Cells In Rheumatoid Arthritis Patients Research Articles

The changes of intestinal flora and its relevance with memory Tfh and B cells in rheumatoid arthritis patients treated with csDMARDs and csDMARDs + bDMARDs

BackgroundA growing body of experimental and clinical evidence has implicated gut microbiota in the onset and course of rheumatoid arthritis (RA). The imbalance of intestinal flora in RA patients may lead to abnormal expression of immune cells and related cytokines. PurposeConventional synthetic disease-modifying antirheumatic drugs (csDMARDs) and conventional synthetic disease-modifying antirheumatic drugs combined with biological disease-modifying antirheumatic drugs (csDMARDs + bDMARDs) are widely used to treat RA, but the characteristics of gut microbiota before and after treatment and their relationship with memory Tfh/B cells and cytokines remain unclear. MethodsStool samples were collected from 50 RA patients and 25 healthy controls (HCs) for 16SrRNA gene sequencing. We examined the proportion of lymphocyte subsets in healthy controls and RA patients. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of related cytokines in serum. The α and β diversity of intestinal flora, and the correlation between intestinal flora and clinical indicators, lymphocyte subsets, cytokines were analyzed. ResultAt the genus level, Ruminococcaceae_Ruminococcus was decreased in the csDMARDs and csDMARDs + bDMARDs treatment group, whereas Faecalibacterium was reduced in the csDMARDs treatment group, compared to untreated group. CD4+CD45RO+CCR7+CXCR5+central memory Tfh cells and CD4+CD45RO+CCR7-CXCR5+effector memory Tfh cells were significantly lower in the csDMARDs + bDMARDs treatment group than in untreated group. CD19+CD27+IgD+pre-switched memory B cells were higher in the csDMARDs and csDMARDs + bDMARDs treatment groups, whereas CD19+CD27+IgD-switched memory B cells were significantly lower than in untreated group. Ruminococcaceae_Ruminococcus was negatively correlated with CD19+CD27+IgD+ pre-switched memory B cells but positively correlated with CD4+CD45RO+CCR7-CXCR5+effector memory Tfh and CD19+CD27+IgD-switched memory B cells in patients with RA treated with DMARDs. ConclusionThe gut microbiota, memory Tfh cells, memory B cells, and cytokines of patients with RA changed significantly under different treatment regimens and had certain correlations with the clinical indicators of RA.

Peptide Derivatives of Human and Rabbit Cathelicidin Reduce Inflammatory Cytokines in Peripheral Blood Mononuclear Cells of Rheumatoid Arthritis Patients

Peptide Derivatives of Human and Rabbit Cathelicidin Reduce Inflammatory Cytokines in Peripheral Blood Mononuclear Cells of Rheumatoid Arthritis Patients

Altered CD39 and CD73 Expression in Rheumatoid Arthritis: Implications for Disease Activity and Treatment Response.

In rheumatoid arthritis (RA) synovium, ATP, and ADP are released, sparking inflammation. Ectoenzymes CD39 and CD73 metabolize these purine nucleotides, generating anti-inflammatory adenosine. Therefore, dysregulated CD39 and CD73 expression may impact RA development. We assessed CD39 and CD73 expression in peripheral blood from 15 healthy controls (Cs) and 35 RA patients at baseline and after 3 and 6 months of tocilizumab treatment using flow cytometry. Additionally, ectoenzyme expression was examined on cultured T cells to understand activation and IL-6 effects. At baseline, RA patients exhibited a lower CD8+CD39-CD73+ cell percentage, which inversely correlated with DAS28. Additionally, they had lower percentages of Treg CD39+CD73+ and CD39-CD73- cells. Good responders tended to have lower B CD39+CD73+ cell percentages at baseline and 3 months. Additionally, Treg, CD8+ T and B cells inversely correlated with DAS28. T-cell activation increased CD39 and decreased CD73 expression, regardless of IL-6. IL-6 reduced IFNγ-secreting CD4+ T-cell percentage in Cs, but increased the percentage of IFNγ-secreting CD4+ and CD8+ T cells in RA patients. These findings indicate differing CD39 and CD73 expression in RA and Cs, influenced by T-cell activation and IL-6. Correlations between these molecules and RA activity suggest their role in dysregulated inflammation in RA.

