Frederique M. Moret

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity

IntroductionMyeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)+ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients.MethodsCD1c+ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4+ T cells was measured.ResultsCD1c+ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4+ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production.ConclusionsThis study suggests that increased numbers of CD1c+ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.

Thymic Stromal Lymphopoietin, a Novel Proinflammatory Mediator in Rheumatoid Arthritis That Potently Activates CD1c+ Myeloid Dendritic Cells to Attract and Stimulate T Cells

To determine the levels of thymic stromal lymphopoietin (TSLP) and the numbers of TSLP receptor (TSLPR)–expressing CD1c+ (blood dendritic cell antigen 1–positive) myeloid dendritic cells (MDCs) in the joints as compared with the peripheral blood (PB) of patients with rheumatoid arthritis (RA), as well as to determine the capacity of TSLP to induce MDC‐dependent T cell activation.

IL-7 drives Th1 and Th17 cytokine production in patients with primary SS despite an increase in CD4 T cells lacking the IL-7Rα

OBJECTIVE To study the phenotypic characteristics of and the balance between systemic IL-7 receptor (IL-7R)α+ and IL-7Rα- Tregs in primary SS (pSS) patients as compared with control subjects and to assess the functional consequences this has for (IL-7-induced) T-cell activation. METHODS The functional properties of IL-7Rα+ and IL-7Rα- (CD25+) CD4 T cells from pSS patients were tested in vitro. Expression of CD25 and FoxP3 by IL-7Rα+ and IL-7Rα- CD4 T cells from pSS patients and healthy controls (HCs) were assessed. Also, the net ex vivo T-cell cytokine production and the capacity of IL-7 to activate total CD4 T cells from pSS patients compared with HCs in vitro was tested. RESULTS IL-7Rα+ T cells from pSS patients strongly proliferated and their numbers were slightly reduced compared with HCs. This reduced number was caused by an increase in both anergic and suppressive IL-7Rα- CD25+ T cells expressing high levels of FoxP3, but also by increases in IL-7Rα- CD25- CD4 T cells that only moderately expressed FoxP3. This altered balance in IL-7Rα+ and IL-7Rα- CD4 T cells was accompanied by unchanged ex vivo Th1, Th2 and Th17 cytokine production of total CD4 T cells. Furthermore, the increased numbers of IL-7Rα- CD25+ T cells did not prevent specific IL-7-induced Th1 and Th17 cytokine production by IL-7Rα+ T cells. CONCLUSION IL-7Rα+ cells are highly proliferating cells that respond strongly to IL-7 despite an increased number of IL-7Rα- T cells that express FoxP3 and CD25. The recent finding that IL-7 and IL-7Rα+ T cells were both found to be increased in exocrine glands of pSS patients indicates that IL-7 could contribute to glandular inflammation by activation of IL-7Rα+ responder T cells despite the increased numbers of Tregs.

Synovial T cell hyporesponsiveness to myeloid dendritic cells is reversed by preventing PD-1/ PD-L1 interactions

IntroductionThe aim of this study was to investigate PD-1/PD-L1 involvement in the hyporesponsiveness of rheumatoid arthritis (RA) synovial fluid (SF) CD4 T cells upon stimulation by thymic stromal lymphopoietin (TSLP)–primed CD1c myeloid dendritic cells (mDCs).MethodsExpression of PD-1 on naïve (Tn), central memory (Tcm) and effector memory (Tem) CD4 T cell subsets was assessed by flow cytometry. PD-L1 expression and its regulation upon TSLP stimulation of mDCs from peripheral blood (PB) and SF of RA patients were investigated by quantitative RT-PCR and flow cytometry. The involvement of PD-1/PD-L1 interactions in SF T cell hyporesponsiveness upon (TSLP-primed) mDC activation was determined by cell culture in the presence of PD-1 blocking antibodies, with or without interleukin 7 (IL-7) as a recognized suppressor of PD-1 expression.ResultsPD-1 expression was increased on CD4 T cells derived from SF compared with PB of RA patients. TSLP increased PD-L1 mRNA expression in both PB and SF mDCs. PD-L1 protein expression was increased on SF mDCs compared with PB mDCs and was associated with T cell hyporesponsiveness. Blockade of PD-1, as well as IL-7 stimulation, during cocultures of memory T cells and (TSLP-primed) mDCs from RA patients significantly recovered T cell proliferation.ConclusionSF T cell hyporesponsiveness upon (TSLP-primed) mDC stimulation in RA joints is partially dependent on PD-1/PD-L1 interactions, as PD-1 and PD-L1 are both highly expressed on SF T cells and mDCs, respectively, and inhibiting PD-1 availability restores T cell proliferation. The potential of IL-7 to robustly reverse this hyporesponsiveness suggests that such proinflammatory cytokines in RA joints strongly contribute to memory T cell activation.

