144.

Multiple type I interferons and interferon-γ (IFN-γ) are expressed under physiological conditions and are increased by stress and infections, and in autoinflammatory and autoimmune diseases. Interferons activate the Jak–STAT signaling pathway and induce overlapping patterns of expression, called ‘interferon signatures’, of canonical interferon-stimulated genes (ISGs) encoding molecules important for antiviral responses, antigen presentation, autoimmunity and inflammation. It has now become clear that interferons also induce an ‘interferon epigenomic signature’ by activating latent enhancers and ‘bookmarking’ chromatin, thus reprogramming cell responses to environmental cues. The interferon epigenomic signature affects ISGs and other gene sets, including canonical targets of the transcription factor NF-κB that encode inflammatory molecules, and is involved in the priming of immune cells, tolerance and the training of innate immune memory. Here we review the mechanisms through which interferon signatures and interferon epigenomic signatures are generated, as well as the expression and functional consequences of these signatures in homeostasis and autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis.

Activation of macrophage proinflammatory and antimicrobial phenotypes is regulated by IFN-γ and LPS via synergistic induction of canonical, inflammatory NF-κB target genes. However, whether IFN-γ negatively regulates components of the LPS response, and how this may affect macrophage activation, is still unclear. Here we use combined transcriptomic and epigenomic approaches to find that IFN-γ selectively abrogates LPS-induced feedback and alters macrophage metabolic pathways by suppressing TLR4-mediated gene activation. In contrast to superinduction of inflammatory genes via enhancers that bind IRF1 and STAT1, IFN-γ represses target enhancers that bind STAT3. TLR4-activated but IFN-γ-suppressed enhancers comprise two subsets discernable by differential regulation of histone acetylation and recruitment of STAT3, CDK8 and cohesin. Our findings thus show that IFN-γ suppresses feedback inhibitory and metabolic components of TLR responses to enhance macrophage activation; they also provide insights for IFN-γ-mediated selective inhibition of TLR4-induced transcription. Such inhibition can contribute to severe and sustained inflammatory responses., Macrophage activation is synergistically controlled by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Here the authors show that IFN-γ promotes macrophage activation not only by activating STAT1-dependent genes, but also by suppressing STAT3-dependent negative feedback regulation downstream of LPS signaling.

Aims It is increasingly appreciated that coordinated regulation of angiogenesis and osteogenesis is needed for bone formation. How this regulation is achieved during peri-implant bone healing, such as osseointegration, is largely unclear. This study examined the relationship between angiogenesis and osteogenesis in a unique model of osseointegration of a mouse tibial implant by pharmacologically blocking the vascular endothelial growth factor (VEGF) pathway. Materials and Methods An implant was inserted into the right tibia of 16-week-old female C57BL/6 mice (n = 38). Mice received anti-VEGF receptor-1 (VEGFR-1) antibody (25 mg/kg) and VEGF receptor-2 (VEGFR-2) antibody (25 mg/kg; n = 19) or an isotype control antibody (n = 19). Flow cytometric (n = 4/group) and immunofluorescent (n = 3/group) analyses were performed at two weeks post-implantation to detect the distribution and density of CD31hiEMCNhi endothelium. RNA sequencing analysis was performed using sorted CD31hiEMCNhi endothelial cells (n = 2/group). Osteoblast lineage cells expressing osterix (OSX) and osteopontin (OPN) were also detected with immunofluorescence. Mechanical pull-out testing (n = 12/group) was used at four weeks post-implantation to determine the strength of the bone-implant interface. After pull-out testing, the tissue attached to the implant surface was harvested. Whole mount immunofluorescent staining of OSX and OPN was performed to determine the amount of osteoblast lineage cells. Results Flow cytometry revealed that anti-VEGFR treatment decreased CD31hiEMCNhi vascular endothelium in the peri-implant bone versus controls at two weeks post-implantation. This was confirmed by the decrease of CD31 and endomucin (EMCN) double-positive cells detected with immunofluorescence. In addition, treated mice had more OPN-positive cells in both peri-implant bone and tissue on the implant surface at two weeks and four weeks, respectively. More OSX-positive cells were present in peri-implant bone at two weeks. More importantly, anti-VEGFR treatment decreased the maximum load of pull-out testing compared with the control. Conclusion VEGF pathway controls the coupling of angiogenesis and osteogenesis in orthopaedic implant osseointegration by affecting the formation of CD31hiEMCNhi endothelium. Cite this article: Bone Joint J 2019;101-B(7 Supple C):108–114.

