Francesca Cavaciocchi

X chromosome gene methylation in peripheral lymphocytes from monozygotic twins discordant for scleroderma

Scleroderma (SSc) is a rare connective tissue disease characterized by fibrosis, microvasculopathy and autoimmune features. The role of genetics is limited in SSc, as suggested by similar concordance rates in monozygotic and dizygotic twin pairs, while environmental factors may act through epigenetic changes, as demonstrated for specific genes. Further, sex chromosome changes have been reported in SSc and may explain the female preponderance. In the present study we compared the methylation profile of all X chromosome genes in peripheral blood mononuclear cells from monozygotic twins discordant (n = 7) and concordant (n = 1) for SSc. Methylated DNA immunoprecipitations from each discordant twin pair were hybridized to a custom‐designed array included 998 sites encompassing promoters of all X chromosome genes and randomly chosen autosomal genes. Biostatistical tools identified sites with an elevated probability to be consistently hypermethylated (n = 18) or hypomethylated (n = 25) in affected twins. Identified genes include transcription factors (ARX, HSFX1, ZBED1, ZNF41) and surface antigens (IL1RAPL2, PGRMC1), and pathway analysis suggests their involvement in cell proliferation (PGK1, SMS, UTP14A, SSR4), apoptosis (MTM1), inflammation (ARAF) and oxidative stress (ENOX2). In conclusion, we propose that X chromosome genes with different methylation profiles in monozygotic twin pairs may constitute candidates for SSc susceptibility.

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.

Infectious agents and xenobiotics in the etiology of primary biliary cirrhosis

Primary biliary cirrhosis (PBC)is a chronic autoimmune cholestatic liver disease that manifests a latitudinal gradient in prevalence and incidence. The mechanisms leading to the initiation and perpetuation of PBC remain largely enigmatic, although it is established that a combination of genetic predisposition and environmental stimulation is required. PBC is also characterized by a high concordance rate in monozygotic twins and is considered a model autoimmune disease because of several features common to other conditions and the relatively homogeneous serological and biochemical features. From a diagnostic standpoint, PBC is characterized by the highest specificity of serum autoantibodies directed at mitochondrial proteins. Several risk factors have been suggested to be associated with PBC, including exposure to infectious agents and chemical xenobiotics that will be critically discussed in the present review article.

Serum antinuclear and extractable nuclear antigen antibody prevalence and associated morbidity and mortality in the general population over 15years

The prevalence of ANA and anti-ENA in the general population is not well established, especially their clinical significance in healthy subjects. We herein determined the prevalence and predictive value of serum ANA and anti-ENA for connective tissue diseases (CTD), cancer, and mortality. We took advantage of a randomly selected sample of the 1998 general population (Isola I) consisting of 2828 subjects (53% women, age 43±13 years) from a well-defined Northern Italian area. Serum ANA and anti-ENA were tested on the 2690 samples available in 2012 (Isola II, 50% women, age 58±13 years). Administrative databases were searched for CTD, cancer diagnosis, and death cases occurring between enrollment and December 31, 2013. The hazard ratio (HR) was calculated for incident cases. Serum ANA is positive in 18.1% for any titer and 6.1% for titers ≥1:160, 23% in subjects over 50 years and 13.1% and 6.1% for any titer and titers ≥1:160, respectively, in women. The HR for CTD development was significantly high for all ANA titers, with the highest for ANA ≥1:160 (HR 14.19, 95% CI 3.07-65.68). ANA positivity was not associated with cancer (HR 1.03; 95% CI 0.75-1.43), or with mortality (HR adjusted for age and sex 1.40; 95% CI 0.94-2.09). Serum anti-ENA is positive in a minority of subjects with highest figures for anti-nucleosome (1.9%), -histone (1.6%) and -PM/Scl (1.5%). In conclusion, serum ANA prevalence in the general population is highest in senior subjects and in women, while the female predominance is significantly lower compared to overt CTD. Serum ANA is associated with an increased probability of CTD development over time, but does not influence survival or cancer risk.

