Rheumatoid Arthritis Macrophages Research Articles

The acute phase reactant orosomucoid-2 directly promotes rheumatoid inflammation

Acute phase proteins involved in chronic inflammatory diseases have not been systematically analyzed. Here, global proteome profiling of serum and urine revealed that orosomucoid-2 (ORM2), an acute phase reactant, was differentially expressed in rheumatoid arthritis (RA) patients and showed the highest fold change. Therefore, we questioned the extent to which ORM2, which is produced mainly in the liver, actively participates in rheumatoid inflammation. Surprisingly, ORM2 expression was upregulated in the synovial fluids and synovial membranes of RA patients. The major cell types producing ORM2 were synovial macrophages and fibroblast-like synoviocytes (FLSs) from RA patients. Recombinant ORM2 robustly increased IL-6, TNF-α, CXCL8 (IL-8), and CCL2 production by RA macrophages and FLSs via the NF-κB and p38 MAPK pathways. Interestingly, glycophorin C, a membrane protein for determining erythrocyte shape, was the receptor for ORM2. Intra-articular injection of ORM2 increased the severity of arthritis in mice and accelerated the infiltration of macrophages into the affected joints. Moreover, circulating ORM2 levels correlated with RA activity and radiographic progression. In conclusion, the acute phase protein ORM2 can directly increase the production of proinflammatory mediators and promote chronic arthritis in mice, suggesting that ORM2 could be a new therapeutic target for RA.

Large-scale computational modelling of the M1 and M2 synovial macrophages in rheumatoid arthritis

Macrophages play an essential role in rheumatoid arthritis. Depending on their phenotype (M1 or M2), they can play a role in the initiation or resolution of inflammation. The M1/M2 ratio in rheumatoid arthritis is higher than in healthy controls. Despite this, no treatment targeting specifically macrophages is currently used in clinics. Thus, devising strategies to selectively deplete proinflammatory macrophages and promote anti-inflammatory macrophages could be a promising therapeutic approach. State-of-the-art molecular interaction maps of M1 and M2 macrophages in rheumatoid arthritis are available and represent a dense source of knowledge; however, these maps remain limited by their static nature. Discrete dynamic modelling can be employed to study the emergent behaviours of these systems. Nevertheless, handling such large-scale models is challenging. Due to their massive size, it is computationally demanding to identify biologically relevant states in a cell- and disease-specific context. In this work, we developed an efficient computational framework that converts molecular interaction maps into Boolean models using the CaSQ tool. Next, we used a newly developed version of the BMA tool deployed to a high-performance computing cluster to identify the models’ steady states. The identified attractors are then validated using gene expression data sets and prior knowledge. We successfully applied our framework to generate and calibrate the M1 and M2 macrophage Boolean models for rheumatoid arthritis. Using KO simulations, we identified NFkB, JAK1/JAK2, and ERK1/Notch1 as potential targets that could selectively suppress proinflammatory macrophages and GSK3B as a promising target that could promote anti-inflammatory macrophages in rheumatoid arthritis.

Synovial-tissue resident macrophages play proinflammatory functions in the pathogenesis of RA while maintaining the phenotypes in the steady state

Synovial tissue-resident macrophages (STRMs) maintain normal joint homeostasis in a steady state. However, it is unclear whether STRMs still play homeostatic roles or change the functions in the joint of rheumatoid arthritis (RA), where infiltrating peripheral blood monocyte-derived macrophages (PBMoMs) play proinflammatory roles. In the present study, we examined changes in the phenotypes and functions of STRMs in response to RA-related stimuli in vitro. STRMs were prepared from non-inflammatory osteoarthritis (OA) joint synovium, which is histologically indistinguishable from normal joint synovium. PBMoMs were prepared and used for comparison. After stimulation with plate-bound IgG, which mimics anti-citrullinated protein antibody immunocomplex formed in RA joints, or with combinations of RA-related inflammatory mediators, namely tumor necrosis factor-α (TNF-α) and prostaglandin E2 or interferon-γ, PBMoMs downregulated surface markers and genes associated with anti-inflammatory macrophages, and upregulated cytokine and marker genes of proinflammatory macrophages in RA. On the other hand, STRMs hardly changed the expression of surface molecules and marker genes but altered the pattern of cytokine gene expression after stimulation like PBMoMs. Furthermore, in vitro stimulated STRMs promote proinflammatory functions of cocultured synovial fibroblasts. Thus, STRMs might play proinflammatory roles in RA joints, while maintaining their phenotypes in the steady state.

