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ObjectivesTo identify key disease pathways driving conventional dendritic cell (cDC) alterations in Systemic Sclerosis (SSc).MethodsTranscriptomic profiling was performed on peripheral blood CD1c+ cDCs (cDC2s) isolated from 12 healthy donors and 48 SSc patients with all major disease subtypes. Differential expression analysis comparing the different SSc subtypes and healthy donors was performed to uncover genes dysregulated in SSc. To identify biologically relevant pathways, a gene co-expression network was built using Weighted Gene Correlation Network Analysis. We validated the role of key transcriptional regulators using ChIP-sequencing and in vitro functional assays.ResultsWe identified 17 modules of co-expressed genes in cDC2s that correlated with SSc subtypes and key clinical traits including auto-antibodies, skin score, and occurrence of interstitial lung disease. A module of immune regulatory genes was markedly down regulated in patients with the diffuse SSc subtype characterized by severe fibrosis. Transcriptional regulatory network analysis performed on this module predicted NR4A (nuclear receptor 4A) subfamily (NR4A1, NR4A2, NR4A3) genes as the key transcriptional mediators of inflammation. Indeed, ChIP-sequencing analysis supported that these NR4A members target numerous differentially expressed genes in SSc cDC2s. Inclusion of NR4A receptor agonists in culture-based experiments provided functional proof that dysregulation of NR4As affects cytokine production by cDC2s and modulates downstream T-cell activation.ConclusionsNR4A1, NR4A2 and NR4A3 are important regulators of immunosuppressive and fibrosis-associated pathways in SSc cDC2s. Thus, the NR4A family represent novel potential targets to restore cDC homeostasis in SSc.KEY MESSAGESWhat is already known about this subject?CD1c+ conventional dendritic cells (cDC2s) are implicated as key players in Systemic Sclerosis (SSc), but key molecular mechanisms underlying their dysregulation were unknown.What does this study add?Transcriptomic analysis and network analysis identified modules of coexpressed genes in cDC2s that correlated with SSc subtypes and key clinical traits.The NR4A (nuclear receptor 4A) subfamily (NR4A1, NR4A2, NR4A3) genes act as master regulators of key immune regulatory genes dysregulated in SSc cDC2s, as shown by multi-omics integration analysis using transcriptomics and targeted ChIP-sequencing.Pharmacological activation of NR4As inhibits pro-inflammatory cytokine production and CD4+ T-cell activation by cDC2s.How might this impact on clinical practice or future developments?NR4As are attractive candidates for novel treatment options to attenuate pro-inflammatory and pro-fibrotic responses in SSc patients.

RNA sequencing and DNA methylomic profiling were performed after differentiating monocytes for 6 days into moDCs with/without CXCL4 presence. We show that CXCL4 downregulates genes associated with tolerogenicity in DCs including C1Q. Expression profiles of C1Q genes were negatively correlated with their DNA methylation profiles and with immunogenic genes.

The T-cell receptor (TCR) is a highly polymorphic surface receptor that allows T-cells to recognize antigenic peptides presented on the major histocompatibility complex (MHC). Changes in the TCR repertoire have been observed in several autoimmune conditions, and these changes are suggested to predispose autoimmunity. Multiple lines of evidence have implied an important role for T-cells in the pathogenesis of Systemic Sclerosis (SSc), a complex autoimmune disease. One of the major questions regarding the roles of T-cells is whether expansion and activation of T-cells observed in the diseases pathogenesis is antigen driven. To investigate the temporal TCR repertoire dynamics in SSc, we performed high-throughput sequencing of CD4+ and CD8+ TCRβ chains on longitudinal samples obtained from four SSc patients collected over a minimum of two years. Repertoire overlap analysis revealed that samples taken from the same individual over time shared a high number of TCRβ sequences, indicating a clear temporal persistence of the TCRβ repertoire in CD4+ as well as CD8+ T-cells. Moreover, the TCRβs that were found with a high frequency at one time point were also found with a high frequency at the other time points (even after almost four years), showing that frequencies of dominant TCRβs are largely consistent over time. We also show that TCRβ generation probability and observed TCR frequency are not related in SSc samples, showing that clonal expansion and persistence of TCRβs is caused by antigenic selection rather than convergent recombination. Moreover, we demonstrate that TCRβ diversity is lower in CD4+ and CD8+ T-cells from SSc patients compared with memory T-cells from healthy individuals, as SSc TCRβ repertoires are largely dominated by clonally expanded persistent TCRβ sequences. Lastly, using “Grouping of Lymphocyte Interactions by Paratope Hotspots” (GLIPH2), we identify clusters of TCRβ sequences with homologous sequences that potentially recognize the same antigens and contain TCRβs that are persist in SSc patients. In conclusion, our results show that CD4+ and CD8+ T-cells are highly persistent in SSc patients over time, and this persistence is likely a result from antigenic selection. Moreover, persistent TCRs form high similarity clusters with other (non-)persistent sequences that potentially recognize the same epitopes. These data provide evidence for an antigen driven expansion of CD4+/CD8+ T-cells in SSc.

