DNA methylation in peripheral blood mononuclear cells (PBMCs) from patients with autoimmune hepatitis (AH) and primary biliary cholangitis (PBC)

Autoimmne hepatitis (AIH) and primary biliary cholangitis (PBC) are the two major autoimmune liver diseases, characterized by chronic inflammation of liver parenchyma that leads to end stage liver disease, if untreated. Their aetiopathognesis has not yet been completely elucidated. Since genetic factors are insufficient to explain the observed risk, the role of potential epigenetic modifications in diseases’ pathogenesis has to be clarified. The aim of the present study was to assess the contribution of epigenetic modifications in AIH and PBC pathogenesis. For this purpose, PBMCs from 10 AIH patients at diagnosis, 8 AIH patients at disease remission, 9 PBC patients and 10 healthy controls (HC) were used for further isolation of CD19(+) B and CD4(+) T lymphocytes with flow cytometry. Global DNA methylation (5mC)/hydroxymethylation (5hmC) was determined by ELISA, while the transcriptional levels of DNA methylation (DNMT1, DNMT3A, DNMT3B)/hydroxymethylation (TET1, TET2, TET3) enzymes were determined by quantitative PCR. Further analysis of the methylation level of specific CpG sites across the whole genome was performed by 850k array of Illumina. According to our findings, CD19(+) and CD4(+) lymphocytes from AIH patients at diagnosis are characterized by increased DNMT3A and reduced TET1 mRNA levels compared to PBC patients and HC respectively. DNMT3A mRNA levels are negatively associated with IgG serum levels, indicating a correlation between DNMT3A anddisease activity. Immunosuppression reduces DNMT3A levels in both CD19(+) and CD4(+) lymphocytes from AIH patients at disease remission. Interestingly, changes in DNMT3A and TET1 expression have no effect on global 5mC/5hmC levels in CD19(+) and CD4(+) cells from AIH patients, supporting the probable presence of differential methylation of isolated genes. This fact is futher supported by the methylation analysis across the whole genome, which revealed hypomethylation of several genes in AIH, in contrast to the hypermethylation of the majority of the differentially methylated genes in PBC. In addition, the hypomethylation of certain genes in AH patients at diagnosis is reversed after treatment administration. Some of the most differentially methylated genes were involved in pathways of innate immunity, development of cholestatis and of hepatocellular carcinoma. Last but not least, our study highlights the role of the X chromosome in PBC pathogenesis, a finding that is not verified in AIH. In conclusion, our study supports that AIH is characterized by altered expression of DNA methylation/hydroxymethylation enzymes, which is associated with disease activity, but also by hypomethylation of specific genes involved in immune pathways, which is reversed by immunosuppressive treatment. These findings open new insights in understanding disease’s pathogenesis and establishing novel, targeted therapeutic interventions.