Cells In Rheumatoid Arthritis Patients Research Articles

Correlation of interleukin 6 and transforming growth factor β1 with peripheral blood regulatory T cells in rheumatoid arthritis patients: a potential biomarker.

IntroductionProinflammatory cytokines and regulatory T cells (Tregs) are considered as important factors involved in autoimmunity development especially in rheumatoid arthritis (RA).Aim of the studyTo investigate the frequency of peripheral blood Tregs and related cytokines in RA patients and to determine the possible correlation between Treg percentage and interleukin 6 (IL-6) and transforming growth factor β1 (TGF-β1) as indicators in assessment of Treg function and mechanisms preceding autoimmunity in RA.Material and methodsThirty-seven Iranian RA patients with a moderate (3.2-5.1) disease activity score (DAS) and the same number of healthy age- and sex-matched individuals were enrolled. Frequency of peripheral blood Tregs (CD4+FoxP3+CD25high) was determined by flow cytometry. Serum levels of IL-6 and TGF-β1 and their expression levels in peripheral blood mononuclear cells (PBMCs) were evaluated by ELISA and Q-PCR, respectively.ResultsRheumatoid arthritis patients showed significantly lower peripheral blood Treg frequencies compared to healthy individuals. Additionally, Treg (%) showed a significant inverse correlation between serum concentrations of IL-6 and mRNA expression of PBMCs, whereas there was no significant correlation between Treg (%) and TGF-β1 levels.ConclusionsThe current study revealed that Treg numbers were reduced in peripheral blood of RA patients. This reduction inversely correlated with IL-6 levels, which may lead to persistent autoimmune and inflammatory conditions in RA patients.

Peripheral blood CD8+ T-cell profiles in patients with psoriatic arthritis: a cross-sectional case-control study.

While CD4+ T-cells are traditionally regarded as the main pathogenic T-cell subpopulation in psoriatic arthritis (PsA), the role of circulating CD8+ T-cells remains poorly characterized. We evaluated the differential representation of CD8+ T-cell subpopulations in peripheral blood (PB) of PsA patients. CD8+IL-17+, CD8+IFNγ+ and CD8+IL-17-IL-22+ T-cells were evaluated by flow-cytometry in 25 consecutive PsA patients, 7 rheumatoid arthritis (RA) patients, 16 patients with psoriasis, and 26 healthy controls (HC). We observed a significant expansion of circulating IFN-γ producing CD8+ T-cells in PsA when compared to psoriasis [21.2 (6.9-55.8)% vs. 3.8 (0.7-11.8)%, p < 0.0001] and HC samples [21.2 (6.9-55.8)% vs. 4.05 (0.44-19.8)%, p < 0.0001]. A frequency of circulating IFN-γ producing CD8+T-cells ≥ 9% distinguished PsA from psoriasis patients with a specificity of 84% and a sensitivity of 87.5% [AUC = 0.9 (0.80-0.99), p < 0.0001]. In addition, we found a significant expansion of circulating IL-17 producing CD8+ T cells in RA patients when compared to PsA, psoriasis and HC samples. By contrast, there were no significant between-group differences in the prevalence of circulating IL-22 producing CD8+ T-cells. In PsA patients there was a significant correlation between number of swollen joints and frequency of circulating IFN-γ producing CD8+ T-cells, and between extent and severity of psoriasis and frequency of circulating IL-17 producing CD8+ T-cells. Circulating IFNγ-producing CD8+ T-cells are raised in PsA when compared to psoriasis, suggesting a potential pathogenetic involvement of CD8+ T-cells and IFNγ production in chronic joint inflammation and damage. The significant enrichment of circulating IL-17 producing CD8+ T-cells in RA when compared to PsA warrants functional characterization and confirmation in larger studies. We found no significant enrichment of circulating IL-22 producing CD8+ T-cells in PsA, RA and psoriasis.

