9 research outputs found
Standardization procedure for flow cytometry data harmonization in prospective multicenter studies
One of the most challenging objective for clinical cytometry in prospective multicenter
immunomonitoring trials is to compare frequencies, absolute numbers of leukocyte populations and
further the mean fluorescence intensities of cell markers, especially when the data are generated from
different instruments. Here, we describe an innovative standardization workflow to compare all data
to carry out any large-scale, prospective multicentric flow cytometry analysis whatever the duration,
the number or type of instruments required for the realization of such project
Genome-wide whole blood transcriptome profiling in a large European cohort of systemic sclerosis patients
Objectives The analysis of annotated transcripts
from genome-wide
expression studies may help to
understand the pathogenesis of complex diseases, such
as systemic sclerosis (SSc). We performed a whole blood
(WB) transcriptome analysis on RNA collected in the
context of the European PRECISESADS project, aiming
at characterising the pathways that differentiate SSc
from controls and that are reproducible in geographically
diverse populations.
Methods Samples from 162 patients and 252 controls
were collected in RNA stabilisers. Cases and controls
were divided into a discovery (n=79+163; Southern
Europe) and validation cohort (n=83+89; Central-Western
Europe). RNA sequencing was performed by
an Illumina assay. Functional annotations of Reactome
pathways were performed with the Functional Analysis
of Individual Microarray Expression (FAIME) algorithm.
In parallel, immunophenotyping of 28 circulating cell
populations was performed. We tested the presence
of differentially expressed genes/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 Overall, 15 224 genes and 1277 functional
pathways were available; of these, 99 and 225 were
significant in both sets. Among replicated pathways,
we found a deregulation in type-I
interferon, Toll-like
receptor cascade, tumour suppressor p53 protein
function, platelet degranulation and activation. RNA
transcripts or FAIME scores were jointly correlated with
cell subtypes with strong geographical differences;
neutrophils were the major determinant of gene
expression in SSc-WB
samples.
Conclusions We discovered a set of differentially
expressed genes/pathways validated in two independent
sets of patients with SSc, highlighting a number of
deregulated processes that have relevance for the
pathogenesis of autoimmunity and SSc.EU/EFPIA/Innovative Medicines Initiative Joint Undertaking PRECISESADS
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Integrative epigenomics in Sjögren´s syndrome reveals novel pathways and a strong interaction between the HLA, autoantibodies and the interferon signature
Primary Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by lymphocytic infiltration and damage of exocrine salivary and lacrimal glands. The etiology of SS is complex with environmental triggers and genetic factors involved. By conducting an integrated multi-omics study, we confirmed a vast coordinated hypomethylation and overexpression effects in IFN-related genes, what is known as the IFN signature. Stratified and conditional analyses suggest a strong interaction between SS-associated HLA genetic variation and the presence of Anti-Ro/SSA autoantibodies in driving the IFN epigenetic signature and determining SS. We report a novel epigenetic signature characterized by increased DNA methylation levels in a large number of genes enriched in pathways such as collagen metabolism and extracellular matrix organization. We identified potential new genetic variants associated with SS that might mediate their risk by altering DNA methylation or gene expression patterns, as well as disease-interacting genetic variants that exhibit regulatory function only in the SS population. Our study sheds new light on the interaction between genetics, autoantibody profiles, DNA methylation and gene expression in SS, and contributes to elucidate the genetic architecture of gene regulation in an autoimmune population
Molecular Characterization of Monocyte Subsets Reveals Specific and Distinctive Molecular Signatures Associated With Cardiovascular Disease in Rheumatoid Arthritis
Objectives: This study, developed within the Innovative Medicines Initiative Joint Undertaking project PRECISESADS framework, aimed at functionally characterize the monocyte subsets in RA patients, and analyze their involvement in the increased CV risk associated with RA.Methods: The frequencies of monocyte subpopulations in the peripheral blood of 140 RA patients and 145 healthy donors (HDs) included in the PRECISESADS study were determined by flow cytometry. A second cohort of 50 RA patients and 30 HDs was included, of which CD14+ and CD16+ monocyte subpopulations were isolated using immuno-magnetic selection. Their transcriptomic profiles (mRNA and microRNA), proinflammatory patterns and activated pathways were evaluated and related to clinical features and CV risk. Mechanistic in vitro analyses were further performed.Results: CD14++CD16+ intermediate monocytes were extended in both cohorts of RA patients. Their increased frequency was associated with the positivity for autoantibodies, disease duration, inflammation, endothelial dysfunction and the presence of atheroma plaques, as well as with the CV risk score. CD14+ and CD16+ monocyte subsets showed distinctive and specific mRNA and microRNA profiles, along with specific intracellular signaling activation, indicating different functionalities. Moreover, that specific molecular profiles were interrelated and associated to atherosclerosis development and increased CV risk in RA patients. In vitro, RA serum promoted differentiation of CD14+CD16− to CD14++CD16+ monocytes. Co-culture with RA-isolated monocyte subsets induced differential activation of endothelial cells.Conclusions: Our overall data suggest that the generation of inflammatory monocytes is associated to the autoimmune/inflammatory response that mediates RA. These monocyte subsets, -which display specific and distinctive molecular signatures- might promote endothelial dysfunction and in turn, the progression of atherosclerosis through a finely regulated process driving CVD development in RA
Standardization procedure for flow cytometry data harmonization in prospective multicenter studies.
One of the most challenging objective for clinical cytometry in prospective multicenter immunomonitoring trials is to compare frequencies, absolute numbers of leukocyte populations and further the mean fluorescence intensities of cell markers, especially when the data are generated from different instruments. Here, we describe an innovative standardization workflow to compare all data to carry out any large-scale, prospective multicentric flow cytometry analysis whatever the duration, the number or type of instruments required for the realization of such projects
Integrative epigenomics in Sjögren´s syndrome reveals novel pathways and a strong interaction between the HLA, autoantibodies and the interferon signature
Primary Sjögren’s syndrome (SS) is a systemic autoimmune disease characterized by lymphocytic infiltration and damage of exocrine salivary and lacrimal glands. The etiology of SS is complex with environmental triggers and genetic factors involved. By conducting an integrated multi-omics study, we confirmed a vast coordinated hypomethylation and overexpression effects in IFN-related genes, what is known as the IFN signature. Stratified and conditional analyses suggest a strong interaction between SS-associated HLA genetic variation and the presence of Anti-Ro/SSA autoantibodies in driving the IFN epigenetic signature and determining SS. We report a novel epigenetic signature characterized by increased DNA methylation levels in a large number of genes enriched in pathways such as collagen metabolism and extracellular matrix organization. We identified potential new genetic variants associated with SS that might mediate their risk by altering DNA methylation or gene expression patterns, as well as disease-interacting genetic variants that exhibit regulatory function only in the SS population. Our study sheds new light on the interaction between genetics, autoantibody profiles, DNA methylation and gene expression in SS, and contributes to elucidate the genetic architecture of gene regulation in an autoimmune population. © 2021, The Author(s)
Genomic Risk Score impact on susceptibility to systemic sclerosis.
Genomic Risk Scores (GRS) successfully demonstrated the ability of genetics to identify those individuals at high risk for complex traits including immune-mediated inflammatory diseases (IMIDs). We aimed to test the performance of GRS in the prediction of risk for systemic sclerosis (SSc) for the first time. Allelic effects were obtained from the largest SSc Genome-Wide Association Study (GWAS) to date (9 095 SSc and 17 584 healthy controls with European ancestry). The best-fitting GRS was identified under the additive model in an independent cohort that comprised 400 patients with SSc and 571 controls. Additionally, GRS for clinical subtypes (limited cutaneous SSc and diffuse cutaneous SSc) and serological subtypes (anti-topoisomerase positive (ATA+) and anti-centromere positive (ACA+)) were generated. We combined the estimated GRS with demographic and immunological parameters in a multivariate generalised linear model. The best-fitting SSc GRS included 33 single nucleotide polymorphisms (SNPs) and discriminated between patients with SSc and controls (area under the receiver operating characteristic (ROC) curve (AUC)=0.673). Moreover, the GRS differentiated between SSc and other IMIDs, such as rheumatoid arthritis and Sjögren's syndrome. Finally, the combination of GRS with age and immune cell counts significantly increased the performance of the model (AUC=0.787). While the SSc GRS was not able to discriminate between ATA+ and ACA+ patients (AUC GRS was successfully implemented in SSc. The model discriminated between patients with SSc and controls or other IMIDs, confirming the potential of GRS to support early and differential diagnosis for SSc
Integrative epigenomics in Sjögren´s syndrome reveals novel pathways and a strong interaction between the HLA, autoantibodies and the interferon signature
Funding for the preparation of this manuscript has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement no 115,565, resources composed of the financial contribution from the European Union's Seventh Framework Program (FP7/2007-2013) and the EFPIA companies' in kind contribution. MT is supported by a Spanish grant from Health Department, Junta de Andalucia (PI/0017/2016) and through the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 806975. This Joint Undertaking receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA. EC-M was funded by the Postdoctoral Training Subprogramme Juan de la Cierva-Ministry of Economy and Competitiveness (FJCI_2014_20652). We thank Ralf Lesche for the production of RNASeq data and Marc Torres Ciuro for design support.Primary Sjögren’s syndrome (SS) is a systemic autoimmune disease characterized by lymphocytic
infiltration and damage of exocrine salivary and lacrimal glands. The etiology of SS is complex with
environmental triggers and genetic factors involved. By conducting an integrated multi-omics study,
we confirmed a vast coordinated hypomethylation and overexpression effects in IFN-related genes,
what is known as the IFN signature. Stratified and conditional analyses suggest a strong interaction
between SS-associated HLA genetic variation and the presence of Anti-Ro/SSA autoantibodies in
driving the IFN epigenetic signature and determining SS. We report a novel epigenetic signature
characterized by increased DNA methylation levels in a large number of genes enriched in pathways
such as collagen metabolism and extracellular matrix organization. We identified potential new
genetic variants associated with SS that might mediate their risk by altering DNA methylation or
gene expression patterns, as well as disease-interacting genetic variants that exhibit regulatory
function only in the SS population. Our study sheds new light on the interaction between genetics,
autoantibody profiles, DNA methylation and gene expression in SS, and contributes to elucidate the
genetic architecture of gene regulation in an autoimmune population.Innovative Medicines Initiative Joint Undertaking from the European Union's Seventh Framework Program (FP7/2007-2013) 115,565EFPIA companiesJunta de Andalucia PI/0017/2016Innovative Medicines Initiative 2 Joint Undertaking 806975
European Union's Horizon 2020 research and innovation programmeEFPIAPostdoctoral Training Subprogramme Juan de la Cierva-Ministry of Economy and Competitiveness FJCI_2014_2065
Molecular characterization of monocyte subsets reveals specific and distinctive molecular signatures associated with cardiovascular disease in rheumatoid arthritis
Objectives: This study, developed within the Innovative Medicines Initiative Joint Undertaking project PRECISESADS framework, aimed at functionally characterize the monocyte subsets in RA patients, and analyze their involvement in the increased CV risk associated with RA. Methods: The frequencies of monocyte subpopulations in the peripheral blood of 140 RA patients and 145 healthy donors (HDs) included in the PRECISESADS study were determined by flow cytometry. A second cohort of 50 RA patients and 30 HDs was included, of which CD14+ and CD16+ monocyte subpopulations were isolated using immuno-magnetic selection. Their transcriptomic profiles (mRNA and microRNA), proinflammatory patterns and activated pathways were evaluated and related to clinical features and CV risk. Mechanistic in vitro analyses were further performed. Results: CD14++CD16+ intermediate monocytes were extended in both cohorts of RA patients. Their increased frequency was associated with the positivity for autoantibodies, disease duration, inflammation, endothelial dysfunction and the presence of atheroma plaques, as well as with the CV risk score. CD14+ and CD16+ monocyte subsets showed distinctive and specific mRNA and microRNA profiles, along with specific intracellular signaling activation, indicating different functionalities. Moreover, that specific molecular profiles were interrelated and associated to atherosclerosis development and increased CV risk in RA patients. In vitro, RA serum promoted differentiation of CD14+CD16- to CD14++CD16+ monocytes. Co-culture with RA-isolated monocyte subsets induced differential activation of endothelial cells. Conclusions: Our overall data suggest that the generation of inflammatory monocytes is associated to the autoimmune/inflammatory response that mediates RA. These monocyte subsets, -which display specific and distinctive molecular signatures- might promote endothelial dysfunction and in turn, the progression of atherosclerosis through a finely regulated process driving CVD development in RA