Metabolic signature and proteasome activity controls synovial migration of CDC42hiCD14+ cells in rheumatoid arthritis.

Objective: Activation of Rho-GTPases in macrophages causes inflammation and severe arthritis in mice. In this study, we explore if Rho-GTPases define the joint destination of pathogenic leukocytes, the mechanism by which they perpetuate rheumatoid arthritis (RA), and how JAK inhibition mitigates these effects. Methods: CD14+ cells of 136 RA patients were characterized by RNA sequencing and cytokine measurement to identify biological processes and transcriptional regulators specific for CDC42 hiCD14+ cells, which were summarized in a metabolic signature (MetSig). The effect of hypoxia and IFN-γ signaling on the metabolic signature of CD14+ cells was assessed experimentally. To investigate its connection with joint inflammation, the signature was translated into the single-cell characteristics of CDC42 hi synovial tissue macrophages. The sensitivity of MetSig to the RA disease activity and the treatment effect were assessed experimentally and clinically. Results: CDC42 hiCD14+ cells carried MetSig of genes functional in the oxidative phosphorylation and proteasome-dependent cell remodeling, which correlated with the cytokine-rich migratory phenotype and antigen-presenting capacity of these cells. Integration of CDC42 hiCD14+ and synovial macrophages marked with MetSig revealed the important role of the interferon-rich environment and immunoproteasome expression in the homeostasis of these pathogenic macrophages. The CDC42 hiCD14+ cells were targeted by JAK inhibitors and responded with the downregulation of immunoproteasome and MHC-II molecules, which disintegrated the immunological synapse, reduced cytokine production, and alleviated arthritis. Conclusion: This study shows that the CDC42-related MetSig identifies the antigen-presenting CD14+ cells that migrate to joints to coordinate autoimmunity. The accumulation of CDC42 hiCD14+ cells discloses patients perceptive to the JAKi treatment.

Survivin prevents the polycomb repressor complex 2 from methylating histone 3 lysine 27.

This study investigates the role of survivin in epigenetic control of gene transcription through interaction with the polycomb repressive complex 2 (PRC2). PRC2 is responsible for silencing gene expression by trimethylating lysine 27 on histone 3. We observed differential expression of PRC2 subunits in CD4+ T cells with varying levels of survivin expression, and ChIP-seq results indicated that survivin colocalizes with PRC2 along DNA. Inhibition of survivin resulted in a significant increase in H3K27 trimethylation, implying that survivin prevents PRC2 from functioning. Peptide microarray showed that survivin interacts with peptides from PRC2 subunits, and machine learning revealed that amino acid composition contains relevant information for predicting survivin interaction. NMR and BLI experiments supported the interaction of survivin with PRC2 subunit EZH2. Finally, protein-protein docking revealed that the survivin-EZH2 interaction interface overlaps with catalytic residues of EZH2, potentially inhibiting its H3K27 methylation activity. These findings suggest that survivin inhibits PRC2 function.

MicroRNA and interleukin 6 interplay in the adipose tissue of rheumatoid arthritis patients.

OBJECTIVES: MicroRNAs (miRs) are non-translated RNA sequences that elicit negative control over protein expression. The adipose tissue (AT) is considered the major producer of miRs and inflammatory interleukin 6 (IL-6). This study aims to investigate the relationship between production of IL-6 and miRs in AT. METHODS: IL-6 gene expression was analysed in RNA extracts from subcutaneous AT of 75 patients with rheumatoid arthritis (RA), with qPCR. Genome-wide profile of human miRs (2565 miRs, 96.6%) was analysed in 35 AT samples on 3D microarray. The miR-processing proteins Dicer, Drosha and DGCR8 were analysed with qPCR. In silico prediction of protein targets for the differentially expressed (DE) miRs (p<0.05; log2FC >±0.5) was conducted by DIANA software. Seven AT samples were stimulated in vitro with IL-6 or IL-6+IL-6R antibody tocilizumab and analysed for the miR processing proteins. RESULTS: We identified 30 DE miRs between AT with high and low IL-6 mRNA, of which 26 miRs were inversely related with IL-6 levels. DE miRs were predicted to interfere in oestrogen (p=0.001), FoxO (p=0.006) and insulin (p=0.03) signalling pathways. High expression of IL-6 in AT was associated with significantly higher expression of Dicer (p=0.04) and Drosha (p=0.04), while inhibition of IL-6 signalling with tocilizumab decreased the levels of total miRs processing enzymes (p=0.003). CONCLUSIONS: IL-6 mRNA production in AT has a negative effect on the miRs expression profile and it increases miR-production capacity.