Macrophage Polarization and Osteoclast Differentiation.

Macrophages are a key player to regulate rheumatoid arthritis pathogenesis from onset to remission. They can alter innate functions under microenvironmental conditions. To understand heterogeneous functions of macrophages in rheumatoid arthritis, several activated statuses of macrophages should be mimicked in vitro. Here, we describe basic protocols for macrophage polarization and osteoclast differentiation.

Sulfated hyaluronic acid gel for the treatment of rheumatoid arthritis in rats

Rheumatoid arthritis (RA) is a chronic autoimmune disease. NSAIDS, cyclophosphamide and glucocorticoid were commonly used to treat RA in clinical application, which long-term administration of these drugs caused serious adverse reactions. Therefore, sulfated hyaluronic acid (sHA) gel (SG) was prepared to firstly treat the RA and avoid the problem of toxic side effect caused by long-term application. In vitro evaluation showed that sHA inhibited the level of reactive oxygen species and TNF-α, IL-1β, and IL-6, and decreased the ratio of macrophage M1/M2 type, which exerted better anti-inflammatory capacity. In vivo studies showed that the injection of SG into the joint cavity of collagen-induced rheumatoid arthritis (CIA) rats could effectively treat joint swelling and reduce the level of inflammatory factors in the serum. Immunofluorescence showed that SG exerted its anti-inflammatory activity by decreasing the ratio of M1/M2 type macrophages in synovial tissue. Cartilage tissue sections showed that SG reduced bone erosion and elevated chondrocyte expression. These results confirmed that sHA is expected to be developed as a drug to treat or relieve RA, which could effectively regulate the level of macrophages in rat RA, alleviate the physiological state of inflammatory over-excitation, and improve its anti-inflammatory and antioxidant capacity.

Disease activity drives transcriptomic heterogeneity in early untreated rheumatoid synovitis

ObjectivesTranscriptomic profiling of synovial tissue from patients with early, untreated rheumatoid arthritis (RA) was used to explore the ability of unbiased, data-driven approaches to define clinically relevant subgroups.MethodsRNASeq was performed...

The effects of IL-1β stimulated human umbilical cord mesenchymal stem cells on polarization and apoptosis of macrophages in rheumatoid arthritis

Macrophages play an important role in the pathogenesis of rheumatoid arthritis (RA), in which the functions of pro-inflammatory macrophages (M1) and anti-inflammatory macrophages (M2) are different. Our previous studies have demonstrated that interleukin-1β (IL-1β) stimulated human umbilical cord mesenchymal stem cells (hUCMSCs) increase the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and initiate breast cancer cell apoptosis via ligand to death receptor 4 (DR4) and DR5. In this study, we examined the effect of IL-1β stimulated hUCMSCs (IL-1β-hUCMSCs) on immunoregulation of M1 and M2 macrophages in vitro and in the RA mouse model. The results showed that IL-1β-hUCMSCs increased macrophage polarization into M2 macrophages and enhanced apoptosis of M1 macrophages in vitro. Moreover, the intravenous injected IL-1β-hUCMSCs in RA mice rehabilitated the imbalance of M1/M2 ratio and thus demonstrated the potential to reduce inflammation in RA. This study advances our knowledge of the underlying immunoregulatory mechanisms involved in IL-1β-hUCMSCs to induce M1 macrophage apoptosis and promote the anti-inflammatory polarization of M2 macrophages and demonstrates the potential of IL-1β-hUCMSCs to reduce inflammation in RA.

