Rheumatoid Arthritis CD4 Research Articles

Mitochondrial Control of Proteasomal Psmb5 Drives the Differentiation of Tissue-Resident Memory T Cells in Patients with Rheumatoid Arthritis.

To explore T cell-intrinsic mechanisms underpinning the mal-differentiation of tissue-resident memory T (Trm) cells in patients with rheumatoid arthritis (RA). Circulating T cells from patient with RA and healthy individuals were used for Trm cell differentiation. The role of Hobit in Trm differentiation was investigated through targeted silencing experiments. Psmb5 expression regulation was explored by identifying BRD2 as a key transcription factor, with the interaction validated through chromatin immunoprecipitation-quantitative polymerase chain reaction. The impact of BRD2 succinylation on Trm differentiation was examined by manipulating succinyl-CoA levels in T cells. Humanized NSG chimeras representing synovitis provided insights into Trm infiltration in RA synovitis and were used for translational experiments. In patients with RA, a notable predisposition of CD4+ T cells toward differentiation into Trm cells was observed, demonstrating a positive correlation with the disease activity score 28. Remarkably, Hobit was a pivotal facilitator in the formation of RA CD4+ Trm cells. Mechanistic studies unveiled the dysregulation of proteasomal Psmb5 in T cells of patients with RA as the key factor contributing to elevated Hobit protein levels. The deficiency of proteasomal Psmb5 was intricately linked to BRD2, with succinylation exerting a significant impact on Psmb5 transcription and Trm cell differentiation. This heightened BRD2 succinylation was attributed to elevated levels of mitochondrial succinyl-CoA in RA T cells. Consequently, targeting succinyl-CoA within CD4+ T cells controlled the inflammation of synovial tissues in humanized chimeras. Mitochondrial succinyl-CoA fosters the succinylation of BRD2, resulting in compromised transcription of proteasomal Psmb5 and the differentiation of Trm cells in RA.

ALOX5 drives the pyroptosis of CD4+ T cells and tissue inflammation in rheumatoid arthritis.

Pyroptosis, an inflammatory form of programmed cell death, is linked to the pathology of rheumatoid arthritis (RA). Here, we investigated the molecular mechanism underlying pyroptosis in T cells isolated from patients with RA. Compared with healthy individuals, patients with RA had more pyroptotic CD4+ T cells in blood and synovia, which correlated with clinical measures of disease activity. Moreover, the mRNA expression and protein abundance of arachidonate 5-lipoxygenase (ALOX5), which converts arachidonic acid to leukotriene A4 (LTA4), were increased in CD4+ T cells from patients with RA and, among patients with RA, were lowest in those in clinical remission. Knockdown or pharmacological inhibition of ALOX5 suppressed CD4+ T cell pyroptosis and improved symptoms in two rodent models of RA. Mechanistically, the increase in ALOX5 activity in RA CD4+ T cells enhanced the production of the LTA4 derivative LTB4, which stimulated Ca2+ influx through ORAI3 channels, leading to the activation of NLRP3 inflammasomes and pyroptosis. Our findings reveal a role for ALOX5 in RA and provide a molecular basis for further exploring the clinical utility of ALOX5 inhibition in RA and for using ALOX5 as a biomarker to distinguish active disease and remission in RA.

Therapeutic Effects of Mesenchymal/Stromal Stem Cells and Their Derived Extracellular Vesicles in Rheumatoid Arthritis.

Currently available therapies for rheumatoid arthritis (RA) are inadequate to alleviate the inflammation and reduce joint damage. While the immune-regulatory effect of human mesenchymal/stromal stem cells (MSCs) extracellular vesicles (EVs) has been tested in many inflammation-related diseases, little is known regarding their effect on patients with RA. Thus, we assessed the effect of human MSCs and MSC-EVs (from naïve or IFN-β-primed MSCs) on CD4+ T cells from patients with RA. Moreover, we investigated the effect of MSC-EVs on RA patients-derived synovial fibroblasts (FLS). MSC-EVs were prepared using a PEG precipitation followed by ultracentrifugation-based protocol. Applied to RA CD4+ T cells, EVs from IFN-β-primed MSCs, suppressed the expression of more key RA-associated cytokines (IL-4, GM-CSF IFN-γ, IL-2, TNF-α), and decreased CD4+ T-cell polyfunctionality than MSCs or EVs from naïve MSCs. MSCs mediated a slight decrease in the frequency of T-regulatory cells, while MSC-EVs rescued the frequency of T-regulatory cells. MSCs significantly inhibited CD4+ T-cell proliferation (P < .05), while no inhibition was observed in response to EV preparations. EVs from IFN-β-primed MSCs inhibited (P < .01) RA FLS migration and downregulated (P < .05) RA FLS surface markers CD34 and HLA-DR. Collectively, we demonstrated the immune-modulatory function of MSCs and their derived EVs in RA CD4+ T cells, which could be further enhanced by priming MSCs with IFN-β. Moreover, EVs from IFN-β-primed MSCs more efficiently inhibit RA FLS migration, and expression of RA FLS-related surface markers, suggesting these EVs as a potent therapy for RA.

