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1.  IFNα enhances the production of IL-6 by human neutrophils activated via TLR8 
Scientific Reports  2016;6:19674.
Recently, we reported that human neutrophils produce biologically active amounts of IL-6 when incubated with agonists activating TLR8, a receptor recognizing viral single strand RNA. In this study, we demonstrate that IFNα, a cytokine that modulates the early innate immune responses toward viral and bacterial infections, potently enhances the production of IL-6 in neutrophils stimulated with R848, a TLR8 agonist. We also show that such an effect is not caused by an IFNα-dependent induction of TLR7 and its consequent co-activation with TLR8 in response to R848, but, rather, it is substantially mediated by an increased production and release of endogenous TNFα. The latter cytokine, in an autocrine manner, leads to an augmented synthesis of the IkBζ co-activator and an enhanced recruitment of the C/EBPβ transcription factor to the IL-6 promoter. Moreover, we show that neutrophils from SLE patients with active disease state, hence displaying an IFN-induced gene expression signature, produce increased amounts of both IL-6 and TNFα in response to R848 as compared to healthy donors. Altogether, data uncover novel effects that type I IFN exerts in TLR8-activated neutrophils, which therefore enlarge our knowledge on the various biological actions which type I IFN orchestrates during infectious and autoimmune diseases.
doi:10.1038/srep19674
PMCID: PMC4726390  PMID: 26790609
3.  Gene Expression Profiling in Peripheral Blood Cells and Synovial Membranes of Patients with Psoriatic Arthritis 
PLoS ONE  2015;10(6):e0128262.
Background
Psoriatic arthritis (PsA) is an inflammatory arthritis whose pathogenesis is poorly understood; it is characterized by bone erosions and new bone formation. The diagnosis of PsA is mainly clinical and diagnostic biomarkers are not yet available. The aim of this work was to clarify some aspects of the disease pathogenesis and to identify specific gene signatures in paired peripheral blood cells (PBC) and synovial biopsies of patients with PsA. Moreover, we tried to identify biomarkers that can be used in clinical practice.
Methods
PBC and synovial biopsies of 10 patients with PsA were used to study gene expression using Affymetrix arrays. The expression values were validated by Q-PCR, FACS analysis and by the detection of soluble mediators.
Results
Synovial biopsies of patients showed a modulation of approximately 200 genes when compared to the biopsies of healthy donors. Among the differentially expressed genes we observed the upregulation of Th17 related genes and of type I interferon (IFN) inducible genes. FACS analysis confirmed the Th17 polarization. Moreover, the synovial trascriptome shows gene clusters (bone remodeling, angiogenesis and inflammation) involved in the pathogenesis of PsA. Interestingly 90 genes are modulated in both compartments (PBC and synovium) suggesting that signature pathways in PBC mirror those of the inflamed synovium. Finally the osteoactivin gene was upregulared in both PBC and synovial biopsies and this finding was confirmed by the detection of high levels of osteoactivin in PsA sera but not in other inflammatory arthritides.
Conclusions
We describe the first analysis of the trancriptome in paired synovial tissue and PBC of patients with PsA. This study strengthens the hypothesis that PsA is of autoimmune origin since the coactivity of IFN and Th17 pathways is typical of autoimmunity. Finally these findings have allowed the identification of a possible disease biomarker, osteoactivin, easily detectable in PsA serum.
doi:10.1371/journal.pone.0128262
PMCID: PMC4473102  PMID: 26086874
4.  Characterization of CD30/CD30L+ Cells in Peripheral Blood and Synovial Fluid of Patients with Rheumatoid Arthritis 
Journal of Immunology Research  2015;2015:729654.
The CD30/CD30L signalling system has been implicated in the pathogenesis of several autoimmune and inflammatory conditions. In rheumatoid arthritis (RA), soluble CD30 (sCD30) levels reflect the recruitment of CD30+ T cells into the inflamed joints and correlate with a positive response to immunosuppressive therapy. The aim of our report was to clarify the role of CD30/CD30L signalling system in the pathogenesis of RA. Our analysis of the CD30L+ T cell subsets in peripheral blood (PB) and synovial fluid (SF) of RA patients and of the related cytokine profiles suggests the involvement of CD30/CD30L signalling in polarization of T cells towards a Th17 phenotype with proinflammatory features. Moreover, in RA SF nearly 50% of Treg cells express CD30, probably as an attempt to downmodulate the ongoing inflammation. We also show here that the engagement of CD30L on neutrophils stimulated with CD30/Fc chimera may play a crucial role in RA inflammation since activated neutrophils release IL-8, thus potentially amplifying the local inflammatory damage. In conclusion, the results obtained suggest that the complex CD30/CD30L signalling pathway is implicated in the pathogenesis and progression of RA synovitis through a concerted action on several immune effector cells.
doi:10.1155/2015/729654
PMCID: PMC4452350  PMID: 26090498
5.  Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjögren Syndrome 
Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX), chimeric virus particles, and Cowpea mosaic virus, empty virus-like particles to display a linear peptide (lipo) derived from human lipocalin, which is immunodominant in Sjögren’s syndrome (SjS) and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.
doi:10.3389/fpls.2015.01080
PMCID: PMC4664701  PMID: 26648961
molecular farming; Sjögren’s syndrome; diagnosis; ELISA; VPN; PVX; CVP; lipocalin
6.  Crossreactive Autoantibodies Directed against Cutaneous and Joint Antigens Are Present in Psoriatic Arthritis 
PLoS ONE  2014;9(12):e115424.
Background
Psoriatic arthritis (PsA) is a chronic inflammatory disease of unknown origin, characterized by erosions and new bone formation. Diagnosis of PsA is mainly clinical and there are no biomarkers available. Moreover in PsA autoantibodies have not been described so far. Indeed an autoimmune origin has been suggested but never proven. Aim of the study was to investigate the possible presence of autoantibodies typically associated with PsA.
Methods
We used pooled IgG immunoglobulins derived from 30 patients with PsA to screen a random peptide library in order to identify disease relevant autoantigen peptides.
Results
Among the selected peptides, one was recognised by nearly all the patients’ sera. The identified peptide (PsA peptide: TNRRGRGSPGAL) shows sequence similarities with skin autoantigens, such as fibrillin 3, a constituent of actin microfibrils, desmocollin 3, a constituent of the desmosomes and keratin 78, a component of epithelial cytoskeleton. Interestingly the PsA peptide shares homology with the nebulin-related anchoring protein (N-RAP), a protein localized in the enthesis (point of insertion of a tendon or ligament to the bone), which represents the first affected site during early PsA. Antibodies affinity purified against the PsA peptide recognize fibrillin, desmocollin, keratin and N-RAP. Moreover antibodies directed against the PsA peptide are detectable in 85% of PsA patients. Such antibodies are not present in healthy donors and are present in 13/100 patients with seroposive rheumatoid arthritis (RA). In seronegative RA these antibodies are detectable only in 3/100 patients.
Conclusions
Our results indicate that PsA is characterized by the presence of serum autoantibodies crossreacting with an epitope shared by skin and joint antigens.
doi:10.1371/journal.pone.0115424
PMCID: PMC4267814  PMID: 25514237
7.  A Candidate Gene Approach Identifies an IL33 Genetic Variant as a Novel Genetic Risk Factor for GCA 
PLoS ONE  2014;9(11):e113476.
Introduction
Increased expression of IL-33 and its receptor ST2, encoded by the IL1RL1 gene, has been detected in the inflamed arteries of giant cell arteritis (GCA) patients. The aim of the present study was to investigate for the first time the potential influence of the IL33 and IL1RL1 loci on GCA predisposition.
Methods
A total of 1,363 biopsy-proven GCA patients and 3,908 healthy controls from four European cohorts (Spain, Italy, Germany and Norway) were combined in a meta-analysis. Six genetic variants: rs3939286, rs7025417 and rs7044343, within the IL33 gene, and rs2058660, rs2310173 and rs13015714, within the IL1RL1 gene, previously associated with immune-related diseases, were genotyped using predesigned TaqMan assays.
Results
A consistent association between the rs7025417 polymorphism and GCA was evident in the overall meta-analysis, under both allele (PMH = 0.041, OR = 0.88, CI 95% 0.78–0.99) and recessive (PMH = 3.40E-03, OR = 0.53, CI 95% 0.35–0.80) models. No statistically significant differences between allele or genotype frequencies for the other IL33 and IL1RL1 genetic variants were detected in this pooled analysis.
Conclusions
Our results clearly evidenced the implication of the IL33 rs7025417 polymorphism in the genetic network underlying GCA.
doi:10.1371/journal.pone.0113476
PMCID: PMC4237421  PMID: 25409453
8.  A systemic sclerosis and systemic lupus erythematosus pan-meta-GWAS reveals new shared susceptibility loci 
Human Molecular Genetics  2013;22(19):4021-4029.
Systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are two archetypal systemic autoimmune diseases which have been shown to share multiple genetic susceptibility loci. In order to gain insight into the genetic basis of these diseases, we performed a pan-meta-analysis of two genome-wide association studies (GWASs) together with a replication stage including additional SSc and SLE cohorts. This increased the sample size to a total of 21 109 (6835 cases and 14 274 controls). We selected for replication 19 SNPs from the GWAS data. We were able to validate KIAA0319L (P = 3.31 × 10−11, OR = 1.49) as novel susceptibility loci for SSc and SLE. Furthermore, we also determined that the previously described SLE susceptibility loci PXK (P = 3.27 × 10−11, OR = 1.20) and JAZF1 (P = 1.11 × 10−8, OR = 1.13) are shared with SSc. Supporting these new discoveries, we observed that KIAA0319L was overexpressed in peripheral blood cells of SSc and SLE patients compared with healthy controls. With these, we add three (KIAA0319L, PXK and JAZF1) and one (KIAA0319L) new susceptibility loci for SSc and SLE, respectively, increasing significantly the knowledge of the genetic basis of autoimmunity.
doi:10.1093/hmg/ddt248
PMCID: PMC3766185  PMID: 23740937
9.  Low-dose oral imatinib in the treatment of systemic sclerosis interstitial lung disease unresponsive to cyclophosphamide: a phase II pilot study 
Arthritis Research & Therapy  2014;16(4):R144.
Introduction
Pulmonary involvement represents a major cause of death of systemic sclerosis (SSc) patients. Recent data suggest that tyrosine kinase inhibitors, such as imatinib, may be a therapeutic option for SSc patients. However, preliminary published clinical trials were inconclusive about imatinib efficacy and showed side effects. The purpose of this study was to verify efficacy and tolerability of low-dose imatinib on interstitial lung disease in a cohort of SSc patients unresponsive to cyclophosphamide therapy.
Methods
Thirty consecutive SSc patients with active pulmonary involvement, unresponsive to cyclophosphamide, were treated with imatinib 200 mg/day for 6 months followed by a 6-month follow-up. A “good response” was defined as an increase of forced vital capacity (FVC) by more of 15% and/or increase of diffusing capacity of carbon monoxide (DLCO) >15% and PaO2 > 90% of initial value and high-resolution computed tomography (HRCT)-scan pattern unchanged or improved.
Results
Twenty-six patients completed the study. Three patients died and one patient was lost to follow-up. Four patients (15.32%) had a good response, 7 worsened and 15 had a stabilized lung disease. Overall, 19 (73.07%) patients had an improved or stabilized lung disease. After a 6-month follow-up, 12 (54.5%) of the 22 patients showed an improved or stabilized lung disease.
Conclusions
Lung function was stabilized in a large proportion of patients unresponsive to cyclophosphamide therapy and a beneficial outcome emerged from the analysis of HRCT lung scans. There was no significant improvement of skin involvement, and the low dose was well tolerated. These data provide useful suggestions to design future randomized clinical trials for SSc therapeutics.
Trial registration
ClinicalTrials.gov NCT00573326. Registered 13 December 2007.
doi:10.1186/ar4606
PMCID: PMC4227120  PMID: 25007944
10.  Implication of IL-2/IL-21 region in systemic sclerosis genetic susceptibility 
Annals of the rheumatic diseases  2012;72(7):10.1136/annrheumdis-2012-202357.
Objective
The interleukin 2 (IL-2) and interleukin 21 (IL-21) locus at chromosome 4q27 has been associated with several autoimmune diseases, and both genes are related to immune system functions. The aim of this study was to evaluate the role of the IL-2/IL-21 locus in systemic sclerosis (SSc).
Patients and methods
The case control study included 4493 SSc Caucasian patients and 5856 healthy controls from eight Caucasian populations (Spain, Germany, The Netherlands, USA, Italy, Sweden, UK and Norway). Four single nucleotide polymorphisms (rs2069762, rs6822844, rs6835457 and rs907715) were genotyped using TaqMan allelic discrimination assays.
Results
We observed evidence of association of the rs6822844 and rs907715 variants with global SSc (pc=6.6E-4 and pc=7.2E-3, respectively). Similar statistically significant associations were observed for the limited cutaneous form of the disease. The conditional regression analysis suggested that the most likely genetic variation responsible for the association was the rs6822844 polymorphism. Consistently, the rs2069762A-rs6822844T-rs6835457G-rs907715T allelic combination showed evidence of association with SSc and limited cutaneous SSc subtype (pc=1.7E-03 and pc=8E-4, respectively).
Conclusions
These results suggested that the IL-2/IL-21 locus influences the genetic susceptibility to SSc. Moreover, this study provided further support for the IL-2/IL-21 locus as a common genetic factor in autoimmune diseases.
doi:10.1136/annrheumdis-2012-202357
PMCID: PMC3887514  PMID: 23172754
11.  Gene Expression Profiling in Peripheral Blood Mononuclear Cells of Patients with Common Variable Immunodeficiency: Modulation of Adaptive Immune Response following Intravenous Immunoglobulin Therapy 
PLoS ONE  2014;9(5):e97571.
Background
Regular intravenous immunoglobulin treatment is used to replace antibody deficiency in primary immunodeficiency diseases; however the therapeutic effect seems to be related not only to antibody replacement but also to an active role in the modulation of the immune response. Common variable immunodeficiency is the most frequent primary immunodeficiency seen in clinical practice.
Methods
We have studied the effect of intravenous immunoglobulin replacement in patients with common variable immunodeficiency by evaluating the gene-expression profiles from Affimetrix HG-U133A. Some of the gene array results were validated by real time RT-PCR and by the measurement of circulating cytokines and chemokines by ELISA. Moreover we performed FACS analysis of blood mononuclear cells from the patients enrolled in the study.
Results
A series of genes involved in innate and acquired immune responses were markedly up- or down-modulated before therapy. Such genes included CD14, CD36, LEPR, IRF-5, RGS-1, CD38, TNFRSF25, IL-4, CXCR4, CCR3, IL-8. Most of these modulated genes showed an expression similar to that of normal controls after immunoglobulin replacement. Real time RT-PCR of selected genes and serum levels of IL-4, CXCR4 before and after therapy changed accordingly to gene array results. Interestingly, serum levels of IL-8 remained unchanged, as the corresponding gene, before and after treatment. FACS analysis showed a marked decrease of CD8+T cells and an increase of CD4+T cells following treatment. Moreover we observed a marked increase of CD23−CD27−IgM−IgG− B cells (centrocytes).
Conclusions
Our results are in accordance with previous reports and provide further support to the hypothesis that the benefits of intravenous immunoglobulin therapy are not only related to antibody replacement but also to its ability to modulate the immune response in common variable immunodeficiency.
doi:10.1371/journal.pone.0097571
PMCID: PMC4022614  PMID: 24831519
12.  Confirmation of TNIP1 but not RHOB and PSORS1C1 as systemic sclerosis risk factors in a large independent replication study 
Annals of the rheumatic diseases  2012;72(4):10.1136/annrheumdis-2012-201888.
Introduction
A recent genome-wide association study in European systemic sclerosis (SSc) patients identified three loci (PSORS1C1, TNIP1 and RHOB) as novel genetic risk factors for the disease. The aim of this study was to replicate the previously mentioned findings in a large multicentre independent SSc cohort of Caucasian ancestry.
Methods
4389 SSc patients and 7611 healthy controls from different European countries and the USA were included in the study. Six single nucleotide polymorphisms (SNP): rs342070, rs13021401 (RHOB), rs2233287, rs4958881, rs3792783 (TNIP1) and rs3130573 (PSORS1C1) were analysed. Overall significance was calculated by pooled analysis of all the cohorts. Haplotype analyses and conditional logistic regression analyses were carried out to explore further the genetic structure of the tested loci.
Results
Pooled analyses of all the analysed SNPs in TNIP1 revealed significant association with the whole disease (rs2233287 pMH=1.94×10−4, OR 1.19; rs4958881 pMH=3.26×10−5, OR 1.19; rs3792783 pMH=2.16×10−4, OR 1.19). These associations were maintained in all the subgroups considered. PSORS1C1 comparison showed association with the complete set of patients and all the subsets except for the anti-centromere-positive patients. However, the association was dependent on different HLA class II alleles. The variants in the RHOB gene were not associated with SSc or any of its subsets.
Conclusions
These data confirmed the influence of TNIP1 on an increased susceptibility to SSc and reinforced this locus as a common autoimmunity risk factor.
doi:10.1136/annrheumdis-2012-201888
PMCID: PMC3887516  PMID: 22896740
13.  A genome-wide association study follow-up suggests a possible role for PPARG in systemic sclerosis susceptibility 
Introduction
A recent genome-wide association study (GWAS) comprising a French cohort of systemic sclerosis (SSc) reported several non-HLA single-nucleotide polymorphisms (SNPs) showing a nominal association in the discovery phase. We aimed to identify previously overlooked susceptibility variants by using a follow-up strategy.
Methods
Sixty-six non-HLA SNPs showing a P value <10-4 in the discovery phase of the French SSc GWAS were analyzed in the first step of this study, performing a meta-analysis that combined data from the two published SSc GWASs. A total of 2,921 SSc patients and 6,963 healthy controls were included in this first phase. Two SNPs, PPARG rs310746 and CHRNA9 rs6832151, were selected for genotyping in the replication cohort (1,068 SSc patients and 6,762 healthy controls) based on the results of the first step. Genotyping was performed by using TaqMan SNP genotyping assays.
Results
We observed nominal associations for both PPARG rs310746 (PMH = 1.90 × 10-6, OR, 1.28) and CHRNA9 rs6832151 (PMH = 4.30 × 10-6, OR, 1.17) genetic variants with SSc in the first step of our study. In the replication phase, we observed a trend of association for PPARG rs310746 (P value = 0.066; OR, 1.17). The combined overall Mantel-Haenszel meta-analysis of all the cohorts included in the present study revealed that PPARG rs310746 remained associated with SSc with a nominal non-genome-wide significant P value (PMH = 5.00 × 10-7; OR, 1.25). No evidence of association was observed for CHRNA9 rs6832151 either in the replication phase or in the overall pooled analysis.
Conclusion
Our results suggest a role of PPARG gene in the development of SSc.
doi:10.1186/ar4432
PMCID: PMC3978735  PMID: 24401602
14.  Identification of CSK as a systemic sclerosis genetic risk factor through Genome Wide Association Study follow-up 
Human Molecular Genetics  2012;21(12):2825-2835.
Systemic sclerosis (SSc) is complex autoimmune disease affecting the connective tissue; influenced by genetic and environmental components. Recently, we performed the first successful genome-wide association study (GWAS) of SSc. Here, we perform a large replication study to better dissect the genetic component of SSc. We selected 768 polymorphisms from the previous GWAS and genotyped them in seven replication cohorts from Europe. Overall significance was calculated for replicated significant SNPs by meta-analysis of the replication cohorts and replication-GWAS cohorts (3237 cases and 6097 controls). Six SNPs in regions not previously associated with SSc were selected for validation in another five independent cohorts, up to a total of 5270 SSc patients and 8326 controls. We found evidence for replication and overall genome-wide significance for one novel SSc genetic risk locus: CSK [P-value = 5.04 × 10−12, odds ratio (OR) = 1.20]. Additionally, we found suggestive association in the loci PSD3 (P-value = 3.18 × 10−7, OR = 1.36) and NFKB1 (P-value = 1.03 × 10−6, OR = 1.14). Additionally, we strengthened the evidence for previously confirmed associations. This study significantly increases the number of known putative genetic risk factors for SSc, including the genes CSK, PSD3 and NFKB1, and further confirms six previously described ones.
doi:10.1093/hmg/dds099
PMCID: PMC3368627  PMID: 22407130
15.  In Type 1 Diabetes a Subset of Anti-Coxsackievirus B4 Antibodies Recognize Autoantigens and Induce Apoptosis of Pancreatic Beta Cells 
PLoS ONE  2013;8(2):e57729.
Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells. The role played by autoantibodies directed against beta cells antigens in the pathogenesis of the disease is still unclear. Coxsackievirus B infection has been linked to the onset of type 1 diabetes; however its precise role has not been elucidated yet. To clarify these issues, we screened a random peptide library with sera obtained from 58 patients with recent onset type 1 diabetes, before insulin therapy. We identified an immunodominant peptide recognized by the majority of individual patients’sera, that shares homology with Coxsackievirus B4 VP1 protein and with beta-cell specific autoantigens such as phogrin, phosphofructokinase and voltage-gated L-type calcium channels known to regulate beta cell apoptosis. Antibodies against the peptide affinity-purified from patients’ sera, recognized the viral protein and autoantigens; moreover, such antibodies induced apoptosis of the beta cells upon binding the L-type calcium channels expressed on the beta cell surface, suggesting a calcium dependent mechanism. Our results provide evidence that in autoimmune diabetes a subset of anti-Coxsackievirus antibodies are able to induce apoptosis of pancreatic beta cells which is considered the most critical and final step in the development of autoimmune diabetes without which clinical manifestations do not occur.
doi:10.1371/journal.pone.0057729
PMCID: PMC3585221  PMID: 23469060
16.  A GWAS follow-up study reveals the association of the IL12RB2 gene with systemic sclerosis in Caucasian populations 
Human Molecular Genetics  2011;21(4):926-933.
A single-nucleotide polymorphism (SNP) at the IL12RB2 locus showed a suggestive association signal in a previously published genome-wide association study (GWAS) in systemic sclerosis (SSc). Aiming to reveal the possible implication of the IL12RB2 gene in SSc, we conducted a follow-up study of this locus in different Caucasian cohorts. We analyzed 10 GWAS-genotyped SNPs in the IL12RB2 region (2309 SSc patients and 5161 controls). We then selected three SNPs (rs3790567, rs3790566 and rs924080) based on their significance level in the GWAS, for follow-up in an independent European cohort comprising 3344 SSc and 3848 controls. The most-associated SNP (rs3790567) was further tested in an independent cohort comprising 597 SSc patients and 1139 controls from the USA. After conditional logistic regression analysis of the GWAS data, we selected rs3790567 [PMH= 1.92 × 10−5 odds ratio (OR) = 1.19] as the genetic variant with the firmest independent association observed in the analyzed GWAS peak of association. After the first follow-up phase, only the association of rs3790567 was consistent (PMH= 4.84 × 10−3 OR = 1.12). The second follow-up phase confirmed this finding (Pχ2 = 2.82 × 10−4 OR = 1.34). After performing overall pooled-analysis of all the cohorts included in the present study, the association found for the rs3790567 SNP in the IL12RB2 gene region reached GWAS-level significant association (PMH= 2.82 × 10−9 OR = 1.17). Our data clearly support the IL12RB2 genetic association with SSc, and suggest a relevant role of the interleukin 12 signaling pathway in SSc pathogenesis.
doi:10.1093/hmg/ddr522
PMCID: PMC3298110  PMID: 22076442
17.  The Systemic Lupus Erythematosus IRF5 Risk Haplotype Is Associated with Systemic Sclerosis 
PLoS ONE  2013;8(1):e54419.
Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P  = 1.34×10−8, OR  = 1.22, CI 95%  = 1.14–1.30; rs2004640: P  = 4.60×10−7, OR  = 0.84, CI 95%  = 0.78–0.90; rs10488631: P  = 7.53×10−20, OR  = 1.63, CI 95%  = 1.47–1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P  = 0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P  = 9.04×10−22, OR  = 1.75, CI 95%  = 1.56–1.97) better explained the observed association (likelihood P-value  = 1.48×10−4), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific.
doi:10.1371/journal.pone.0054419
PMCID: PMC3553151  PMID: 23372721
18.  KCNA5 gene is not confirmed as a systemic sclerosis-related pulmonary arterial hypertension genetic susceptibility factor 
Arthritis Research & Therapy  2012;14(6):R273.
Introduction
Potassium voltage-gated channel shaker-related subfamily member 5 (KCNA5) is implicated in vascular tone regulation, and its inhibition during hypoxia produces pulmonary vasoconstriction. Recently, a protective association of the KCNA5 locus with systemic sclerosis (SSc) patients with pulmonary arterial hypertension (PAH) was reported. Hence, the aim of this study was to replicate these findings in an independent multicenter Caucasian SSc cohort.
Methods
The 2,343 SSc cases (179 PAH positive, confirmed by right-heart catheterization) and 2,690 matched healthy controls from five European countries were included in this study. Rs10744676 single-nucleotide polymorphism (SNP) was genotyped by using a TaqMan SNP genotyping assay.
Results
Individual population analyses of the selected KCNA5 genetic variant did not show significant association with SSc or any of the defined subsets (for example, limited cutaneous SSc, diffuse cutaneous SSc, anti-centromere autoantibody positive and anti-topoisomerase autoantibody positive). Furthermore, pooled analyses revealed no significant evidence of association with the disease or any of the subsets, not even the PAH-positive group. The comparison of PAH-positive patients with PAH-negative patients showed no significant differences among patients.
Conclusions
Our data do not support an important role of KCNA5 as an SSc-susceptibility factor or as a PAH-development genetic marker for SSc patients.
doi:10.1186/ar4124
PMCID: PMC3674598  PMID: 23270786
19.  Analysis of the association between CD40 and CD40 ligand polymorphisms and systemic sclerosis 
Arthritis Research & Therapy  2012;14(3):R154.
Introduction
The aim of the present study was to investigate the possible role of CD40 and CD40 ligand (CD40LG) genes in the susceptibility and phenotype expression of systemic sclerosis (SSc).
Methods
In total, 2,670 SSc patients and 3,245 healthy individuals from four European populations (Spain, Germany, The Netherlands, and Italy) were included in the study. Five single-nucleotide polymorphisms (SNPs) of CD40 (rs1883832, rs4810485, rs1535045) and CD40LG (rs3092952, rs3092920) were genotyped by using a predesigned TaqMan allele-discrimination assay technology. Meta-analysis was assessed to determine whether an association exists between the genetic variants and SSc or its main clinical subtypes.
Results
No evidence of association between CD40 and CD40LG genes variants and susceptibility to SSc was observed. Similarly, no significant statistical differences were observed when SSc patients were stratified by the clinical subtypes, the serologic features, and pulmonary fibrosis.
Conclusions
Our results do not suggest an important role of CD40 and CD40LG gene polymorphisms in the susceptibility to or clinical expression of SSc.
doi:10.1186/ar3890
PMCID: PMC3446540  PMID: 22731751
20.  A multicenter study confirms CD226 gene association with systemic sclerosis-related pulmonary fibrosis 
Introduction
CD226 genetic variants have been associated with a number of autoimmune diseases and recently with systemic sclerosis (SSc). The aim of this study was to test the influence of CD226 loci in SSc susceptibility, clinical phenotypes and autoantibody status in a large multicenter European population.
Methods
A total of seven European populations of Caucasian ancestry were included, comprising 2,131 patients with SSc and 3,966 healthy controls. Three CD226 single nucleotide polymorphisms (SNPs), rs763361, rs3479968 and rs727088, were genotyped using Taqman 5'allelic discrimination assays.
Results
Pooled analyses showed no evidence of association of the three SNPs, neither with the global disease nor with the analyzed subphenotypes. However, haplotype block analysis revealed a significant association for the TCG haplotype (SNP order: rs763361, rs34794968, rs727088) with lung fibrosis positive patients (PBonf = 3.18E-02 OR 1.27 (1.05 to 1.54)).
Conclusion
Our data suggest that the tested genetic variants do not individually influence SSc susceptibility but a CD226 three-variant haplotype is related with genetic predisposition to SSc-related pulmonary fibrosis.
doi:10.1186/ar3809
PMCID: PMC3446459  PMID: 22531499
21.  Correction: Identification of Novel Genetic Markers Associated with Clinical Phenotypes of Systemic Sclerosis through a Genome-Wide Association Strategy 
Gorlova, Olga | Martin, Jose-Ezequiel | Rueda, Blanca | Koeleman, Bobby P. C. | Ying, Jun | Teruel, Maria | Diaz-Gallo, Lina-Marcela | Broen, Jasper C. | Vonk, Madelon C. | Simeon, Carmen P. | Alizadeh, Behrooz Z. | Coenen, Marieke J. H. | Voskuyl, Alexandre E. | Schuerwegh, Annemie J. | van Riel, Piet L. C. M. | Vanthuyne, Marie | van 't Slot, Ruben | Italiaander, Annet | Ophoff, Roel A. | Hunzelmann, Nicolas | Fonollosa, Vicente | Ortego-Centeno, Norberto | González-Gay, Miguel A. | García-Hernández, Francisco J. | González-Escribano, María F. | Airo, Paolo | van Laar, Jacob | Worthington, Jane | Hesselstrand, Roger | Smith, Vanessa | de Keyser, Filip | Houssiau, Fredric | Chee, Meng May | Madhok, Rajan | Shiels, Paul G. | Westhovens, Rene | Kreuter, Alexander | de Baere, Elfride | Witte, Torsten | Padyukov, Leonid | Nordin, Annika | Scorza, Raffaella | Lunardi, Claudio | Lie, Benedicte A. | Hoffmann-Vold, Anna-Maria | Palm, Øyvind | García de la Peña, Paloma | Carreira, Patricia | Varga, John | Hinchcliff, Monique | Lee, Annette T. | Gourh, Pravitt | Amos, Christopher I. | Wigley, Frederick M. | Hummers, Laura K. | Nelson, J. Lee | Riemekasten, Gabriella | Herrick, Ariane | Beretta, Lorenzo | Fonseca, Carmen | Denton, Christopher P. | Gregersen, Peter K. | Agarwal, Sandeep | Assassi, Shervin | Tan, Filemon K. | Arnett, Frank C. | Radstake, Timothy R. D. J. | Mayes, Maureen D. | Martin, Javier
PLoS Genetics  2011;7(8):10.1371/annotation/3aeebb2e-64e5-4548-8d65-1f2d5dfeb073.
doi:10.1371/annotation/3aeebb2e-64e5-4548-8d65-1f2d5dfeb073
PMCID: PMC3166261
22.  Correction: Identification of Novel Genetic Markers Associated with Clinical Phenotypes of Systemic Sclerosis through a Genome-Wide Association Strategy 
Gorlova, Olga | Martin, Jose-Ezequiel | Rueda, Blanca | Koeleman, Bobby P. C. | Ying, Jun | Teruel, Maria | Diaz-Gallo, Lina-Marcela | Broen, Jasper C. | Vonk, Madelon C. | Simeon, Carmen P. | Alizadeh, Behrooz Z. | Coenen, Marieke J. H. | Voskuyl, Alexandre E. | Schuerwegh, Annemie J. | van Riel, Piet L. C. M. | Vanthuyne, Marie | van 't Slot, Ruben | Italiaander, Annet | Ophoff, Roel A. | Hunzelmann, Nicolas | Fonollosa, Vicente | Ortego-Centeno, Norberto | González-Gay, Miguel A. | García-Hernández, Francisco J. | González-Escribano, María F. | Airo, Paolo | van Laar, Jacob | Worthington, Jane | Hesselstrand, Roger | Smith, Vanessa | de Keyser, Filip | Houssiau, Fredric | Chee, Meng May | Madhok, Rajan | Shiels, Paul G. | Westhovens, Rene | Kreuter, Alexander | de Baere, Elfride | Witte, Torsten | Padyukov, Leonid | Nordin, Annika | Scorza, Raffaella | Lunardi, Claudio | Lie, Benedicte A. | Hoffmann-Vold, Anna-Maria | Palm, Øyvind | García de la Peña, Paloma | Carreira, Patricia | Varga, John | Hinchcliff, Monique | Lee, Annette T. | Gourh, Pravitt | Amos, Christopher I. | Wigley, Frederick M. | Hummers, Laura K. | Nelson, J. Lee | Riemekasten, Gabriella | Herrick, Ariane | Beretta, Lorenzo | Fonseca, Carmen | Denton, Christopher P. | Gregersen, Peter K. | Agarwal, Sandeep | Assassi, Shervin | Tan, Filemon K. | Arnett, Frank C. | Radstake, Timothy R. D. J. | Mayes, Maureen D. | Martin, Javier
PLoS Genetics  2011;7(8):10.1371/annotation/7a52649c-0942-4bd8-a5d3-3cdacca03cd8.
doi:10.1371/annotation/7a52649c-0942-4bd8-a5d3-3cdacca03cd8
PMCID: PMC3166262
23.  Identification of Novel Genetic Markers Associated with Clinical Phenotypes of Systemic Sclerosis through a Genome-Wide Association Strategy 
Gorlova, Olga | Martin, Jose-Ezequiel | Rueda, Blanca | Koeleman, Bobby P. C. | Ying, Jun | Teruel, Maria | Diaz-Gallo, Lina-Marcela | Broen, Jasper C. | Vonk, Madelon C. | Simeon, Carmen P. | Alizadeh, Behrooz Z. | Coenen, Marieke J. H. | Voskuyl, Alexandre E. | Schuerwegh, Annemie J. | van Riel, Piet L. C. M. | Vanthuyne, Marie | van 't Slot, Ruben | Italiaander, Annet | Ophoff, Roel A. | Hunzelmann, Nicolas | Fonollosa, Vicente | Ortego-Centeno, Norberto | González-Gay, Miguel A. | García-Hernández, Francisco J. | González-Escribano, María F. | Airo, Paolo | van Laar, Jacob | Worthington, Jane | Hesselstrand, Roger | Smith, Vanessa | de Keyser, Filip | Houssiau, Fredric | Chee, Meng May | Madhok, Rajan | Shiels, Paul G. | Westhovens, Rene | Kreuter, Alexander | de Baere, Elfride | Witte, Torsten | Padyukov, Leonid | Nordin, Annika | Scorza, Raffaella | Lunardi, Claudio | Lie, Benedicte A. | Hoffmann-Vold, Anna-Maria | Palm, Øyvind | García de la Peña, Paloma | Carreira, Patricia | Varga, John | Hinchcliff, Monique | Lee, Annette T. | Gourh, Pravitt | Amos, Christopher I. | Wigley, Frederick M. | Hummers, Laura K. | Hummers, J. | Nelson, J. Lee | Riemekasten, Gabriella | Herrick, Ariane | Beretta, Lorenzo | Fonseca, Carmen | Denton, Christopher P. | Gregersen, Peter K. | Agarwal, Sandeep | Assassi, Shervin | Tan, Filemon K. | Arnett, Frank C. | Radstake, Timothy R. D. J. | Mayes, Maureen D. | Martin, Javier
PLoS Genetics  2011;7(7):e1002178.
The aim of this study was to determine, through a genome-wide association study (GWAS), the genetic components contributing to different clinical sub-phenotypes of systemic sclerosis (SSc). We considered limited (lcSSc) and diffuse (dcSSc) cutaneous involvement, and the relationships with presence of the SSc-specific auto-antibodies, anti-centromere (ACA), and anti-topoisomerase I (ATA). Four GWAS cohorts, comprising 2,296 SSc patients and 5,171 healthy controls, were meta-analyzed looking for associations in the selected subgroups. Eighteen polymorphisms were further tested in nine independent cohorts comprising an additional 3,175 SSc patients and 4,971 controls. Conditional analysis for associated SNPs in the HLA region was performed to explore their independent association in antibody subgroups. Overall analysis showed that non-HLA polymorphism rs11642873 in IRF8 gene to be associated at GWAS level with lcSSc (P = 2.32×10−12, OR = 0.75). Also, rs12540874 in GRB10 gene (P = 1.27 × 10−6, OR = 1.15) and rs11047102 in SOX5 gene (P = 1.39×10−7, OR = 1.36) showed a suggestive association with lcSSc and ACA subgroups respectively. In the HLA region, we observed highly associated allelic combinations in the HLA-DQB1 locus with ACA (P = 1.79×10−61, OR = 2.48), in the HLA-DPA1/B1 loci with ATA (P = 4.57×10−76, OR = 8.84), and in NOTCH4 with ACA P = 8.84×10−21, OR = 0.55) and ATA (P = 1.14×10−8, OR = 0.54). We have identified three new non-HLA genes (IRF8, GRB10, and SOX5) associated with SSc clinical and auto-antibody subgroups. Within the HLA region, HLA-DQB1, HLA-DPA1/B1, and NOTCH4 associations with SSc are likely confined to specific auto-antibodies. These data emphasize the differential genetic components of subphenotypes of SSc.