Circular RNA expression profiles of peripheral blood mononuclear cells in rheumatoid arthritis patients, based on microarray chip technology.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic synovial inflammation and finally leads to variable degrees of bone and cartilage erosion. The diagnosis of RA is not an accurate indicator, but a series of scores and the mechanisms underlying it remain only partially understood. The present study explored whether circular RNAs (circRNAs) contribute to the RA pathophysiological mechanism. Total RNA from peripheral blood mononuclear cells of 10 RA patients and 10 healthy controls were extracted and circRNA expression profiling was followed by microarray analysis. In addition, circRNA interactions with microRNAs were performed and microRNA response elements were listed to identify differentially expressed binding site targets in RA. Reverse transcription-quantitative polymerase chain reaction amplification (RT-qPCR) was used to verify the differential expression of circRNAs. A total of 584 circRNAs were differentially expressed in RA patients vs. healthy controls, by circRNA microarray, including 255 circRNAs which were significantly upregulated and 329 downregulated among the RA samples. RT-qPCR validation demonstrated that the expression levels of hsa_circRNA_104194, hsa_circRNA_104593, hsa_circRNA_103334, hsa_circRNA_101407 and hsa_circRNA_102594 were consistent with the results from the microarray analysis. The current study presented differentially expressed circRNAs and their corresponding microRNA binding sites in RA. circRNAs may exhibit a role in the regulation of expression of symbol genes that influence the occurrence and development of RA.

Effector T helper cell populations are elevated in the bone marrow of rheumatoid arthritis patients and correlate with disease severity

This study is to investigate the frequencies of T-helper (Th)22, Th17 and Th1 cells and the levels of related cytokines in subchondral bone marrow in patients with rheumatoid arthritis (RA). Bone marrow and peripheral blood samples were collected from RA, osteoarthritis (OA) patients and healthy controls. The frequencies of Th22, Th17, and Th1 cells were examined by flow cytometry. Levels of interleukin (IL)-22, IL-17 and IFN-γ were examined by ELISA. Disease Activity Score in 28 joints (DAS28) of RA patients were analyzed. Bone marrow Th22, Th17 and Th1 cells in RA patients were markedly increased comparing to OA or healthy controls. Each T cell subset in bone marrow was elevated comparing with that in the peripheral blood in RA patients. Consistently, plasma levels of IL-22 and IL-17 were elevated in RA patients, and the elevation was more notable in the bone marrow than in the peripheral blood. Additionally, the percentages of Th22, Th17 and Th1 cells as well as the levels of IL-22 and IL-17 in bone marrow were positively correlated with DAS28. These results suggest that local pro-inflammatory Th cells are elevated in bone marrow, which may play an important role in situ in RA and contribute to the pathogenesis of in RA.

Therapeutic effect of a novel histone deacetylase 6 inhibitor, CKD-L, on collagen-induced arthritis in vivo and regulatory T cells in rheumatoid arthritis in vitro

BackgroundHistone deacetylase (HDAC) inhibitor has recently been reported to have a therapeutic effect as an anti-inflammatory agent in collagen-induced arthritis (CIA). We investigated the therapeutic effect of a new selective HDAC6 inhibitor, CKD-L, compared to ITF 2357 or Tubastatin A on CIA and regulatory T (Treg) cells in patients with rheumatoid arthritis (RA).MethodsCIA was induced by bovine type II collagen (CII) in DBA/1 J mice. Mice were treated with HDAC inhibitor for 18 days. Arthritis score was assessed and histological analysis was performed by hematoxylin and eosin (H&E) stain. Cytotoxic T-lymphocyte associated protein (CTLA)-4 expression in induced Treg cells was analyzed and suppression assay was analyzed using Treg cells and effector T (Teff) cells isolated from naive C57BL/6 mice by flow cytometry. Cytokines were analyzed in peripheral blood mononuclear cells (PBMC) of five patients with RA by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Tumor necrosis factor (TNF) was analyzed using PMA- activated THP-1 cells by ELISA. Suppression assay was analyzed using Treg cells and Teff cells isolated from RA patients by flow cytometry.ResultsIn the CIA model, CKD-L and Tubastatin A significantly decreased the arthritis score. CKD-L increased CTLA-4 expression in Foxp3+ T cells and inhibited the proliferation of Teff cells in the suppression assay. In RA PBMC, CKD-L significantly inhibited TNF and interleukin (IL)-1β, and increased IL-10. CKD-L and Tubastatin A inhibited TNF secretion from PMA-activated THP-1 cells. CKD-L and ITF 2357 inhibited the proliferation of Teff cells in RA patients in the suppression assay. Tubastatin A had no effect on inhibition of proliferation.ConclusionCKD-L decreased the arthritis score in CIA, reduced the expression of TNF and IL-1β, and increased the expression of IL-10 in PBMC from RA patients. CKD-L increased CTLA-4 expression and the suppressive function of Treg cells. These results suggest that CKD-L may have a beneficial effect in the treatment of RA.