PR-957 retards rheumatoid arthritis progression and inflammation by inhibiting LMP7-mediated CD4+ T cell imbalance

ObjectiveLow molecular mass polypeptide 7 (LMP7) is an immunoproteasome subunit that regulates T cell amplification, differentiation, and inflammation and is involved in rheumatoid arthritis (RA) progression. This study intended to apply PR-957 (an anti-LMP7 agent) for RA treatment in vitro and in vivo and evaluate its interaction with LMP7-mediated CD4+ T cell imbalance. MethodsPeripheral blood mononuclear cells (PBMCs) were obtained from 30 RA patients and 30 healthy controls. RA fibroblast-like synoviocytes (RA-FLSs) and CD4+ T cells were isolated from RA patients and then cocultured with PR-957 and/or LMP7 overexpression adenovirus (Ad-LMP7). Collagen-induced arthritis (CIA) mice were constructed and then treated with PR-957 and/or Ad-LMP7. ResultsLMP7 was higher in RA patients (versus healthy controls) and positively correlated with T helper (Th)1 cells, the Th1/Th2 ratio, Th17 cells, and the Th17/Treg ratio but not with Th2 or T regulatory (Treg) cells. PR-957 reduced Th1 and Th17 cells but increased Th2 and Treg cells in RA-CD4+ T cells, and this effect was partially reversed by Ad-LMP7 transfection. Interestingly, when cocultured with RA-CD4+ T cells, PR-957 increased RA-FLS apoptosis and decreased its invasive ability, viability, and inflammation, as suggested by IL-6, CCL2, MMP1, and MMP3; however, these phenomena were weakened in RA-FLSs without RA-CD4+ T cell coculture. In addition, Ad-LMP7 transfection attenuated the above effects of PR-957. In CIA mice, PR-957 decreased the arthritis score, synovial hyperproliferation and articular injury, inflammation in the synovium and serum, and the imbalance of Th1/Th2 and Th17/Treg in the spleen, and these effects were attenuated by Ad-LMP7. ConclusionPR-957 ameliorates RA progression and inflammation by repressing LMP7-mediated CD4+ T cell imbalance.

MALT1 regulates Th2 and Th17 differentiation via NF-κB and JNK pathways, as well as correlates with disease activity and treatment outcome in rheumatoid arthritis

ObjectiveMALT1 regulates immunity and inflammation in multiple ways, while its role in rheumatoid arthritis (RA) is obscure. This study aimed to investigate the relationship of MALT1 with disease features, treatment outcome, as well as its effect on Th1/2/17 cell differentiation and underlying molecule mechanism in RA.MethodsTotally 147 RA patients were enrolled. Then their blood Th1, Th2, and Th17 cells were detected by flow cytometry. Besides, PBMC MALT1 expression was detected before treatment (baseline), at week (W) 6, W12, and W24. PBMC MALT1 in 30 osteoarthritis patients and 30 health controls were also detected. Then, blood CD4+ T cells were isolated from RA patients, followed by MALT1 overexpression or knockdown lentivirus transfection and Th1/2/17 polarization assay. In addition, IMD 0354 (NF-κB antagonist) and SP600125 (JNK antagonist) were also added to treat CD4+ T cells.ResultsMALT1 was increased in RA patients compared to osteoarthritis patients and healthy controls. Meanwhile, MALT1 positively related to CRP, ESR, DAS28 score, Th17 cells, negatively linked with Th2 cells, but did not link with other features or Th1 cells in RA patients. Notably, MALT1 decreased longitudinally during treatment, whose decrement correlated with RA treatment outcome (treatment response, low disease activity, or disease remission). In addition, MALT1 overexpression promoted Th17 differentiation, inhibited Th2 differentiation, less affected Th1 differentiation, activated NF-κB and JNK pathways in RA CD4+ T cells; while MALT1 knockdown exhibited the opposite effect. Besides, IMD 0354 and SP600125 addition attenuated MALT1’s effect on Th2 and Th17 differentiation.ConclusionMALT1 regulates Th2 and Th17 differentiation via NF-κB and JNK pathways, as well as correlates with disease activity and treatment outcome in RA.

Cell division control protein 42 correlates with lower disease risk and its elevation predicts better treatment response, and inhibits T-helper 17 cell differentiation in rheumatoid arthritis.