Rheumatoid arthritis macrophages are primed for inflammation and display bioenergetic and functional alterations.

Myeloid cells with a monocyte/macrophage phenotype are present in large numbers in the RA joint, significantly contributing to disease; however, distinct macrophage functions have yet to be elucidated. This study investigates the metabolic activity of infiltrating polarized macrophages and their impact on pro-inflammatory responses in RA. CD14+ monocytes from RA and healthy control (HC) bloods were isolated and examined ex vivo or following differentiation into 'M1/M2' macrophages. Inflammatory responses and metabolic analysis ± specific inhibitors were quantified by RT-PCR, western blot, Seahorse XFe technology, phagocytosis assays and transmission electron microscopy along with RNA-sequencing (RNA-seq) transcriptomic analysis. Circulating RA monocytes are hyper-inflammatory upon stimulation, with significantly higher expression of key cytokines compared with HC (P < 0.05) a phenotype which is maintained upon differentiation into mature ex vivo polarized macrophages. This induction in pro-inflammatory mechanisms is paralleled by cellular bioenergetic changes. RA macrophages are highly metabolic, with a robust boost in both oxidative phosphorylation and glycolysis in RA along with altered mitochondrial morphology compared with HC. RNA-seq analysis revealed divergent transcriptional variance between pro- and anti-inflammatory RA macrophages, revealing a role for STAT3 and NAMPT in driving macrophage activation states. STAT3 and NAMPT inhibition results in significant decrease in pro-inflammatory gene expression observed in RA macrophages. Interestingly, NAMPT inhibition specifically restores macrophage phagocytic function and results in reciprocal STAT3 inhibition, linking these two signalling pathways. This study demonstrates a unique inflammatory and metabolic phenotype of RA monocyte-derived macrophages and identifies a key role for NAMPT and STAT3 signalling in regulating this phenotype.

Comprehensive Analysis and Functional Characteristics of Differential Expression of N6-Methyladenosine Methylation Modification in the Whole Transcriptome of Rheumatoid Arthritis.

N6-methyladenosine (m6A) modification is the most prevalent chemical modification in eukaryotic mRNA and is associated with the development of various immune diseases. However, the role of m6A methylation in rheumatoid arthritis (RA) development is unclear. We preliminarily explored the role of m6A methylation-related mRNAs in RA for its clinical application. The discovery of m6A methylation-modifying genes in this study may provide a fresh perspective on the development of drugs for RA treatment. High-throughput sequencing combined with methylated RNA immunoprecipitation (MeRIP-seq) and RNA sequencing were used to assess whole-transcriptome m6A modifications in the synovium of patients with RA. The relationship between m6A-modified target genes and RA inflammation and macrophages was determined. The expression of the m6A-modified significant transcript-enriched inflammatory signaling pathway was assessed through animal experiments. Differentially expressed m6A genes were correlated with macrophage activation involved in immune response, vascular endothelium, MAPK signaling pathway, PI3K - Akt signaling pathway, and other inflammatory processes. Furthermore, combined analysis with m6A-seq and RNA-seq revealed 120 genes with significant changes in both m6A modification and mRNA expression. We selected the top 3 candidate mRNAs that were upregulated and downregulated simultaneously. The expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) mRNA and protein in RA patients was lower than that in healthy control (HC). SHC-binding protein 1 (SHCBP1) and neurexophilin-3 (NXPH3) mRNA expressions were increased in RA patients. The expression of M1 macrophages was increased in RA patients. RA markers are such as rheumatoid factor (RF) and peptide containing citrulline (CCP). Further animal experiments showed that the expression of synovial MAPK, PI3K, and Akt1 proteins in the RA model was increased, and the PTEN, p-PTEN protein expression was decreased. PI3K, Akt1, PTEN, and p-PTEN were correlated to RA joint inflammation. This study revealed a unique pattern of differential m6A methylation modifications in RA and concluded that m6A modification is related to the occurrence of RA synovial inflammation.