Increased gene expression of CCR6 and RORγt in peripheral blood cells of rheumatoid arthritis patients and their correlation with anti-cyclic citrullinated peptide and disease activity.

The significance of T helper 17 (Th17) cells in the pathogenesis of rheumatoid arthritis (RA) has recently been demonstrated in many studies. Retinoic acid receptor-related orphan receptor γt (RORγt) is a transcription factor that is specifically involved in the generation of Th17 cells. Besides, the chemokine receptor CCR6, the receptor for CCL20, is characteristically expressed by these cells. Considering the pivotal roles of Th17 cells in RA pathogenesis, in this study, we assessed the gene expression of CCR6 and RORγt in the peripheral blood leukocytes of new case RA patients. Also, we evaluated their association with anticyclic citrullinated peptide (anti-CCP) antibodies and disease activity. Forty-five new case RA patients and 45 healthy persons have been recruited in this investigation. The gene expression of CCR6 and RORγt was evaluated by quantitative real-time PCR (qRT-PCR), and anti-CCP antibodies plasma levels were measured using the enzyme-linked immunosorbent assay (ELISA) technique. Disease activity was measured according to the disease activity score-28 (DAS-28) formula. The gene expression of CCR6 and RORγt increased remarkably in new case RA patients compared to healthy controls (p < .05 and p < .01, respectively). Moreover, there was a positive correlation between RORγt gene expression and parameters, including gene expression of CCR6 (p = .001, r = .461), plasma levels of CCL20 (p = .0009, r = .477), ESR (p = .004, r = .419), DAS-28 (p = .006, r = .402), anti-CCP (p = .019, r = .346), and RF (p = .001, r = .451). Also, CCR6 gene expression was positively associated with the DAS-28 (p = .037, r = .310), plasma levels of anti-CCP (p = .037, r = .312), and ESR (p = .029, r = .327). Increased gene expression of CCR6 and RORγt in peripheral blood leukocytes of new case RA patients may contribute to the exacerbation and pathogenesis of RA.

Identification of biomarkers in patients with rheumatoid arthritis responsive to DMARDs but with progressive bone erosion.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that may cause joint destruction and disability. The pharmacological treatment of RA aims at obtaining disease remission by effectively ceasing joint inflammation and arresting progressive bone erosions. Some patients present bone lesions accrual even after controlling joint inflammation with current therapies. Our study aimed to analyze lymphocyte subsets and levels of circulating cytokines in patients with RA with progressive bone erosions. We enrolled 20 patients with a diagnosis of RA and 12 healthy donors (HD). Patients with RA were divided into patients with bone erosions (RA-BE+) and without bone erosions (RA-BE-). Lymphocyte subsets in peripheral blood were evaluated by flow cytometry. Circulating cytokines levels were evaluated by protein array. The distribution of lymphocyte subsets was not able to separate HD from AR patients and RA-BE+ and RA-BE- in cluster analysis. We observed a significant expansion of CXCR5- PD1+ T peripheral helper cells (Tph cells) and a reduction in both total memory B cells and switched memory B cells in RA patients compared to HD. We observed an expansion in the frequency of total B cells in RA-BE+ patients compared to RA-BE- patients. Unsupervised hierarchical clustering analysis of 39 cytokines resulted in a fairly good separation of HD from RA patients but not of RA-BE+ patients from RA-BE- patients. RA-BE+ patients showed significantly higher levels of IL-11 and IL-17A than RA-BE- patients. We show that patients with progressive erosive disease are characterized by abnormalities in B cells and in cytokines with a proven role in bone reabsorption. Understanding the role played by B cells and the cytokine IL-11 and IL-17A in progressive erosive disease can help identify novel biomarkers of erosive disease and design treatment approaches aimed at halting joint damage in RA.

Circulating Th17/Treg as a promising biomarker for patients with rheumatoid arthritis in indicating comorbidity with atherosclerotic cardiovascular disease.

Immune and inflammatory responses have a pivotal role in the pathogenesis of rheumatoid arthritis (RA) and atherosclerotic cardiovascular disease (ASCVD). This study aims to explore the change of peripheral lymphocytes, especially the absolute and relative changes in peripheral T cells in RA patients with and without ASCVD. The changes in the lymphocyte subsets were assessed to provide a novel insight in diagnosing and preventing ASCVD in patients with RA. A propensity score matching system (1:1) was conducted to perform a matched case-control study with 169 pairs RA-ASCVD and RA participants. Univariate and multivariate analyses were performed to determine the association between peripheral lymphocytes and RA-ASCVD. Multivariate logistic regression analysis demonstrated that Th17 cell absolute, Th17 cell Ratio, Th17/Treg were associated with a significantly higher risk of ASCVD after model adjustment. Then we focused on Th17/Treg, multivariate logistic analyses in tri-sectional Th17/Treg groups showed that the odds of ASCVD is gradually increasing with Th17/Treg rank's rising after model adjustment. Finally, the restricted cubic spline of Th17/Treg and odds ratio of RA-ASCVD was conducted. Interestingly, we found a critical point of Th17/Treg (critical point = 0.2399). Th17/Treg shows a protective role in the odds of ASCVD when Th17/Treg < 0.2399. With smaller Th17/Treg, the protective efficiency is more obvious when Th17/Treg < 0.2399. Our study suggested that increasing absolute and percentage of Th17 cells in the peripheral blood of patients with RA was associated with the development of ASCVD. And Th17/Treg may be a promising biomarker for patients with RA in indicating comorbidity with ASCVD.

Fibroblast growth factor receptor 1 as a potential marker of terminal effector peripheral T helper cells in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by destructive polyarthritis. CD4+ T cells are pivotal to its pathogenesis, and our previous study revealed the expression of fibroblast growth factor receptor 1 (FGFR1) is modulated by methotrexate treatment in CD4+ T cells of RA patients; however, the roles of FGFR1 in CD4+ T cells in the pathogenesis of RA is unclear. Therefore, in this study, we aimed to characterize FGFR1-positive CD4+ T cells in RA patients. The abundance of FGFR1-positive CD4+ T cells in peripheral blood and synovium was determined. Single-cell RNA sequencing (scRNA-seq) was performed on synovial CD4+ T cells to characterize FGFR1-positive cells. In addition, T cell activation status and cytokine production were determined using flow cytometry. The percentage of FGFR1-positive CD4+ T cells in the peripheral blood was higher in RA patients than in healthy controls (P= 0.0035). They were also present in the synovium of active RA patients. The results of scRNA-seq revealed that peripheral T helper (Tph) cells preferentially expressed FGFR1. Additionally, these FGFR1-positive Tph cells displayed a terminal effector cell phenotype. Consistent with this finding, FGFR1-positive CD4+ T cells in peripheral blood expressed interleukin-21 and interferon-γ. Our study provides evidence that FGFR1 marks terminal effector Tph cells in patients with RA.

Diminished LAG3+ B cells correlate with exacerbated rheumatoid arthritis

Background Lymphocyte activation gene-3 (LAG3) positive B cells have been identified as a novel regulatory B cell subset, while the role of LAG3+ B cells in the pathogenesis of rheumatoid arthritis (RA) remains elusive. Materials and methods Peripheral blood mononuclear cells (PBMCs) from RA, osteoarthritis (OA) patients and healthy volunteers were collected for flow cytometry staining of LAG3+ B cells. Their correlation with RA patient clinical and immunological features were analyzed. Moreover, the frequencies of LAG3+ B cells in collagen-induced arthritis (CIA) mice and naive mice were also detected. Results A significant decrease of LAG3+ B cells was observed in RA patients as compared with healthy individuals and OA patients. Notably, the frequencies of LAG3+ B cells were negatively correlated with tender joint count (r = −0.4301, p = .0157) and DAS28-ESR (r = −0.4018, p = .025) in RA patients. In CIA mice, LAG3+ B cell frequencies were also decreased and negatively correlated with the CIA arthritis score. Conclusions Impairment of LAG3+ B cells potentially contributes to RA development. Reconstituting LAG3+ B cells might provide novel therapeutic strategies for the persistent disease. Key messages LAG3+ B cells have been identified as a novel regulatory B cell subset. However, its role in the pathogenesis of RA remains unknown. This study revealed the decreased frequency of LAG3+ B cells in RA patients. Notably, LAG3+ B cells were negatively correlated with RA disease activity including the tender joint count and DAS28-ESR. In CIA mice, LAG3+ B cell frequencies were also decreased and negatively correlated with the CIA arthritis score. Reconstitution of LAG3+ B cells might provide novel therapeutic strategies for disease perpetuation.