Association of MicroRNA-618 Expression With Altered Frequency and Activation of Plasmacytoid Dendritic Cells in Patients With Systemic Sclerosis

Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFNα than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)–mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc.

Expression of miR-618 in plasmacytoid dendritic cells from systemic sclerosis patients is associated with their altered frequency and activation

Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFNα than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)–mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc.

The efficacy of abatacept in reducing synovial T cell activation by CD1c myeloid dendritic cells is overruled by the stimulatory effects of T cell-activating cytokines

To investigate whether the potential of abatacept to inhibit vigorous CD1c myeloid dendritic cell (MDC)–driven activation of naive and memory CD4 T cells is abrogated in the presence of T cell–activating cytokines.

Targeting Th2-typified immune responses to prevent immunopathology in rheumatic diseases: belittled therapeutic strategies?

Although Th2-associated immune responses have been well defined in atopic diseases and parasitic infections, their potential contribution to immunopathology in rheumatic diseases has scarcely been recognised. This is probably related to the clear predominance of Th1/Th17 over Th2 cells in a number of rheumatic diseases, such as rheumatoid arthritis (RA), psoriatic arthritis and primary Sjogrens syndrome (pSS). Also, whereas a clear role for Th1/Th17 activity in immunopathology has been clearly demonstrated in these diseases, Th2-related phenomena such as IgE production are hardly detected. Indeed, Th2-associated atopic conditions and Th2 cytokines like interleukin (IL)-4 and IL-10 were shown to inhibit Th1-induced inflammatory responses in these diseases.1 ,2 As a consequence the understanding of Th2-associated mediators, other than IL-4 and IgE, in these and other rheumatic conditions and the potential to target these have been underevaluated. However, recent data show that immune activation by typical Th2-associated pathways, such as mast cell activation and histamine-induced responses, or Th2-typified key regulatory molecules, such as thymic stromal lymphopoietin (TSLP), IL-33 and IL-13, contribute to inflammation and immunopathology in several rheumatic diseases. Mast cells have been shown to have a detrimental role in allergic reactions and parasitic infections. In addition, mast cells have been shown to be increased at inflammatory sites in a plethora of rheumatic conditions, including RA, pSS, systemic sclerosis (SSc), spondyloarthropathies (SpA), dermatomyositis and eosinophilia-myalgia syndromes.3–8 Mast cells have also been shown to be important mediators in different experimental inflammatory (arthritis) models by secretion of proinflammatory mediators such as tumour necrosis factor α (TNFα), IL-6, IL-8 and IL-17. In addition, inhibitors of histamine release such as salbutamol and cromolyn were shown to prevent joint destruction in immune complex-mediated arthritis.9 ,10 Recent data suggest that targeting of mast cell-associated pathways prevents inflammatory responses driven by the innate and acquired …

Targeting CD1c-expressing classical dendritic cells to prevent thymus and activation-regulated chemokine (TARC)-mediated T-cell chemotaxis in rheumatoid arthritis