Autologous blood-derived products such as platelet-rich plasma (PRP) are widely used to treat musculoskeletal conditions, including knee osteoarthritis (OA). However, the clinical outcomes after PRP administration are often variable, and there is limited information about the specific characteristics of PRP that impact bioactivity and clinical responses. In this study, we aimed to develop an integrative workflow to evaluate responses to PRP in vitro, and to assess if the in vitro responses to PRP are associated with the PRP composition and clinical outcomes in patients with knee OA. To do this, we used a coculture system of macrophages and fibroblasts paired with transcriptomic analyses to comprehensively characterize the modulation of inflammatory responses by PRP in vitro. Relying on patient-reported outcomes and achievement of minimal clinically important differences in OA patients receiving PRP injections, we identified responders and non-responders to the treatment. Comparisons of PRP from these patient groups allowed us to identify differences in the composition and in vitro activity of PRP. We believe that our integrative workflow may enable the development of targeted approaches that rely on PRP and other orthobiologics to treat musculoskeletal pathologies.

The number of total joint replacements (TJRs) in the United States is increasing annually. Cementless implants are intended to improve upon traditional cemented implants by allowing bone growth directly on the surface to improve implant longevity. One major complication of TJR is implant loosening, which is related to deficient osseointegration in cementless TJRs. Although poor osseointegration in aged patients is typically attributed to decreased basal bone mass, little is known about the molecular pathways that compromise the growth of bone onto porous titanium implants. To identify the pathways important for osseointegration that are compromised by aging, we developed an approach for transcriptomic profiling of peri-implant tissue in young and aged mice using our murine model of osseointegration. Based on previous findings of changes of bone quality associated with aging, we hypothesized that aged mice have impaired activation of bone anabolic pathways at the bone-implant interface. We found that pathways most significantly downregulated in aged mice relative to young mice are related to angiogenic, Notch and Wnt signaling. Downregulation of these pathways is associated with markedly increased expression of inflammatory and immune genes at the bone-implant interface in aged mice. These results identify osseointegration pathways affected by aging and suggest that an increased inflammatory response in aged mice may compromise peri-implant bone healing. Targeting the Notch and Wnt pathways, promoting angiogenesis, or modulating the immune response at the peri-implant site may enhance osseointegration and improve the outcome of joint replacement in older patients.

Epigenetic and transcriptional analyses of B cell subsets from patients with systemic lupus erythematosus reveal extensive effects of the disease environment and identify previously unknown connections and regulators of DN2 B cells and ABC-like cells.

Osteoporosis is a metabolic bone disease with dysregulated coupling between bone resorption and bone formation, which results in decreased bone mineral density. The MEF2C locus, which encodes the transcription factor MADS box transcription enhancer factor 2, polypeptide C (MEF2C), is strongly associated with adult osteoporosis and osteoporotic fractures. Although the role of MEF2C in bone and cartilage formation by osteoblasts, osteocytes, and chondrocytes has been studied, the role of MEF2C in osteoclasts, which mediate bone resorption, remains unclear. In this study, we identified MEF2C as a positive regulator of human and mouse osteoclast differentiation. While decreased MEF2C expression resulted in diminished osteoclastogenesis, ectopic expression of MEF2C enhanced osteoclast generation. Using transcriptomic and bioinformatic approaches, we found that MEF2C promotes the RANKL-mediated induction of the transcription factors c-FOS and NFATc1, which play a key role in osteoclastogenesis. Mechanistically, MEF2C binds to FOS regulatory regions to induce c-FOS expression, leading to the activation of NFATC1 and downstream osteoclastogenesis. Inducible deletion of Mef2c in mice resulted in increased bone mass under physiological conditions and protected mice from bone erosion by diminishing osteoclast formation in K/BxN serum induced arthritis, a murine model of inflammatory arthritis. Our findings reveal direct regulation of osteoclasts by MEF2C, thus adding osteoclasts as a cell type in which altered MEF2C expression or function can contribute to pathological bone remodeling.

