OP0137 GENOME-WIDE WHOLE-BLOOD TRANSCRIPTOME PROFILING IN A LARGE EUROPEAN COHORT OF SYSTEMIC SCLEROSIS PATIENTS

Background:The analysis of annotated transcripts from genome-wide expression studies data is of paramount importance to understand the molecular phenomena underlying the occurrence of complex diseases, such as systemic sclerosis (SSc).Objectives:To perform whole-blood transcriptome and pathway analysis on whole-blood (WB) RNA collected in two cohorts of European SSc patients. Via a discovery and validation strategy we aimed at characterizing the molecular pathways that differentiate SSc from controls and that are reproducible in geographically diverse populations.Methods:WB samples from 252 controls and 162 SSc patients were collected in RNA stabilizers. Patients were divided into a discovery (n=79; Southern Europe) and validation cohort (n=83; Central-Western Europe). RNA sequencing was performed by an Illumina assay. Functional annotations of Reactome pathways were performed with the FAIME algorithm. In parallel, a immunophenotyping analysis on 28 circulating cell populations was assessed. We then tested: the presence of differentially expressed genes or pathways and the correlation between absolute cell counts and RNA transcripts/FAIME scores in regression models. Results significant in both populations were considered as replicated.Results:A total of 15224 genes and 1277 related functional pathways were available for analysis. Among these, 99 genes and 225 pathways were significant in both sets. The heatmap in figure shows the relative expression of replicated pathways and the distribution of cases and controls (red and green bars). Among the significant pathways we found a deregulation in: type-I IFN, TLR-cascade and signalling, function of the tumor suppressor p53 protein, platelet degranulation and activation. Correlation analysis showed that the count of several cell subtypes is jointly associated with RNA transcripts or FAIME scores with strong differences in relation to the geographical origin of samples; neutrophils emerged as the major determinant of gene expression in SSc-whole-blood samples.Conclusion:We discovered a set of differentially expressed genes and pathways that could be validated in two independent sets of SSc patients highlighting a number of deregulated molecular processes that have relevance for the pathogenesis of autoimmunity and SSc.Acknowledgments:This work was supported by EU/EFPIA/Innovative Medicines Initiative Joint Undertaking PRECISESADS grant No. 115565.Disclosure of Interests:Lorenzo Beretta Grant/research support from: Pfizer, Guillermo Barturen: None declared, Barbara Vigone: None declared, Chiara Bellocchi: None declared, Nicolas Hunzelmann: None declared, Ellen Delanghe: None declared, László Kovács: None declared, Ricard Cervera: None declared, Maria Gerosa: None declared, Rafaela Ortega Castro: None declared, Isabel Almeida: None declared, Divi Cornec: None declared, Carlo Chizzolini Consultant of: Boehringer Ingelheim, Roche, Jacques-Olivier Pers: None declared, Zuzanna Makowska Employee of: Bayer AG, Anne buttgereit Employee of: Bayer AG, Ralf Lesche Employee of: Bayer, Martin Kerick: None declared, Marta Alarcon-Riquelme: None declared, Javier Martin Ibanez: None declare

In Vivo Dioxin Favors Interleukin-22 Production by Human CD4+ T Cells in an Aryl Hydrocarbon Receptor (AhR)-Dependent Manner

