Francesca Meda

The epigenetics of autoimmunity

The etiology of autoimmune diseases remains largely unknown. Concordance rates in monozygotic twins are lower than 50% while genome-wide association studies propose numerous significant associations representing only a minority of patients. These lines of evidence strongly support other complementary mechanisms involved in the regulation of genes expression ultimately causing overt autoimmunity. Alterations in the post-translational modification of histones and DNA methylation are the two major epigenetic mechanisms that may potentially cause a breakdown of immune tolerance and the perpetuation of autoimmune diseases. In recent years, several studies both in clinical settings and experimental models proposed that the epigenome may hold the key to a better understanding of autoimmunity initiation and perpetuation. More specifically, data support the impact of epigenetic changes in systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and other autoimmune diseases, in some cases based on mechanistical observations. We herein discuss what we currently know and what we expect will come in the next future. Ultimately, epigenetic treatments already being used in oncology may soon prove beneficial also in autoimmune diseases.

Expansion and de novo generation of potentially therapeutic regulatory T cells in patients with autoimmune hepatitis

CD4+CD25+ regulatory T cells (T‐regs) are central to the maintenance of immune tolerance and represent an immune intervention candidate in autoimmune hepatitis (AIH), a condition characterized by impaired T‐reg number and function. We investigated whether T‐regs can be expanded from the existing CD4+CD25+ T cell pool and generated de novo from CD4+CD25− T cells in AIH patients and healthy controls. Purified CD4+CD25+ and CD4+CD25− T cells from 24 patients with type 1 AIH and 22 healthy controls were cultured for up to 5 weeks with anti‐CD3/anti‐CD28 T cell expander and high‐dose interleukin‐2 (IL‐2). Cell phenotypes, suppressor ability, forkhead winged/helix transcription factor box P3 (FOXP3) gene, and protein expression were assessed weekly by cytofluorimetry, proliferation assay, real‐time polymerase chain reaction (PCR), and immunoblot. During culture, the number of CD4+CD25+ T cells derived from the existing T‐reg pool (expanded T‐regs) and generated de novo from CD4+CD25− T cells (newly generated T‐regs) increased constantly up to week 4 in both healthy controls and, to a lesser extent, in AIH patients. Expanded T‐regs retained conventional T‐reg phenotype and, compared with baseline, demonstrated more vigorous suppressive function and increased FOXP3 gene and protein expression. Newly generated T‐regs not only acquired T‐reg phenotype but underwent greater growth and were more resistant to apoptosis than expanded T‐regs. Their suppressive function augmented throughout culture, reaching a peak at week 4, preceded by a peak FOXP3 gene and protein expression at week 2. Suppressor function and FOXP3 expression of both expanded and newly generated T‐regs were higher in normal controls than in AIH patients. Conclusion: Functionally enhanced T‐regs can be expanded and generated de novo in patients with AIH. This finding may assist in reconstituting impaired immune regulation and restoring peripheral tolerance through T‐reg infusion in this condition. (HEPATOLOGY 2008;47:581–591.)

Genome-Wide Analysis of DNA Methylation, Copy Number Variation, and Gene Expression in Monozygotic Twins Discordant for Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is an uncommon autoimmune disease with a homogeneous clinical phenotype that reflects incomplete disease concordance in monozygotic (MZ) twins. We have taken advantage of a unique collection consisting of genomic DNA and mRNA from peripheral blood cells of female MZ twins (n = 3 sets) and sisters of similar age (n = 8 pairs) discordant for disease. We performed a genome-wide study to investigate differences in (i) DNA methylation (using a custom tiled four-plex array containing tiled 50-mers 19,084 randomly chosen methylation sites), (ii) copy number variation (CNV) (with a chip including markers derived from the 1000 Genomes Project, all three HapMap phases, and recently published studies), and/or (iii) gene expression (by whole-genome expression arrays). Based on the results obtained from these three approaches we utilized quantitative PCR to compare the expression of candidate genes. Importantly, our data support consistent differences in discordant twins and siblings for the (i) methylation profiles of 60 gene regions, (ii) CNV of 10 genes, and (iii) the expression of 2 interferon-dependent genes. Quantitative PCR analysis showed that 17 of these genes are differentially expressed in discordant sibling pairs. In conclusion, we report that MZ twins and sisters discordant for PBC manifest particular epigenetic differences and highlight the value of the epigenetic study of twins.