Molecular signature of methotrexate response among rheumatoid arthritis patients.

Methotrexate (MTX) is the first line treatment for rheumatoid arthritis (RA), but failure of satisfying treatment response occurs in a significant proportion of patients. Here we present a longitudinal multi-omics study aimed at detecting molecular and cellular processes in peripheral blood associated with a successful methotrexate treatment of rheumatoid arthritis. Eighty newly diagnosed patients with RA underwent clinical assessment and donated blood before initiation of MTX, and 3 months into treatment. Flow cytometry was used to describe cell types and presence of activation markers in peripheral blood, the expression of 51 proteins was measured in serum or plasma, and RNA sequencing was performed in peripheral blood mononuclear cells (PBMC). Response to treatment after 3 months was determined using the EULAR response criteria. We assessed the changes in biological phenotypes during treatment, and whether these changes differed between responders and non-responders with regression analysis. By using measurements from baseline, we also tried to find biomarkers of future MTX response or, alternatively, to predict MTX response. Among the MTX responders, (Good or Moderate according to EULAR treatment response classification, n = 60, 75%), we observed changes in 29 partly overlapping cell types proportions, levels of 13 proteins and expression of 38 genes during treatment. These changes were in most cases suppressions that were stronger among responders compared to non-responders. Within responders to treatment, we observed a suppression of FOXP3 gene expression, reduction of immunoglobulin gene expression and suppression of genes involved in cell proliferation. The proportion of many HLA-DR expressing T-cell populations were suppressed in all patients irrespective of clinical response, and the proportion of many IL21R+ T-cells were reduced exclusively in non-responders. Using only the baseline measurements we could not detect any biomarkers or prediction models that could predict response to MTX. We conclude that a deep molecular and cellular phenotyping of peripheral blood cells in RA patients treated with methotrexate can reveal previously not recognized differences between responders and non-responders during 3 months of treatment with MTX. This may contribute to the understanding of MTX mode of action and explain non-responsiveness to MTX therapy.

Efficacy of PF-06651600 in alleviating the pro-inflammatory capacity of CD4+ T cells in rheumatoid arthritis patients.

PF-06651600 is a highly specific inhibitor of Janus-activated kinase 3 and the Tec family of kinases. Regarding its dual function in the inhibition of both γc cytokine receptors and T cell receptor signaling, the present study aimed at evaluating the impact of PF-06651600 on the status of T-helper cells (Th) as the central game players in the pathogenesis of rheumatoid arthritis (RA). TCD4+ cells were isolated from 34 RA patients and 15 healthy control individuals and were evaluated after treatment with PF-06651600. RA patients had higher percentages of TCD4+ cells, CD4+ PD-1+ cells, and CD4+ PD-1+ TIGIT+ cells compared to a healthy control group and the TCD4+ cells of these patients showed higher interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-17 secretion along with higher messenger RNA (mRNA) expressions of T-bet. The percentage of CD4+ PD-1+ TIGIT+ cells showed a reverse correlation with the Disease Activity Score of 28 joints of the RA patients. PF-06651600 caused a significant decrease in the mRNA expressions of T-bet and RAR-related orphan receptor γt and the secretion of interferon (IFN)-γ and TNF-α in TCD4+ cells of RA patients. On the other hand, the population of CD4+ PD-1+ TIGIT+ cells was expanded under the influence of PF-06651600. This treatment also reduced the proliferation of TCD4+ cells. PF-06651600 demonstrated a potential to modulate the activity of TCD4+ cells in RA patients and to reduce the commitment of Th cells to the pathogenic Th1 and Th17 subsets. Further, it caused TCD4+ cells to gain an exhausted phenotype which is associated with better prognosis in RA patients.