Η αυτοάνοση ηπατίτιδα (ΑΗ) και η πρωτοπαθής χολική χολαγγειίτιδα (ΠΧΧ) είναι τα δύο κύρια αυτοάνοσα νοσήματα του ήπατος τα οποία χαρακτηρίζονται από χρόνια φλεγμονή, η οποία οδηγεί σε κίρρωση αν αφεθεί χωρίς θεραπεία. Καθώς οι γενετικοί παράγοντες, μόνοι τους, αδυνατούν να εξηγήσουν την αιτιοπαθογένειά των αυτοανόσων νόσων του ήπατος, η μελέτη των επιγενετικών παραγόντων θα μπορούσε να διαφωτίσει μέρος των μηχανισμών που σχετίζονται με την παθογένεση και την εξέλιξή τους. Σκοπός της παρούσας διατριβής είναι η μελέτη της συμμετοχής των επιγενετικών μηχανισμών στην αιτιοπαθογένεια της ΑΗ και της ΠΧΧ με στόχο την ανάπτυξη νέων θεραπευτικών στρατηγικών. Για το σκοπό αυτό περιφερικά μονοκύτταρα (PBMCs) 10 ασθενών με ΑΗ κατά τη διάγνωση (σε 8 από αυτούς έγινε μελέτη και κατά την επίτευξη πλήρους ανταπόκρισης μετά από χορήγηση ανοσοκαταστολης), 9 ασθενών με ΠΧΧ και 10 υγιών μαρτύρων (ΥΜ) χρησιμοποιήθηκαν για την απομόνωση CD19(+) B και CD4(+) T λεμφοκυττάρων με κυτταρομετρία ροής. Στη συνέχεια έγινε προσδιορισμός της ολικής μεθυλίωσης (5mC)/υδροξυμεθυλίωσης (5hmC) του DNA με τη χρήση ELISA, ενώ η έκφραση των ενζύμων μεθυλίωσης (DNMT1, DNMT3A, DNMT3B)/υδρόξυμεθυλίωσης (ΤΕΤ1, ΤΕΤ2, ΤΕΤ3) σε μεταγραφικό επίπεδο, έγινε με τη χρήση ποσοτικής αντίδρασης πολυμεράσης (qPCR). Περαιτέρω ανάλυση της μεθυλίωσης των επιμέρους CpG αλληλουχιών κατά μήκος του γονιδιώματος έγινε με την αλληλουχία 850k της Illumina.Αυξημένη DNMT3A και χαμηλή ΤΕΤ1 χαρακτηρίζει τα CD19(+) και CD4(+) λεμφοκύτταρα των ασθενών με ΑΗ κατά τη διάγνωση συγκριτικά με τους ασθενείς με ΠΧΧ και τους ΥΜ αντίστοιχα. Τα επίπεδα mRNA της DNMT3A σχετιζόταν αρνητικά με την IgG ανοσοσφαιρίνη κατά τη διάγνωση της ΑΗ υποδηλώνοντας τη συσχέτισή της DNMT3A με τη δραστηριότητα της νόσου. Η χορήγηση ανοσοκατασταλτικής αγωγής βρέθηκε ότι μειώνει τα επίπεδα της DNMT3A στους ασθενείς με ΑΗ κατά την ύφεση της νόσου τόσο στα CD19(+) όσο και στα CD4(+) λεμφοκύτταρα. Οι αλλαγές στα επίπεδα των DNMT3A και ΤΕΤ1 δε συμβάδιζαν με σημαντικές διαφορές στο επίπεδο ολικής 5mC/5hmC, παραπέμποντας στην παρουσία εντοπισμένων επιγενετικών αλλαγών που επιδρούν στην έκφραση συγκεκριμένων γονιδίων. Υπέρ του ευρήματος αυτού, συνηγορούν τα αποτελέσματα της ανάλυσης της μεθυλίωσης του συνόλου του γονιδιώματος των CD4(+) T λεμφοκυττάρων, που ανέδειξαν σημαντικές διαφορές στη μεθυλίωση μεμονωμένων γονιδίων μεταξύ ασθενών με ΑΗ έναντι των ΥΜ και των ασθενών με ΠΧΧ. Συγκεκριμένα, η πλειοψηφία των διαφορετικά μεθυλιωμένων γονιδίων στους ασθενείς με ΑΗ κατά τη διάγνωση βρέθηκαν υπομεθυλιωμένα, ενώ στους ασθενείς με ΠΧΧ παρατηρήθηκε υπερμεθυλίωση του πλείστου των διαφορετικά μεθυλιωμένων γονιδίων. Επιπλέον, η υπομεθυλίωση των γονιδίων στην ΑΗ επηρεαζόταν από την ανοσοκατασταλτική αγωγή. Μεταξύ των πλέον διαφορετικά μεθυλιωμένων γονιδίων αναδειχθηκαν γονίδια, που εμπλέκονται σε μονοπάτια φυσικής ανοσίας καθώς και γονίδια που συμμετέχουν σε μηχανισμούς ανάπτυξης χολόστασης και ηπατοκαρκινογένεσης. Επιπλέον, στην παρούσα μελέτη επιβεβαιώθηκε η σημασία του χρωμοσώματος Χ στην παθογένεια της ΠΧΧ, ενώ στην ΑΗ δεν υπήρχε αντίστοιχο εύρημα (τα περισσότερα διαφορετικά μεθυλιωμένα γονίδια εδράζονταν σε αυτοσωμικά χρωμοσώματα). Συμπερασματικά, η μελέτη μας δείχνει για πρώτη φορά, ότι η ΑΗ χαρακτηρίζεται από αλλαγές στην έκφραση των ενζύμων μεθυλίωσης/υδροξυμεθυλίωσης, που σχετίζονται με τη δραστηριότητα της νόσου, αλλά και από αλλαγές στη μεθυλίωση μεμονωμένων γονιδίων, που επηρεάζονται από τη χορήγηση ανοσοκαταστολτικής αγωγής. Αντίθετα, η ΠΧΧ χαρακτηρίζεται από υπερμεθυλίωση των περισσότερων διαφορετικά μεθυλιωμένων γονιδίων. Τα ευρήματα αυτά ανοίγουν νέους δρόμους για την κατανόηση της αιτιοπαθογένειας των αυτοανόσων νοσημάτων του ήπατος και την εύρεση νέων, στοχευμένων θεραπειών

Postinfantile Giant Cell Hepatitis with Features of Acute Severe Autoimmune Hepatitis Probably Triggered by Diclofenac in a Patient with Primary Myelofibrosis

Giant cell hepatitis (GCH) is commonly reported in neonatal and infantile liver diseases but rarely in adults where the term postinfantile GCH (PIGCH) is used. PIGCH is associated with many diseases, including drugs toxicity, viruses, and autoimmune liver diseases, with autoimmune hepatitis (AIH) being the most prevalent. We present a case of PIGCH in a 76-year-old female without known history of liver disease who suffered from an acute severe episode of hepatitis. After careful exclusion of other hepatitis causes by imaging, virological, immunological, and microbiological investigations, a diagnosis of acute severe AIH (AS-AIH) was established. The patient was started on corticosteroids but she did not respond and died 3 days later because of advanced acute liver failure. Postmortem liver biopsy showed typical PIGCH lesions. Physicians must keep this catastrophic entity in mind in cases of unexplained acute liver injury as, contrary to our case, prompt rescue therapy with corticosteroids may be life-saving

Interplay between genetic and epigenetic factors in primary Sjögren’s syndrome.

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JAK Inhibitors and Oxidative Stress Control

International audiencePrimary Sjögren's syndrome (SjS) is a complex autoimmune epithelitis, with few treatment options, but the use of Janus kinase (JAK) inhibitors is promising because suppression of the JAK/signal transducer and activator of transcription (STAT) pathway improves sicca manifestations. Playing a primary and pathogenic role in disease development, the oxidative stress response is upregulated in activated salivary gland epithelial cells (SGECs) from patients with SjS. Therefore, the aim of this study was to investigate whether JAK inhibitors would suppress SGEC activation in response to an oxidative stress. For this purpose, the human salivary gland (HSG) cell line was used, and cells were treated with the reactive oxygen species (ROS) inducer hydrogen peroxide (H2O2) or with interferons (IFN Type I and Type II), used as positive controls, to mimic activated SGECs as observed in SjS patients. Afterward, the levels of the intracellular adhesion molecule-1 (ICAM-1) and the regulatory programmed-death ligand-1 (PD-L1) were measured by real-time PCR and flow cytometry, and the STAT1/3 phosphorylation status was assessed by Western blotting. Using the HSG cell line, our results showed that both ICAM-1 and PD-L1 are induced by ROS through pSTAT3, and that this activation pathway is reversed by the use of JAK inhibitors, AG490 and ruxolitinib, as well as by N-acetylcysteine, which is a direct inhibitor of ROS. These findings open new perspectives regarding the pathogenesis and therapeutic possibilities for SjS

JAK Inhibitors Suppress Innate Epigenetic Reprogramming: a Promise for Patients with Sjögren’s Syndrome

International audiencePathogenesis of primary Sjögren's syndrome (SjS) remains obscure. However, recent data demonstrate the implication of epigenetic alterations in the DNA methylation/hydroxymethylation process in SjS mostly affecting genes regulated by two innate cytokines, interferon α (IFNα) and IFNγ as well as the oxidative stress pathways. The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is known to be activated by IFN and reactive oxygen species (ROS). This prompts us to test the potential implication of JAK/STAT signaling on DNA methylation/hydroxymethylation alterations in SjS. For this purpose, the human salivary gland (HSG) cell line was used and cells were treated with both types of IFNs and H2O2 to mimic activated salivary gland epithelial cells (SGEC) as observed in SjS patients. Afterwards, the global DNA level of methylcytosine and hydroxymethylcytosine, the expression of the DNA methylating enzymes (DNMTs) and ten-eleven translocation (TETs) methyl cytosine dioxygenase that controls DNA hydroxymethylation, both at transcriptional and at protein level, as well as STAT phosphorylation and ROS status were determined. Our results showed that expression of TET3 and in turn global DNA hydroxymethylation is controlled through the induction of STAT3 mediated by IFNα, IFNγ, and H2O2. On the other hand, treatment with JAK inhibitors (AG490 and ruxolitinib) reverses this process, suggesting a novel treatment pathway for patients with autoimmune diseases and Sjögren's syndrome

Epigenetic Modifications in Generalized Autoimmune Epithelitis: Sjögren’s Syndrome and Primary Biliary Cholangitis

Sjögren’s syndrome (SjS) and primary biliary cholangitis (PBC) can be classified as a model of generalized autoimmune epithelitis based on their frequent coexistence in clinical practice and the highly specific immune mediated injury of target epithelial cells. Both of these autoimmune diseases are characterized by female predominance, highly specific circulating autoantibodies, and immune-mediated destruction of the salivary and lachrymal glands and the biliary epithelial cells, respectively. Although the genetic predisposition has been well described for both diseases, genetic studies have failed to completely elucidate their pathogenesis. The recent integration of epigenetic data, analyzing the different cellular partners, opens new perspectives and allows for better understanding of these complex and still incurable diseases. Epigenetic studies on SjS have elucidated the role of DNA methylation alterations in disease pathogenesis, while epigenetic changes that influence expression of genes on the X chromosome have been implicated in the geo-variability and occurrence of PBC. The aim of this review is to describe the advances in epigenetics in the field of autoimmune epithelitis as well as to highlight how epigenetic changes could contribute to better understanding of disease pathogenesis and progression. These advances could yield insights on novel therapeutic interventions

FibroMeter scores for the assessment of liver fibrosis in patients with autoimmune liver diseases

Introduction and Objectives: We assessed FibroMeter virus (FMvirus) and FibroMeter vibration-controlled transient elastography (FMVCTE) in 134 patients with autoimmune liver diseases [ALD, autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC)], in order to assess new potential non-invasive biomarkers of liver fibrosis in patients with ALD, as similar data are missing. Patients and Methods: The following groups were included: group 1: n = 78 AIH; group 2: n = 56 PBC. FMvirus and FMVCTE were determined in all 134 patients who underwent liver biopsy and TE the same day with sera collection. In addition, APRI and FIB-4 scores were calculated. Results: The AUCs for TE and FMVCTE were significantly better (0.809; p < 0.001 and 0.772; p = 0.001, respectively for AIH and 0.997; p < 0.001 and 1; p < 0.001, for PBC) than the other three markers in predicting ≥ F3 fibrosis irrespective of the biochemical activity. FMVCTE and TE had good diagnostic accuracy (75.6% and 73%, respectively) for predicting severe fibrosis in AIH and performed even better in PBC (94.6% and 96.4%, respectively). The cut-offs of TE and FMVCTE had the best sensitivity and specificity in predicting ≥ F3 fibrosis in both AIH and PBC. Conclusions: FMVCTE seems to detect severe fibrosis equally to TE in patients with ALD but with better specificity. Biochemical disease activity did not seem to affect their diagnostic accuracy in ALD and therefore, could be helpful for the assessment of fibrosis, especially if they are performed sequentially (first TE with the best sensitivity and then FMVCTE with the best specificity)

Linking genetic variation with epigenetic profiles in Sjögren's syndrome

International audienceDNA methylation represents an important regulatory event governing gene expression that is dysregulated in Sjögren's syndrome (SjS) and a number of autoimmune/inflammatory diseases. As disease-associated single-nucleotide polymorphisms (SNPs) have relevance in controlling DNA methylation, 94 non-HLA SjS-SNPs were investigated, among them 57 (60.6%) with widespread effects on 197 individual DNA methylation quantitative trait loci (meQTL) were selected. Typically, these SNPs are intronic, possess an active promoter histone mark, and control cis-meQTLs located around transcription start sites. Interplay is independent of the physical distance between SNPs and meQTLs. Using epigenome-wide association study datasets, SjS-meQTLs were characterized (41 genes and 13 DNA methylation CpG motifs) and for the most part map to a pro-inflammatory cytokine pathway, which is important for the control of DNA methylation in autoimmune diseases. In conclusion, exploring meQTLs represents a valuable tool to predict and investigate downstream effects of genetic factors in complex diseases such as SjS