Author Summary
Scleroderma or systemic sclerosis is a complex autoimmune disease affecting one individual of every 100,000 in Caucasian populations. Even though current genetic studies have led to better understanding of the pathogenesis of the disease, much remains unknown. Scleroderma is a heterogeneous disease, which can be subdivided according to different criteria, such as the involvement of organs and the presence of specific autoantibodies. Such subgroups present more homogeneous genetic groups, and some genetic associations with these manifestations have already been described. Through reanalysis of a genome-wide association study data, we identify three novel genes containing genetic variations which predispose to subphenotypes of the disease (IRF8, GRB10, and SOX5). Also, we better characterize the patterns of associated loci found in the HLA region. Together, our findings lead to a better understanding of the genetic component of scleroderma.
doi:10.1371/journal.pgen.1002178
PMCID: PMC3136437  PMID: 21779181
24.  Gene expression profiling in circulating endothelial cells from systemic sclerosis patients shows an altered control of apoptosis and angiogenesis that is modified by iloprost infusion 
Arthritis Research & Therapy  2010;12(4):R131.
Introduction
Circulating endothelial cells are increased in patients affected by systemic sclerosis (SSc) and their number strongly correlates with vascular damage. The effects of iloprost in systemic sclerosis are only partially known. We aimed at studying the gene expression profile of circulating endothelial cells and the effects of iloprost infusion and gene expression in patients with systemic sclerosis.
Methods
We enrolled 50 patients affected by systemic sclerosis, 37 patients without and 13 patients with digital ulcers. Blood samples were collected from all patients before and 72 hours after either a single day or five days eight hours iloprost infusion. Blood samples were also collected from 50 sex- and age-matched healthy controls. Circulating endothelial cells and endothelial progenitors cells were detected in the peripheral blood of patients with systemic sclerosis by flow cytometry with a four-colour panel of antibodies. Statistical analysis was performed with the SPSS 16 statistical package.Circulating endothelial cells were then isolated from peripheral blood by immunomagnetic CD45 negative selection for the gene array study.
Results
The number of both circulating endothelial cells and progenitors was significantly higher in patients affected by systemic sclerosis than in controls and among patients in those with digital ulcers than in patients without them. Circulating endothelial cells and progenitors number increased after iloprost infusion. Gene array analysis of endothelial cells showed a different transcriptional profile in patients compared to controls. Indeed, patients displayed an altered expression of genes involved in the control of apoptosis and angiogenesis. Iloprost infusion had a profound impact on endothelial cells gene expression since the treatment was able to modulate a very high number of transcripts.
Conclusions
We report here that circulating endothelial cells in patients with systemic sclerosis show an altered expression of genes involved in the control of apoptosis and angiogenesis. Moreover we describe that iloprost infusion has a strong effect on endothelial cells and progenitors since it is able to modulate both their number and their gene expression profile.
doi:10.1186/ar3069
PMCID: PMC2945021  PMID: 20609215
25.  Endothelial Cells' Activation and Apoptosis Induced by a Subset of Antibodies against Human Cytomegalovirus: Relevance to the Pathogenesis of Atherosclerosis 
PLoS ONE  2007;2(5):e473.
Background
Human cytomegalovirus (hCMV) is involved in the pathogenesis of atherosclerosis. We have previously shown in patients with atherosclerosis that antibodies directed against the hCMV-derived proteins US28 and UL122 are able to induce endothelial cell damage and apoptosis of non-stressed endothelial cells through cross-rection with normally expressed surface molecules. Our aim was to dissect the molecular basis of such interaction and to investigate mechanisms linking innate immunity to atherosclerosis.
Methodology/Principal Findings
We analysed the gene expression profiles in endothelial cells stimulated with antibodies affinity-purified against either the UL122 or the US28 peptides using the microarray technology. Microarray results were validated by quantitative PCR and by detection of proteins in the medium. Supernatant of endothelial cells incubated with antibodies was analysed also for the presence of Heat Shock Protein (HSP)60 and was used to assess stimulation of Toll-Like Receptor-4 (TLR4). Antibodies against UL122 and US28 induced the expression of genes encoding for adhesion molecules, chemokines, growth factors and molecules involved in the apoptotis process together with other genes known to be involved in the initiation and progression of the atherosclerotic process. HSP60 was released in the medium of cells incubated with anti-US28 antibodies and was able to engage TLR4.
Conclusions/Significance
Antibodies directed against hCMV modulate the expression of genes coding for molecules involved in activation and apoptosis of endothelial cells, processes known to play a pivotal role in the pathogenesis of atherosclerosis. Moreover, endothelial cells exposed to such antibodies express HSP60 on the cell surface and release HSP60 in the medium able to activate TLR4. These data confirm that antibodies directed against hCMV-derived proteins US28 and UL122 purified from patients with coronary artery disease induce endothelial cell damage and support the hypothesis that hCMV infection may play a crucial role in mediating the atherosclerotic process.
doi:10.1371/journal.pone.0000473
PMCID: PMC1868596  PMID: 17534423

Results 1-25 (27)