Clinical Significance of Diabetes-Mellitus-Associated Antibodies in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a canonical autoimmune disease that shares numerous risk factors with diabetes mellitus (DM). The production of autoantibodies is a characteristic feature in both diseases. To determine the frequency and specificity of DM-related antibodies (DMab) in RA patients and to study whether DMab associates with new DM cases in RA patients, we measured DMab defined as IgG against glutamic acid decarboxylase (GADA), tyrosine phosphatase (IA2-ab), and zinc transporter (ZnT8-ab) in a cohort of 290 RA patients (215 women and 75 men, median disease duration 11 years). Of those, 21 had a DM diagnosis at baseline. The development of new DM cases and mortality were traced in a 10-year prospective follow-up. Predictive analyses for DM and mortality were carried out by the Mantel-Cox regression. We found that 27 of the patients (9.3%) had DMab, equally often men and women. The presence of DMab was more frequent in patients with DM (p = 0.027. OR 4.01, 95%CI [1.20; 11.97]), suggesting their specificity for the disease. Men had more prevalent incidental DM at the baseline (12% vs. 5%, p = 0.030) and among the new DM cases (p = 0.012. HR 6.08, 95%CI [1.57; 25]). New DM developed equally frequently in DMab-positive and DMab-negative patients. DM, but not DMab, significantly increased the estimated mortality rate in RA patients (p = 0.021, OR 4.38 [1.2; 13.52]). Taken together, we conclude that DMab are associated with DM in RA patients, but they are not solely enough to predict disease development or mortality in those patients.

Survivin promotes a glycolytic switch in CD4+ T cells by suppressing the transcription of PFKFB3 in rheumatoid arthritis.

In this study, we explore the role of nuclear survivin in maintaining the effector phenotype of IFNγ-producing T cells acting through the transcriptional control of glucose utilization. High expression of survivin in CD4+T cells was associated with IFNγ-dependent phenotype and anaerobic glycolysis. Transcriptome of CD4+ cells and sequencing of survivin-bound chromatin showed that nuclear survivin had a genome-wide and motif-specific binding to regulatory regions of the genes controlling cell metabolism. Survivin coprecipitates with transcription factors IRF1 and SMAD3, which repressed the transcription of the metabolic check-point enzyme phosphofructokinase 2 gene PFKFB3 and promoted anaerobic glycolysis. Combining transcriptome analyses of CD4+ cells and functional studies in glucose metabolism, we demonstrated that the inhibition of survivin reverted PFKFB3 production, inhibited glucose uptake, and reduces interferon effects in CD4+ cells. These results present a survivin-dependent mechanism in coordinating the metabolic adaptation of CD4+T cells and propose an attractive strategy to counteract IFNγ-dependent inflammation in autoimmunity.

IGF1R signalling is a guardian of self-tolerance restricting autoantibody production.

Objective: Insulin-like growth factor 1 receptor (IGF1R) acts at the crossroad between immunity and cancer, being an attractive therapeutic target in these areas. IGF1R is broadly expressed by antigen-presenting cells (APC). Using mice immunised with the methylated albumin from bovine serum (BSA-immunised mice) and human CD14+ APCs, we investigated the role that IGF1R plays during adaptive immune responses. Methods: The mBSA-immunised mice were treated with synthetic inhibitor NT157 or short hairpin RNA to inhibit IGF1R signalling, and spleens were analysed by immunohistology and flow cytometry. The levels of autoantibody and cytokine production were measured by microarray or conventional ELISA. The transcriptional profile of CD14+ cells from blood of 55 patients with rheumatoid arthritis (RA) was analysed with RNA-sequencing. Results: Inhibition of IGF1R resulted in perifollicular infiltration of functionally compromised S256-phosphorylated FoxO1+ APCs, and an increased frequency of IgM+CD21+ B cells, which enlarged the marginal zone (MZ). Enlargement of MHCII+CD11b+ APCs ensured favourable conditions for their communication with IgM+ B cells in the MZ. The reduced expression of ICOSL and CXCR5 by APCs after IGF1R inhibition led to impaired T cell control, which resulted in autoreactivity of extra-follicular B cells and autoantibody production. In the clinical setting, the low expression of IGF1R on CD14+ APCs was associated with an involuted FOXO pathway, non-inflammatory cell metabolism and a high IL10 production characteristic for tolerogenic macrophages. Furthermore, autoantibody positivity was associated with low IGF1R signalling in CD14+ APCs. Conclusions: In experimental model and in patient material, this study demonstrates that IGF1R plays an important role in preventing autoimmunity. The study raises awareness of that immune tolerance may be broken during therapeutic IGF1R targeting.

