Long-Term Dominance of Carbapenem-Non-Susceptible Pseudomonas aeruginosa ST111 in Hematologic Malignancy Patients and Hematopoietic Cell Transplant Recipients.

An epidemiological study uncovered that fluoroquinolone (FQ) neutropenic prophylaxis in hematopoietic cell transplant and hematologic malignancy (HCT/HM) patients was associated with breakthrough Pseudomonas aeruginosa bloodstream infections (BSIs) with isolates non-susceptible to both FQs and meropenem. The molecular epidemiology of the FQ/meropenem-non-susceptible P. aeruginosa isolates causing FQ-breakthrough BSIs in the HCT/HM patients remains unclear. Through whole genome sequencing on 57 P. aeruginosa isolates from 54 patients diagnosed with HM or receiving an HCT, we found that ST111 strains predominated, accounting for 22 (38.6%) of the isolates. 17 of 33 (51.5%) FQ-breakthrough BSIs were caused by ST111 strains, of which 15 (88.2%) were meropenem non-susceptible. ST111 strains, but not other oprD-deficient, meropenem-non-susceptible clinical strains, were found to have a colonization advantage over P. aeruginosa strain PA14 in C. elegans and to outcompete PA14 in in vitro co-culture assays. Together, we found that breakthrough P. aeruginosa BSIs during FQ prophylaxis in HCT/HM patients are dominated by clonally-related FQ/meropenem non-susceptible strains, predominantly ST111 type, and that the dominance of ST111 strains may be explained by a relative fitness advantage over other clinical strains. Additional work is necessary to better understand the factors driving the dominance and persistence of these ST111 strains.

Identification of an Association of TNFAIP3 Polymorphisms With Matrix Metalloproteinase Expression in Fibroblasts in an Integrative Study of Systemic Sclerosis-Associated Genetic and Environmental Factors.

OBJECTIVE: Systemic sclerosis (SSc) is a fibrotic disease attributed to both genetic susceptibility and environmental factors. This study was undertaken to investigate the associations between SSc-associated genetic variants and the expression of extracellular matrix (ECM) genes in human fibroblasts stimulated with silica particles in time-course and dose-response experiments. METHODS: A total of 200 fibroblast strains were examined for ECM gene expression after stimulation with silica particles. The fibroblasts were genetically profiled using Immunochip assays and then subjected to whole-genome genotype imputation. Associations of genotypes and gene expression were first analyzed in a Caucasian cohort and then validated in a meta-analysis combining the results from Caucasian, African American, and Hispanic subjects. A linear mixed model for longitudinal data analysis was used to identify genetic variants associated with the expression of ECM genes, and the associations were validated by using a haplotype-based longitudinal association test on regions that included the loci identified. RESULTS: The single-nucleotide polymorphism rs58905141 in TNFAIP3 was consistently associated with time-course and/or dose-response expression of MMP3 and MMP1 in the fibroblasts stimulated with silica particles in both the analysis of Caucasian subjects only and the meta-analysis. Results of the haplotype-based analysis validated the association signals. CONCLUSION: Our findings indicate that a genetic variant of TNFAIP3 is strongly associated with the silica-induced profibrotic response of fibroblasts. In silico functional analysis based on the ENCODE database revealed that rs58905141 might affect the binding activities of the transcription factors for TNFAIP3. This is the first genome-wide study of interactions between genetic and environmental factors in a complex SSc fibroblast model.

Confirmation of CCR6 as a risk factor for anti-topoisomerase I antibodies in systemic sclerosis.

OBJECTIVES: The current knowledge of the influence of systemic sclerosis (SSc) risk loci in the clinical sub-phenotypes is still limited. The main limitation lies in the low frequency of some sub-phenotypes which could be solved by replication studies in independent cohorts and meta-analysis between studies. In this regard, CCR6 gene variants have been recently associated with anti-topoisomerase I positive (ATA+) production in SSc patients in a candidate gene study. This gene has been proposed to have a critical role in IL-17-driven autoimmunity in human diseases. METHODS: In order to confirm the association between CCR6 and ATA+ SSc patients, we performed an independent replication study in populations of European ancestry. We studied two CCR6 genetic variants (rs968334 and rs3093024) in a total of 901 ATA+ SSc cases, 3,258 ATA- SSc cases and 7,865 healthy controls and compared allelic frequencies for those SNPs in ATA+ SSc with healthy controls and also with ATA- SSc patients. RESULTS: The comparison performed between ATA+ SSc patients and healthy controls showed significant association with SNP rs968334 (p=4.88x10(-2), OR=1.11). When we compared ATA+ SSc cases with ATA- SSc, both SNPs, rs3093024 and rs968334, showed significant associations (p=2.89x10(-2), OR=1.13; p=1.69x10(-2), OR=1.15). Finally, in order to increase even more sample size and statistical power, we meta-analysed our study with the previous reported and found a significant association between SNP rs3093024 and ATA+ SSc patients (p=1.00x10(-4), OR=1.16) comparing with healthy controls. CONCLUSIONS: Our work confirms the association of CCR6 gene and ATA+ SSc patients.

Dissecting the heterogeneity of skin gene expression patterns in systemic sclerosis.

OBJECTIVE: To examine the heterogeneity of global transcriptome patterns in systemic sclerosis (SSc) skin in a large sample of patients with SSc and control subjects. METHODS: Skin biopsy specimens obtained from 61 patients enrolled in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort and 36 unaffected control subjects with a similar demographic background were examined by Illumina HumanHT-12 bead arrays. Followup experiments using quantitative polymerase chain reaction and immunohistochemical analysis were also performed. RESULTS: We identified 2,754 differentially expressed transcripts in SSc patients compared with controls. Clustering analysis revealed 2 prominent transcriptomes in SSc patients: the keratin and fibroinflammatory signatures. Higher keratin transcript scores were associated with shorter disease duration and interstitial lung disease, while higher fibroinflammatory scores were associated with diffuse cutaneous involvement, a higher skin score at the biopsy site, and a higher modified Rodnan skin thickness score. A subgroup of patients with significantly longer disease duration had a normal-like transcript pattern. Analysis of cell type-specific signature scores revealed remarkable heterogeneity across patients. Significantly higher scores were calculated for fibroblasts (72% of patients), microvascular cells (61%), macrophages (54%), and dendritic cells (DCs) (49%). The majority of samples with significantly higher fibroblast scores (35 of 44 [80%]) had significantly increased macrophage and/or DC scores. Further analysis and immunohistochemical staining indicated that the keratin signature was not a general marker of keratinocyte activation but was in fact associated with an activation pattern in hair and adnexal structures. CONCLUSION: Prominent fibroinflammatory and keratin signatures are present in SSc skin. Expression profiles of SSc skin show significant heterogeneity, and this finding might be useful for stratifying patients for targeted therapies or predicting the response to immunosuppression.

Unique temporal and spatial expression patterns of IL-33 in ovaries during ovulation and estrous cycle are associated with ovarian tissue homeostasis.

Ovaries are among the most active organs. Frequently occurring events such as ovulation and ovarian atresia are accompanied with tissue destruction and repairing. Critical roles of immune cells or molecules in those events have been well recognized. IL-33 is a new member of the IL-1 cytokine gene family. Recent studies suggest its roles beyond immune responses. We systemically examined its expression in ovaries for its potential roles in ovarian functions. During ovulation, a high level of IL-33 was transiently expressed, making it the most significantly upregulated immune gene. During estrous cycle, IL-33 expression levels fluctuated along with numbers of ovarian macrophages and atresia wave. Cells with nuclear form of IL-33 (nIL-33(+) cells) were mostly endothelial cells of veins, either in the inner layer of theca of ovulating follicles during ovulation, or surrounding follicles during estrous cycle. Changes in number of nIL-33(+) cells showed a tendency similar to that in IL-33 mRNA level during estrous cycle. However, the cell number sharply declined before a rapid increase of macrophages and a surge of atresia. The decline in nIL-33(+) cell number was coincident with detection of higher level of the cytokine form of IL-33 by Western blot, suggesting a release of cytokine form of IL-33 before the surge of macrophage migration and atresia. However, IL-33 Ab, either by passive transfer or immunization, showed a limited effect on ovulation or atresia. It raises a possibility of IL-33's role in tissue homeostasis after ovarian events, instead of a direct involvement in ovarian functions.

Identification of cadherin 11 as a mediator of dermal fibrosis and possible role in systemic sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a chronic autoimmune disease clinically manifesting as progressive fibrosis of the skin and internal organs. Recent microarray studies demonstrated that cadherin 11 (Cad-11) expression is increased in the affected skin of patients with SSc. The purpose of this study was to examine our hypothesis that Cad-11 is a mediator of dermal fibrosis. METHODS: Biopsy samples of skin from SSc patients and healthy control subjects were used for real-time quantitative polymerase chain reaction analysis to assess Cad-11 expression and for immunohistochemistry to determine the expression pattern of Cad-11. To determine whether Cad-11 is a mediator of dermal fibrosis, Cad-11-deficient mice and anti-Cad-11 monoclonal antibodies (mAb) were used in the bleomycin-induced dermal fibrosis model. In vitro studies with dermal fibroblasts and bone marrow-derived macrophages were used to determine the mechanisms by which Cad-11 contributes to the development of tissue fibrosis. RESULTS: Levels of messenger RNA for Cad-11 were increased in skin biopsy samples from patients with SSc and correlated with the modified Rodnan skin thickness scores. Cad-11 expression was localized to dermal fibroblasts and macrophages in SSc skin. Cad-11-knockout mice injected with bleomycin had markedly attenuated dermal fibrosis, as quantified by measurements of skin thickness, collagen levels, myofibroblast accumulation, and profibrotic gene expression, in lesional skin as compared to the skin of wild-type mice. In addition, anti-Cad-11 mAb decreased fibrosis at various time points in the bleomycin-induced dermal fibrosis model. In vitro studies demonstrated that Cad-11 regulated the production of transforming growth factor ß (TGFß) by macrophages and the migration of fibroblasts. CONCLUSION: These data demonstrate that Cad-11 is a mediator of dermal fibrosis and TGFß production and suggest that Cad-11 may be a therapeutic target in SSc.

Secondary use of existing public microarray data to predict outcome for hepatocellular carcinoma.

BACKGROUND: Since 1990, numerous public repositories of microarray data have been created to store vast genomic data sets. Our hypothesis is that a secondary analysis of an available hepatocellular carcinoma (HCC) public data set could generate new findings and additional hypotheses. METHODS: The Gene Expression Omnibus at the National Center for Biotechnology Information was queried for available data sets specific for 'HCC' and 'clinical data.' Genes that passed filtering and normalization criteria were analyzed using the class comparison and prediction functions in BRB-ArrayTools. Ingenuity pathway analysis software was used to identify potential gene networks up- or down-regulated. RESULTS: The file GDS274, which measured gene expression in primary HCC lesions with or without hepatic metastases from a cohort of Chinese patients, was identified as an appropriate data set and was imported into BRB-ArrayTools. 9984 genes passed filtering criteria. Clinical data demonstrated alpha fetoprotein (AFP) >100 ng/mL predictive of worse survival (HR 5.87, 95% confidence interval: 1.11-31.0). A class comparison between patients with an AFP >100 and those with AFP <100 demonstrated 92 genes to be differentially expressed. Ingenuity pathway analyses demonstrated the top networks associated with the observed gene expression. CONCLUSIONS: Using available HCC microarray data, we identified genes differentially expressed based on AFP >100. Canonical pathway analysis demonstrated functional gene pathways and associated upstream regulators. This study maximizes the use of publicly available data by generating new findings. Secondary analyses of these data sets should be considered by investigators before embarking on new genomic experiments.

Immunochip analysis identifies multiple susceptibility loci for systemic sclerosis.

In this study, 1,833 systemic sclerosis (SSc) cases and 3,466 controls were genotyped with the Immunochip array. Classical alleles, amino acid residues, and SNPs across the human leukocyte antigen (HLA) region were imputed and tested. These analyses resulted in a model composed of six polymorphic amino acid positions and seven SNPs that explained the observed significant associations in the region. In addition, a replication step comprising 4,017 SSc cases and 5,935 controls was carried out for several selected non-HLA variants, reaching a total of 5,850 cases and 9,401 controls of European ancestry. Following this strategy, we identified and validated three SSc risk loci, including DNASE1L3 at 3p14, the SCHIP1-IL12A locus at 3q25, and ATG5 at 6q21, as well as a suggested association of the TREH-DDX6 locus at 11q23. The associations of several previously reported SSc risk loci were validated and further refined, and the observed peak of association in PXK was related to DNASE1L3. Our study has increased the number of known genetic associations with SSc, provided further insight into the pleiotropic effects of shared autoimmune risk factors, and highlighted the power of dense mapping for detecting previously overlooked susceptibility loci.

Skin gene expression correlates of severity of interstitial lung disease in systemic sclerosis.

OBJECTIVE: We undertook this hypothesis-generating study to identify skin transcripts correlating with severity of interstitial lung disease (ILD) in systemic sclerosis (SSc). METHODS: Skin biopsy samples from 59 patients enrolled in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort or an open-label imatinib study (baseline visit) were examined by global gene expression analysis using Illumina HT-12 arrays. Skin transcripts correlating with concomitantly obtained forced vital capacity (FVC) values and the modified Rodnan skin thickness score (MRSS) were identified by quantitative trait analysis. Also, immunofluorescence staining for selected transcripts was performed in affected skin and lung tissue. Plasma levels of CCL2, soluble SELP, and soluble P-selectin glycoprotein ligand 1 (sPSGL-1) were examined in all patients enrolled in the GENISOS cohort (n = 266). RESULTS: Eighty-two skin transcripts correlated significantly with FVC. This gene list distinguished patients with more severe ILD (FVC <70% predicted) in unsupervised hierarchical clustering analysis (P < 0.001). These genes included SELP, CCL2, and matrix metalloproteinase 3, which are involved in extravasation and adhesion of inflammatory cells. Among the FVC correlates, 8 genes (CCL2, HAPLN3, GPR4, ADCYAP1, WARS, CDC25B, PLP1, and STXBP6) also correlated with the MRSS. Immunofluorescence staining revealed that SELP and CCL2 were also overexpressed in affected skin and lung tissue from SSc patients compared to those from controls. Plasma levels of CCL2 and sPSGL-1 correlated with concomitantly obtained FVC values (r = -0.22, P = 0.001 and r = 0.17, P = 0.015, respectively). This relationship was independent of potential confounders (age, sex, ethnicity, smoking status, anti-topoisomerase I positivity, treatment with immunosuppressive agents, MRSS, disease type, and disease duration). CONCLUSION: A limited number of skin transcripts including genes involved in extravasation and adhesion of inflammatory cells correlate with severity of ILD.

A systemic sclerosis and systemic lupus erythematosus pan-meta-GWAS reveals new shared susceptibility loci.

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.

New insight on the Xq28 association with systemic sclerosis.

OBJECTIVE: To evaluate whether the systemic sclerosis (SSc)-associated IRAK1 non-synonymous single-nucleotide polymorphism rs1059702 is responsible for the Xq28 association with SSc or whether there are other independent signals in the nearby methyl-CpG-binding protein 2 gene (MECP2). METHODS: We analysed a total of 3065 women with SSc and 2630 unaffected controls from five independent Caucasian cohorts. Four tag single-nucleotide polymorphisms of MECP2 (rs3027935, rs17435, rs5987201 and rs5945175) and the IRAK1 variant rs1059702 were genotyped using TaqMan predesigned assays. A meta-analysis including all cohorts was performed to test the overall effect of these Xq28 polymorphisms on SSc. RESULTS: IRAK1 rs1059702 and MECP2 rs17435 were associated specifically with diffuse cutaneous SSc (PFDR=4.12×10(-3), OR=1.27, 95% CI 1.09 to 1.47, and PFDR=5.26×10(-4), OR=1.30, 95% CI 1.14 to 1.48, respectively), but conditional logistic regression analysis showed that the association of IRAK1 rs1059702 with this subtype was explained by that of MECP2 rs17435. On the other hand, IRAK1 rs1059702 was consistently associated with presence of pulmonary fibrosis (PF), because statistical significance was observed when comparing SSc patients PF+ versus controls (PFDR=0.039, OR=1.30, 95% CI 1.07 to 1.58) and SSc patients PF+ versus SSc patients PF- (p=0.025, OR=1.26, 95% CI 1.03 to 1.55). CONCLUSIONS: Our data clearly suggest the existence of two independent signals within the Xq28 region, one located in IRAK1 related to PF and another in MECP2 related to diffuse cutaneous SSc, indicating that both genes may have an impact on the clinical outcome of the disease.

Correlation of interferon-inducible chemokine plasma levels with disease severity in systemic sclerosis.

OBJECTIVE: To measure interferon (IFN)-inducible chemokines in the plasma of patients with systemic sclerosis (SSc) and investigate whether the chemokine levels are correlated with disease severity. METHODS: Plasma levels of the IFN-inducible chemokines IFNγ-inducible protein 10 (IP-10/CXCL10), IFN-inducible T cell α chemoattractant (I-TAC/CXCL11), and monocyte chemoattractant protein 1 (CCL2) were measured in SSc patients and examined for correlation with the IFN gene expression signature. A composite IFN-inducible chemokine score was generated for chemokines showing a correlation with the IFN gene signature (IP-10 and I-TAC), and this score was compared between 266 patients with SSc enrolled in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort and 97 matched control subjects. Subsequently, the correlation between the IFN-inducible chemokine score at baseline and markers of disease severity was assessed. In addition, the course of the IFN-inducible chemokine score over time was examined. RESULTS: The plasma IFN-inducible chemokine score correlated with the IFN gene expression signature, and this score was higher in SSc patients compared to controls. The IFN-inducible chemokine score was also associated with the absence of anti-RNA polymerase III antibodies and presence of anti-U1 RNP antibodies, but not with disease duration, disease type, or other autoantibodies. The chemokine score correlated with concomitantly obtained scores on the Medsger Severity Index for muscle, skin, and lung involvement in SSc, as well as the forced vital capacity, diffusing capacity for carbon monoxide, and creatine kinase levels. The association of the chemokine score with disease severity was independent of the presence of anti-U1 RNP or other potential confounders (age, sex, ethnicity, disease duration, and treatment with immunosuppressive agents). Finally, there was not a significant change in the IFN-inducible chemokine score over time. CONCLUSION: The IFN-inducible chemokine score is a stable serologic marker of a more severe form of SSc and may be useful for risk stratification of patients, regardless of disease type (limited or diffuse) or duration of disease.

Confirmation of TNIP1 but not RHOB and PSORS1C1 as systemic sclerosis risk factors in a large independent replication study.

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 p(MH)=1.94×10(-4), OR 1.19; rs4958881 p(MH)=3.26×10(-5), OR 1.19; rs3792783 p(MH)=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.

Nucleic acid-containing amyloid fibrils potently induce type I interferon and stimulate systemic autoimmunity.

The immunopathophysiologic development of systemic autoimmunity involves numerous factors through complex mechanisms that are not fully understood. In systemic lupus erythematosus, type I IFN (IFN-I) produced by plasmacytoid dendritic cells (pDCs) critically promotes the autoimmunity through its pleiotropic effects on immune cells. However, the host-derived factors that enable abnormal IFN-I production and initial immune tolerance breakdown are largely unknown. Previously, we found that amyloid precursor proteins form amyloid fibrils in the presence of nucleic acids. Here we report that nucleic acid-containing amyloid fibrils can potently activate pDCs and enable IFN-I production in response to self-DNA, self-RNA, and dead cell debris. pDCs can take up DNA-containing amyloid fibrils, which are retained in the early endosomes to activate TLR9, leading to high IFNα/ß production. In mice treated with DNA-containing amyloid fibrils, a rapid IFN response correlated with pDC infiltration and activation. Immunization of nonautoimmune mice with DNA-containing amyloid fibrils induced antinuclear serology against a panel of self-antigens. The mice exhibited positive proteinuria and deposited antibodies in their kidneys. Intriguingly, pDC depletion obstructed IFN-I response and selectively abolished autoantibody generation. Our study reveals an innate immune function of nucleic acid-containing amyloid fibrils and provides a potential link between compromised protein homeostasis and autoimmunity via a pDC-IFN axis.

IRF5 polymorphism predicts prognosis in patients with systemic sclerosis.

OBJECTIVE: The first genome-wide association study (GWAS) of systemic sclerosis (SSc) demonstrated three non-major histocompatibility complex (MHC) susceptibility loci. The goal of this study was to investigate the impact of these gene variants on survival and severity of interstitial lung disease (ILD) in SSc. METHODS: The authors examined 1443 Caucasian SSc patients enrolled in the Genetics versus Environment In Scleroderma Outcome Study (GENISOS) and Scleroderma Family Registry (n = 914 - discovery cohort) and The Johns Hopkins Scleroderma Cohort (n = 529 - replication cohort). Forced vital capacity (FVC)% predicted was used as a surrogate for ILD severity. Five single nucleotide polymorphisms, IRF5 (rs10488631, rs12537284, rs4728142), STAT4 (rs3821236), CD247 (rs2056626) reached genome-wide significance in the SSc-GWAS and were examined in the current study. RESULTS: Overall, 15.5% of the patients had died over the follow-up period of 5.5 years. The IRF5 rs4728142 minor allele was predictive of longer survival in the discovery cohort (p = 0.021) and in the independent replication cohort (p = 0.047) and combined group (HR: 0.75, 95% CI 0.62 to 0.90, p = 0.002). The association of this SNP with survival was independent of age at disease onset, disease type and autoantibody profile (anticentromere and antitopoisomerase antibodies). The minor allele frequency of IRF5 rs4728142 was 49.4%. Moreover, IRF5 rs4728142 minor allele correlated with higher FVC% predicted at enrolment (p = 0.019). Finally, the IRF5 rs4728142 minor allele was associated with lower IRF5 transcript expression in patients and controls (p = 0.016 and p = 0.034, respectively), suggesting that the IRF5, rs4728142 SNP, may be functionally relevant. CONCLUSION: An SNP in the IRF5 promoter region (rs4728142), associated with lower IRF5 transcript levels, was predictive of longer survival and milder ILD in patients with SSc.

Osteopontin in systemic sclerosis and its role in dermal fibrosis.

Osteopontin (OPN) is a matricellular protein with proinflammatory and profibrotic properties. Previous reports demonstrate a role for OPN in wound healing and pulmonary fibrosis. Here, we determined whether OPN levels are increased in a large cohort of patients with systemic sclerosis (SSc) and whether OPN contributes to the development of dermal fibrosis. The plasma OPN levels were increased in SSc patients, including patients with limited and diffuse disease, compared with healthy controls. Immunohistology demonstrated OPN on fibroblast-like and inflammatory cells in SSc skin and lesional skin from mice in the bleomycin (bleo)-induced dermal fibrosis model. OPN-deficient (OPN(-/-)) mice developed less dermal fibrosis compared with wild-type (WT) mice in the bleo-induced dermal fibrosis model. Additional in vivo studies have demonstrated that lesional skin from OPN(-/-)mice had fewer Mac-3-positive cells, fewer myofibroblasts, decreased transforming growth factor (TGF)-ß and genes in the TGF-ß pathway, and decreased numbers of cells expressing phosphorylated SMAD2 (pSMAD) and extracellular signal-regulated kinase. In vitro, OPN(-/-) dermal fibroblasts had decreased migratory capacity but similar phosphorylation of SMAD2 by TGF-ß. Finally, TGF-ß production by OPN-deficient macrophages was reduced compared with WT. These data demonstrate an important role for OPN in the development of dermal fibrosis and suggest that it may be a new therapeutic target in SSc.

Identification of CSK as a systemic sclerosis genetic risk factor through Genome Wide Association Study follow-up.

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.

A GWAS follow-up study reveals the association of the IL12RB2 gene with systemic sclerosis in Caucasian populations.

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 [P(MH)= 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 (P(MH)= 4.84 × 10(-3) OR = 1.12). The second follow-up phase confirmed this finding (P(χ2) = 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 (P(MH)= 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.