Compelling evidence shows the involvement of plasmacytoid dendritic cells (pDCs) in systemic sclerosis (SSc) pathogenesis. This study investigated whether microRNAs (miRNAs) are involved in the dysregulation of pDCs in SSc patients already at early stages. RNA from circulating pDCs was isolated from two independent cohorts of SSc patients with different disease phenotypes, and individuals with Raynaud’s phenomenon, for microRNA profiling and RNA-sequencing analysis. Proteomic analysis was exploited to identify novel direct miRNA targets at the protein level. Twelve and fifteen miRNAs were differentially expressed in at least one group of patients compared to healthy controls in discovery cohort I and II, respectively. Of note, miR-126 and miR-139-5p were upregulated in both preclinical and definite SSc patients and correlated with the expression of type I interferon (IFN)-responsive genes. Toll-like receptor 9 (TLR9) stimulation of healthy pDCs upregulated the expression of both miRNAs, similarly to what was observed in patients. The proteomic analysis identified USP24 as a novel target of miR-139-5p. The expression level of USP24 was inversely correlated with miR-139-5p expression in SSc patients and induced by TLR9 stimulation in healthy pDCs. These findings demonstrated that the miRNA profile is altered in pDCs of SSc patients already at early stages of the disease and indicate their potential contribution to pDC activation observed in patients.

OBJECTIVE: 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 IFNalpha 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. METHODS: We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs. RESULTS: Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFNalpha, mimicking the PDC phenotype observed in SSc patients. CONCLUSION: Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFNalpha, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions.

Fibrosis is a condition shared by numerous inflammatory diseases. Our incomplete understanding of the molecular mechanisms underlying fibrosis has severely hampered effective drug development. CXCL4 is associated with the onset and extent of fibrosis development in systemic sclerosis, a prototypic inflammatory and fibrotic disease. Here, we integrated 65 paired longitudinal transcriptional and methylation profiles from monocyte-derived cells with/without CXCL4 exposure. Using data-driven gene regulatory network analyses, we demonstrate that CXCL4 dramatically alters the trajectory of monocyte differentiation, inducing a novel pro-inflammatory and pro-fibrotic phenotype mediated via key transcriptional regulators including CIITA. CXCL4 exposed monocyte-derived cells directly trigger a fibrotic cascade by producing ECM molecules and inducing myofibroblast differentiation. Inhibition of CIITA mimicked CXCL4 in inducing a pro-inflammatory and pro-fibrotic phenotype, validating the relevance of the gene regulatory network. Our study unveils CXCL4 as a key secreted factor driving innate immune training and forming the long-sought link between inflammation and fibrosis.

Systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and primary Sjogrens syndrome (pSS) are clinically distinct systemic autoimmune diseases (SADs) that share molecular pathways. We quantified the frequency of circulating immune-cells in 169 patients with these SADs and 44 healty controls (HC) using mass-cytometry and assessed the diagnostic value of these results. Alterations in the frequency of immune-cell subsets were present in all SADs compared to HC. Most alterations, including a decrease of CD56hi NK-cells in SSc and IgM+ Bcells in pSS, were disease specific; only a reduced frequency of plasmacytoid dendritic cells was common between all SADs Strikingly, hierarchical clustering of SSc patients identified 4 clusters associated with different clinical phenotypes, and 9 of the 12 cell subset-alterations in SSc were also present during the preclinical-phase of the disease. Additionally, we found a strong association between the use of prednisone and alterations in B-cell subsets. Although differences in immune-cell frequencies between these SADs are apparent, the discriminative value thereof is too low for diagnostic purposes. Within each disease, mass cytometry analyses revealed distinct patterns between endophenotypes. Given the lack of tools enabling early diagnosis of SSc, our results justify further research into the value of cellular phenotyping as a diagnostic aid.