Chronic hyperinsulinemia promotes human hepatocyte senescence.

OBJECTIVE: Cellular senescence, an irreversible proliferative cell arrest, is caused by excessive intracellular or extracellular stress/damage. Increased senescent cells have been identified in multiple tissues in different metabolic and other aging-related diseases. Recently, several human and mouse studies emphasized the involvement of senescence in development and progression of NAFLD. Hyperinsulinemia, seen in obesity, metabolic syndrome, and other conditions of insulin resistance, has been linked to senescence in adipocytes and neurons. Here, we investigate the possible direct role of chronic hyperinsulinemia in the development of senescence in human hepatocytes. METHODS: Using fluorescence microscopy, immunoblotting, and gene expression, we tested senescence markers in human hepatocytes subjected to chronic hyperinsulinemia in vitro and validated the data in vivo by using liver-specific insulin receptor knockout (LIRKO) mice. The consequences of hyperinsulinemia were also studied in senescent hepatocytes following doxorubicin as a model of stress-induced senescence. Furthermore, the effects of senolytic agents in insulin- and doxorubicin-treated cells were analyzed. RESULTS: Results showed that exposing the hepatocytes to prolonged hyperinsulinemia promotes the onset of senescence by increasing the expression of p53 and p21. It also further enhanced the senescent phenotype in already senescent hepatocytes. Addition of insulin signaling pathway inhibitors prevented the increase in cell senescence, supporting the direct contribution of insulin. Furthermore, LIRKO mice, in which insulin signaling in the liver is abolished due to deletion of the insulin receptor gene, showed no differences in senescence compared to their wild-type counterparts despite having marked hyperinsulinemia indicating these are receptor-mediated effects. In contrast, the persistent hyperinsulinemia in LIRKO mice enhanced senescence in white adipose tissue. In vitro, senolytic agents dasatinib and quercetin reduced the prosenescent effects of hyperinsulinemia in hepatocytes. CONCLUSION: Our findings demonstrate a direct link between chronic hyperinsulinemia and hepatocyte senescence. This effect can be blocked by reducing the levels of insulin receptors or administration of senolytic drugs, such as dasatinib and quercetin.

Rho-GTPase dependent leukocyte interaction generates pro-inflammatory thymic Tregs and causes arthritis.

Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the ß-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.

Cohesin-Mediated Chromatin Interactions and Autoimmunity.

Proper physiological functioning of any cell type requires ordered chromatin organization. In this context, cohesin complex performs important functions preventing premature separation of sister chromatids after DNA replication. In partnership with CCCTC-binding factor, it ensures insulator activity to organize enhancers and promoters within regulatory chromatin. Homozygous mutations and dysfunction of individual cohesin proteins are embryonically lethal in humans and mice, which limits in vivo research work to embryonic stem cells and progenitors. Conditional alleles of cohesin complex proteins have been generated to investigate their functional roles in greater detail at later developmental stages. Thus, genome regulation enabled by action of cohesin proteins is potentially crucial in lineage cell development, including immune homeostasis. In this review, we provide current knowledge on the role of cohesin complex in leukocyte maturation and adaptive immunity. Conditional knockout and shRNA-mediated inhibition of individual cohesin proteins in mice demonstrated their importance in haematopoiesis, adipogenesis and inflammation. Notably, these effects occur rather through changes in transcriptional gene regulation than through expected cell cycle defects. This positions cohesin at the crossroad of immune pathways including NF-kB, IL-6, and IFNγ signaling. Cohesin proteins emerged as vital regulators at early developmental stages of thymocytes and B cells and after antigen challenge. Human genome-wide association studies are remarkably concordant with these findings and present associations between cohesin and rheumatoid arthritis, multiple sclerosis and HLA-B27 related chronic inflammatory conditions. Furthermore, bioinformatic prediction based on protein-protein interactions reveal a tight connection between the cohesin complex and immune relevant processes supporting the notion that cohesin will unearth new clues in regulation of autoimmunity.