Previous research reports that cell division control protein 42 (CDC42) is dysregulated in rheumatoid arthritis (RA). This study aimed to further explore the linkage of CDC42 with T-helper (Th) 1 and Th17 cell differentiation, and its implication in RA management. After enrolling 80 RA patients, their blood CDC42, Th1 and Th17 cells were detected by RT-qPCR and flow cytometry, respectively, IFN-γ and IL-17A were detected by ELISA. Based on the treatment response at week 12, the patients were classified as response patients and no response patients. In addition, blood CDC42 was also detected after enrolling 40 healthy controls. Subsequently, naïve CD4+ T cells from RA patients were transfected with control, CDC42 overexpression and knockdown lentivirus, followed by differentiation assay. CDC42 was reduced in RA patients versus healthy controls (P < 0.001). In RA patients, CDC42 was negatively correlated with IFN-γ (P = 0.023), Th17 cells (P = 0.011) and IL-17A (P = 0.003) but not Th1 cells (P = 0.200). CDC42 presented an increasing trend after treatment (P < 0.001); besides, CDC42 at week 8 (P = 0.027) and week 12 (P < 0.001) were increased in response patients versus no response patients. Subsequent experiment showed that in RA CD4+ T cells, CDC42 overexpression reduced IFN-γ and IL-17A (both P < 0.05), while CDC42 knockdown elevated IL-17A (P < 0.05) but not IFN-γ (P > 0.05). Moreover, CDC42 overexpression inhibited, while CDC42 knockdown increased Th17 cell percentage (P < 0.05) but not Th1 cell percentage (P > 0.05). CDC42 negatively correlates with disease risk and its elevation predicts better treatment response; it also inhibits Th17 but not Th1 cell differentiation in RA.

Effect of High-Intensity Interval Training on Rheumatoid Arthritis CD4+ T Cell Oxidative Metabolism

Persons with rheumatoid arthritis (RA) have poor cardiorespiratory fitness and accelerated biological aging driven by systemic impairments in metabolism and inflammation. In this study of older RA participants, our goal was to identify the effects of a high-intensity interval training (HIIT) program on cardiorespiratory fitness and peripheral CD4+ T cell metabolism. We isolated CD4+ T cells from peripheral blood mononuclear cells in sedentary female RA participants (n=6; age=64.0+/-6.3 years) who underwent cardiopulmonary exercise testing and phlebotomy before and after 10 weeks of HIIT. HIIT improved RA cardiorespiratory fitness by 6.5+/-6.0% (pre-HIIT VO2 peak=25.1+/-5.1 ml/kg/min, post-HIIT VO2 peak=26.7+/-5.0; p=0.05). As measured by Seahorse XF Mito Stress Test, there were no significant mean changes in CD4+ T cell oxidative (oxygen consumption rate (OCR); pmol/min) or glycolytic (extracellular acidification rate (ECAR); mpH/min) metabolism, however there was large interindividual variability. RA peripheral CD4+ T cells preferred glycolytic metabolism (pre-HIIT mean basal OCR/ECAR ratio=0.78+/-0.13 pmol/mpH), while HIIT non-significantly shifted cellular preference toward oxidative metabolism (post-HIIT mean basal OCR/ECAR ratio=0.86+/-0.16; p=0.30). Increases in RA cardiorespiratory fitness following HIIT were significantly associated with increases in RA peripheral CD4+ T cell OCR/ECAR ratio (Spearman’s rho=1.0, p<0.001) and basal and maximal respiration (rho=0.89, p=0.02 for both). Additionally, increases in CD4+ T cell mitochondrial ATP-linked respiration were significantly associated with increased quantities of circulating naïve CD4+CCR7+CD45RA+ T cells (rho=0.89, p=0.02). Our findings suggest that targeting cardiorespiratory fitness may be key in modulating T cell specific oxidative metabolism and function to prevent immunosenescence in older patients with chronic inflammatory diseases.

Optimization of Human Mesenchymal Stem Cells for Rheumatoid Arthritis: Implications for Improved Therapeutic Outcomes.

ObjectiveSeropositive rheumatoid arthritis (RA) is a chronic autoimmune disease that is rarely “cured.” Human mesenchymal stem cells (hMSCs) are known to reduce inflammation and restore immune homeostasis. However, methods for predicting therapeutic hMSC potency have not been established. The goal of these studies was to use and refine an ex vivo functional assay that determines potency of hMSCs and can then be validated in clinical trials as a potency measure of hMSCs used therapeutically to treat RA.MethodsAllogeneic hMSCs were cytokine‐stimulated, and a conditioned medium (CM) was harvested. The CM was tested for the potential to attenuate RA CD4+ T cell proliferation using suppression assays. Indoleamine 2, 3‐dioxygenase (IDO) mRNA, and protein were quantified in hMSCs as a measure to compare hMSCs across (prior) studies.ResultsTo mimic a proinflammatory environment that resembles that in RA, interleukin‐1(IL1β), tumor necrosis factor α (TNFα), and interferon γ (IFNγ) (alone or in combination) were used to precondition hMSCs. Treating hMSCs with a combination of these cytokines generated a CM “secretome” that suppressed T cell proliferation between 70 and 83%. Forty‐eight hours of cytokine preconditioning hMSCs was required to maximize this effect. T cell suppression positively correlated with increases in hMSC cellular IDO mRNA and protein.ConclusionBy standardizing assays to measure hMSC effects, their potency on T cell suppression can be quantified. These studies demonstrate that hMSCs can be compared functionally to identify optimal preparation(s) for therapeutic use in RA and that the potency of hMSC‐dependent T cell suppression may differ between hMSC donors. Clinical studies are warranted to validate the hypothesis that ex vivo potency in suppressing T cells will positively correlate with a reduction in RA disease activity and increase in immunological quiescence.

Pim Kinases as Therapeutic Targets in Early RheumatoidArthritis.

As well as being an established oncoprotein and therapeutic target in cancer, proviral integration site for Moloney murine leukemia virus 1 (Pim-1) is implicated in human autoimmunity. This study was undertaken to investigate Pim-1 and its family members as potential therapeutic targets in early rheumatoid arthritis (RA). A flow cytometry assay for PIM1 transcript measurement in peripheral blood mononuclear cells from patients with early arthritis was validated and applied as a biomarker of Pim-1 activity at the cellular level. Synovial protein expression was similarly determined by multiplex immunofluorescence in tissue samples from untreated RA patients and non-RA disease controls. Functional consequences of Pim kinase family manipulation in freshly isolated CD4+ T cells from these individuals were ascertained, along with the impact of Pim inhibition on mice with collagen-induced arthritis (CIA). The percentage of circulating CD4+ T cells positive for PIM1 transcript by flow cytometry proved a faithful surrogate for gene expression and was significantly higher in patients with early RA than in those with other diseases. Pim-1 protein levels were similarly up-regulated in synovial CD4+ T cells from patients with early RA. Ex vivo, exposure of T cell receptor-stimulated early RA CD4+ T cells to Pim kinase inhibitors restrained their activation and proliferative capacity. Diminished production of proinflammatory cytokines (interferon-γ and interleukin-17) and an expanded CD25high FoxP3+ Treg cell fraction were also observed in exposed versus unexposed cells. Finally, administration of Pim inhibitors robustly limited arthritis progression and cartilage destruction in CIA. Our findings indicate that Pim kinases are plausible therapeutic targets in a readily identifiable subgroup of patients with early RA. Repurposing of Pim inhibitors for this disease should be considered.

P148 Differential methylation of the TNF gene promoter, potential for the development of a diagnostic biomarker for RA

Abstract Background/Aims Diagnosing rheumatoid arthritis (RA) early is difficult despite the use of anti-citrullinated protein antibodies in the new EULAR-2010 classification criteria, particularly for antibody-negative patients. Alterations in epigenetic patterns have been associated with RA CD4+T-cells at the TNF gene locus (1). The second aim of my PhD project was to select a CpG site to develop a quantitative methylation sensitive PCR (qMSP) assay as a diagnostic biomarker test for RA classification. Methods Patients attending an early arthritis clinic were used (with full ethical approval). An 450K Illumina methylation genome-wide dataset analysing DNA from CD4+T-cells identified CpG on TNF promoter region as a differentially methylated CpG between controls and RA patients (1). A qMSP assay was designed using a TaqMan approach (primers and probe), using a reference gene (GAPHD) for normalisation. The assay was validated in DNA extracted from PBMC. The performance of the assay for RA classification was accessed using binary logistic regression. Results We noted differences in levels of methylation in CD4+T-cells in RA, notably affecting the TNF gene promoter in about 15% of cells. We validated this using bisulphite sequencing showing about 30% of cells with differential methylation in early RA compared to controls. This allowed us to select cg11484872, cg21370522, and cg01569083 as candidates. We optimised a qMSP assay using fully methylated/un-methylated DNA. PBMC samples were retrieved from our tissue bank for 65 early RA, and 64 non-RA patients (including 11 reactive arthritis, 37 undifferentiated arthritis, and 16 psoriatic arthritis). The methylation levels (%) detected by the TNF-qMSP assay were significantly lower in RA (Δ-DM =-10.28%, p = 7x10-7). The loss of TNF promoter gene methylation was associated with RA classification (p &amp;lt; 0.0001, unadjusted OR = 0.900 (95% CI: 0.85-0.94)) with a good classification performance (AUROC = 0.76 (95% CI: 0.67, 0.84)). An adjusted regression model using the demographic and clinical parameters associated with RA classification in this group of patients (age, smoking, counts for tender and swollen joints) showed that adding TNF to the clinical model improved the model’s performance (increasing AUROC from 0.86 , to 0.91). Comparing classification accuracy, adding the TNF-qMSP resulted in an increased correct classification of 5.7 % compared to the clinical model. A similar result was obtained in the antibody negative patients, with TNF-qMSP assay improving the model’s classification from AUROC 0.91 to 0.96 and 3.3% more patients correctly classified. Conclusion The loss of DNA methylation on the TNF gene promoter identified in early RA patients provided a valuable target for a biomarker assay development. The results will need to be confirmed in a larger cohort but the TNF-qMSP assay offers good opportunities for RA versus non-RA classification particular in antibody negative disease. Disclosure R. Pitaksalee: Grants/research support; ROYAL THAI GOVERNMENT SCHOLARSHIP. P. Emery: None. R. Hodgett: None. F. Ponchel: None.

Decreased Levels of STAT1 and Interferon-γ-Induced STAT1 Phosphorylation in Rheumatoid Arthritis CD4 and CD8 T Cells.

ObjectiveWe investigated whether a previously reported association of IFNGR expression with rheumatoid arthritis (RA) and its radiographic severity reflects differences in proximal interferon‐γ (IFN‐γ) signaling in T cells from patients with RA compared with healthy controls (HC).MethodsUsing phosphoflow cytometry, we compared IFN‐γ–stimulated signal transducer and activator of transcription 1 (STAT1) activation in CD4+ and CD8+ T‐cell populations from patients with RA and HC.ResultsCompared with controls, patients with RA had a higher proportion of CD4+ T cells, associated with expansion of the CD4+ effector memory subset. Several CD4+ T‐cell types exhibited reduced IFN‐γ–induced phosphoSTAT1Y701 (pSTAT1Y701) in patients with RA compared with HC. Engaging the T‐cell receptor (TCR) complex on CD4+ T cells during IFN‐γ stimulation abrogated the reduction in STAT1 activation in patients with RA but had no effect in HC. The phosphorylation of STAT1S727 was similar in CD4+ T cells from patients with RA and HC. In contrast to CD4+ T cells, IFN‐γ–induced pSTAT1Y701 levels in CD8+ T cells were equivalent or higher in patients with RA compared with HC. Total STAT1 levels (phosphorylated + unphosphorylated) were lower in CD4+ and CD8+ T cells from patients with RA compared with HC.ConclusionWe report diminished IFN‐γ–induced pSTAT1Y701 levels in CD4+ T cells in patients with RA, which were restored by TCR engagement. There were lower levels of total STAT1 in patients with RA compared with HC, but this likely does not explain diminished IFN‐γ–induced pSTAT1Y701 levels in CD4+ T cells because activation in CD8+ T cells was higher or equivalent to that seen in HC. The enhanced IFNGR expression in patients with RA reported previously may reflect a compensatory mechanism to overcome deficiency in IFN‐γ responsiveness.

Immunometabolism in the development of rheumatoid arthritis.

In rheumatoid arthritis (RA), breakdown of self-tolerance and onset of clinical disease are separated in time and space, supporting a multi-hit model in which emergence of autoreactive T cells is a pinnacle pathogenic event. Determining factors in T cell differentiation and survival include antigen recognition, but also the metabolic machinery that provides energy and biosynthetic molecules for cell building. Studies in patients with RA have yielded a disease-specific metabolic signature, which enables naive CD4 T cells to differentiate into pro-inflammatory helper T cells that are prone to invade into tissue and elicit inflammation through immunogenic cell death. A typifying property of RA CD4 T cells is the shunting of glucose away from glycolytic breakdown and mitochondrial processing toward the pentose phosphate pathway, favoring anabolic over catabolic reactions. Key defects have been localized to the mitochondria and the lysosome; including instability of mitochondrial DNA due to the lack of the DNA repair nuclease MRE11A and inefficient lysosomal tethering of AMPK due to deficiency of N-myristoyltransferase 1 (NMT1). The molecular taxonomy of the metabolically reprogrammed RA T cells includes glycolytic enzymes (glucose-6-phosphate dehydrogenase, phosphofructokinase), DNA repair molecules (MRE11A, ATM), regulators of protein trafficking (NMT1), and the membrane adapter protein TSK5. As the mechanisms determining abnormal T cell behavior in RA are unraveled, opportunities will emerge to interject autoimmune T cells by targeting their metabolic checkpoints.