Characteristics of the (Auto)Reactive T Cells in Rheumatoid Arthritis According to the Immune Epitope Database

T cells are known to be involved in the pathogenesis of rheumatoid arthritis (RA). Accordingly, and to better understand T cells' contribution to RA, a comprehensive review based on an analysis of the Immune Epitope Database (IEDB) was conducted. An immune CD8+ T cell senescence response is reported in RA and inflammatory diseases, which is driven by active viral antigens from latent viruses and cryptic self-apoptotic peptides. RA-associated pro-inflammatory CD4+ T cells are selected by MHC class II and immunodominant peptides, which are derived from molecular chaperones, host extra-cellular and cellular peptides that could be post-translationally modified (PTM), and bacterial cross-reactive peptides. A large panel of techniques have been used to characterize (auto)reactive T cells and RA-associated peptides with regards to their interaction with the MHC and TCR, capacity to enter the docking site of the shared epitope (DRB1-SE), capacity to induce T cell proliferation, capacity to select T cell subsets (Th1/Th17, Treg), and clinical contribution. Among docking DRB1-SE peptides, those with PTM expand autoreactive and high-affinity CD4+ memory T cells in RA patients with an active disease. Considering original therapeutic options in RA, mutated, or altered peptide ligands (APL) have been developed and are tested in clinical trials.

Hypoxia-inducible factor-1α regulates the interleukin-6 production by B cells in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a disease characterised by bone destruction and systemic inflammation, and interleukin-6 (IL-6) is a therapeutic target for treating it. The study aimed at investigating the sources of IL-6 and the influence of hypoxia-inducible factor-1α (HIF-1α) on IL-6 production by B cells in RA patients. The phenotype of IL-6-producing cells in the peripheral blood of RA patients was analysed using flow cytometry. Bioinformatics, real-time polymerase chain reaction, Western blot and immunofluorescence staining were used to determine the IL-6 production and HIF-1α levels in B cells. A dual-luciferase reporter assay and chromatin immunoprecipitation were used to investigate the regulatory role of HIF-1α on IL-6 production in human and mouse B cells. Our findings revealed that B cells are major sources of IL-6 in the peripheral blood of RA patients, with the proportion of IL-6-producing B cells significantly correlated with RA disease activity. The CD27-IgD+ naïve B cell subset was identified as the typical IL-6-producing subset in RA patients. Both HIF-1α and IL-6 were co-expressed by Bcells in the peripheral blood and synovium of RA patients, and HIF-1α was found to directly bind to the IL6 promoter and enhance its transcription. This study highlights the role of B cells in producing IL-6 and the regulation of this production by HIF-1α in patients with RA. Targeting HIF-1α might provide a new therapeutic strategy for treating RA.

Dynamics of circulating follicular helper T cell subsets and follicular regulatory T cells in rheumatoid arthritis patients according to HLA-DRB1 locus.

B cells, follicular helper T (Tfh) cells and follicular regulatory T (Tfr) cells are part of a circuit that may play a role in the development or progression of rheumatoid arthritis (RA). With the aim of providing further insight into this topic, here we evaluated the frequency of different subsets of Tfh and Tfr in untreated and long-term treated RA patients from a cohort of Argentina, and their potential association with particular human leukocyte antigen (HLA) class-II variants and disease activity. We observed that the frequency of total Tfh cells as well as of particular Tfh subsets and Tfr cells were increased in seropositive untreated RA patients. Interestingly, when analyzing paired samples, the frequency of Tfh cells was reduced in synovial fluid compared to peripheral blood, while Tfr cells levels were similar in both biological fluids. After treatment, a decrease in the CCR7loPD1hi Tfh subset and an increase in the frequency of Tfr cells was observed in blood. In comparison to healthy donors, seropositive patients with moderate and high disease activity exhibited higher frequency of Tfh cells while seropositive patients with low disease activity presented higher Tfr cell frequency. Finally, we observed that HLA-DRB1*09 presence correlated with higher frequency of Tfh and Tfr cells, while HLA-DRB1*04 was associated with increased Tfr cell frequency. Together, our results increase our knowledge about the dynamics of Tfh and Tfr cell subsets in RA, showing that this is altered after treatment.