Impact of the Uncoupling Protein 1 on Cardiovascular Risk in Patients with Rheumatoid Arthritis.

Adiposity is strongly associated with cardiovascular (CV) morbidity. Uncoupling protein 1 (UCP1) increases energy expenditure in adipocytes and may counteract adiposity. Our objective was to investigate a connection between UCP1 expression and cardiovascular health in patients with rheumatoid arthritis (RA) in a longitudinal observational study. Transcription of UCP1 was measured by qPCR in the subcutaneous adipose tissue of 125 female RA patients and analyzed with respect to clinical parameters and the estimated CV risk. Development of new CV events and diabetes mellitus was followed for five years. Transcription of UCP1 was identified in 89 (71%) patients. UCP1 positive patients had often active RA disease (p = 0.017), high serum levels of IL6 (p = 0.0025) and were frequently overweight (p = 0.015). IL-6hiBMIhi patients and patients treated with IL6 receptor inhibitor tocilizumab had significantly higher levels of UCP1 compared to other RA patients (p < 0.0001, p = 0.032, respectively). Both UCP1hi groups displayed unfavorable metabolic profiles with high plasma glucose levels and high triglyceride-to-HDL ratios, which indicated insulin resistance. Prospective follow-up revealed no significant difference in the incidence of new CV and metabolic events in the UCP1hi groups and remaining RA patients. The study shows that high transcription of UCP1 in adipose tissue is related to IL6-driven processes and reflects primarily metabolic CV risk in female RA patients.

Prognostic Significance of BIRC5/Survivin in Breast Cancer: Results from Three Independent Cohorts.

Breast cancer (BC) histological and molecular classifications significantly improved the treatment strategy and prognosis. Inhibitor of apoptosis BIRC5/survivin is often overexpressed in cancers, however, indications of its importance in BC are inconsistent. We integrate BIRC5 protein and mRNA measures with clinical associates and long-term outcome in three independent cohorts Protein levels of BIRC5 were measured in primary lysates of 845 patients of the West Swedish BC cohort (VGR-BC) and linked to 5- and 27-years survival. The results were externally validated in transcriptomic data from METABRIC and SCAN-B cohorts. Survival analysis showed that high levels of BIRC5 were consistently associated with a poor probability of 5-year overall survival. High BIRC5 in VGR-BC contributed negatively to the disease-specific survival at 5 and 27 years. Subsets with different status by ER (estrogen receptor) expression and presence of nodal metastasis supported independent association of high BIRC5 with poor prognosis in all cohorts. In METABRIC and SCAN-B cohorts, high levels of BIRC5 mRNA were associated with the basal-like and luminal B molecular BC subtypes and with increasing histologic grade. BIRC5 is a sensitive survival marker that acts independent of ER and nodal status, and its levels need to be considered when making treatment decisions.

Nicotine Changes the microRNA Profile to Regulate the FOXO Memory Program of CD8+ T Cells in Rheumatoid Arthritis.

Objective: Smoking suppresses PD-1 expression in patients with rheumatoid arthritis (RA). In this study, we assess if smoking changed the epigenetic control over CD8+ T cell memory formation through a microRNA (miR) dependent mechanism. Methods: Phenotypes of CD8+ T cells from smokers and non-smokers, RA and healthy, were analyzed by flow cytometry. A microarray analysis was used to screen for differences in miR expression. Sorted CD8+ cells were in vitro stimulated with nicotine and analyzed for transcription of miRs and genes related to memory programming by qPCR. Results: CD27+CD107a-CD8+ T cells, defining a naïve-memory population, had low expression of PD-1. Additionally, the CD27+ population was more frequent in smokers (p = 0.0089). Smokers were recognized by differential expression of eight miRs. Let-7c-5p, let-7d-5p and let-7e-5p, miR-92a-3p, miR-150-5p, and miR-181-5p were up regulated, while miR-3196 and miR-4723-5p were down regulated. These miRs were predicted to target proteins within the FOXO-signaling pathway involved in CD8+ memory programming. Furthermore, miR-92a-3p was differentially expressed in CD8+ cells with naïve-memory predominance. Nicotine exposure of CD8+ cells induced the expression of miR-150-5p and miR-181a-5p in the naïve-memory cells in vitro. Additionally, nicotine exposure inverted the ratio between mRNAs of proteins in the FOXO pathway and their targeting miRs. Conclusions: Smokers have a high prevalence of CD8+ T cells with a naïve-memory phenotype. These cells express a miR profile that interacts with the memory programming conducted through the FOXO pathway.