ER-stressed MSC displayed more effective immunomodulation in RA CD4+CXCR5+ICOS+ follicular helper-like T cells through higher PGE2 binding with EP2/EP4

Objectives: To analyze the further immunomodulatory effects of endoplasmic reticulum (ER)-stressed umbilical cord-derived mesenchymal stem cells MSCs (UC-MSCs) on rheumatoid arthritis (RA) CD4+CXCR5+ICOS+ T (follicular helper-like T, Tfh) cells.Methods: MSCs were isolated from umbilical cord and surface markers were identified by flow cytometry. CD4+ T cells were purified from RA patients’ peripheral blood mononuclear cells (PBMCs) using immunomagnetic beads. Thapsigargin (Tg)-stimulated or unstimulated MSCs were co-cultured with RA CD4+ T cells. CD4+CXCR5+ICOS+ T cells were analyzed with fluorescence activating cell sorter (FACS) and major soluble factors secreted by MSCs were detected by qRT-PCR as well as ELISA. Receptors of prostanoid E2 (PGE2), known as EP1-4, on CD4+ T cells were tested with RT-PCR and FACS. Proportion of CD4+CXCR5+ICOS+ T cells was determined after EP2/EP4 antagonists and anti-IL-6R antibody was added into co-cultured system, respectively.Results: ER-stressed MSCs further down-regulated peripheral CD4+CXCR5+ICOS+ T cells compared with Tg-stimulated MSCs and CD4+ T co-cultured group. PGE2 and IL-6 increased obviously in the supernatants. EP2/EP4 could be detected on CD4+ T cells and frequencies of CD4+CXCR5+ICOS+ T cells were upregulated when EP2 and/or EP4 antagonists rather than anti-IL-6R antibody were added.Conclusions: ER-stressed MSCs exhibited better inhibition effect on RA CD4+CXCR5+ICOS+ T cells by releasing PGE2, indicating the immunosuppressive effect of MSCs could be enhanced by induction of ER stress.

Leptin Upregulates Peripheral CD4+CXCR5+ICOS+ T Cells via Increased IL-6 in Rheumatoid Arthritis Patients.

CD4+CXCR5+ICOS+ T cells, known as Tfh (T Follicular helper) cells, are required for antibody production. Abnormal production and differentiation of Tfh cells are involved in many autoimmune diseases, including rheumatoid arthritis (RA). Leptin has the property of modulating immune system. Here, we explored the effect of leptin on CD4+CXCR5+ICOS+ T cells production in RA patients. Serum leptin levels were measured by enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMC) stimulated with CD3/CD28 were cultured in the presence and absence of leptin and with or without anti-IL-6 receptor (anti-IL-6R), anti-IL-21R, and anti-IL-12R antibody respectively. IL-6, IL-21, and IL-12 levels were determined by ELISA. Bcl-6 was detected by reverse transcription-polymerase chain reaction. STAT1, pSTAT1, STAT3, and pSTAT3 were examined by western blot. We found that leptin levels were higher in RA patients than healthy controls. Leptin-stimulated RA PBMC upregulated CD4+CXCR5+ICOS+ T cells, along with increased IL-6, IL-21, and IL-12.CD4+CXCR5+ICOS+ T cells, Bcl-6 mRNA expression, pSTAT1, and pSTAT3 obviously declined when anti-IL-6R antibody was added into leptin-treated RA PBMC, which suggested that leptin upregulated RA CD4+CXCR5+ICOS+ T cells via increased IL-6 by activation of STAT1 and STAT3. We presented an innovative mechanism on how leptin participated in RA pathogenesis.

Deficient Activity of the Nuclease MRE11A Induces T Cell Aging and Promotes Arthritogenic Effector Functions in Patients with Rheumatoid Arthritis

Immune aging manifests with a combination of failing adaptive immunity and insufficiently restrained inflammation. In patients with rheumatoid arthritis (RA), Tcell aging occurs prematurely, but the mechanisms involved and their contribution to tissue-destructive inflammation remain unclear. We found that RA CD4+ Tcells showed signs of aging during their primary immune responses and differentiated into tissue-invasive, proinflammatory effector cells. RA Tcells had low expression of the double-strand-break repair nuclease MRE11A, leading to telomeric damage, juxtacentromeric heterochromatin unraveling, and senescence marker upregulation. Inhibition of MRE11A activity in healthy Tcells induced the aging phenotype, whereas MRE11A overexpression in RA Tcells reversed it. In human-synovium chimeric mice, MRE11Alow Tcells were tissue-invasive and pro-arthritogenic, and MRE11A reconstitution mitigated synovitis. Our findings link premature Tcell aging and tissue-invasiveness to telomere deprotection and heterochromatin unpacking, identifying MRE11A as a therapeutic target to combat immune aging and suppress dysregulated tissue inflammation.

Enhanced IFN-γ signals and STAT5-phosphatase dependent attenuated IL-2 signals in the pathogenesis of rheumatoid arthritis.

Abstract The importance of type I interferons as a driver of pathogenesis in autoimmunity, including rheumatoid arthritis (RA) is well recognized. However, in this context the contribution of type II interferons (IFN-γ) has been equivocal. This study developed from our recent discovery that the expression of IFN-γ receptor gene Ifngr1 strongly associates with RA disease and expression of Ifngr2 associates with radiographic severity. In this study we show that IFN-γ induced activation is significantly greater in naïve, central memory, Tfh and Treg subsets of CD4+ T cell populations from RA patients compared to healthy controls (HC). Remarkably, such enhanced IFN-γ induced STAT1 activation was not observed in CD8 T cell populations from RA patients. The activation of STAT1 and consequently, IFN-γ signals are regulated by tyrosine phosphatases. Treatment with phenyl-arsine oxide (PAO), a broadly active phosphatase inhibitor, had no effect on IFN-γ induced STAT1 activation in any T cell population from RA or HC. In contrast, such treatment with PAO greatly enhanced IL-2 induced activation of STAT5 in RA CD4 T cell populations (naïve, central memory effector memory, Treg, Tfh) but not from HC or in CD8 T cell populations. This result suggests that the regulatory activity of IL-2 in RA is dampened by a STAT5-specific phosphatase. We further determined that such a STAT5-phosphatase was also expressed and active in regulatory and effector CD4 T cell populations from the spontaneously autoimmune BXD2 mice. Overall our results indicate that CD4 T cell subpopulation dependent enhanced IFN-γ signals and dampened IL2-STAT5 signals contributes to pathogenesis in RA.

Subpopulations of regulatory T-lymphocytes in the peripheral blood of patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory rheumatic disease associated with a dysfunction of the T cell-mediated tolerance and leading to the disability of working population. The regulatory CD4' T cells play an important role in the regulation of autoimmunity and can suppress immune responses. With that, there is no consensus on the content of these lymphocytes and their role in the pathogenesis of RA. Objective: The aim of the study was to assess the content ofperipheral blood regulatory Tcells (Treg) according to the expression of membrane markers CD4, CD25, CD127 and intracellular FOXP3 marker, as well as the expression of two functional molecules (CTLA-4 and CCR4) in Treg cells of patients with RA. Peripheral blood samples of RA patients (median age 65,5 years [54;68,3) and healthy controls (median age 58 years [44; 66]) were analyzed. Cell count and the expression level of molecules were assessed by flow cytometry. Peripheral blood samples of 36 RA patients and 20 healthy donors were analyzed. The number of the cells with Treg-associated phenotypes CD4⁺CD25hi and CD4⁺CD25hiCD127low/⁻ was higher in RA patients in comparison with healthy donors. Increased levels of RA CD4⁺ T cells expressing FOXP3 were also observed. This may be due to increasing in the number of CD4⁺FOXP3⁺CD25⁻ lymphocytes, whereas the content of RA CD4⁺FOXP3⁺CD25⁺ Treg cells was at the level of the control. The expression of the functional molecule CTLA-4 in Treg cells of patients with RA was not different from the control, while the expression level of the chemokine receptor CCR4 which provides migration of lymphocytes at sites of inflammation and barrier tissues was significantly increased in RA patients. Increase in the levels of certain Treg-associated lymphocyte populations were detected in peripheral blood of RA patients. During the natural course of RA, alterations in the level of the chemokine receptor CCR4 might indicate the enhanced lymphocyte migration.

Roles of 1,25(OH)2D3 and Vitamin D Receptor in the Pathogenesis of Rheumatoid Arthritis and Systemic Lupus Erythematosus by Regulating the Activation of CD4+ T Cells and the PKCδ/ERK Signaling Pathway

Background/Aims: The study aims to elucidate the roles of 1,25(OH)₂D₃ and vitamin D receptor (VDR) in the pathogenesis of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) by regulating the activation of CD4⁺ T cells and the PKCδ/ERK signaling pathway. Methods: From January 2013 to December 2015, a total of 130 SLE patients, 137 RA patients and 130 healthy controls were selected in this study. Serum levels of 1,25(OH)₂D₃ and VDR mRNA expression were detected by ELISA and real-time fluorescence quantitative PCR (RT-qPCR). Density gradient centrifugation was performed to separate peripheral blood mononuclear cells (PBMCs). CD4⁺ T cells were separated using magnetic activated cell sorting (MACS). CD4⁺T cells in logarithmic growth phase were collected and assigned into 9 groups: the normal control group, the normal negative control (NC) group, the VDR siRNA group, the RA control group, the RA NC group, the VDR over-expressed RA group, the SLE control group, the SLE NC group, and the VDR over-expressed SLE group. The mRNA and protein expressions of VDR, PKCδ, ERK1/2, CD11a, CD70 and CD40L were detected by RT-qPCR and Western blotting. Bisulfite genomic sequencing was conducted to monitor the methylation status of CD11a, CD70 and CD40L. Results: Compared with healthy controls, serum 1,25(OH)₂D₃ level and VDR mRNA expression in peripheral blood were decreased in SLE patients and RA patients. With the increase of concentrations of 1,25(OH)₂D₃ treatment, the VDR mRNA expression and DNA methylation levels of CD11a, CD70 and CD40L were declined, while the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L were elevated in SLE, RA and normal CD4⁺T cells. Compared with the SLE contro, RA control, SLE NC and RA NC groups, the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L decreased but DNA methylation levels of CD11a, CD70 and CD40L increased in the VDR over-expressed SLE group and VDR over-expressed RA group. However, compared with the normal control and normal NC groups, the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L increased, but DNA methylation levels of CD11a, CD70 and CD40L decreased in the VDR siRNA group. Compared with the normal control group, the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L increased, but DNA methylation levels of CD11a, CD70 and CD40L decreased in the SLE control and RA control groups. Conclusion: Our study provide evidence that 1,25(OH)₂D₃ and VDR could inhibit the activation of CD4⁺ T cells and suppress the immune response of SLE and RA through inhibiting PKCδ/ERK pathway and promoting DNA methylation of CD11a, CD70 and CD40L.

