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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.

Multi-omics, meaning “multiple omics,” became accessible because of the revolution in high-throughput technologies. Multi-omics approaches have been integrated and applied by many studies to a wide range of biological problems, including disease mechanism revealing, biomarker identification, and patient classification. The multi-omics involved in this thesis are epigenomics (DNA methylomics), transcriptomics, and proteomics. This thesis divides into two major parts, aiming at using multi-omics to provide a better understanding of autoimmune diseases, including systemic sclerosis (SSc), psoriatic arthritis (PsA), psoriasis (Pso), Ankylosing spondylitis (AS), and rheumatoid arthritis (RA), even to depict a paradigm to study other diseases in general, ultimately facilitating personalized medicine. The first part focuses on using multi-omics to reveal disease mechanisms from the perspective of studying cytokine stimulation of cells. The first part of this thesis provides not only the potential mechanisms of CXCL4 involved in SSc using multi-omics but also an R package called RegEnrich to reveal the mechanisms of other diseases and biological processes. The second part mainly describes the (multi-)omic commonality and the distinction between patients with different autoimmune diseases (PsA, Pso, and AS) or with different subtypes of the same disease (RA). In this part, we have revealed shared serum proteomic signatures between patients with Pso and PsA and divergent multi-omic signatures between responders and non-responders to TNFi using machine learning techniques. All of these findings may ultimately facilitate personalized medicine, either from a better understanding of diseases or simply from providing informative drug response predictors.

Systemic sclerosis (SSc) is characterized by skin/internal organ fibrosis, vasculopathy and autoimmunity. Chemokine (C-X-C motif) ligand 4 (CXCL4) is an SSc biomarker, predicting unfavorable prognosis and lung fibrosis. CXCL4 binds DNA/RNA and favors interferon (IFN)-&alpha, production by plasmacytoid dendritic cells (pDCs), contributing to the type I IFN (IFN-I) signature in SSc patients. However, whether CXCL4 is an autoantigen in SSc is unknown. Here, we show that at least half of SSc patients show consistent antibody reactivity to CXCL4. T-cell proliferation to CXCL4, tested in a limited number of patients, correlates with anti-CXCL4 antibody reactivity. Antibodies to CXCL4 mostly correlate with circulating IFN-&alpha, levels and are significantly higher in patients with lung fibrosis in two independent SSc cohorts. Antibodies to CXCL4 implement the CXCL4&ndash, DNA complex&rsquo, s effect on IFN-&alpha, production by pDCs, CXCL4&ndash, DNA/RNA complexes stimulate purified human B-cells to become antibody-secreting plasma cells in vitro. These data indicate that CXCL4 is indeed an autoantigen in SSc and suggest that CXCL4, and CXCL4-specific autoantibodies, can fuel a harmful loop: CXCL4&ndash, DNA/RNA complexes induce IFN-&alpha, in pDCs and direct B-cell stimulation, including the secretion of anti-CXCL4 antibodies. Anti-CXCL4 antibodies may further increase pDC stimulation and IFN-&alpha, release in vivo, creating a vicious cycle which sustains the SSc IFN-I signature and general inflammation.

Systemic sclerosis (SSc) is a rare autoimmune disease with heterogeneous symptoms and characterized by immune alteration, vascular abnormalities, fibrosis and hypoxia. SSc is a poorly understood disease with no available effective cure and an unknown pathophysiology. Recent studies identified chemokine (C-X-C-motif) ligand-4 (CXCL4) as a potential biomarker produced by pro-inflammatory immune-cells. Besides, alteration in fat metabolism has been shown to elicit immune-cell polarisation in SSc. The aim of this thesis is to explore the augmented production of CXCL4 and its role in aberrant fibrogenesis in patients with SSc. Moreover, the metabolic status of fat biology in pathophysiology of SSc was further inquired. In healthy immune-cells CXCL4-production depends on mitochondrial reactive oxygen species and hypoxia inducible factor (HIF)-2 stabilization. We observed that Toll-Like receptor (TLR) triggered upon hypoxic environment is responsible for augmented CXCL4-production in SSc. Also, we pinpointed the potential beneficial role of HIF-2 stabilization on cytokine production and confirmed its potential features as a therapeutic target in SSc treatment. Besides, we demonstrated that fatty acids and carnitine composition were altered in SSc and demonstrated that inhibiting fatty acid oxidation abates pro-inflammatory cytokine production in immune-cells of SSc patients. Finally, we observed that inhibition of mammalian target of rapamycin (mTOR), with metformin reduces pro-inflammatory cytokine production, signifying the potential therapeutic effect of metabolic mediators in immune cells. Taken together, targeting TLR stimulation, HIF-2 stabilization and fatty acid oxidation inhibits aberrant cytokine and CXCL4-production in immune cells and metformin treatment can contribute in reducing pro-inflammatory cytokine production in patients with SSc.