Objectives: Thymus and activation-regulated chemokine (TARC) attracts cells that express the C-C chemokine receptor type 4 (CCR4), including CD4 T cells. As expression of CCR4 is increased on peripheral T cells and intra-articular interleukin (IL)-17-producing cells in patients with rheumatoid arthritis (RA), we investigated whether TARC plays a role in the attraction of T cells to the synovial compartment. In addition, we assessed the role of classical dendritic cells (cDCs) in the production of TARC in RA. Method: TARC was measured in synovial fluid (SF) samples from RA and osteoarthritis (OA) patients. Spontaneous and thymic stromal lymphopoietin (TSLP)-induced TARC production by mononuclear cells (MCs) and CD1c cDCs from peripheral blood (PB) and SF was assessed. The role of TARC in CD4 T-cell migration towards cDCs was assessed and the contribution of CD1c-expressing cells to TARC production was studied. Results: TARC concentrations were higher in SF of RA patients compared to OA patients. MCs from SF produced TARC spontaneously and produced more TARC upon stimulation than paired PBMCs. Blocking TARC strongly inhibited CD4 T-cell chemotaxis by TSLP-stimulated cDCs, associated with decreased production of tumour necrosis factor (TNF)-α. Depletion of cDCs from SFMCs strongly reduced TARC production. Conclusions: TARC levels are increased in RA SF and our data indicate that this results from production by SFMCs and in particular CD1c cDCs. TARC attracts T cells and TARC secretion by MCs is crucially dependent on the presence of CD1c cDCs. Considering the potential of SF cDCs to activate T cells and induce pro-inflammatory cytokine secretion, targeting intra-articular cDCs constitutes a novel therapeutic approach in RA.

THU0234 Epigenetic cell counting: a novel tool to quantify immune cells in salivary glands detects robust correlations of tfh cells with immunopathology

Background Histological analysis of salivary glands for decades has been a valuable tool in the characterization of patients with primary Sjögrens syndrome (pSS) and non-Sjögrens sicca (nSS) patients. Importantly, it has helped in understanding the immunopathology of sicca patients. Nonetheless, standardization of histological assessments, e.g. to quantify lymphocytic foci or germinal centers is lacking, contributing to improper classification of disease and assessment of risk of lymphoma for example. Also, detailed and reproducible quantification of the heterogeneity of inflammatory cells and their contribution to immunopathology is lacking. Recent progress in epigenetics has revealed that cell-specific DNA methylation profiles can be applied to reliably quantify numbers of cells in blood and tissues. Objectives To investigate whether epigenetic cell counting can serve as a novel reliable tool to quantify immune cells in salivary glands of sicca patients. Methods DNA was isolated from frozen tissue sections of 13 nSS, 12 probable SS, 29 pSS and 7 overlap SS patients. Bisulfite conversion of demethylated DNA sites was followed by cell specific qPCR that was used to calculate the percentage of cell subsets related to the total number of cells quantified by housekeeping gene expression. Percentages of epigenetically counted cells were correlated to gene expression generated by RNA-Seq analysis of matched salivary gland tissue and histological and clinical parameters (LFS, %IgA+ plasma cells, serum IgG, SSA positivity). Results Strongly increased percentages of epigenetically quantified percentages of CD3, CD4, CD8, B cells, T follicular helper (Tfh) cells and Treg cells in pSS vs nSS patients were observed (all p<0.001, CD8 p<0.01). These inflammatory cell types all strongly correlated with LFS (all at least p<0.001, CD8 p=0.014), local B cell hyperactivity (% IgA+ cells, all p<0.001, except CD8 p=0.06 and B cells, p=0.127) and systemic B cell hyperactivity (all at least p<0.01, except CD8 p=0.051). Th17 cells were not significantly different between nSS and pSS patients. Only CD8 T cells were significantly increased in probable SS patients as compared to nSS patients (p<0.05). Percentages of CD3 and B cells positively correlated with CD3 and CD19 RNA expression (r=0.608, p<0.0001; r=0.597, p<0.0001, resp.). Interestingly, percentages of Tfh cells correlated with CXCL13 (r=0.789, p<0.0001), IL7R, CXCR5 and ICOS RNA expression (all p≤0.001) and were strongly associated with autoimmunity (SSA positivity, p<0.001). Conclusions Epigenetic cell counting is a promising novel tool to reproducibly and easily quantify immune cells in the (inflamed) labial salivary gland of sicca patients with relatively low amount of tissue needed (<1 mm3). Considering the potential of this technique to include a huge number of (cell-specific) biomarkers we believe this opens up new standardized ways for salivary gland analysis with high relevance for patient classification, understanding of immunopathology and clinical trials. Disclosure of Interest None declared