Immunosenescence of T cells: a key player in rheumatoid arthritis.

The incidence of rheumatoid arthritis (RA) and its complications are expected to increase with age. Remarkably, RA patients were identified features of accelerated aging, particularly in immunosenescence. As is known, T cells in RA patients readily differentiate into pro-inflammatory phenotypes that maintain chronic and persistent inflammatory changes in joints and many other organ systems. Recent evidence suggests that T cells are most sensitive to aging, and aged CD4+ T cells contribute to inflammaging, which plays a crucial role in accelerating the disease process. In recent years, the molecular mechanisms of T cell immunosenescence were beginning to be understood. Immune aging in RA T cells is associated with thymus insufficiency, metabolic abnormalities, shortened telomere length, and chronic energy stress. Therefore, we summarized the role and mechanism of T cell immunosenescence in RA. A computer-based online search was performed using the PubMed database for published articles concerning T cells aging and rheumatoid arthritis. In this review, we assess the roles of CD4+ T cells in the center of inflammaging especially in RA and emphasize arthritogenic effector functions of senescent T cell; also we discuss the possible molecular mechanisms of senescent T cells and therapeutic targets to intervene T cells immunosenescence for improvement of RA.

Phenotypic features of innate lymphoid cells in rheumatoid arthritis

Currently, rheumatoid arthritis (RA) is considered among the most common autoimmune diseases worldwide, being associated with progressing disability, systemic organ and tissue lesions, as well as social and economic losses for the state. Studies of innate lymphoid cells (ILS) seems to be actual and significant when studying development of autoimmune inflammation in RA, in particular, the issues of the cell plasticity. ILC represent tissue resident lymphoid cells that display functional diversity, like as T cells. Moreover, ILC regulate orientation of immune response by means of cytokine production. Small amounts of ILCs are present in the bloodstream, presumably for migration to target organs and tissues. Accordingly, the study of ILC in RA will promote understanding of the RA pathogenesis. It is also possible in the future to develop new therapeutic strategies based on the impact on the immunological balance, as well as reducing the inflammatory process in RA. The aim of this study was to determine the subpopulation composition and phenotypic features of ILCs in RA.&#x0D; We have isolated and studied peripheral blood mononuclear cells (PBMC) from 7 patients with RA and 6 healthy donors. The isolated blood MNCs were stained with monoclonal antibodies conjugated to fluorochromes: lineage-specific (CD3/14/16/19/20/56) and anti-FceR1 alpha-FITC, anti-CD294-APC/Cy7, anti-CD127-PerCP/Cy5.5, anti-CD336-PE, anti-CD117-APC. ILCs were defined as Lin-CD127+. The numbers of CD294+ILCs (ILC2) were estimated in the general population, CD117-CD294-ILCs were defined as ILC1, and CD117+CD294-ILCs, as ILC3. The cell phenotype was analyzed with a FACS Canto II flow cytometer (BD Biosciences, USA).&#x0D; We determined relative numbers of different ILC subpopulations (ILC1, ILC2, and ILC3) among the total blood MNCs. It was shown, that the number of ILC2 cells in RA patients was statistically significantly reduced compared to healthy donors, whereas no significant differences in percentage of ILC1 and ILC3 were revealed between donors and patients. We also evaluated the amount of c-Kit+ILC2; there were no significant differences in the proportion of these cells between donors and patients.&#x0D; ILCs represent a population of cells that contribute to the RA pathogenesis. The role of ILC2 in RA is, presumably, protective. The ILC imbalance may contribute to the development of RA. For a better understanding of the RA pathogenesis, further studies of the subpopulation profile, phenotypic and functional characteristics of ILC are required in this disorder.