Systemic sclerosis (SSc) is a rare autoimmune disease with heterogeneous symptoms and characterized by immune alteration, vascular abnormalities, fibrosis and hypoxia. SSc is a poorly understood disease with no available effective cure and an unknown pathophysiology. Recent studies identified chemokine (C-X-C-motif) ligand-4 (CXCL4) as a potential biomarker produced by pro-inflammatory immune-cells. Besides, alteration in fat metabolism has been shown to elicit immune-cell polarisation in SSc. The aim of this thesis is to explore the augmented production of CXCL4 and its role in aberrant fibrogenesis in patients with SSc. Moreover, the metabolic status of fat biology in pathophysiology of SSc was further inquired. In healthy immune-cells CXCL4-production depends on mitochondrial reactive oxygen species and hypoxia inducible factor (HIF)-2 stabilization. We observed that Toll-Like receptor (TLR) triggered upon hypoxic environment is responsible for augmented CXCL4-production in SSc. Also, we pinpointed the potential beneficial role of HIF-2 stabilization on cytokine production and confirmed its potential features as a therapeutic target in SSc treatment. Besides, we demonstrated that fatty acids and carnitine composition were altered in SSc and demonstrated that inhibiting fatty acid oxidation abates pro-inflammatory cytokine production in immune-cells of SSc patients. Finally, we observed that inhibition of mammalian target of rapamycin (mTOR), with metformin reduces pro-inflammatory cytokine production, signifying the potential therapeutic effect of metabolic mediators in immune cells. Taken together, targeting TLR stimulation, HIF-2 stabilization and fatty acid oxidation inhibits aberrant cytokine and CXCL4-production in immune cells and metformin treatment can contribute in reducing pro-inflammatory cytokine production in patients with SSc.

Systemic sclerosis (SSc) is a heterogeneous autoimmune disorder characterised by vasculopathy, immune cell dysfunction, antibodies production and aberrant deposition of extracellular matrix components (ECM), such as fibronectin. Changes in frequency and activation of platelets and immune cells are implicated in aberrant inflammatory and fibrotic responses associated with the pathology of SSc. CXCL4 is a chemokine released by activated platelets and immune cells. Our group and others have shown that CXCL4 levels are increased of in circulation and in skin of SSc patients. In this thesis we investigated whether the exposure to CXCL4 potentiates pro-inflammatory and pro-fibrotic responses by monocyte-derived dendritic cells (moDCs). Additionally we explored the underlying molecular mechanisms implicated in the reprogramming of moDC phenotype and function by CXCL4. We found that CXCL4 primes moDCs to a semi-mature phenotype and morphology, amplifies aberrant TLR3-mediated responses and potentiates the activation of autologous and antigen specific T-cell responses. We unrevealed that CXCL4-mediated immunogenic responses are regulated at the transcriptional, post-transcriptional and epigenetic level. Using data driven gene regulatory network analyses, we showed that CXCL4 modulates pro-inflammatory and pro-fibrotic responses via key transcription regulators, such as CIITA (Class II Major Histocompatibility Complex Transactivator) and IRF8 (Interferon Regulatory Factor 8). Additionally, we unrevealed that aberrant pro-inflammatory cytokine production upon TLR3-mediated stimulation relies in part on enhanced cytokine mRNA stability dictated by the inactivation of tristetrapolin (TTP), an AU-rich element binding protein (ARE-BP). Altogether, this thesis highlights the role of CXCL4 in reprogramming moDC phenotype and function and opens new avenues for therapeutic intervention

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.

Contains fulltext : 136617.pdf (Publisher’s version ) (Open Access) BACKGROUND: Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. METHODS: We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. RESULTS: Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (+/-SD) level of CXCL4 in patients with systemic sclerosis was 25,624+/-2652 pg per milliliter, which was significantly higher than the level in controls (92.5+/-77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346+/-1011 pg per milliliter), ankylosing spondylitis (1368+/-1162 pg per milliliter), or liver fibrosis (1668+/-1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. CONCLUSIONS: Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).