Immune Effects of Macrophages in Rheumatoid Arthritis: A Bibliometric Analysis From 2000 to 2021.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by macrophage activation. The current characteristics, hotspots, and research frontiers of macrophage-related RA were analyzed using bibliometric analysis. Relatedpapers published from 2000 to 2021 in the Web of Science database were retrieved. The diagrams were generated and analyzed using the bibliometric software package. VOSviewer and CiteSpace were used to evaluate and visualize the research trends and hotspots in macrophage-related RA. A total of 7253 original articles were obtained. Global research on macrophage-related RA is in an advanced stage of development, with core authors, teams and research institutions emerging. United States has published the most papers, received the most citations, and had the highest H-index over the last 22 years. The University of Amsterdam and the journal of Arthritis and Rheumatism are the most productive research institutions and journals. Tak PP’s (St Vincent’s Hospital) paper has the highest publication and citation scores. The keywords “bone loss” and “polarization” have the highest frequency. Additionally, the study of macrophage polarization in RA has been research focus in recent years. This study demonstrates that research on macrophages in RA will continue. China is a significant producer, whereas the United States is an influential nation in this regard. In the last decade, most studies have concentrated on fundamental research. Recent studies have shown how macrophages play a role in controlling and weakening inflammation, and drug delivery and mechanism have come to the fore.

Extracellular sulfatase-2 is overexpressed in rheumatoid arthritis and mediates the TNF-α-induced inflammatory activation of synovial fibroblasts.

Extracellular sulfatase-2 (Sulf-2) influences receptor-ligand binding and subsequent signaling by chemokines and growth factors, yet Sulf-2 remains unexplored in inflammatory cytokine signaling in the context of rheumatoid arthritis (RA). In the present study, we characterized Sulf-2 expression in RA and investigated its potential role in TNF-α-induced synovial inflammation using primary human RA synovial fibroblasts (RASFs). Sulf-2 expression was significantly higher in serum and synovial tissues from patients with RA and in synovium and serum from hTNFtg mice. RNA sequencing analysis of TNF-α-stimulated RASFs showed that Sulf-2 siRNA modulated ~2500 genes compared to scrambled siRNA. Ingenuity Pathway Analysis of RNA sequencing data identified Sulf-2 as a primary target in fibroblasts and macrophages in RA. Western blot, ELISA, and qRT‒PCR analyses confirmed that Sulf-2 knockdown reduced the TNF-α-induced expression of ICAM1, VCAM1, CAD11, PDPN, CCL5, CX3CL1, CXCL10, and CXCL11. Signaling studies identified the protein kinase C-delta (PKCδ) and c-Jun N-terminal kinase (JNK) pathways as key in the TNF-α-mediated induction of proteins related to cellular adhesion and invasion. Knockdown of Sulf-2 abrogated TNF-α-induced RASF proliferation. Sulf-2 knockdown with siRNA and inhibition by OKN-007 suppressed the TNF-α-induced phosphorylation of PKCδ and JNK, thereby suppressing the nuclear translocation and DNA binding activity of the transcription factors AP-1 and NF-κBp65 in human RASFs. Interestingly, Sulf-2 expression positively correlated with the expression of TNF receptor 1, and coimmunoprecipitation assays demonstrated the binding of these two proteins, suggesting they exhibit crosstalk in TNF-α signaling. This study identified a novel role of Sulf-2 in TNF-α signaling and the activation of RA synoviocytes, providing the rationale for evaluating the therapeutic targeting of Sulf-2 in preclinical models of RA.