Expression of non-receptor protein tyrosine phosphatases in peripheral blood mononuclear cells of patient with rheumatoid arthritis

Objective To study the expression and clinical importance of non-receptor protein tyrosine phosphatases (PTPN) in patients with rheumatoid arthritis (RA). Methods Thirty-two RA patients enrolled in our hospital from February 2015 to October 2015 and 29 healthy controls were recruited. Peripheral blood was obtained and mononuclear cells were prepared using Ficoll density gradient centrifugation. The expression of protein tyrosine phosphatases (PTPs) mRNA and protein was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunoblot respectively. Meanwhile, the levels of inflammatory factors like interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and biochemical indexes like erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were evaluated, with the calculation of DAS28, a measure of disease activity in RA. The correlation between PTPs and inflammatory factors, or biochemical indexes, or DAS28 was analyzed. Results In comparison with the controls, the expression of PTPN1, PTPN2, PTPN3, PTPN4, PTPN7, PTPN9, PTPN13 was reduced, while that of PTPN11 (2.530±0.743 vs. 1.000±0.034) and PTPN14 (2.269±0.548 vs. 1.000±0.057) was augmented in RA patients. The expression of PTPN11 was positively correlated with TNF-α levels [(5.95±1.57) ng/ml, r=0.669, P=0.000], CRP levels [(28.58±6.72) mg/L, r=0.795, P=0.000] and DAS28 score [(4.20±0.73) points, r=0.703, P=0.000]. PTPN14 was positively correlated with TNF-α (r=0.633, P=0.000) and CRP level (r=0.435, P=0.013), but not with DAS28 score (r=0.033, P=0.856). Conclusion PTPs were expressed differently in peripheral mononuclear cells of RA patients. Among them, PTPN11 expression was correlated with inflammatory factors, CRP level and DAS28, which may serve as novel markers in RA diagnosis. Key words: Non-receptor protein tyrosine phosphatases; Rheumatoid arthritis; Mononuclear cells; Inflammatory factor; DAS28

Tim3+ Foxp3 + Treg Cells Are Potent Inhibitors of Effector T Cells and Are Suppressed in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disorder. Earlier studies have demonstrated that regulatory T (Treg) cells, the main cell type mediating immune tolerance, appeared to be enriched in the inflamed synovial tissues. It is still unclear why the Treg cells in RA patients are unable to limit exacerbated inflammation. Here, we found that the frequency of Tim3+Foxp3+ Treg cells, which were potent suppressors of proinflammatory responses, was downregulated in RA patients. Reduction in Tim3+Foxp3+ Treg frequency was correlated with increased RA disease activity. Furthermore, we observed that Tim3+Foxp3+ Tregs were expressed more interleukin (IL)-10 than Tim3-Foxp3+ Tregs. CD4+CD25+Tim3+ T cells had higher capability of inhibiting interferon (IFN)-γ and tumor necrosis factor (TNF)-α secretion from T cells and peripheral blood mononuclear cells (PBMCs) than CD4+CD25+Tim3- T cells. Compared to that in healthy individuals, CD4+CD25+ T cells in RA patients were less potent in suppressing IFN-γ and TNF-α production from PBMCs. Blocking Tim3 on CD4+CD25+ T cells from healthy controls resulted in an elevation of IFN-γ and TNF-α production from PBMCs, suggesting that Tim3 expression on CD4+CD25+ T cells was required for optimal Treg function. However, this phenomenon was not observed in RA patients. In conclusion, our study suggested that the CD4+CD25+Foxp3+ Treg cells from RA patients demonstrated a reduction of Tim3 and were less functional than Treg cells from healthy controls in a Tim3-related manner.

Direct recognition of LPS drive TLR4 expressing CD8+ T cell activation in patients with rheumatoid arthritis

Aberrant immune responses characterize autoimmune disorders like Rheumatoid Arthritis (RA) wherein lymphocytes are recognized as key players. Role of CD8+ T cells in RA has been less defined however we found that these cells are activated in RA patients with increased expression of cytolytic granules and inflammatory mediators thereby modulating immune responses contributing to disease severity. Though unconventional expression of different Toll Like Receptors (TLRs) on CD8+ T cells has been proposed but their expression and role in T cell activation and differentiation in RA still remains obscure. Herein we report, for the first time, an increased expression of TLR4 on peripheral CD8+ T cells of RA patients and its role in skewing CD8+ T cells towards activated and inflammatory phenotype thereby playing a significant role in pathogenesis and progression of RA. We found that the surface expression of TLR4 on CD8+ T cells directly correlates with disease severity. Moreover, these CD8+ T cells respond to the TLR4 ligand LPS and express robust amounts of cytotolytic and inflammatory molecules including TNFα and IFNγ. Our study hence identifies an important role for CD8+ T cells in orchestrating RA through TLR4 mediated activation and differentiation.

Induction and Differentiation of IL-10-Producing Regulatory B Cells from Healthy Blood Donors and Rheumatoid Arthritis Patients.

The most important feature of B cells is the production of Abs upon activation; additionally, B cells produce pro- and anti-inflammatory cytokines in response to certain stimuli. IL-10-producing B cells represent a major subset of regulatory B cells (Bregs) that suppress autoimmune and inflammatory responses. B cells play a crucial role in the development and maintenance of the chronic inflammatory autoimmune disease rheumatoid arthritis (RA); however, controversial data are available on IL-10- producing Bregs in RA. Our aim was to identify the optimal conditions that induce IL-10+ Bregs and, furthermore, to shed light on the signaling pathways that are responsible for their expansion. The results show that dual stimulation by CpG and CD40L for 48 h is optimal for IL-10 induction, and this can be synergistically boosted by IL-21. We identified the CD19+CD27+ memory B cell population as the major source of IL-10+ Bregs. We detected significantly fewer CD19+CD27+IL-10+ cells in RA patients compared with healthy controls, and these were functionally defective in suppressing IFN-γ production by CD4+ T cells in coculture. IL-21 drastically increased the number of IL-10+ Bregs within the CD19+CD27+ and CD19+CD27- populations; furthermore, it induced the appearance of IL-10+Blimp-1+ plasmablasts. Monitoring the phosphorylation of key signaling molecules revealed that activation of ERK, p38, and CREB is indispensable for the induction of IL-10 production, whereas phosphorylation of STAT3 further enhances IL-10 expression in human Bregs. We conclude that CREB and STAT3 are the key transcription factors responsible for the expansion and differentiation of human IL-10-producing Bregs.

A molecular signature of preclinical rheumatoid arthritis triggered by dysregulated PTPN22.