Autoantibodies targeting malondialdehyde-modifications in rheumatoid arthritis regulate osteoclasts via inducing glycolysis and lipid biosynthesis

Proteins subjected to post-translational modifications, such as citrullination, carbamylation, acetylation or malondialdehyde (MDA)-modification are targeted by autoantibodies in seropositive rheumatoid arthritis (RA). Epidemiological and experimental studies have both suggested the pathogenicity of such humoral autoimmunity, however, molecular mechanisms triggered by anti-modified protein antibodies have remained to be identified.Here we describe in detail the pathways induced by anti-MDA modified protein antibodies that were obtained from synovial B cells of RA patients and that possessed robust osteoclast stimulatory potential and induced bone erosion in vivo. Anti-MDA antibodies boosted glycolysis in developing osteoclasts via an FcγRI, HIF-1α and MYC-dependent mechanism and subsequently increased oxidative phosphorylation. Osteoclast development required robust phosphoglyceride and triacylglyceride biosynthesis, which was also enhanced by anti-MDA by modulating citrate production and expression of the glycerol-3-phosphate dehydrogenase 1 (GPD1) and glycerol-3-phosphate acyltransferase 2 (GPAT2) genes. In summary, we described novel metabolic pathways instrumental for osteoclast differentiation, which were targeted by anti-MDA antibodies, accelerating bone erosion, a central component of RA pathogenesis.

Increased frequency of TIGIT+ CD4 T Cell subset in autoantibody-positive first-degree relatives of patients with rheumatoid arthritis.

BackgroundDespite immune cell dysregulation being an important event preceding the onset of rheumatoid arthritis (RA), the phenotype of T and B cells in preclinical RA is less understood. The aim of this study was to characterize T and B cell populations in RA patients and their autoantibody (aAb) negative and positive first-degree relatives (FDR).MethodsCryopreserved peripheral blood mononuclear cells (PBMCs) collected at scheduled visits from aAb-(n=25), and aAb+ FDR (n=10) and RA patients (n=13) were thawed and stained using optimized antibody cocktails as per a specific 13-color T or B cell panel. Immunophenotyping was performed using a Cytoflex LX (Beckman-Coulter) flow cytometer and FlowJo software was used for analyzing the frequency of immune cell populations.ResultsMulticolor flow cytometry experiments identified an increased TIGIT expression in circulating lymphocytes of aAb+ FDR and RA patients, relative to aAb- FDR (P<0.01). These TIGIT+ T cells exhibited a memory phenotype and expressed high levels of PD-1, ICOS, HLA-DR, CXCR3 and CXCR5. Moreover, increased TIGIT+ CD4 T cell frequency correlated with the frequency of PD-1+ CD4 T cells (r = 0.4705: P = 0.0043) and circulating levels of ACPA and RF. We also identified a decreased frequency of CD27+IgD- switched memory B cells in RA patients (P < 0.01), while increased frequency of TIGIT+ CD4 T cells in FDR correlated with the frequency of PD1+PTEN+ B cells (r = 0.6838, P = 0.0004) and autoantibody positivity (P = 0.01).ConclusionWe demonstrate TIGIT as a distinct CD4 T cell marker for differentiating aAb- FDR from aAb+FDR and might play a critical role in regulating T and B cell crosstalk in preclinical RA.

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.

Altered Transcriptional Regulation of Glycolysis in Circulating CD8+ T Cells of Rheumatoid Arthritis Patients.

Peripheral T lymphocytes of rheumatoid arthritis (RA) patients show pathological changes in their metabolic pathways, especially glycolysis. These changes may drive the increased proliferation and tissue invasiveness of RA T cells. In order to study the transcriptional regulation underlying these alterations, we analysed publicly available RNA sequencing data from circulating T lymphocyte subsets of healthy individuals, untreated RA patients, and patients undergoing treatment for RA. Differential co-expression networks were created using sample-wise edge weights from an analysis called “linear interpolation to obtain network estimates for single sample” (lionessR), and annotated using the Gene Transcription Regulation Database (GTRD). Genes with high centrality scores were identified. CD8+ effector memory cells (Tem) and CD8+CD45RA+ effector memory cells (Temra) showed large changes in the transcriptional regulation of glycolysis in untreated RA. PFKFB3 and GAPDH were differentially regulated and had high centrality scores in CD8+ Tem cells. PFKFB3 downregulation may be due to HIF1A post transcriptional inhibition. Tocilizumab treatment partially reversed the RA-associated differential expression of several metabolic and regulatory genes. MYC was upregulated and had high centrality scores in RA CD8+ Temra cells; however, its glycolysis targets were unaltered. The upregulation of the PI3K-AKT and mTOR pathways may explain MYC upregulation.

OP0265 COMPARISON OF THE EFFECT OF DIFFERENT JANUS KINASE INHIBITORS ON ACTIVATION, FUNCTION AND PROPERTY OF NK CELLS TO CONTROL CANCER CELL LINES PROLIFERATION: AN EX VIVO AND IN VITRO STUDY.

BackgroundJanus kinase inhibitors (JAKi) are effective targeted synthetic DMARDs licensed in rheumatoid arthritis (RA) and in other immune-mediated diseases. Among the adverse events described, the increased risk of herpes zoster is specific of this therapeutic class but variable depending on the drugs, especially lower with filgotinib (FILGO)1. Recently the ORAL Surveillance study highlighted an increased risk of lymphoma and lung cancer with tofacitinib (TOFA) in RA patients older than 50 years with cardio-vascular risk factors2. Because of their antitumor and antiviral role, especially against herpes viruses, effect of JAKi on NK cells may play a causal role in these adverse events. Our working hypothesis is that the different JAKi could have a differential impact on the activation and function of NK cells leading to the variability of the tolerance profiles observed between the molecules.ObjectivesTo evaluate the phenotypic and functional impact of JAKi on NK cells.MethodsWe first performed ex vivo phenotypic analyses of NK cells in RA patients treated with TOFA or baricitinib (BARI) in comparison with patients treated with methotrexate (MTX). To go deeper, we phenotyped sorted NK cells from healthy donors after 6 days of culture with 4 JAKi (TOFA, BARI, upadacitinib (UPA) and FILGO) or control (DMSO). We used 3 different doses carefully chosen to mimic in vivo exposure [therapeutic concentration (therap) defined as mean concentration in patients at the licensed dose, infra therapeutic (infra, mean -2DS) and supra therapeutic concentration (supra, mean +2Ds)]. Thirdly, we stimulated sorted NK cells from healthy donors exposed to TOFA or BARI at therap and supra concentrations with anti-NKp30 crosslinking to assess NK cells function: intra-cellular IFNγ/TNF production and degranulation (CD107a). Finally, we challenged sorted NK cells from healthy donors exposed in vitro to TOFA or BARI at therap and supra doses with 2 different tumor cells line to assess degranulation and cytotoxicity: a lung cancer line A549 and a lymphoma cell line SU-DHL-4.ResultsTwenty-eight RA patients were included in the ex vivo assay (12 MTX, 6 TOFA, and 10 BARI). Patients under TOFA and not under BARI had a significant reduced CD69 (an activation marker) expression on NK cells (p&lt;0.05) compared to MTX. After in vitro culture of NK cells with JAKi, we confirmed the negative impact of JAKi on NK cells activation (CD69), maturation markers (CD57, Tim3) and activating receptor (NKp30), more pronounced with TOFA and UPA (Figure 1A). After crosslinking with anti-NKp30, NK cells exposed to TOFA produced significantly less IFNγ/TNF and expressed less CD107a compared to DMSO (p&lt;0,005). There was a non-significant trend for a dose-effect (Figure 1B). BARI did not induce any significant effect. Lastly, when NK cells were co-cultured with the cancer cell lines, previous exposition to TOFA but not to BARI led to a significant reduction of CD107a expression on NK cells (p&lt;0.005) and to reduced cytotoxicity (p&lt;0.05) of NK cells versus A549 at therap dose. Studies regarding SU-DHL-4 are still in progress.Figure 1.Impact of in vitro exposure to JAKi at therap and supra dose on NK cells regarding phenotype (A) and function after crosslinking (B)ConclusionJAKi have a phenotypic and functional dose-effect impact on NK cells activation, both in ex vivo and in vitro experiments. TOFA has more impact than other JAKi on NK cells phenotype and function and has the property to impair the control of proliferation of lung cancer and lymphoma cell lines by NK cells. The question remains open if this mechanism could explain the increased risk of lung cancer and lymphoma observed with TOFA in the ORAL surveillance trial.