Background Aberrant accumulation of extracellular matrix (ECM) in multiple organs or fibrosis is one of the three hallmarks that characterises the pathogenesis of systemic sclerosis (SSc), together with immune dysregulation and small vessel vasculopathy. Recent studies have shown that CXCL4 (Chemokine CXC motif ligand 4) levels are increased in patients with SSc and correlated with skin and lung fibrosis.1 CXCL4 plays key role in physiological processes, although it has been also implicated in several pathological conditions such as autoimmune diseases and cancer. We and others have shown that CXCL4 modulates the phenotype and function of immune cells 2, suggesting a critical role of this chemokine in innate and adaptive immune responses. However, how CXCL4 exactly modulates immune cell responses remains unclear. Objectives Here we investigated the impact of CXCL4 exposure on the transcriptome and DNA methylation of monocyte-derived dendritic cells (moDCs), and the consequence on their function. Methods We differentiated human moDCs in the presence of CXCL4. After 6 days differentiation, cells were stimulated with a TLR3 ligand (polyI:C) as described in our previous study.2 RNA sequencing and DNA methylation profiling was performed at various time points during differentiation and stimulation. Results Integration of high-throughput analyses of RNA sequencing and DNA methylation reveals that CXCL4 drives to dramatic changes on the transcriptome and epigenome levels. This is reflected in the dysregulation of critical innate and adaptive immune pathways, like antigen presentation, and cytokine signalling. For the first time, we show that CXCL4 potentiates a novel function to dendritic cells, namely, the production of ECM molecules, such as fibronectin (FN1) and osteopontin (OPN). Furthermore, we also found that CXCL4 exposure results in epigenetic imprinting during moDC differentiation. Using novel bioinformatic methods, we have found that CXCL4 mediates the altered cell function via key transcriptional regulators. Conclusions This study provides better understanding how CXCL4 affects moDCs through several immune and non-immune pathways and shows for the first time the direct implication of CXCL4 on the production of ECM by inflammatory cells, thereby underscoring the pivotal role of CXCL4 in inflammatory and fibrotic conditions such as SSc. References [1] L. van Bon, Affandi AJ, et al. Proteome-wide analysis and CXCL4 as a biomarker in systemic sclerosis. N. Engl. J. Med2014. doi:10.1056/NEJMoa1114576 [2] Silva-Cardoso SC, Affandi AJ, et al. CXCL4 Exposure Potentiates TLR-Driven Polarization of Human Monocyte-Derived Dendritic Cells and Increases Stimulation of T Cells. J. Immunol2017. doi:10.4049/jimmunol.1602020 Acknowledgements This study was supported by the: PhD fellowship SFRH/BD/89643/2012 from the Portuguese Fundaxxo para a Cixncia e a Tecnologia (FCT) to Sandra C. Silva-Cardoso; China Scholarship Council (CSC) fellowship No. 201606300050 to Weiyang Tao; ERC starting grant (CIRCUMVENT) and Arthritis foundation grant to Timothy R.D.J. Radstake. Disclosure of Interest None declared

Objective MicroRNAs (miRNAs) are regulatory molecules, which have been addressed as potential biomarkers and therapeutic targets in rheumatic diseases. Here, we investigated the miRNA signature in the serum of systemic sclerosis (SSc) patients and we further assessed their expression in early stages of the disease. Methods The levels of 758 miRNAs were evaluated in the serum of 26 SSc patients as compared to 9 healthy controls by using an Openarray platform. Three miRNAs were examined in an additional cohort of 107 SSc patients and 24 healthy donors by single qPCR. MiR-483-5p expression was further analysed in the serum of patients with localized scleroderma (LoS) (n = 22), systemic lupus erythematosus (SLE) (n = 33) and primary Sjogren's syndrome (pSS) (n = 23). The function of miR-483-5p was examined by transfecting miR-483-5p into primary human dermal fibroblasts and pulmonary endothelial cells. Results 30 miRNAs were significantly increased in patients with SSc. Of these, miR-483-5p showed reproducibly higher levels in an independent SSc cohort and was also elevated in patients with preclinical-SSc symptoms (early SSc). Notably, miR-483-5p was not differentially expressed in patients with SLE or pSS, whereas it was up-regulated in LoS, indicating that this miRNA could be involved in the development of skin fibrosis. Consistently, miR-483-5p overexpression in fibroblasts and endothelial cells modulated the expression of fibrosis-related genes. Conclusions Our findings showed that miR-483-5p is up-regulated in the serum of SSc patients, from the early stages of the disease onwards, and indicated its potential function as a fine regulator of fibrosis in SSc.