Gamma-delta T lymphocytes and 25-hydroxy vitamin D levels as key factors in autoimmunity and inflammation: The case of zoledronic acid-induced acute phase reaction

Zoledronic acid (ZA) infusion for osteoporosis is frequently associated with the onset of an acute phase reaction (APR) secondary to the activation of γδ T cell receptor (TCR) lymphocytes (γδ T cells) and to low vitamin D levels, similar to what is observed in chronic inflammation and autoimmunity. In this study we investigated whether the phenotype of γδ T cells is associated with APR and 25-OH vitamin D (25-OHvD) levels. For flow-cytometry analysis, peripheral blood samples were obtained from 52 osteoporotic women prior to 5 mg ZA intravenous infusion and from nine women (five with APR) one week later. Twenty-six/52 (50%) patients reported APR and APR+ cases had a higher percentage of central memory Th1-like γδ T cells. One week after ZA infusion, APR was associated with a decreased percentage of central memory Th1-like γδ T cells, an increase in the percentage and activation of effector memory Th1-like γδ T cells, and an increase in Th17-like γδ T cells. Lower 25-OHvD levels were significantly associated with APR, but no correlation was found between 25-OHvD level and γδ T cell percentage or subsets. In conclusion, patients experiencing APR related to ZA infusion have lower 25-OHvD levels and we suggest that the higher percentage of central memory Th1-like γδ T cells and the expansion of effector memory Th1-like and Th17-like γδ T cells are associated with the occurrence of APR.

Effects of type II collagen epitope carbamylation and citrullination in human leucocyte antigen (HLA)-DR4+ monozygotic twins discordant for rheumatoid arthritis

The aim of this study is to investigate the effect of the native, citrullinated or carbamylated type II human collagen T cell‐ and B cell‐epitopes on the adaptive immune response in rheumatoid arthritis (RA). Peripheral blood T and B cells obtained from a human leucocyte D4‐related (antigen DR4− HLA‐DR4)+ woman with early RA, her healthy monozygotic twin and an unrelated HLA‐DR3+ woman with early RA were analysed for activation (CD154/CD69), apoptosis (annexin/7‐aminoactinomycin), cytokine production [interferon (IFN)γ/interleukin (IL)−17/IL‐4/IL‐10/IL‐6] and functional phenotype (CD45Ra/CCR7) after stimulation with the collagen native T cell epitope (T261‐273), the K264 carbamylated T cell epitope (carT261–273), the native B cell epitope (B359–369) or the R360 citrullinated B cell epitope (citB359–369), and the combinations of these. The T cell memory compartment was activated by T cell epitopes in both discordant DR4+ twins, but not in the DR3+ RA. The collagen‐specific activation of CD4+ T cells was induced with both the native and carbamylated T cell epitopes only in the RA twin. Both T cell epitopes also induced IL‐17 production in the RA twin, but a greater IL‐4 and IL‐10 response in the healthy twin. The citrullinated B cell epitope, particularly when combined with the carbamylated T cell epitope, induced B cell activation and an increased IL‐6/IL‐10 ratio in the RA twin compared to a greater IL‐10 production in the healthy twin. Our data suggest that circulating collagen‐specific T and B cells are found in HLA‐DR4+ subjects, but only RA activated cells express co‐stimulatory molecules and produce proinflammatory cytokines. Carbamylation and citrullination further modulate the activation and cytokine polarization of T and B cells.

ACUTE PHASE REACTIONS AFTER ZOLEDRONIC ACID INFUSION: PROTECTIVE ROLE OF 25-HYDROXYVITAMIN D AND PREVIOUS ORAL BISPHOSPHONATE THERAPY

OBJECTIVE The most common adverse reaction to zoledronic acid (ZOL) infusion is the acute phase reaction (APR), characterized by transient, usually mild, flu-like symptoms. Previous treatment with oral amino-bisphosphonates (BPs) was reported as an independent protective factor for APR, and an association between APR and 25-hydroxyvitamin D (25(OH)D) levels in BP-naïve patients treated with ZOL was identified. The aims of our study were to confirm this association and to see if it was different in patients previously treated with oral BPs compared with BP-naïve patients and to investigate the role of 25(OH)D for the time of APR onset. METHODS We included 153 consecutive patients with postmenopausal osteoporosis undergoing their first ZOL infusion. Sixty-eight had been previously treated with oral BPs. Clinical, demographic, and serologic data were recorded. RESULTS 25(OH)D levels were significantly lower in patients experiencing APR compared to patients without APR (26.3 ± 12.7 vs. 37.0 ± 13.5 ng/mL, respectively; P<.0001). Patients with 25(OH)D <30 ng/mL had a significantly higher risk of APR (odds ratio [OR] 4.2 [95% confidence interval [CI] 2.1-8.2]) occurring in 65%. APR was significantly less frequent in patients previously treated with oral BPs than in BP-naïve subjects (33.8% [23/68] vs 52.9% [45/85], P = .018), but only a weak association remained after correction for 25(OH)D (OR 0.5, 95% CI 0.3-1.1, P = .08). CONCLUSION Higher baseline 25(OH)D levels appear to be protective for APR post-ZOL infusion. The role of previous treatment with oral BPs as an independent protective factor for APR should be evaluated in a larger cohort. ABBREVIATIONS APR = acute phase reaction; BPs = amino-bisphosphonates; CI = confidence interval; 25(OH)D = 25-hydroxyvitamin D; OP = osteoporosis; OR = odds ratio; PTH = parathyroid hormone; ROC = receiver operating characteristic; ZOL = zoledronic acid.