Serum autoantibodies: A road map for the clinical hepatologist

The widespread use of serum autoantibodies in the practice of clinical hepatology has led to novel challenges in the interpretation of results obtained with routine techniques, such as indirect immunofluorescence (IIF) or with recombinant antigens. In fact, the laboratory methods are often overlooked factors in the interpretation of data by the bedside clinician despite being critical in the interpretation of data. Importantly, the sensitivity and specificity of these serum hallmarks are not defined in all cases. Taken altogether, these observations point towards the need for a systematic discussion of autoimmune serology in the clinical setting of everyday practice. The target of this review article is therefore, to illustrate the current knowledge and available experimental evidence to guide the diagnostic and prognostic decision making in autoimmune and viral chronic liver diseases.

Disease‐specific autoantibodies in patients with acute liver failure: The King's College London Experience

For example, Bcl-2 repression by TGFrequires C-terminal phosphorylation of Smad3.6 To evade apoptosis, HCC might acquire Bcl-2 overexpression induced by pSmad3L pathway. Escaping apoptosis is a critical step in the progression to full malignancy of cancers, which overcome multiple fail-safe genetic controls. From the viewpoint of TGFsignaling, a key therapeutic aim in cancer would be restoration of the lost tumor suppressor function observed in normal hepatocytes at the expense of effects promoting aggressive behavior in HCC. Our model suggests that specific inhibitors of the pSmad3L-mediated oncogenic/fibrogenic signaling should inhibit progression of HCC.

Serum autoantibodies for the diagnosis and management of autoimmune liver diseases

The spectrum of autoimmune liver diseases (AILD) includes primary biliary cirrhosis, primary sclerosing cholangitis and autoimmune hepatitis. The immunological mechanisms triggering the initiation and perpetuation of AILD remains unknown, while autoantigens are now recognized in most cases, and are generally nontraditional in their widespread distribution. Sensitive and specific methods for the detection of serum autoantibodies in patients affected by AILD represent a challenge for researchers and clinicians who desire to obtain an early and certain diagnosis as well as markers of disease control. To this regard, the use and interpretation of serum autoantibodies in AILD may be seen as paradigmatic for the large gaps in our knowledge based on the lack of true population-based studies. The present review article will critically discuss the available evidence on the use of autoantibody findings in the diagnosis or management of autoimmune liver disease.

FRI0354 Effects of Carbamylation and Citrullination of B and T Epitopes of Human Type II Collagen on Lymphocytes from Dr4+ Monozygotic Twins Discordant for Rheumatoid Arthritis

Background Rheumatoid arthritis (RA) susceptibility is linked to HLA DR4 and DR1. T cells in RA recognize the DR4/DR1-restricted epitope 261-273 of the human type II collagen, whereas B cells recognize the epitope 359-369. These epitopes can undergo post-translational modifications such as carbamylation and citrullination and both processes are involved in RA pathogenesis. Objectives To investigate the role of B and T cell epitopes and their post-translational modifications on the adaptive immune response in the unique model of DR4 carrying monozygotic twins discordant for RA. Methods PBMCs were obtained from a treatment-naïve HLA-DR4 positive woman with early RA (rheumatoid factor +, anti-CCP -, elevated CRP, bilateral wrist arthritis for 3 months) and from her healthy monozygotic twin sister (autoantibody-negative, normal CRP). Cells were cultured for 3 days with the native form of the collagen T epitope, its K264 carbamylated form (homocitT), the native form of the collagen B epitope, its R360 citrullinated form (citB) or a combination of the native and modified epitopes. After the stimulation, cells were analyzed by flow cytometry for activation, apoptosis, and cytokine polarization. Results The activation of CD4 T cells was induced only with the modified B and T epitope stimulation in the RA twin (CD4+CD154+ in RA vs healthy; unstimulated: 1 vs 0.4%; T: 0.9 vs 1.6%; homocitT: 1.5 vs 0.7; B: 1 vs 0.6%; citB: 1.7 vs 0.1%, T + B: 0,5 vs 0,3%; homocitT + citB: 0,9 vs 0,4%). The antigen-activated CD4 T cells in the RA twin were significantly less apoptotic compared to unstimulated cells and the healthy twin (RA vs healthy; unstimulated: 37.5 vs 40%; T: 25 vs 24.1%; homocitT: 17 vs 50%; B: 30.8 vs 28%; citB: 13.9 vs 83.3%; T + B: 12.3 vs 63.6%; homocitT + citB: 19 vs 50%). Opposite from what observed in RA, the healthy twin manifested a higher increase in Th2 cells (CD4+CD154+IL4+ in RA vs healthy; unstimulated: 1.7 vs 2.1%; T: 2.9 vs 5.7%; homocitT: 3.6 vs 5.8%; T + B: 4 vs 4.9%; homocitT + citB: 3.9 vs 2.9%) compared to Th17 cells (CD4+CD154+IL17+ in RA vs healthy; unstimulated: 2.7 vs 1.7%; T: 4.1 vs 3.1%; homocitT: 4.5 vs 3.2%; T + B: 4.8 vs 3.5%; homocitT + citB: 5.5 vs 3%). In the healthy twin the Th2 increase was observed also in the antigen-activated CD8 T cells while B cell activation increased after different stimulations, but in all conditions activated B cells were apoptotic in 80% of the cases. Cytokine B cell production was not substantially altered with the epitope stimulations in the RA twin, while in the healthy twin there was a general increase in all cytokines. Conclusions Our data obtained in the unique model of discordant monozygotic twins suggest that the exposure of collagen self-epitopes to lympocytes induces changes with pathogenic or protective effects that appear to be independent on DR4 and on post-translational antigen modifications. Further, this is associated with a greater Th17 shift in the antigen-activated CD4 T cells and with their enhanced resistance to spontaneous apoptosis in RA, while a greater Th2 shift is observed in the healthy twin. Carbamylation and citrullination of collagen epitopes may promote the activation of CD4 T cells. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4278

THU0132 Prevalence and Predictive Value of Serum Autoantibodies in the General Population: Results of a Longitudinal Study on 2690 Subjects with a 13-Year Observation

Background The prevalence of serum autoantibodies in the general population is generally obtained from large datasets including blood donors or employees and are thus poorly representative. Similarly, it is challenging to determine the risk of developing an autoimmune disease in subjects found to be positive for serum autoantibodies in the absence of clinical suspicion. Objectives To determine the prevalence and predictive value of ANA, ENA, and ACPA antibodies in the general population participating in a 13-year longitudinal study. Methods The general population of a Northern Italian area was enrolled in 1998/1999 in a study to determine the prevalence of viral hepatitis infections. Resident subjects aged 18-75 were randomized 1:4 and 71% of subjects took part in the study (mean age 42, range 18-75, female/male 1.15). In 2011 serum samples (n=2690) were blindly tested for ANA, ENA, anti-CCP using commercially available indirect immunofluorescence and ELISA (Aesku.Diagnostics). For all subjects we sought for exemptions (i.e. the fiscal mechanism that allows subjects with a chronic condition to waive copayments for visits, medications, and blood tests) specific for autoimmune diseases (including connective tissue disease, CTD, and rheumatoid arthritis, RA) over the following 13 years. We should note that this approach does not account for 11% of the population which, for economic limits or advanced age, have a waiver overcoming the disease-specific exemptions. Administrative data were ultimately analyzed to determine the long-term predictive value of serum autoantibodies in terms of odd ratios (OR). Results Serum ANA were detected in 18.1% (for titers ≥1:80) and 5.7% (for titers ≥1:160) of tested samples and the observed pattern was speckled in 71% of cases. The prevalence of specific anti-ENA autoantibodies ranged between 0.1% (Jo-1) and 1.9% (anti-nucleosome) while ACPA were positive in 4.7% of subjects (0.9% considering only medium-to-high titers). In all cases, prevalence rates were higher in female individuals but the predominance was lower than classically reported for CTD and RA. The OR for developing any autoimmune disease for ANA positive individuals over 13 years was 2.77 (95% confidence interval -CI- 1.73-4.44). The OR of developing a CTD for individuals with high-titer ANA was 12.20 (95% CI 2.41-61.59) while the OR of developing RA with positive ACPA was 10.86 (95% CI 1.96-59.98). Conclusions We report that the prevalence of serum ANA, ENA, and ACPA may be higher than previously reported when a general unselected population is studied. Further, the serum positivity confers a significant risk of developing an autoimmune disease when subjects are observed for a sufficient period of time. Disclosure of Interest None Declared