Chemokines have been shown to play immune-modulatory functions unrelated to steering cell migration. CXCL4 is a chemokine abundantly produced by activated platelets and immune cells. Increased levels of circulating CXCL4 are associated with immune-mediated conditions, including systemic sclerosis. Considering the central role of dendritic cells (DCs) in immune activation, in this article we addressed the effect of CXCL4 on the phenotype and function of monocyte-derived DCs (moDCs). To this end, we compared innate and adaptive immune responses of moDCs with those that were differentiated in the presence of CXCL4. Already prior to TLR- or Ag-specific stimulation, CXCL4-moDCs displayed a more matured phenotype. We found that CXCL4 exposure can sensitize moDCs for TLR-ligand responsiveness, as illustrated by a dramatic upregulation of CD83, CD86, and MHC class I in response to TLR3 and TLR7/8-agonists. Also, we observed a markedly increased secretion of IL-12 and TNF-α by CXCL4-moDCs exclusively upon stimulation with polyinosinic-polycytidylic acid, R848, and CL075 ligands. Next, we analyzed the effect of CXCL4 in modulating DC-mediated T cell activation. CXCL4-moDCs strongly potentiated proliferation of autologous CD4+ T cells and CD8+ T cells and production of IFN-γ and IL-4, in an Ag-independent manner. Although the internalization of Ag was comparable to that of moDCs, Ag processing by CXCL4-moDCs was impaired. Yet, these cells were more potent at stimulating Ag-specific CD8+ T cell responses. Together our data support that increased levels of circulating CXCL4 may contribute to immune dysregulation through the modulation of DC differentiation.

The chemokine CXCL4 has been implicated in several immune diseases. Exposure of monocyte-derived dendritic cells (moDCs) to CXCL4 potentiates the production of inflammatory cytokines in the presence of TLR3 or TLR7/8 agonists. Here we investigated the transcriptional and post-transcriptional events underlying the augmented inflammatory responses in CXCL4-moDCs. Our results indicate that CXCL4-moDCs display an increased expression and secretion of IL-12, IL-23, IL-6 and TNF upon TLR3 activation. Analysis of the cytokine transcripts for the presence of AU-rich elements (ARE), motifs necessary for ARE-mediated mRNA decay, revealed that all these cytokine transcripts are, at least in silico, possibly regulated at the level of mRNA stability. In vitro assays confirmed that mRNA stability of IL6 and TNF, but not IL12B and IL23A, is increased in CXCL4-moDCs. We next screened the expression of ARE-binding proteins (ARE-BPs) and found that TLR stimulation of CXCL4-moDCs induced tristetraprolin (TTP or ZFP36). Increased TTP mRNA expression was found to be a consequence of TTP phospho-mediated inactivation, which over time causes the protein to degrade its own mRNA. Concomitantly with TTP inactivation, we observed increased MAPK p38 signalling, upstream of TTP, in stimulated CXCL4-moDCs. P38 inhibition restored TTP activation and subsequently reduced the production of inflammatory cytokines. Finally, TTP knockdown in moDCs resulted in an increased production of IL6 and TNF after TLR stimulation. Overall, our study shows that the pro-inflammatory phenotype of CXCL4-moDCs relies in part on enhanced cytokine mRNA stability dictated by TTP inactivation.