A unique feature of rheumatoid arthritis (RA) is the presence of anti-citrullinated protein antibodies (ACPA). Several risk factors for RA are known to increase the expression or activity of peptidyl arginine deiminases (PADs), which catalyze citrullination and, when dysregulated, can result in hypercitrullination. However, the consequence of hypercitrullination is unknown and the function of each PAD has yet to be defined. Th cells of RA patients are hypoglycolytic and hyperproliferative due to impaired expression of PFKFB3 and ATM, respectively. Here, we report that these features are also observed in peripheral blood mononuclear cells (PBMCs) from healthy at-risk individuals (ARIs). PBMCs of ARIs are also hypercitrullinated and produce more IL-2 and Th17 cytokines but fewer Th2 cytokines. These abnormal features are due to impaired induction of PTPN22, a phosphatase that also suppresses citrullination independently of its phosphatase activity. Attenuated phosphatase activity of PTPN22 results in aberrant expression of IL-2, ATM, and PFKFB3, whereas diminished nonphosphatase activity of PTPN22 leads to hypercitrullination mediated by PADs. PAD2- or PAD4-mediated hypercitrullination reduces the expression of Th2 cytokines. By contrast, only PAD2-mediated hypercitrullination can increase the expression of Th17 cytokines. Taken together, our data depict a molecular signature of preclinical RA that is triggered by impaired induction of PTPN22.

Increased autophagy in CD4+ T cells of rheumatoid arthritis patients results in T-cell hyperactivation and apoptosis resistance.

Rheumatoid arthritis (RA) is an autoimmune disease hallmarked by aberrant cellular homeostasis, resulting in hyperactive CD4+ T cells that are more resistant to apoptosis. Both hyperactivation and resistance to apoptosis may contribute to the pathogenicity of CD4+ T cells in the autoimmune process. A better knowledge of the mechanisms determining such impaired homeostasis could contribute significantly to both the understanding and the treatment of the disease. Here we investigated whether autophagy, is dysregulated in CD4+ T cells of RA patients, resulting in disturbed T-cell homeostasis. We demonstrate that the rate of autophagy is significantly increased in CD4+ T cells from RA patients, and that increased autophagy is also a feature of in vitro activated CD4+ T cells. The increased apoptosis resistance observed in CD4+ T cells from RA patients was significantly reversed upon autophagy inhibition. These mechanisms may contribute to RA pathogenesis, as autophagy inhibition reduced both arthritis incidence and disease severity in a mouse collagen induced arthritis mouse model. Conversely, in Atg5flox/flox -CD4-Cre+ mice, in which all T cells are autophagy deficient, T cells showed impaired activation and proliferation. These data provide novel insight into the pathogenesis of RA and underscore the relevance of autophagy as a promising therapeutic target.

The Proportion of Regulatory T Cells in Patients with Rheumatoid Arthritis: A Meta-Analysis.

BackgroundRegulatory T cells (Tregs) have important functions in peripheral immune tolerance. Dysfunction of Tregs is considered to be a pivotal cause of autoimmune diseases, including rheumatoid arthritis (RA). However, previous reports describing the proportion of Tregs among CD4+ T cells in RA patients were controversial because a range of markers are used to identify Tregs with little consensus. To clarify the status of Tregs in RA, we investigated the proportion of Tregs with focusing on the definitions of them.MethodsWe identified the studies reporting the proportion of Tregs in RA patients using PubMed and Google Scholar. We performed a systematic review of them and a meta-analysis to evaluate the proportion of Tregs (FOXP3-positive and/or CD25-positive) among CD4+ T cells in peripheral blood (PB) and synovial fluid (SF) of RA patients and control subjects.ResultsA total 31 studies were selected. The proportion of Tregs defined by all definitions among CD4+ T cells in PB was not significantly different between RA patients and control subjects (-0.65, [-1.30, 0.01]). Then we performed sub-analyses based on individual definitions. The proportion of Tregs defined by either CD25 or FOXP3 alone did not differ between RA patients and control subjects. The proportion of Tregs defined by both FOXP3 and CD25 was lower in RA patients than that in control subjects (-2.42 [-3.49, -1.34]). The proportion of Tregs defined by both FOXP3 and CD25 was higher in SF than that in PB among RA patients (3.27 [0.40, 6.14]).ConclusionThe status of Tregs varied according to the definition system. The proportion of Tregs defined by stricter and functionally validated methods decreased in PB and increased in SF among RA patients. If the proportion of Tregs differs in RA, accurate and functionally relevant definitions of Tregs are necessary to elucidate their status in RA.

Generation of Functional Cardiomyocytes from the Synoviocytes of Patients with Rheumatoid Arthritis via Induced Pluripotent Stem Cells.

Cardiovascular disease is a leading cause of morbidity in rheumatoid arthritis (RA) patients. This study aimed to generate and characterise cardiomyocytes from induced pluripotent stem cells (iPSCs) of RA patients. Fibroblast-like synoviocytes (FLSs) from patients with RA and osteoarthritis (OA) were successfully reprogrammed into RA-iPSCs and OA-iPSCs, respectively. The pluripotency of iPSCs was confirmed by quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining. Established iPSCs were differentiated into cardiomyocytes using a small molecule-based monolayer differentiation protocol. Within 12 days of cardiac differentiation from patient-specific and control-iPSCs, spontaneously beating cardiomyocytes (iPSC-CMs) were observed. All iPSC-CMs exhibited a reliable sarcomeric structure stained with antibodies against cardiac markers and similar expression profiles of cardiac-specific genes. Intracellular calcium signalling was recorded to compare calcium-handling properties among cardiomyocytes differentiated from the three groups of iPSCs. RA-iPSC-CMs had a lower amplitude and a shorter duration of calcium transients than the control groups. Peak tangential stress and the maximum contractile rate were also decreased in RA-iPSC-CMs, suggesting that contractility was reduced. This study demonstrates the successful generation of functional cardiomyocytes from pathogenic synovial cells in RA patients through iPSC reprogramming. Research using RA-iPSC-CMs might provide an opportunity to investigate the pathophysiology of cardiac involvement in RA.

A proliferation inducing ligand (APRIL) promotes IL-10 production and regulatory functions of human B cells

B cells may have a negative regulatory role, mainly mediated by interleukin 10 (IL-10). We recently showed that regulatory B-cell functions are impaired in patients with rheumatoid arthritis (RA) and that mice transgenic for a proliferation-inducing ligand (APRIL) are protected against collagen-induced arthritis. We aimed to explore the effect of APRIL on human B-cell IL-10 production, in healthy subjects and in patients with RA. The IL-10 production of B-cell was greater with APRIL than with BLyS or control medium, in a dose dependent manner. TACI expression was greater in IL-10 producing B cells (B10) than non-IL-10–producing B cells whereas BAFF-R expression was lower. TNF-α and IFN-γ secretion of T-cells were decreased by APRIL-stimulated B cells. APRIL stimulated STAT3 and STAT3 inhibition decreased B10 cells. APRIL also promoted B10 cells in RA patients. In conclusion, APRIL but not BLyS promotes IL-10 production by CpG-activated B cells and enhances the regulatory role of B cells on T cells. B10 cells in RA patients are responsive to APRIL, which suggests a possible therapeutic application of APRIL to expand B10 cells. This could also explain the difference of clinical efficacy observed between belimumab and atacicept in RA.

The Expression of BTLA Was Increased and the Expression of HVEM and LIGHT Were Decreased in the T Cells of Patients with Rheumatoid Arthritis [corrected

BackgroundCurrently, the pathogenesis of rheumatoid arthritis (RA) is not clearly understood. The LIGHT/HVEM/BTLA co-signaling pathway may be involved in the pathogenesis of RA, although reports on the expression levels of LIGHT, HVEM and BTLA in T lymphocytes from RA patients are limited.MethodIn this study, we recruited 30 healthy controls and 21 RA patients. Clinical characteristics were collected for RA patients. The levels of LIGHT, HVEM and BTLA expressed on the surface of circulating T cells of RA patients and healthy controls were measured by flow cytometry.ResultThe percentages of CD3+, CD4+ and CD8+ T lymphocytes that expressed BTLA from RA patients were all higher than those of the controls (all p < 0.05), while the percentages of CD3+, CD4+ and CD8+ T lymphocytes that expressed HVEM and LIGHT were all lower than those of the controls (all p < 0.05). The rheumatoid factor and the percentage of HVEM+CD4+ T lymphocytes showed a statistically significant negative correlation in RA patients (r = -0.453, p = 0.039), as did the swollen joint count and the percentage of BTLA+CD8+ T lymphocytes (r = -0.501, p = 0.021).ConclusionHere, we provide the first report on the increased expression of BTLA in T lymphocytes and on the decreased expression of HVEM and LIGHT in RA patients. BTLA, HVEM and LIGHT might be involved in the pathogenesis of RA and have the potential to be new clinically useful characteristics of RA.

Latent Cytomegalovirus Infection in Rheumatoid Arthritis and Increased Frequencies of Cytolytic LIR-1+CD8+ T Cells.

ObjectiveLeukocyte immunoglobulin‐like receptor 1 (LIR‐1) is up‐regulated by cytomegalovirus (CMV), which in turn, has been associated with premature aging and more severe joint disease in patients with rheumatoid arthritis (RA). The aim of this study was to investigate the expression and functional significance of LIR‐1 in CMV‐positive RA patients.MethodsWe determined the phenotype, cytolytic potential, CMV‐specific proliferation, and HLA–G–triggered, LIR‐1–mediated inhibition of interferon‐γ secretion of LIR‐1+ T cells in RA patients and healthy controls.ResultsWe found increased frequencies of CD8+ T cells with CMV pp65–specific T cell receptors in CMV‐positive RA patients as compared to CMV‐positive healthy controls. CMV‐specific CD8+ T cells in these patients were preferentially LIR‐1+ and exhibited a terminally differentiated polyfunctional phenotype. The numbers of LIR‐1+CD8+ T cells increased with age and disease activity, and showed high levels of reactivity to CMV antigens. Ligation of LIR‐1 with soluble HLA–G molecules in vitro confirmed an inhibitory role of the molecule when expressed on CD8+ T cells in RA patients.ConclusionWe propose that latent CMV infection in the context of a chronic autoimmune response induces the recently described “chronic infection phenotype” in CD8+ T cells, which retains anti‐infectious effector features while exhibiting autoreactive cytolytic potential. This response is likely dampened by LIR‐1 to avoid overwhelming immunopathologic changes in the setting of the autoimmune disease RA. The known deficiency of soluble HLA–G in RA and the observed association of LIR‐1 expression with disease activity suggest, however, that LIR‐1+ T cells are insufficiently controlled in RA and are still likely to be involved in the pathogenesis of the disease.

Correlation Between the Expression of MicroRNA-301a-3p and the Proportion of Th17 Cells in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation and subsequent joint destruction. Previous studies have confirmed that Th17 cells play a critical role in the pathogenesis of RA. MicroRNA (miR)-301a-3p is a regulatory factor for Th17cells differentiation that contributes to the pathogenesis of autoimmune diseases. The purposes of this study were to identify the alteration of Th17 cells and analyze the correlation between the expression of the miR-301a-3p and the proportion of Th17 cells in RA patients. The results showed that the frequency of Th17 cells and the expression of transcription factors (RORγt and STAT3) significantly increased in the peripheral blood mononuclear cells (PBMCs) from RA patients, and the associated proinflammatory cytokines were also upregulated. We also observed that the expression of protein inhibitor of activated STAT3 (PIAS3), the main cellular inhibitor of STAT3, was attenuated in RA patients and negatively correlated with the percentage of Th17 cells in RA. Interestingly, miR-301a-3p, an inhibitor of PIAS3 expression, was overexpressed in the PBMCs from RA patients and positively correlated with the frequency of Th17 cells in patients with RA. Taken together, these data indicated that miR-301a-3p and Th17 cells were augmented in peripheral blood, which may play an important role in the process of RA.

Tumor Necrosis Factor Blockade Stimulates Circulating Osteoblastic Lineage Cells Activity while Reducing Circulating Osteoclasts

Objective. This study examines the effects of tumor necrosis factor (TNF) blockade on markers of bone metabolism in peripheral blood from active rheumatoid arthritis (RA) patients. Methods. Eighteen patients (16 women, 2 men) aged 50 years (range 37-63 years), with persistently active RA (mean disease duration 7 years) were studied. Most took methotrexate (mean dose 12.5 mg) and all except one received corticosteroid (mean dose 5.7 mg). Four were treated with etanercept, eight received adalimumab and six received infliximab. Before and six months after taking TNF blockers, blood was sampled to obtain peripheral blood mononuclear cells (PBMCs), and serum bone turnover markers and acute phase reactants were measured. PBMCs were seeded and cultured to produce osteoblastic lineage cells and osteoclasts. Results. The formation of calcified nodules by osteoblastic lineage cells from PBMC increased from 205.7±196.3 μmol/well at the baseline to 752.5±671.9 μmol/well after TNF blockade (p＜0.024). The serum levels of bone formation markers, including bone specific alkaline phosphatase and osteocalcin also increased. The number of circulating osteoclasts and area of bone resorption pits made by osteoclasts were reduced after TNF blockade. Conclusion. The activity of circulating osteoblastic lineage cells increased after TNF blockade, whereas peripheral osteoclastogenesis tended to be suppressed. This is the first study of cultured human peripheral osteoblastic lineage cells in RA patients. Given that peripheral bone formation is difficult to study using radiologic methods, culture of these cells may provide a new modality for studying bone metabolism in RA. (J Rheum Dis 2016;23:356-362)

ID: 38: Plasmacytoid and myeloid dendritic cells in whole peripheral blood producing intracellular cytokines in patients with rheumatoid arthritis

Background Rheumatoid arthritis (RA) is characterized by active cellular reactions, in which formation dendritic cells (DC) are directly involved in particular by of various cytokines production [1] . Objectives To estimate intracellular cytokine production by different subsets of DCs in peripheral blood (PB) in patients with RA. Methods Monoclonal antibody for flow cytometry CD3, CD16, CD19, HLA-DR, CD11c, CD123, IL-10, IL-4, IFN- α , IL-12 (BD, USA) were added to the PB of 9 patients with RA and 14 healthy donors. Results Estimated low expression of IL-4, IL-10 as the key Th2 cytokines in myeloid (mDC) and plasmacytoid (pDC) dendritic cells in RA patients confirms the hypothesis of altered immune response towards Th1 pathway in this disease. Significant level of IL- 12 intracellular production was determined only in RA pDC that indicate their active involvement and participation in pathological process. This fact also confirms the presence of the cytokine imbalance as IFN- α plays an important immunoregulatory role in enhancing the Th1 cell-mediated immunity. Conclusions mDC and pDC in PB in patients with RA compared to healthy donors shows increase of IL-12 and IFN- α producing cells and decrease of IL-4 and IL-10 producing cells that indicate prevalence ability of DC in cellular immune response forming in patients with RA.

The in vitro modulatory effect of TNFα on the mRNA expression and protein levels of zinc finger protein ZNF334 in CD4(+) lymphocytes of healthy people.

We have shown before that the expression of ZNF334 gene, coding for a newly described zinc finger protein of as yet unknown function, is extremely reduced in CD4(+) lymphocytes of rheumatoid arthritis (RA) patients regardless of their age, and thus can be considered a new molecular marker of the disease. Based on the promoter sequence of the gene we speculated that it might be regulated by TNFα. Here we have tested that hypothesis, studying the in vitro influence of TNFα on the ZNF334 gene expression and protein levels in resting and stimulated CD4(+) cells of healthy volunteers. We have confirmed that treatment with TNFα modifies the levels of ZNF334 expression in the CD4(+) cells ex vivo; however, the effect varied for different individuals and reduction of expression was seen only for those cell samples that initially exhibited high transcriptional activity of the gene, while for those exhibiting initially very low expression, some increase in the transcriptional activity was observed. Incubation with TNFα significantly reduced the amounts of two isoforms of ZNF334 protein (initially high in all subjects) in parallel to the reduced transcription. Finally, the expression of ZNF334 in CD4(+) lymphocytes isolated after various periods of anti-CD3 stimulation generally increased with longer culture times, and the effect of TNFα treatment was negligible. Concluding, our results obtained in vitro for helper lymphocytes of healthy individuals seem to mimic the regulatory effect of TNFα on the expression of ZNF334 in the cells of RA patients.