Effects of type II collagen epitope carbamylation and citrullination in HLA‐DR4+ monozygotic twins discordant for rheumatoid arthritis

The aim of this study is to investigate the effect of the native, citrullinated or carbamylated type II human collagen T cell‐ and B cell‐epitopes on the adaptive immune response in rheumatoid arthritis (RA). Peripheral blood T and B cells obtained from a human leucocyte D4‐related (antigen DR4− HLA‐DR4)+ woman with early RA, her healthy monozygotic twin and an unrelated HLA‐DR3+ woman with early RA were analysed for activation (CD154/CD69), apoptosis (annexin/7‐aminoactinomycin), cytokine production [interferon (IFN)γ/interleukin (IL)−17/IL‐4/IL‐10/IL‐6] and functional phenotype (CD45Ra/CCR7) after stimulation with the collagen native T cell epitope (T261‐273), the K264 carbamylated T cell epitope (carT261–273), the native B cell epitope (B359–369) or the R360 citrullinated B cell epitope (citB359–369), and the combinations of these. The T cell memory compartment was activated by T cell epitopes in both discordant DR4+ twins, but not in the DR3+ RA. The collagen‐specific activation of CD4+ T cells was induced with both the native and carbamylated T cell epitopes only in the RA twin. Both T cell epitopes also induced IL‐17 production in the RA twin, but a greater IL‐4 and IL‐10 response in the healthy twin. The citrullinated B cell epitope, particularly when combined with the carbamylated T cell epitope, induced B cell activation and an increased IL‐6/IL‐10 ratio in the RA twin compared to a greater IL‐10 production in the healthy twin. Our data suggest that circulating collagen‐specific T and B cells are found in HLA‐DR4+ subjects, but only RA activated cells express co‐stimulatory molecules and produce proinflammatory cytokines. Carbamylation and citrullination further modulate the activation and cytokine polarization of T and B cells.

Effects of type II collagen epitope carbamylation and citrullination in human leucocyte antigen (HLA)-DR4+monozygotic twins discordant for rheumatoid arthritis: collagen epitopes in rheumatoid arthritis

The aim of this study is to investigate the effect of the native, citrullinated or carbamylated type II human collagen T cell‐ and B cell‐epitopes on the adaptive immune response in rheumatoid arthritis (RA). Peripheral blood T and B cells obtained from a human leucocyte D4‐related (antigen DR4− HLA‐DR4)+ woman with early RA, her healthy monozygotic twin and an unrelated HLA‐DR3+ woman with early RA were analysed for activation (CD154/CD69), apoptosis (annexin/7‐aminoactinomycin), cytokine production [interferon (IFN)γ/interleukin (IL)−17/IL‐4/IL‐10/IL‐6] and functional phenotype (CD45Ra/CCR7) after stimulation with the collagen native T cell epitope (T261‐273), the K264 carbamylated T cell epitope (carT261–273), the native B cell epitope (B359–369) or the R360 citrullinated B cell epitope (citB359–369), and the combinations of these. The T cell memory compartment was activated by T cell epitopes in both discordant DR4+ twins, but not in the DR3+ RA. The collagen‐specific activation of CD4+ T cells was induced with both the native and carbamylated T cell epitopes only in the RA twin. Both T cell epitopes also induced IL‐17 production in the RA twin, but a greater IL‐4 and IL‐10 response in the healthy twin. The citrullinated B cell epitope, particularly when combined with the carbamylated T cell epitope, induced B cell activation and an increased IL‐6/IL‐10 ratio in the RA twin compared to a greater IL‐10 production in the healthy twin. Our data suggest that circulating collagen‐specific T and B cells are found in HLA‐DR4+ subjects, but only RA activated cells express co‐stimulatory molecules and produce proinflammatory cytokines. Carbamylation and citrullination further modulate the activation and cytokine polarization of T and B cells.

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