Objective CD8+ T cells are abundant in rheumatoid arthritis (RA). However, their role in disease pathogenesis is poorly defined. This study was undertaken to investigate the relationship between disease activity and CD8+ T cell phenotypes, production of cytokines, and production of cytotoxic molecules in the peripheral blood (PB) and synovial fluid (SF) of patients with RA. Methods CD8+ T cell phenotypes were determined in 96 patients with RA (44 with disease in remission, 34 with active disease, 18 with low disease activity) and in 64 sex- and age-matched healthy controls. Ten paired PB and SF samples from patients with active RA were analyzed. The expression of surface markers, cytokines, and proteolytic enzymes in CD8+ T cells was evaluated using flow cytometry. Results PB CD8+ T cells from RA patients with active disease exhibited an effector (CD27−CD62L−) phenotype (P = 0.005), with elevated expression of proinflammatory cytokines (tumor necrosis factor α [TNFα], interferon-γ [IFNγ], interleukin-6 [IL-6], IL-17A) when compared to healthy controls. In a state of remission, the same phenotype observed in patients with active disease persisted, including a significant increase in the frequency of CD69 (P < 0.001), but lower cytokine production was observed. SF CD8+ T cells from RA patients expressed more robust effector memory (CD27+CD62L−) and activated (CD69+) profiles compared to the T cell subsets in paired PB samples. Production of cytokines (IL-6, IL-17A, and IFNγ) by CD8+ T cells from RA PB was positively correlated within individual donors. Moreover, production of cytokines (TNFα, IFNγ, and IL-17A) by CD8+ T cells from RA PB positively correlated with the Disease Activity Score in 28 joints. Conclusion The activation status and proinflammatory potential of CD8+ T cell subsets observed in the RA patients in this study strongly suggest that a phenotype of local and systemic cytotoxic effector T cells plays a role in this disease.

Background and Objectives CD8 + T cells are known to be present in the synovial fluid and synovium of rheumatoid arthritis. However, their role in the disease pathogenesis remains largely unexplored. This work intended to characterise blood and synovial fluid CD8 + T cell functional subsets found in patients with rheumatoid arthritis and explore their importance in regulating the activity of the disease. Methods The functional phenotypes of CD8 + T cells were determined in seventy-eight patients with rheumatoid arthritis (forty-four in remission (DAS28 3.2)) and the results were compared to those of sixty-four sex and age-matched healthy controls (HC). Ten paired samples of blood and synovial fluid (SF) from patients with active disease were also analysed, and the expression of surface markers, cytokines and proteolytic enzymes of CD8 + T cells were determined. Results Data shows that peripheral blood CD8 + T cells from both patients with active disease and in remission present an activated phenotype with a marked pro-inflammatory profile. Remission is associated with a lower production of proinflammatory mediators, such as IL-6 and IFNγ, but the changes in cell subsets observed in active disease persist or are even accentuated, with the frequency of the central memory CD27 + CD62L + CCR7 + CD8 + T cells significantly reduced, and the short-term effector CD27 - CD62L - CD8 + and the activated subset CD69 + CD8 + T cell subsets significantly increased when compared to healthy controls. The CD8 + T cells present in the SF of active RA present a more exacerbated effector memory (CD27 + CD62L - ) and activated (CD25 + and CD69 + ) CD8 + T cell phenotype than the ones in the paired blood samples. Finally, we report that the production of cytokines by CD8 + T cells from the synovial fluid is correlated with that observed in peripheral blood samples, with increased production of proinflammatory cytokines such as IL-6 and TNFα. Conclusions Our data demonstrate major shifts in CD8 + T cells subsets and in their cytokine production in rheumatoid arthritis, which are related to disease activity. This suggests that these cells play relevant roles in the pathogenesis of the disease.

Background and objectives Detailed information on CD8 + T cells in rheumatoid arthritis (RA) is still reduced. However, studies on animal models of arthritis from the authors9 team and others suggest a major potential role of CD8 + T cells in the pathogenesis of chronic polyarthritis. In the present study the authors characterised the phenotype and cytokine-production profile of CD8 + T cells from peripheral blood (PB) and synovial fluid (SF) of RA patients. Materials and methods Unstimulated CD8 + T cells from the PB and SF of 40 patients with established RA were analysed by flow-cytometry for cell surface markers expression and intracellular cytokine production, and compared to the ones present in the PB of 15 healthy donors. Results 40% of the total T cells infiltrating the SF were CD8 + . The SF was characterised by a significant (p + T cells infiltrating the SF presented an effector phenotype (CD62L − CD27 − short-term effector, or CD62L − CD27 + central effector), and the presence of the activation markers CD69 and CD25 were significantly (p − CD27 − CD8 + T cells and in the SF central memory CD62L + CD27 + CD8 + T cells when compared to RA PB. The intracellular expression of the pro-inflammatory cytokines IFN-γ, interleukin-6, interleukin-17 and tumour necrosis factor-α in central memory CD62L + CD8 + T cells were significantly (p Conclusions The present study presents strong evidence for a marked role of CD8 + T cells in RA pathogenesis. The authors propose that activated effector CD8 + T cells, home into the SF, where they might contribute to articular destruction and maintenance of a chronic pro-inflammatory environment through the production of cytokines and cytotoxic agents.