Rheumatic diseases have complex aetiologies that are not fully understood, which makes the study of pathogenic mechanisms in these diseases a challenge for researchers. Next-generation sequencing (NGS) and related omics technologies, such as transcriptomics, epigenomics and genomics, provide an unprecedented genome-wide view of gene expression, environmentally responsive epigenetic changes and genetic variation. The integrated application of NGS technologies to samples from carefully phenotyped clinical cohorts of patients has the potential to solve remaining mysteries in the pathogenesis of several rheumatic diseases, to identify new therapeutic targets and to underpin a precision medicine approach to the diagnosis and treatment of rheumatic diseases. This Review provides an overview of the NGS technologies available, showcases important advances in rheumatic disease research already powered by these technologies and highlights NGS approaches that hold particular promise for generating new insights and advancing the field. Next-generation sequencing (NGS) technologies are being developed at a rapid pace. This Review highlights advances in our understanding of rheumatic diseases that have been gained from NGS and offers guidance to rheumatology researchers using these technologies.

Macrophages tailor their function to the signals found in tissue microenvironments, taking on a wide spectrum of phenotypes. In human tissues, a detailed understanding of macrophage phenotypes is limited. Using single-cell RNA-sequencing, we define distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF+inflammatory macrophages is enriched in RA tissues and shaped by resident fibroblasts and the cytokine TNF. These macrophages promote fibroblast invasiveness in an EGF receptor dependent manner, indicating that inflammatory intercellular crosstalk reshapes both cell types and contributes to fibroblast-mediated joint destruction. In anex vivotissue assay, the HBEGF+inflammatory macrophage is targeted by several anti-inflammatory RA medications, however, COX inhibition redirects it towards a different inflammatory phenotype that is also expected to perpetuate pathology. These data highlight advances in understanding the pathophysiology and drug mechanisms in chronic inflammatory disorders can be achieved by focusing on macrophage phenotypes in the context of complex interactions in human tissues.One Sentence SummaryA newly identified human macrophage phenotype from patients with the autoimmune condition RA is found to promote joint tissue invasiveness and demonstrates variable sensitivities to anti-inflammatory medications used to treat the disease.

Increased osteoclastogenesis is responsible for osteolysis, which is a severe consequence of inflammatory diseases associated with bone destruction, such as rheumatoid arthritis and periodontitis. The mechanisms that limit osteoclastogenesis under inflammatory conditions are largely unknown. We previously identified transcription factor RBP-J as a key negative regulator that restrains TNF-α–induced osteoclastogenesis and inflammatory bone resorption. In this study, we tested whether RBP-J suppresses inflammatory osteoclastogenesis by regulating the expression of microRNAs (miRNAs) important for this process. Using high-throughput sequencing of miRNAs, we obtained the first, to our knowledge, genome-wide profile of miRNA expression induced by TNF-α in mouse bone marrow–derived macrophages/osteoclast precursors during inflammatory osteoclastogenesis. Furthermore, we identified miR-182 as a novel miRNA that promotes inflammatory osteoclastogenesis driven by TNF-α and whose expression is suppressed by RBP-J. Downregulation of miR-182 dramatically suppressed the enhanced osteoclastogenesis program induced by TNF-α in RBP-J–deficient cells. Complementary loss- and gain-of-function approaches showed that miR-182 is a positive regulator of osteoclastogenic transcription factors NFATc1 and B lymphocyte–induced maturation protein-1. Moreover, we identified that direct miR-182 targets, Foxo3 and Maml1, play important inhibitory roles in TNF-α–mediated osteoclastogenesis. Thus, RBP-J–regulated miR-182 promotes TNF-α–induced osteoclastogenesis via inhibition of Foxo3 and Maml1. Suppression of miR-182 by RBP-J serves as an important mechanism that restrains TNF-α–induced osteoclastogenesis. Our results provide a novel miRNA-mediated mechanism by which RBP-J inhibits osteoclastogenesis and suggest that targeting of the newly described RBP-J–miR-182–Foxo3/Maml1 axis may represent an effective therapeutic approach to suppress inflammatory osteoclastogenesis and bone resorption.

Osteoclasts are resorptive cells that are important for homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role for epigenetic mechanisms in osteoclastogenesis. A recent study showed that epigenetic silencing of the negative regulator of osteoclastogenesis Irf8 by DNA methylation is required for osteoclast differentiation. In this study, we investigated the role of EZH2, which epigenetically silences gene expression by histone methylation, in osteoclastogenesis. Inhibition of EZH2 by the small molecule GSK126, or decreasing its expression using antisense oligonucleotides, impeded osteoclast differentiation. Mechanistically, EZH2 was recruited to the IRF8 promoter after RANKL stimulation to deposit the negative histone mark H3K27me3 and downregulate IRF8 expression. GSK126 attenuated bone loss in the ovariectomy mouse model of postmenopausal osteoporosis. Our findings provide evidence for an additional mechanism of epigenetic IRF8 silencing during osteoclastogenesis that likely works cooperatively with DNA methylation, further emphasizing the importance of IRF8 as a negative regulator of osteoclastogenesis.

ObjectiveWe investigated genome-wide changes in gene expression and chromatin remodelling induced by tumour necrosis factor (TNF) in fibroblast-like synoviocytes (FLS) and macrophages to better understand the contribution of FLS to the pathogenesis of rheumatoid arthritis (RA).MethodsFLS were purified from patients with RA and CD14+ human monocyte-derived macrophages were obtained from healthy donors. RNA-sequencing, histone 3 lysine 27 acetylation (H3K27ac), chromatin immunoprecipitation-sequencing (ChIP-seq) and assay for transposable accessible chromatin by high throughput sequencing (ATAC-seq) were performed in control and TNF-stimulated cells.ResultsWe discovered 280 TNF-inducible arthritogenic genes which are transiently expressed and subsequently repressed in macrophages, but in RA, FLS are expressed with prolonged kinetics that parallel the unremitting kinetics of RA synovitis. 80 out of these 280 fibroblast-sustained genes (FSGs) that escape repression in FLS relative to macrophages were desensitised (tolerised) in macrophages. Epigenomic analysis revealed persistent H3K27 acetylation and increased chromatin accessibility in regulatory elements associated with FSGs in TNF-stimulated FLS. The accessible regulatory elements of FSGs were enriched in binding motifs for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interferon-regulatory factors (IRFs) and activating protein-1 (AP-1). Inhibition of bromodomain and extra-terminal motif (BET) proteins, which interact with histone acetylation, suppressed sustained induction of FSGs by TNF.ConclusionOur genome-wide analysis has identified the escape of genes from transcriptional repression in FLS as a novel mechanism potentially contributing to the chronic unremitting synovitis observed in RA. Our finding that TNF induces sustained chromatin activation in regulatory elements of the genes that escape repression in RA FLS suggests that altering or targeting chromatin states in FLS (eg, with inhibitors of BET proteins) is an attractive therapeutic strategy.

Complete polarization of macrophages towards an M1-like proinflammatory and antimicrobial state requires combined action of IFN-γ and LPS. Synergistic activation of canonical inflammatory NF-κB target genes by IFN-γ and LPS is well appreciated, but less is known about whether IFN-γ negatively regulates components of the LPS response, and how this affects polarization. A combined transcriptomic and epigenomic approach revealed that IFN-γ selectively abrogates LPS-induced feedback and select metabolic pathways by suppressing TLR4-mediated activation of gene enhancers. In contrast to superinduction of inflammatory genesviaenhancers that harbor IRF sequences and bind STAT1, IFN-γ-mediated repression targeted enhancers with STAT sequences that bound STAT3. TLR4-activated IFN-γ-suppressed enhancers comprised two subsets distinguished by differential regulation of histone acetylation and recruitment of STAT3, CDK8 and cohesin, and were functionally inactivated by IFN-γ. These findings reveal that IFN-γ suppresses feedback inhibitory and metabolic components of the TLR response to achieve full M1 polarization, and provide insights into mechanisms by which IFN-γ selectively inhibits TLR4-induced transcription.

IFNγ is a cytokine with important roles in tissue homeostasis, immune and inflammatory responses and tumour immunosurveillance. Signalling by the IFNγ receptor activates the Janus kinase (JAK)–signal transducer and activator of transcription 1 (STAT1) pathway to induce the expression of classical interferon-stimulated genes that have key immune effector functions. This Review focuses on recent advances in our understanding of the transcriptional, chromatin-based and metabolic mechanisms that underlie IFNγ-mediated polarization of macrophages to an ‘M1-like’ state, which is characterized by increased pro-inflammatory activity and macrophage resistance to tolerogenic and anti-inflammatory factors. In addition, I describe the newly discovered effects of IFNγ on other leukocytes, vascular cells, adipose tissue cells, neurons and tumour cells that have important implications for autoimmunity, metabolic diseases, atherosclerosis, neurological diseases and immune checkpoint blockade cancer therapy.

Macrophages tailor their function according to the signals found in tissue microenvironments, assuming a wide spectrum of phenotypes. A detailed understanding of macrophage phenotypes in human tissues is limited. Using single-cell RNA sequencing, we defined distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF

Background Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers. Electronic supplementary material The online version of this article (10.1186/s13075-018-1631-y) contains supplementary material, which is available to authorized users.

Objective In this study, we sought to refine histologic scoring of rheumatoid arthritis (RA) synovial tissue by training with gene expression data and machine learning. Methods Twenty histologic features were assessed in 129 synovial tissue samples (n = 123 RA patients and n = 6 osteoarthritis [OA] patients). Consensus clustering was performed on gene expression data from a subset of 45 synovial samples. Support vector machine learning was used to predict gene expression subtypes, using histologic data as the input. Corresponding clinical data were compared across subtypes. Results Consensus clustering of gene expression data revealed 3 distinct synovial subtypes, including a high inflammatory subtype characterized by extensive infiltration of leukocytes, a low inflammatory subtype characterized by enrichment in pathways including transforming growth factor β, glycoproteins, and neuronal genes, and a mixed subtype. Machine learning applied to histologic features, with gene expression subtypes serving as labels, generated an algorithm for the scoring of histologic features. Patients with the high inflammatory synovial subtype exhibited higher levels of markers of systemic inflammation and autoantibodies. C-reactive protein (CRP) levels were significantly correlated with the severity of pain in the high inflammatory subgroup but not in the others. Conclusion Gene expression analysis of RA and OA synovial tissue revealed 3 distinct synovial subtypes. These labels were used to generate a histologic scoring algorithm in which the histologic scores were found to be associated with parameters of systemic inflammation, including the erythrocyte sedimentation rate, CRP level, and autoantibody levels. Comparison of gene expression patterns to clinical features revealed a potentially clinically important distinction: mechanisms of pain may differ in patients with different synovial subtypes.

Fibroblasts regulate tissue homeostasis, coordinate inflammatory responses, and mediate tissue damage. In rheumatoid arthritis (RA), synovial fibroblasts maintain chronic inflammation which leads to joint destruction. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to pathology. Here, we show functional and transcriptional differences between fibroblast subsets from human synovial tissues using bulk transcriptomics of targeted subpopulations and single-cell transcriptomics. We identify seven fibroblast subsets with distinct surface protein phenotypes, and collapse them into three subsets by integrating transcriptomic data. One fibroblast subset, characterized by the expression of proteins podoplanin, THY1 membrane glycoprotein and cadherin-11, but lacking CD34, is threefold expanded in patients with RA relative to patients with osteoarthritis. These fibroblasts localize to the perivascular zone in inflamed synovium, secrete proinflammatory cytokines, are proliferative, and have an in vitro phenotype characteristic of invasive cells. Our strategy may be used as a template to identify pathogenic stromal cellular subsets in other complex diseases., Synovial fibroblasts are thought to be central mediators of joint destruction in rheumatoid arthritis (RA). Here the authors use single-cell transcriptomics and flow cytometry to identify synovial fibroblast subsets that are expanded and display distinct tissue distribution and function in patients with RA.

Enhancers regulate gene expression and have been linked with disease pathogenesis. Little is known about enhancers that regulate human disease-associated genes in primary cells relevant for pathogenesis. Here we use BAC transgenics and genome editing to dissect, in vivo and in primary immune cells, enhancers that regulate human TNFAIP3, which encodes A20 and is linked with autoimmune diseases. A20 expression is dependent on a topologically associating subdomain (sub-TAD) that harbors four enhancers, while another >20 enhancers in the A20 locus are redundant. This sub-TAD contains cell- and activation-specific enhancers, including an enhancer (termed TT>A) harboring a proposed causal SLE-associated SNV. Deletion of the sub-TAD or the TT>A enhancer results in enhanced inflammatory responses, autoantibody production, and inflammatory arthritis, thus establishing functional importance in vivo and linking enhancers with a specific disease phenotype. These findings provide insights into enhancers that regulate human A20 expression to prevent inflammatory pathology and autoimmunity., The human TNFAIP3 gene, which encodes for A20, is associated with autoimmune diseases. Here, the authors use BAC transgenics combined with CRISPR- and recombineering-mediated genome editing to dissect in vivo and in primary immune cells, the role of enhancers regulating TNFAIP3.