Low Soluble Receptor for Advanced Glycation End Products Precedes and Predicts Cardiometabolic Events in Women With Rheumatoid Arthritis.

Background: Cardiovascular disease (CVD) causes premature mortality in rheumatoid arthritis (RA). Levels of soluble (s)RAGE change with aging, hypertension and hypercholesterolemia. We assessed whether sRAGE was associated with increased risk of CVD in RA patients. Methods: Serum sRAGE was measured in 184 female RA patients and analyzed with respect to CVD risk estimated by the Framingham algorithm (eCVR), metabolic profile and inflammation. Levels of sRAGE in 13 patients with known cardio-metabolic morbidity defined the cut-off for low sRAGE. Prospective 5-year follow-up of new CV and metabolic events was completed. Results: Low sRAGE was significantly associated with previous history and with new imminent cardiometabolic events in the prospective follow-up of RA patients. In both cases, low sRAGE reflected higher estimation of CVR in those patients. Low sRAGE was attributed to adverse metabolic parameters including high fasting plasma glucose and body fat content rather than inflammation. The association of sRAGE and poor metabolic profile was prominent in patients younger than 50 years. Conclusions: This study points at low sRAGE as a marker of metabolic failure developed during chronic inflammation. It highlights the importance for monitoring metabolic health in female RA patients for timely prevention of CVD. Trial registration: ClinicalTrials.gov with ID NCT03449589. Registered 28, February 2018.

Low serum IGF1 is associated with hypertension and predicts early cardiovascular events in women with rheumatoid arthritis.

OBJECTIVES: Since low insulin-like growth factor (IGF) 1 is often linked to inflammation, we analyze whether serum levels of IGF1 are associated with cardiovascular disease (CVD) in rheumatoid arthritis (RA) in a longitudinal observational study. METHODS: A CVD risk was estimated (eCVR) in 184 female RA patients (mean age 52 years) and in 132 female patients after ischemic stroke (mean age 56 years) with no rheumatic disease, using the Framingham algorithm. The median level of IGF1 divided the cohorts in IGF1high and IGF1low groups. A 5-year prospective follow-up for new CVD events was completed in all RA patients. The Mantel-Cox analysis and event-free survival curves were prepared. Unsupervised clustering of proteins within the IGF1 signaling pathway was employed to identify their association with eCVR. RESULTS: Low IGF1 resulted in a higher eCVR in RA patients (7.2% and 3.3%, p = 0.0063) and in stroke (9.3% and 7.1%, p = 0.033). RA had higher rate for new CVD events at prospective follow-up (OR 4.96, p = 0.028). Hypertension was the major risk factor associated with low IGF1 in RA and stroke. In hypertension, IGF1 was no longer responsible for intracellular activation and lost its correlation to IRS1/2 adaptor proteins. The clustering analysis confirmed that combination of low IGF1 and IRS1/2 with high IL6, insulin, and glucose predisposed to high eCVR and emphasized the functional role of serum IGF1. CONCLUSIONS: Low serum IGF1 precedes and predicts development of early CVD events in female RA patients. Hypertension and aberrant IGF1 receptor signaling are highlighted as the important contributors to IGF1-related CVD events.