The transcription factor aryl hydrocarbon receptor (AhR) mediates the effects of a group of chemicals known as dioxins, ubiquitously present in our environment. However, it is poorly known how the in vivo exposure to these chemicals affects in humans the adaptive immune response. We therefore assessed the functional phenotype of T cells from an individual who developed a severe cutaneous and systemic syndrome after having been exposed to an extremely high dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).T cells of the TCDD-exposed individual were studied for their capacity to produce cytokines in response to polyclonal and superantigenic stimulation, and for the expression of chemokine receptors involved in skin homing. The supernatants from T cells of the exposed individual contained a substantially increased amount of interleukin (IL)-22 but not of IL-17A, interferon (IFN)-γ or IL-10 when compared to nine healthy controls. In vitro experiments confirmed a direct, AhR-dependent, enhancing effect of TCDD on IL-22 production by CD4+ T cells. The increased production of IL-22 was not dependent on AhR occupancy by residual TCDD molecules, as demonstrated in competition experiments with the specific AhR antagonist CH-223191. In contrast, it was due to an increased frequency of IL-22 single producing cells accompanied by an increased percentage of cells expressing the skin-homing chemokine receptors CCR6 and CCR4, identified through a multiparameter flow cytometry approach. Of interest, the frequency of CD4+CD25(hi)FoxP3+ T regulatory cells was similar in the TCDD-exposed and healthy individuals.This case strongly supports the contention that human exposure to persistent AhR ligands in vivo induce a long-lasting effect on the human adaptive immune system and specifically polarizes CD4+ T cells to produce IL-22 and not other T cell cytokines with no effect on T regulatory cells

Prospective evaluation of the capillaroscopic skin ulcer risk index in systemic sclerosis patients in clinical practice: a longitudinal, multicentre study.

Nailfold capillaroscopy (NC) is an important tool for the diagnosis of systemic sclerosis (SSc). The capillaroscopic skin ulcer risk index (CSURI) was suggested to identify patients at risk of developing digital ulcers (DUs). This study aims to assess the reliability of the CSURI across assessors, the CSURI change during follow-up and the value of the CSURI in predicting new DUs. This multicentre, longitudinal study included SSc patients with a history of DUs. NC images of all eight fingers were obtained at baseline and follow-up and were separately analysed by two trained assessors. Sixty-one patients were included (median observation time 1.0 year). In about 40% of patients (assessor 1, n = 24, 39%; assessor 2, n = 26, 43%) no megacapillary was detected in any of the baseline or follow-up images; hence the CSURI could not be calculated. In those 34 patients in whom CSURI scores were available from both assessors (26% male; median age 57 years) the median baseline CSURI was 5.3 according to assessor 1 (IQR 2.6-16.3), increasing to 5.9 (IQR 1.3-12.0) at follow-up. According to assessor 2, the CSURI diminished from 6.4 (IQR 2.4-12.5) to 5.0 (IQR 1.7-10.0). The ability of a CSURI ≥ 2.96 category to predict new DUs was low (for both assessors, positive predictive value 38% and negative predictive value 50%) and the inter-assessor agreements for CSURI categories were fair to moderate. In this study, around 40% of patients could not be evaluated with the CSURI due to the absence of megacapillaries. Clinical decisions based on the CSURI should be made with caution. Current Controlled Trials, ISRCTN04371709 . Registered on 18 March 2011

IgM antibodies against malondialdehyde and phosphorylcholine in different systemic rheumatic diseases

IgM antibodies against phosphorylcholine (anti-PC) and malondialdehyde (anti-MDA) may have protective properties in cardiovascular and rheumatic diseases. We here compare these antibodies in systemic rheumatic conditions and study their properties. Anti-PC and anti-MDA was measured using ELISA in patients with SLE (374), RA (354), Mixed connective tissue disease (MCTD, 77), Systemic sclerosis (SSc, 331), Sj\uf6gren\u2019s syndrome (SjS, 324), primary antiphospholipid syndrome (PAPs, 65), undifferentiated connective tissue disease (UCTD, 118) and 515 matched healthy controls (HC). Cardiovascular score (CV) was broadly defined based on clinical disease symptoms. Anti-PC and anti-MDA peptide/protein characterization were compared using a proteomics de novo sequencing approach. anti-MDA and anti-PC were extracted from total IgM. The proportion of Treg cells was determined by flow cytometry. The maximal difference between cases and controls was shown for MCTD: significantly lower IgM Anti-PC but not anti-MDA among patients (median 49.3RU/ml vs 70.4 in healthy controls, p(t-test) = 0.0037). IgM low levels were more prevalent in MCTD, SLE, SjS, SSc and UCTD. IgM anti-PC variable region profiles were different from and more homologous than anti-MDA. Anti-PC but not anti-MDA were significantly negatively correlated with CV in the whole patient group. In contrast to IgM anti-PC, anti-MDA did not promote polarization of Tregs. Taken together, Anti-PC is decreased in MCTD and also in SLE, SjS and SSc but not in other studied diseases. Anti-PC may thus differentiate between these. In contrast, anti-MDA did not show these differences between diseases studied. Anti-PC level is negatively correlated with CV in the patient group cohort. In contrast to anti-PC, anti-MDA did not promote Treg polarization. These findings could have both diagnostic and therapeutic implications, one possibility being active or passive immunization with PC in some rheumatic conditions