An ROS-responsive artesunate prodrug nanosystem co-delivers dexamethasone for rheumatoid arthritis treatment through the HIF-1α/NF-κB cascade regulation of ROS scavenging and macrophage repolarization

The signaling cascade between nuclear factor-kappa B (NF-κB) and hypoxia-inducible factor-1α (HIF-1α) can be activated by proinflammatory M1 macrophages in rheumatoid arthritis (RA), which produces reactive oxygen species (ROS) and enhances M1 macrophage polarization, thus aggravating the development of RA. Therefore, an ROS-responsive artesunate prodrug micellar nanosystem for co-delivery of dexamethasone (DEX/HA-TK-ART micelles, abbreviated as DEX/HTA) was developed for synergistic inhibition of the HIF-1α/NF-κB cascade to regulate ROS scavenging and macrophage repolarization in RA combination therapy. DEX/HTA micelles displayed prolonged circulation in blood and efficiently co-delivered ART&DEX in the inflamed joints of adjuvant-induced arthritis (AIA) rats; moreover, they were specifically recognized and internalized into M1 macrophages through CD44 receptor-mediated endocytosis. ROS-responsive co-released ART&DEX then exerted a synergistic action to efficiently perform ROS scavenging and repolarization of M1 to M2 macrophages by inhibition of the HIF-1α/NF-κB cascade. The intravenous administration of DEX/HTA micelles into AIA rat models significantly alleviated inflammatory cell infiltration and repaired cartilage injury in the joint. Collectively, our study highlights the therapeutic potential of DEX/HTA micelles for treating RA through synergistic inhibition of the HIF-1α/NF-κB signaling cascade to regulate ROS scavenging and macrophage repolarization. Statement of significanceAn ROS-responsive artesunate (ART) prodrug micellar nanosystem for co-delivering dexamethasone (DEX), abbreviated as DEX/HA-TK-ART micelle, was developed for synergistic cascade regulation of the HIF-1α/NF-κB pathway on ROS scavenging and macrophage repolarization in combination therapy for rheumatoid arthritis. The well-designed nanosystem showed prolonged circulation in blood and superior ART&DEX accumulation in the inflamed joints of AIA rats; moreover, the micelles were specifically internalized into M1 macrophages and co-released ART&DEX, subsequently leading to inhibition of the HIF-1α/NF-κB pathway for ROS scavenging and macrophage repolarization, thus generating synergistic anti-inflammatory effects in RAW 264.7 cells and AIA rats. The HIF-1α/NF-κB cascade regulation on ROS scavenging and macrophage repolarization based on ART&DEX combination with smart nanotechnology could serve as a promising approach for rheumatoid arthritis therapy.

Transcription factor KLF4 regulated STAT1 to promote M1 polarization of macrophages in rheumatoid arthritis.

This study aimed to reveal the mechanism of transcription factor Kruppel-like factor 4 (KLF4) in regulating M1 polarization of macrophages in rheumatoid arthritis (RA) in order to induce inflammatory response. The results suggested that KLF4 overexpression promoted the M1 polarization of RAW 264.7 cells, increased STAT1 expression and up-regulated the phosphorylation level. After KLF4 silencing, the M1 polarization level was down-regulated. Besides, the induced M1 macrophages were co-cultured with articular chondrocytes. KLF4 overexpression further aggravated chondrocyte injury, increased the cell apoptosis rate and activated the inflammatory injury. However, pretreatment with STAT1 inhibitor Cerulomycin resisted the effect of KLF4, and significantly suppressed STAT1 expression and M1 polarization of cells. KLF4 overexpression aggravated synovial tissue injury in mouse joints, up-regulated the expression of inflammatory factors, and increased the levels of CD86 and STAT1.It was discovered that transcription factor KLF4 promoted the transcription of STAT1 to regulate the M1 polarization of macrophages, thus aggravating the progression of RA and inflammatory response.

FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

Increasing evidence shows that adipokines play a vital role in the development of rheumatoid arthritis (RA). Fatty acid-binding protein 4 (FABP4), a novel adipokine that regulates inflammation and angiogenesis, has been extensively studied in a variety of organs and diseases. However, the effect of FABP4 on RA remains unclear. Here, we found that FABP4 expression was upregulated in synovial M1-polarized macrophages in RA. The increase in FABP4 promoted synovitis, angiogenesis, and cartilage degradation to exacerbate RA progression in vivo and in vitro, whereas BMS309403 (a FABP4 inhibitor) and anagliptin (dipeptidyl peptidase 4 inhibitor) inhibited FABP4 expression in serum and synovial M1-polarized macrophages in mice to alleviate RA progression. Further studies showed that constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) by TSC1 deletion specifically in the myeloid lineage regulated FABP4 expression in macrophages to exacerbate RA progression in mice. In contrast, inhibition of mTORC1 by ras homolog enriched in brain (Rheb1) disruption specifically in the myeloid lineage reduced FABP4 expression in macrophages to attenuate RA development in mice. Our findings established an essential role of FABP4 that is secreted by M1-polarized macrophages in synovitis, angiogenesis, and cartilage degradation in RA. BMS309403 and anagliptin inhibited FABP4 expression in synovial M1-polarized macrophages to alleviate RA development. Hence, FABP4 may represent a potential target for RA therapy.

Microvesicle-camouflaged biomimetic nanoparticles encapsulating a metal-organic framework for targeted rheumatoid arthritis therapy

BackgroundMethotrexate (MTX) has been highlighted for Rheumatoid arthritis (RA) treatment, however, MTX does not accumulate well at inflamed sites, and long-term administration in high doses leads to severe side effects. In this study, a novel anti-RA nanoparticle complex was designed and constructed, which could improve the targeted accumulation in inflamed joints and reduce side effects.ResultsHere, we prepared a pH-sensitive biomimetic drug delivery system based on macrophage-derived microvesicle (MV)-coated zeolitic imidazolate framework-8 nanoparticles that encapsulated the drug methotrexate (hereafter MV/MTX@ZIF-8). The MV/MTX@ZIF-8 nanoparticles were further modified with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate (polyethylene glycol)-2000] (hereafter FPD/MV/MTX@ZIF-8) to exploit the high affinity of folate receptor β for folic acid on the surface of activated macrophages in RA. MTX@ZIF-8 nanoparticles showed high DLE (~ 70%) and EE (~ 82%). In vitro study showed that effective drug release in an acidic environment could be achieved. Further, we confirmed the activated macrophage could uptake much more FPD/MV/MTX@ZIF-8 than inactivated cells. In vivo biodistribution experiment displayed FPD/MV/MTX@ZIF-8 nanoparticles showed the longest circulation time and best joint targeting. Furthermore, pharmacodynamic experiments confirmed that FPD/MV/MTX@ZIF-8 showed sufficient therapeutic efficacy and safety to explore clinical applications.ConclusionsThis study provides a novel approach for the development of biocompatible drug-encapsulating nanomaterials based on MV-coated metal-organic frameworks for effective RA treatment.Graphical

Attenuation of Rheumatoid Arthritis Through the Inhibition of Tumor Necrosis Factor-Induced Caspase 3/Gasdermin E-Mediated Pyroptosis.

ObjectiveTo determine the role of gasdermin E (GSDME)–mediated pyroptosis in the pathogenesis and progression of rheumatoid arthritis (RA), and to explore the potential of GSDME as a therapeutic target in RA.MethodsThe expression and activation of caspase 3 and GSDME in the synovium, macrophages, and monocytes of RA patients were determined by immunohistochemistry, immunofluorescence, and Western blot analysis. The correlation of activated GSDME with RA disease activity was evaluated. The pyroptotic ability of monocytes from RA patients was tested, and the effect of tumor necrosis factor (TNF) on caspase 3/GSDME‐mediated pyroptosis of monocytes and macrophages was investigated. In addition, collagen‐induced arthritis (CIA) was induced in mice lacking Gsdme, and the incidence and severity of arthritis were assessed.ResultsCompared to cells from healthy controls, monocytes and synovial macrophages from RA patients showed increased expression of activated caspase 3, GSDME, and the N‐terminal fragment of GSDME (GSDME‐N). The expression of GSDME‐N in monocytes from RA patients correlated positively with disease activity. Monocytes from RA patients with higher GSDME levels were more susceptible to pyroptosis. Furthermore, TNF induced pyroptosis in monocytes and macrophages by activating the caspase 3/GSDME pathway. The use of a caspase 3 inhibitor and silencing of GSDME significantly blocked TNF‐induced pyroptosis. Gsdme deficiency effectively alleviated arthritis in a mouse model of CIA.ConclusionThese results support the notion of a pathogenic role of GSDME in RA and provide an alternative mechanism for RA pathogenesis involving TNF, which activates GSDME‐mediated pyroptosis of monocytes and macrophages in RA. In addition, targeting GSDME might be a potential therapeutic approach for RA.

IL-21 impairs pro-inflammatory activity of M1-like macrophages exerting anti-inflammatory effects on rheumatoid arthritis

Objective：Macrophages are the main source of inflammatory mediators and play important roles in the pathogenesis of rheumatoid arthritis (RA). Interleukin-21 (IL-21) regulates both innate and adaptive immune responses and exerts major effects on inflammatory responses that promote the development of RA. However, its effect on macrophage polarisation remains unclear. Methods：CD14+ monocytes of the peripheral blood of Human healthy donors (HD) and RA, and macrophages of RA synovial fluid (RA-SF MΦs) were isolated. IL-21 receptor (IL-21R) was detected by flow cytometry. Cytokine production by MΦs from different sources pre-treated with IL-21 and/or LPS was measured by real-time polymerase chain reaction (RT-PCR) and ELISA. CD14+ monocytes were differentiated into M1-like and M2-like macrophages via stimulation with GM-CSF, interferon-γ (IFN-γ), and LPS or M-CSF, IL-4, and IL-13, respectively. To determine the effect of IL-21 on macrophage polarisation, macrophage phenotypes, gene expression, and cytokine secretion were detected by flow cytometry, RT-PCR, and ELISA. TLR4 and ERK1/2 were determined by western blotting. Results：IL-21 exerted different effects on LPS-mediated inflammatory responses in various derived MΦs, and inhibited macrophages polarisation to M1-like macrophages and promote their polarisation to M2-like macrophages in HD and RA. Moreover, IL-21 inhibited LPS-mediated secretion of inflammatory cytokines, probably by downregulating the ERK1/2, in RA-SF MΦs. Conclusion：For the first time, we indicated that IL-21 inhibits LPS-mediated cytokine production in RA-SF MΦs, and impairs pro-inflammatory activity of M1-like macrophages, hereby exerting anti-inflammatory effects on RA. Thus, IL-21 might not be an appropriate therapeutic target for RA.

M2-type exosomes nanoparticles for rheumatoid arthritis therapy via macrophage re-polarization

Imbalance between the activities of pro-inflammatory M1 and anti-inflammatory M2 macrophages in rheumatoid arthritis (RA) induces synovial inflammation and autoimmunity, leading to joint damage. Here we encapsulated a plasmid DNA encoding the anti-inflammatory cytokine interleukin-10 (IL-10 pDNA) and the chemotherapeutic drug betamethasone sodium phosphate (BSP) into biomimetic vector M2 exosomes (M2 Exo) derived from M2-type macrophages. We demonstrate that the loaded exosomes target and reduce inflammation for combined therapy against RA. The in vitro efficiency of the M2 Exo/pDNA/BSP co-delivery system was attributed to the synergistic effect of IL-10 pDNA and BSP, which also promoted M1-to-M2 macrophage polarization by reducing the secretion of pro-inflammatory cytokines (IL-1β, TNF-α) and increasing the expression of IL-10 cytokine. In a mouse model of RA, M2 Exo/pDNA/BSP showed good accumulation at inflamed joint sites, high anti-inflammatory activity, and potent therapeutic effect. The delivery system was non-toxic both in vitro and in vivo. Thus, this system may serve as a promising biocompatible drug carrier and anti-inflammatory agent for RA treatment based on M1-to-M2 macrophage re-polarization.

Abstract 12924: CCL18 Promotes Effector Functions of Pro-Inflammatory Macrophages by Inactivating GSK-3β

Background: The risk for cardiovascular disease is multifold higher in patients with the autoimmune disease rheumatoid arthritis, but underlying mechanisms remain poorly defined. We have explored whether patients with rheumatoid arthritis (RA) and patients with coronary artery disease (CAD) share pro-inflammatory effector cells that sustain tissue inflammation, thus maintaining synovial inflammation and atherosclerotic plaque formation. Methods: Tissue cytokines expressed in RA synovial lesions were analyzed by cytokine array technology. Cytokine production in macrophages was verified with RT-PCR, immunohistochemistry and flow cytometry. Mitochondrial function was evaluated by Seahorse analyzer. Results: In highly inflamed synovial tissues, 21 of 60 cytokines tested were significantly elevated, with the small cytokine CCL18 being strongly associated with tissue inflammation. Immunohistochemical examination assigned CCL18 to CD68 + tissue macrophages in both the atherosclerotic plaque and in rheumatoid synovitis. In monocyte-derived macrophages (MΦ), CCL18 enhanced production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and stimulated production of the tissue-injurious enzyme cathepsin K. Pro-inflammatory effector functions were linked to CCL18 induced activation of mitochondrial metabolism, quantified by oxygen consumption rates and generation of ATP. Macrophage stimulation with recombinant CCL18 resulted in the inactivating phosphorylation of the metabolic sensor GSK-3β. Immunoprecipitation experiments localized GSK-3β to the mitochondrial surface and imaging studies revealed excessive formation of mitochondria-endoplasmic reticulum connections in patient-derived CCL18 hi macrophages. Conclusions: Pro-inflammatory MΦ in CAD and RA patients share a metabolic profile of increased mitochondrial oxygen consumption and ATP production. Mitochondrial activation depends on inactivation of the metabolic sensor GSK-3β and promote pro-inflammatory effector functions, such as production of cytokines and proteases. Inhibiting GSK-3β activation provides a novel therapeutic strategy to suppress pro-inflammatory macrophages in CAD and RA.

Identification of Candidate Genes Related to Synovial Macrophages in Rheumatoid Arthritis by Bioinformatics Analysis.

ObjectiveRheumatoid arthritis (RA) is one of the most prevalent inflammatory arthritis worldwide. However, the genes and pathways associated with macrophages from synovial fluids in RA patients still remain unclear. This study aims to screen and verify differentially expressed genes (DEGs) related to identifying candidate genes related to synovial macrophages in rheumatoid arthritis by bioinformatics analysis.MethodsWe searched the Gene Expression Omnibus (GEO) database, and GSE97779 and GSE10500 with synovial macrophages expression profiling from multiple RA microarray dataset were selected to conduct a systematic analysis. GSE97779 included nine macrophage samples from synovial fluids of RA patients and five macrophage samples from primary human blood of HC. GSE10500 included five macrophage samples from synovial fluids of RA patients and three macrophage samples from primary human blood of HC. Functional annotation of DEGs was performed, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Protein–protein interaction (PPI) network of DEGs was established using the STRING database. CytoHubba was used to identify hub genes. MCODE was used to determine gene clusters in the interactive network.ResultsThere were 2638 DEGs (1425 upregulated genes and 1213 downregulated ones) and 889 DEGs (438 upregulated genes and 451 downregulated ones) selected from GSE97779 and GSE10500, respectively. Venn diagrams showed that 173 genes were upregulated and 106 downregulated in both two datasets. The top 10 hub genes, including FN1, VEGFA, HGF, SERPINA1, MMP9, PPBP, CD44, FPR2, IGF1, and ITGAM, were identified using the PPI network.ConclusionThis study provides new insights for the potential biomarkers and the relevant molecular mechanisms in RA patients. Our findings suggest that the 10 candidate genes might be used in diagnosis, prognosis, and therapy of RA in the future. However, further studies are required to confirm the expression of these genes in synovial macrophages in RA and control specimen.