Inflammation in the hippocampus affects IGF1 receptor signaling and contributes to neurological sequelae in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an inflammatory joint disease with a neurological component including depression, cognitive deficits, and pain, which substantially affect patients' quality of daily life. Insulin-like growth factor 1 receptor (IGF1R) signaling is one of the factors in RA pathogenesis as well as a known regulator of adult neurogenesis. The purpose of this study was to investigate the association between IGF1R signaling and the neurological symptoms in RA. In experimental RA, we demonstrated that arthritis induced enrichment of IBA1+ microglia in the hippocampus. This coincided with inhibitory phosphorylation of insulin receptor substrate 1 (IRS1) and up-regulation of IGF1R in the pyramidal cell layer of the cornus ammoni and in the dentate gyrus, reproducing the molecular features of the IGF1/insulin resistance. The aberrant IGF1R signaling was associated with reduced hippocampal neurogenesis, smaller hippocampus, and increased immobility of RA mice. Inhibition of IGF1R in experimental RA led to a reduction of IRS1 inhibition and partial improvement of neurogenesis. Evaluation of physical functioning and brain imaging in RA patients revealed that enhanced functional disability is linked with smaller hippocampus volume and aberrant IGF1R/IRS1 signaling. These results point to abnormal IGF1R signaling in the brain as a mediator of neurological sequelae in RA and provide support for the potentially reversible nature of hippocampal changes.

Survivin Measurement improves Clinical Prediction of Transition From Arthralgia to RA-Biomarkers to Improve Clinical Sensitivity of Transition From Arthralgia to RA.

Background: Arthralgia often predates development of rheumatoid arthritis (RA). A set of joint symptoms commonly found in patients during their transition from arthralgia to RA, has been recently proposed. Aim: To combine clinical and serological markers and to improve recognition of imminent rheumatoid arthritis (RA) among patients with arthralgia. Methods: The total of 1,743 first-visit patients attending the rheumatology ward in Gothenburg for joint symptoms were identified during 12 consecutive months. Among those, 63 patients were classified as RA, 73 had undifferentiated arthritis and 180 had unexplained arthralgia. New RA cases, which prospectively developed during 48 months, comprised the preclinical (pre) RA group. The joint symptoms of the first-visit were analyzed aiming to distinguish patients with arthralgia and arthritis, and patients with pre-RA, who later developed the disease. The receiver operating characteristics curves were constructed. In the model, symptoms with the odds ratio >2.0 between the arthralgia and pre-RA were combined with information about RA-specific antibodies, C-reactive protein (CRP), and survivin in serum. Results: The proposed set of clinical symptoms distinguished the arthralgia patients from RA and pre-RA. Presence of survivin in serum showed strong association with clinical joint symptoms in arthralgia. A combination of symptoms in several small joint areas, increasing number of joints with symptoms, and patient's experience of swelling in small hand joints at the first visit identified pre-RA cases with 93% specificity. Grouping those symptoms with information about survivin, RA-specific antibodies, and CRP (or gender) in the final algorithm achieved 91% specificity and 55.2% of positive prediction for transition from arthralgia to RA. Conclusion: Clinical and serological parameters in combination aid recognition of imminent RA among arthralgia patients with appropriate sensitivity.

Smoking Is Associated With Low Levels of Soluble PD-L1 in Rheumatoid Arthritis.

Background: Smoking is a risk factor for developing rheumatoid arthritis (RA), but the mechanism remains uncertain. We previously demonstrated that smoking lowers the T cell activation threshold by limiting programmed death protein 1 (PD-1) expression. Aim: To investigate how smoking influence the levels of soluble PD-1 ligand (sPD-L1). Method: Serum levels of sPD-L1 were measured in 246 RA patients and in 168 healthy subjects. The analysis was done with respect to inflammation, smoking, treatments, and autoantibody status. The effect of therapeutic TNF-inhibiting antibodies (TNFi) on sPD-L1 was studied in 16 RA patients at their first infliximab infusion. The expression of Fcγ-receptor (FcγR) subclass IIB and IIIA was analyzed with quantitative polymerase chain reaction in peripheral blood mononuclear cells (PBMCs) from 12 RA patients and 15 healthy controls, and in healthy PBMC exposed to IgG containing antibodies to cyclic citrullinated peptides (aCCP). Results: The negative association between smoking and sPD-L1 in RA patients was established by multiple logistic regression (OR = 0.52, p = 0.038). Other covariates in the regression model were serum levels of IL-1ß representing inflammation (OR = 1.6, p = 0.0076) and aCCP positivity (OR = 1.9, p = 0.047). First infliximab infusion repressed sPD-L1 (p = 0.023) in patients, and low levels of sPD-L1 were found in patients with early RA treated with TNFi (p = 0.018). Treatment with TNFi was associated with higher sPD-L1 in patients with long disease duration (p = 0.041) and restored levels in smokers. In vitro exposure to aCCP+ IgG suppressed sPD-L1 (p = 0.036), but aCCP+ patients with long disease duration had higher sPD-L1 (p = 0.016). High ratio of the inhibitory FcγR subclass IIB over the stimulatory IIIA resulted in low sPD-L1 release (p = 0.029). Smoking was associated with a higher FcγR IIB/IIIA ratio (p = 0.00062) and lower levels of sPD-L1 (p = 0.013). Conclusion: In RA, serum sPD-L1 was related to systemic inflammation and aCCP positivity. Smoking altered the expression of FcγRs and limited sPD-L1 in RA patients, permitting inappropriate T cell responses. Differential regulation of sPD-L1 during the early and late RA may indicate transposition from acute to chronic inflammation.

Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis.

Objective: Macrophage inflammatory protein 1-alpha (CCL3) is a chemokine that regulates macrophage trafficking to the inflamed joint. The agonistic effect of CCL3 on osteolytic lesions in patients with multiple myeloma is recognized; however, its role in skeletal damage during inflammatory arthritis has not been established. The aim of the study was to explore the role of osteoclast-associated CCL3 upon bone resorption, and to test its pharmacological blockade for protecting against bone pathology during inflammatory arthritis. Methods: CCL3 production was studied during osteoclast differentiation from osteoclast precursor cells: human CD14-positive mononuclear cells. Mice with CIA were treated with an anti-CCL3 antibody. The effect of CCL3 blockade through mAb was studied through osteoclast number, cytokine production and bone resorption on ivory disks, and in vivo through CIA progression (clinical score, paw diameter, synovial inflammation and bone damage). Results: Over time, CCL3 increased in parallel with the number of osteoclasts in culture. Anti-CCL3 treatment achieved a concentration-dependent inhibition of osteoclast fusion and reduced pit formation on ivory disks (P ⩽ 0.05). In CIA, anti-CCL3 treatment reduced joint damage and significantly decreased multinucleated tartrate-resistant acid phosphatase-positive osteoclasts and erosions in the wrists (P < 0.05) and elbows (P < 0.05), while also reducing joint erosions in the hind (P < 0.01) and fore paws (P < 0.01) as confirmed by X-ray. Conclusion: Inhibition of osteoclast-associated CCL3 reduced osteoclast formation and function whilst attenuating arthritis-associated bone loss and controlling development of erosion in murine joints, thus uncoupling bone damage from inflammation. Our findings may help future innovations for the diagnosis and treatment of inflammatory arthritis.

Effects of implant-delivered insulin on bone formation in osteoporotic rats.

Osteoporosis is a major cause of age-related fractures. Healing complications in osteoporotic patients are often associated with increased mortality and morbidity. Stimulation of the implant-adjacent bone could be beneficial in terms of the surgical outcome. Over the past decade, numerous investigations have implicated insulin in normal bone growth, and recent studies have described the advantages of administering insulin locally to increase bone formation. Therefore, we hypothesized that insulin-coated titanium implants would increase bone formation in osteoporotic animals. The aim of this study was to evaluate the effects of insulin delivered from an implant surface on bone-related gene expression and bone formation in osteoporotic rats. Characterizations of the surfaces of insulin-coated and control implants were performed using ellipsometry and interferometry. Forty ovariectomized and four healthy Sprague Dawley rats were used and implants were inserted in the tibias. The systemic effect of insulin was assessed by measuring the blood glucose levels and total body weight. The animals were sacrificed either 1 day or 3 weeks postimplantation. Implant-adherent cells were analyzed by quantitative real-time PCR, and the bone adjacent to the implants was examined by microcomputed tomography and histomorphometry. The insulin-coated implants had no systemic effects. The insulin-coated samples demonstrated significantly lower expression of the gene for interleukin 1ß (p = 0.019) at 1 day, and significantly exhibited more periosteal callus (p = 0.029) at 3 weeks. Locally delivered insulin has potential for promoting bone formation and it exerts potentially anti-inflammatory effects in osteoporotic rats. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2472-2480, 2018.