Novel insights into the regulatory architecture of CD4+ T cells in rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most frequent autoimmune chronic inflammatory disease of the joints and it is characterized by the inflammation of the synovial membrane and the subsequent destruction of the joints. In RA, CD4+ T cells are the main drivers of disease initiation and the perpetuation of the damaging inflammatory process. To date, however, the genetic regulatory mechanisms of CD4+ T cells associated with RA etiology are poorly understood. The genome-wide analysis of expression quantitative trait loci (eQTL) in disease-relevant cell types is a recent genomic integration approach that is providing significant insights into the genetic regulatory mechanisms of many human pathologies. The objective of the present study was to analyze, for the first time, the genome-wide genetic regulatory mechanisms associated with the gene expression of CD4+ T cells in RA. Whole genome gene expression profiling of CD4+ T cells and the genome-wide genotyping (598,258 SNPs) of 29 RA patients with an active disease were performed. In order to avoid the excessive burden of multiple testing associated with genome-wide trans-eQTL analysis, we developed and implemented a novel systems genetics approach. Finally, we compared the genomic regulation pattern of CD4+ T cells in RA with the genomic regulation observed in reference lymphoblastoid cell lines (LCLs). We identified a genome-wide significant cis-eQTL associated with the expression of FAM66C gene (P = 6.51e−9). Using our new systems genetics approach we identified six statistically significant trans-eQTLs associated with the expression of KIAA0101 (P<7.4e−8) and BIRC5 (P = 5.35e−8) genes. Finally, comparing the genomic regulation profiles between RA CD4+ T cells and control LCLs we found 20 genes showing differential regulatory patterns between both cell types. The present genome-wide eQTL analysis has identified new genetic regulatory elements that are key to the activity of CD4+ T cells in RA.

Reciprocal Activation of CD4+ T Cells and Synovial Fibroblasts by Stromal Cell–Derived Factor 1 Promotes RANKL Expression and Osteoclastogenesis in Rheumatoid Arthritis

Stromal cell-derived factor 1 (SDF-1) is a chemokine that is involved in the bone-destructive process in rheumatoid arthritis (RA) and bony metastasis in malignancy. This study was undertaken to determine the role and mechanism of SDF-1 in RA-associated osteoclastogenesis. The expression of SDF-1, tumor necrosis factor α (TNFα), and RANKL in RA synovial tissue was analyzed using confocal microscopy. After synovial fibroblasts and CD4+ T cells were treated with SDF-1, RANKL messenger RNA expression was determined by real-time and reverse transcription polymerase chain reaction. Osteoclastogenesis was assessed by counting tartrate-resistant acid phosphatase-positive multinucleated cells in CD14+ monocytes cultured with SDF-1 in the presence of anticytokine antibodies or signal inhibitors and in monocytes cocultured with SDF-1-pretreated synovial fibroblasts and CD4+ T cells. RANKL, TNFα, and SDF-1 were coexpressed in the lining and sublining of RA synovium. SDF-1 stimulated RANKL expression in RA synovial fibroblasts and CD4+ T cells, and TNFα inhibition reduced this stimulation. When monocytes isolated from human peripheral blood were cultured with SDF-1, they were differentiated into osteoclasts in the absence of RANKL. Monocytes were also differentiated into osteoclasts when they were cocultured with SDF-1-pretreated synovial fibroblasts or CD4+T cells; however, this osteoclastogenesis was reduced by TNFα inhibition. Our findings indicate that SDF-1 induces osteoclastogenesis directly and indirectly via up-regulating RANKL expression in RA synovial fibroblasts and CD4+ T cells, and that this is mediated by TNFα. The axis of SDF-1 and RANKL is a potential therapeutic target for RA-associated bone destruction.