Prostaglandin I2 Signaling Drives Th17 Differentiation and Exacerbates Experimental Autoimmune Encephalomyelitis

BACKGROUND: Prostaglandin I(2) (PGI(2)), a lipid mediator currently used in treatment of human disease, is a critical regulator of adaptive immune responses. Although PGI(2) signaling suppressed Th1 and Th2 immune responses, the role of PGI(2) in Th17 differentiation is not known. METHODOLOGY/PRINCIPAL FINDINGS: In mouse CD4(+)CD62L(+) naïve T cell culture, the PGI(2) analogs iloprost and cicaprost increased IL-17A and IL-22 protein production and Th17 differentiation in vitro. This effect was augmented by IL-23 and was dependent on PGI(2) receptor IP signaling. In mouse bone marrow-derived CD11c(+) dendritic cells (BMDCs), PGI(2) analogs increased the ratio of IL-23/IL-12, which is correlated with increased ability of BMDCs to stimulate naïve T cells for IL-17A production. Moreover, IP knockout mice had delayed onset of a Th17-associated neurological disease, experimental autoimmune encephalomyelitis (EAE), and reduced infiltration of IL-17A-expressing mononuclear cells in the spinal cords compared to wild type mice. These results suggest that PGI(2) promotes in vivo Th17 responses. CONCLUSION: The preferential stimulation of Th17 differentiation by IP signaling may have important clinical implications as PGI(2) and its analogs are commonly used to treat human pulmonary hypertension

Digital ulcers predict a worse disease course in patients with systemic sclerosis

Objective: Systemic sclerosis (SSc) is a systemic autoimmune disease with high morbidity and significant mortality. There is a great need of predictors that would allow risk stratification of patients with SSc and ultimately initiation of treatment early enough to ensure optimal clinical results. In this study, we evaluated whether a history of digital ulcers (HDU) at presentation may be a predictor of vascular outcomes and of overall clinical worsening and death in patients with SSc. Methods: Patients from the EULAR Scleroderma Trials and Research (EUSTAR) database, satisfying at inclusion the 1980 American College of Rheumatology classification criteria for SSc, who had a follow-up of at least 3 years since baseline or who have died, were included in the analysis. HDU at presentation as a predictor of disease worsening or death was evaluated by Cox proportional hazards regression analysis. Results :3196 patients matched the inclusion criteria (male sex 13.2%, 33.4% diffuse subset). At presentation, 1092/3196 patients had an HDU (34.1%). In multivariable analysis adjusting for age, gender and all parameters considered potentially significant, HDU was predictive for the presence of active digital ulcers (DUs) at prospective visits (HR (95% CI)): 2.41(1.91 to 3.03), p<0.001, for an elevated systolic pulmonary arterial pressure on heart ultrasound (US-PAPs):1.36 (1.03 to 1.80), p=0.032, for any cardiovascular event (new DUs, elevated US-PAPs or LV failure):3.56 (2.26 to 5.62), p<0.001, and for death (1.53 (1.16 to 2.02), p=0.003). Conclusions :In patients with SSc, HDU at presentation predicts the occurrence of DUs at follow-up and is associated with cardiovascular worsening and decreased survival

Bacteria Modulate the CD8+ T Cell Epitope Repertoire of Host Cytosol-Exposed Proteins to Manipulate the Host Immune Response

The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested