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1.  Proteomic analysis of synovial fluid from the osteoarthritic knee: comparison with transcriptome analyses of joint tissues 
Arthritis and rheumatism  2013;65(4):981-992.
Objective
The pathophysiology of the most common joint disease, osteoarthritis (OA), remains poorly understood. Since synovial fluid (SF) bathes joint cartilage and synovium, we reasoned that a comparative analysis of its protein constituents in health and OA could identify pathways involved in joint damage. A proteomic analysis of knee SF from OA patients and control subjects was performed and compared to microarray expression data from cartilage and synovium.
Methods
Age-matched knee SF samples from control subjects, and patients with early- and late-stage OA (n=10 per group) were compared using two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A MS with multiplexed peptide selected reaction monitoring (SRM) assay was used to confirm differential expression of a subset of proteins in an independent OA patient cohort. Proteomic results were analyzed by Ingenuity pathway analysis and compared to published synovial tissue and cartilage mRNA profiles.
Results
66 proteins were differentially present in healthy and OA SF. Three major pathways were identified among these proteins: the acute phase response, and the complement and coagulation pathways. Differential expression of 5 proteins was confirmed by SRM assay. A focused analysis of transcripts corresponding to the differentially present proteins indicates that both synovial and cartilage tissues may contribute to the OA SF proteome.
Conclusion
Proteins involved in the acute phase response, complement and coagulation pathways are differentially regulated in SF of OA patients suggesting they contribute to joint damage. Validation of these pathways and their utility as biomarkers or therapeutic targets in OA is warranted.
doi:10.1002/art.37823
PMCID: PMC3618606  PMID: 23400684
2.  Increased IFNα Activity and Differential Antibody Response in Patients with a History of Lyme Disease and Persistent Cognitive Deficits 
Journal of neuroimmunology  2012;255(1-2):85-91.
Following antibiotic treatment for Lyme disease, some patients report persistent or relapsing symptoms of pain, fatigue, and/or cognitive deficits. Factors other than active infection, including immune abnormalities, have been suggested, but few clues regarding mechanism have emerged. Furthermore, the effect of antibiotic treatment on immune response in affected individuals remains unknown. In this study, a longitudinal analysis of specific immune markers of interest was carried out in patients with a history of Lyme disease and persistent objective memory impairment, prior to and following treatment with either ceftriaxone or placebo. IFNα activity was measured by detection of serum-induced changes in specific target genes, using a functional cell-based assay and quantitative real-time PCR. Level and pattern of antibody reactivity to brain antigens and to Borrelia burgdorferi proteins were analyzed by ELISA and immunoblotting. Sera from the patient cohort induced significantly higher expression of IFIT1 and IFI44 target genes than those from healthy controls, indicating increased IFNα activity. Antibody reactivity to specific brain and borrelial proteins was significantly elevated in affected patients. IFNα activity and antibody profile did not change significantly in response to ceftriaxone. The heightened antibody response implies enhanced immune stimulation, possibly due to prolonged exposure to the organism prior to the initial diagnosis and antibiotic treatment of Lyme disease. The increase in IFNα activity is suggestive of a mechanism contributing to the ongoing neuropsychiatric symptoms.
doi:10.1016/j.jneuroim.2012.10.011
PMCID: PMC3557545  PMID: 23141748
Lyme disease; Borrelia burgdorferi; Post-Lyme disease syndrome; Chronic Lyme; IFNα; Cognitive dysfunction; Antibody
3.  Synovial Fluid from Patients with Early Osteoarthritis Modulates Fibroblast-like Synoviocyte Responses to TLR-4 and TLR-2 Ligands via Soluble CD14 
Arthritis and Rheumatism  2012;64(7):2268-2277.
Objective
Synovial inflammation, a feature of both osteoarthritis (OA) and meniscal injury, is hypothesized to be triggered in part via stimulation of Toll-like receptors (TLRs). We tested whether a TLR-2 or TLR-4 stimulating factor in synovial fluid (SF) from early knee OA patients with meniscal injury could lead to inflammatory activation of synoviocytes.
Methods
SF was obtained from patients with early OA cartilage damage undergoing arthroscopic meniscal procedures. SF was used to stimulate cell lines transfected with TLR-2 or TLR-4, and primary cultures of fibroblast-like synoviocytes (FLS). SF was used either alone or in combination with a TLR-2 stimulus (Pam3Cysk4) or a TLR-4 stimulus (LPS). In blocking experiments, SF was pre-incubated with anti-CD14 antibody.
Results
SF from these patients did not stimulate IL-8 release from TLR transfectants. Compared with SF on its own, SF (0.09–25%) in combination with TLR-2 or TLR-4 ligands resulted in significant augmentation of IL-8 release from both transfectants and primary FLS. Soluble CD14 (sCD14), a co-receptor for TLRs, was measured in early OA SF at levels comparable to advanced OA and rheumatoid arthritis. Blockade with anti-CD14 antibody abolished the ability of SF to augment IL-8 production in response to LPS, and diminished Pam3CysK4 responses.
Conclusions
SF augments FLS responses to TLR-2 and TLR-4 ligands. This effect was largely due to sCD14. Our results demonstrate that sCD14 in the setting of OA and meniscal injury sensitizes FLS to respond to inflammatory stimuli such as TLR ligands.
doi:10.1002/art.34495
PMCID: PMC3386375  PMID: 22492243
Osteoarthritis; inflammation; fibroblast-like synoviocytes; synovitis
4.  Interferon-alpha: a therapeutic target in systemic lupus erythematosus 
Summary
The long history of elevated IFNα in association with disease activity in patients with SLE has taken on high significance in the past decade with accumulating data strongly supporting broad activation of the type I IFN pathway in cells of lupus patients, association of IFN pathway activation with significant clinical manifestations of SLE, and increased disease activity based on validated measures. In addition, a convincing association of IFN pathway activation with the presence of autoantibodies specific for RNA-binding proteins has contributed to delineation of an important role for TLR activation by RNA-containing immune complexes in amplifying innate immune system activation and IFN pathway activation. While the primary triggers of SLE and the IFN pathway remain undefined, rapid progress in lupus genetics is helping to define lupus – associated genetic variants with a functional relationship to IFN production or response in lupus patients. Together, the explosion of data and understanding related to the IFN pathway in SLE have readied the lupus community for translation of those insights to improved patient care. Patience will be needed to allow the required collection of clinical data and biologic specimens across multiple clinical centers that will support the required testing of IFN activity, IFN-inducible gene expression or target chemokine gene products as candidate biomarkers. Meanwhile, promising clinical trials are moving forward to test the safety and efficacy of monoclonal antibody inhibitors of IFNα. Other therapeutic approaches to target the IFN pathway may follow close behind.
doi:10.1016/j.rdc.2009.12.008
PMCID: PMC2843146  PMID: 20202598
Systemic lupus erythematosus; interferon-alpha;; innate immune response
5.  Serum Type I Interferon Activity Is Dependent on Maternal Diagnosis in Anti-SSA/Ro–Positive Mothers of Children With Neonatal Lupus 
Arthritis and rheumatism  2008;58(2):541-546.
Objective
The type I interferon (IFN) pathway is activated in many patients with systemic lupus erythematosus (SLE), and high serum levels of IFN are associated with anti-SSA/Ro autoantibodies. To investigate the clinical features associated with type I IFN production in vivo, we compared serum IFN activity in individuals with anti-SSA/Ro antibodies who were asymptomatic with that in individuals with clinical manifestations of SLE or Sjögren's syndrome (SS).
Methods
Antibody-positive sera from 84 mothers of children with manifestations of neonatal lupus were studied for type I IFN activity, using a functional reporter cell assay. Maternal health status was characterized as asymptomatic, SS, SLE, pauci-SLE, or pauci-SS, based on a screening questionnaire, telephone interview, and review of medical records. The prefix “pauci-” indicates symptoms insufficient for a formal classification of the disease.
Results
Only 4% of asymptomatic mothers had high serum type I IFN activity, compared with 73% with pauci-SLE (P = 5.7 × 10−5), 35% with SLE (P = 0.011), and 32% of patients with SS (P = 0.032). One of the 4 patients with pauci-SS had high levels of IFN. The majority of patients for whom longitudinal data were available had stable type I IFN activity over time, and changes in IFN activity were not clearly accompanied by changes in the clinical diagnosis.
Conclusion
Patients with SLE, patients with pauci-SLE, and patients with SS are more likely to have high serum IFN activity than asymptomatic individuals with SSA/Ro autoantibodies, suggesting that these autoantibodies are insufficient for activation of the type I IFN pathway, and that disease-specific factors are important for type I IFN generation in vivo.
doi:10.1002/art.23191
PMCID: PMC2755051  PMID: 18240214
6.  Increased Serum Type I Interferon Activity in Organ-Specific Autoimmune Disorders: Clinical, Imaging, and Serological Associations 
Background: Activation of the type I interferon (IFN) pathway has been implicated in the pathogenesis of systemic autoimmune disorders but its role in the pathogenesis of organ-specific autoimmunity is limited. We tested the hypothesis that endogenous expression of type I IFN functional activity contributes to the pathogenesis of autoimmune thyroid disease (ATD) and type I diabetes (T1DM).
Methods: We studied 39 patients with ATD and 39 age and sex matched controls along with 88 T1DM patients and 46 healthy matched controls respectively. Available clinical and serological parameters were recorded by chart review, and thyroid ultrasound was performed in 17 ATD patients. Type I IFN serum activity was determined in all subjects using a reporter cell assay. The rs1990760 SNP of the interferon-induced helicase 1 gene was genotyped in ATD patients.
Results: Serum type I IFN activity was increased in patients with ATD and T1DM compared to controls (p-values: 0.002 and 0.04, respectively). ATD patients with high type I IFN serum activity had increased prevalence of antibodies against thyroglobulin (anti-Tg) and cardiopulmonary manifestations compared to those with low IFN activity. Additionally, the presence of micronodules on thyroid ultrasound was associated with higher type I IFN levels. In patients with T1DM, high IFN levels were associated with increased apolipoprotein-B levels.
Conclusion: Serum type I IFN activity is increased in ATD and T1DM and is associated with specific clinical, serological, and imaging features. These findings may implicate type I IFN pathway in the pathogenesis of specific features of organ-specific autoimmunity.
doi:10.3389/fimmu.2013.00238
PMCID: PMC3746787  PMID: 23966997
type I interferon; autoimmune thyroid disease; organ-specific autoimmunity; type I diabetes
7.  Autoimmune Disease Risk Variant of IFIH1 Is Associated with Increased Sensitivity to IFN-α and Serologic Autoimmunity in Lupus Patients 
Increased IFN-α signaling is a heritable risk factor for systemic lupus erythematosus (SLE). IFN induced with helicase C domain 1 (IFIH1) is a cytoplasmic dsRNA sensor that activates IFN-α pathway signaling. We studied the impact of the autoimmune-disease–associated IFIH1 rs1990760 (A946T) single nucleotide polymorphism upon IFN-α signaling in SLE patients in vivo. We studied 563 SLE patients (278 African-American, 179 European-American, and 106 Hispanic-American). Logistic regression models were used to detect genetic associations with autoantibody traits, and multiple linear regression was used to analyze IFN-α–induced gene expression in PBMCs in the context of serum IFN-α in the same blood sample. We found that the rs1990760 T allele was associated with anti-dsDNA Abs across all of the studied ancestral backgrounds (meta-analysis odds ratio = 1.34, p = 0.026). This allele also was associated with lower serum IFN-α levels in subjects who had anti-dsDNA Abs (p = 0.0026). When we studied simultaneous serum and PBMC samples from SLE patients, we found that the IFIH1 rs1990760 T allele was associated with increased IFN-induced gene expression in PBMCs in response to a given amount of serum IFN-α in anti-dsDNA–positive patients. This effect was independent of the STAT4 genotype, which modulates sensitivity to IFN-α in a similar way. Thus, the IFIH1 rs1990760 Tallele was associated with dsDNA Abs, and in patients with anti-dsDNA Abs this risk allele increased sensitivity to IFN-α signaling. These studies suggest a role for the IFIH1 risk allele in SLE in vivo.
doi:10.4049/jimmunol.1100857
PMCID: PMC3304466  PMID: 21705624
8.  Synovial inflammation in patients undergoing arthroscopic meniscectomy: molecular characterization and relationship with symptoms 
Arthritis and Rheumatism  2011;63(2):391-400.
Objective
Traumatic and degenerative meniscal tears have different anatomic features and different proposed etiologies, yet both are associated with development or progression of osteoarthritis (OA). In established OA, synovitis is associated with pain and progression, but a relationship between synovitis and symptoms in isolated meniscal disease has not been reported. Accordingly, we sought to characterize synovial pathology in patients with traumatic meniscal injuries and determine the relationships between inflammation, meniscal and cartilage pathology, and symptoms.
Methods
Thirty-three patients without evidence of OA undergoing arthroscopic meniscectomy for meniscal injuries were recruited. Pain and function were assessed preoperatively; meniscal and cartilage abnormalities were documented at the time of surgery. Inflammation in synovial biopsies was scored and associations between inflammation and clinical outcomes determined. Microarray analysis of synovial tissue was performed and gene expression patterns in patients with or without inflammation compared.
Results
Synovial inflammation was present in 43% of patients and was associated with worse pre-operative pain and function scores, independent of age, gender, or cartilage pathology. Microarray analysis and real-time PCR revealed a chemokine signature in synovial biopsies with increased inflammation scores.
Conclusion
In patients with traumatic meniscal injury undergoing arthroscopic meniscectomy without clinical or radiographic evidence of OA, synovial inflammation occurs frequently and is associated with increased pain and dysfunction. Synovia with increased inflammation scores exhibit a unique chemokine signature. Chemokines may contribute to the development of synovial inflammation in patients with meniscal pathology; they also represent potential therapeutic targets for reducing inflammatory symptoms.
doi:10.1002/art.30137
PMCID: PMC3260472  PMID: 21279996
Meniscectomy; meniscal injury; inflammation; synovium; synovitis
9.  Anti-neural antibody reactivity in patients with a history of Lyme borreliosis and persistent symptoms 
Brain, behavior, and immunity  2010;24(6):1018-1024.
Some Lyme disease patients report debilitating chronic symptoms of pain, fatigue, and cognitive deficits despite recommended courses of antibiotic treatment. The mechanisms responsible for these symptoms, collectively referred to as post-Lyme disease syndrome (PLS) or chronic Lyme disease, remain unclear. We investigated the presence of immune system abnormalities in PLS by assessing the levels of antibodies to neural proteins in patients and controls. Serum samples from PLS patients, post-Lyme disease healthy individuals, patients with systemic lupus erythematosus, and normal healthy individuals were analyzed for anti-neural antibodies by immunoblotting and immunohistochemistry. Anti-neural antibody reactivity was found to be significantly higher in the PLS group than in the post-Lyme healthy (p<0.01) and normal healthy (p<0.01) groups. The observed heightened antibody reactivity in PLS patients could not be attributed solely to the presence of cross-reactive anti-borrelia antibodies, as the borrelial seronegative patients also exhibited elevated anti-neural antibody levels. Immunohistochemical analysis of PLS serum antibody activity demonstrated binding to cells in the central and peripheral nervous systems. The results provide evidence for the existence of a differential immune system response in PLS, offering new clues about the etiopathogenesis of the disease that may prove useful in devising more effective treatment strategies.
doi:10.1016/j.bbi.2010.03.002
PMCID: PMC2897967  PMID: 20227484
post-Lyme disease syndrome; chronic Lyme disease; Borrelia burgdorferi; immune dysregulation; antibody
10.  Imatinib mesylate (Gleevec) in the treatment of diffuse cutaneous systemic sclerosis: results of a 1-year, phase IIa, single-arm, open-label clinical trial 
Annals of the Rheumatic Diseases  2011;70(6):1003-1009.
Objective
To assess the safety and effectiveness of imatinib mesylate in the treatment of diffuse cutaneous systemic sclerosis (dcSSc).
Methods
In this phase IIa, open-label, single-arm clinical trial, 30 patients with dcSSc were treated with imatinib 400 mg daily. Patients were monitored monthly for safety assessments. Modified Rodnan skin scores (MRSS) were assessed every 3 months. Pulmonary function testing, chest radiography, echocardiography and skin biopsies were performed at baseline and after 12 months of treatment.
Results
Twenty-four patients completed 12 months of therapy. 171 adverse events (AE) with possible relation to imatinib were identified; 97.6% were grade 1 or 2. Twenty-four serious AE were identified, two of which were attributed to study medication. MRSS decreased by 6.6 points or 22.4% at 12 months (p=0.001). This change was evident starting at the 6-month time point (Δ=−4.5; p<0.001) and was seen in patients with both early and late-stage disease. Forced vital capacity (FVC) improved by 6.4% predicted (p=0.008), and the diffusion capacity remained stable. The improvement in FVC was significantly greater in patients without interstitial lung disease. Health-related quality of life measures improved or remained stable. Blinded dermatopathological analysis confirmed a significant decrease in skin thickness and improvement in skin morphology.
Conclusions
Treatment with imatinib was tolerated by most patients in this cohort. Although AE were common, most were mild to moderate. In this open-label experience, improvements in skin thickening and FVC were observed. Further investigation of tyrosine kinase inhibition for dcSSc in a double-blind randomised placebo controlled trial is warranted.
ClinicalTrials.gov, NCT00555581
doi:10.1136/ard.2010.143974
PMCID: PMC3086082  PMID: 21398330
11.  A loss-of-function variant of the antiviral molecule MAVS is associated with a subset of systemic lupus patients 
EMBO Molecular Medicine  2011;3(3):142-152.
Dysregulation of the antiviral immune response may contribute to autoimmune diseases. Here, we hypothesized that altered expression or function of MAVS, a key molecule downstream of the viral sensors RIG-I and MDA-5, may impair antiviral cell signalling and thereby influence the risk for systemic lupus erythematosus (SLE), the prototype autoimmune disease. We used molecular techniques to screen non-synonymous single nucleotide polymorphisms (SNPs) in the MAVS gene for functional significance in human cell lines and identified one critical loss-of-function variant (C79F, rs11905552). This SNP substantially reduced expression of type I interferon (IFN) and other proinflammatory mediators and was found almost exclusively in the African-American population. Importantly, in African-American SLE patients, the C79F allele was associated with low type I IFN production and absence of anti-RNA-binding protein autoantibodies. These serologic associations were not related to a distinct, functionally neutral, MAVS SNP Q198K. Hence, this is the first demonstration that an uncommon genetic variant in the MAVS gene has a functional impact upon the anti-viral IFN pathway in vivo in humans and is associated with a novel sub-phenotype in SLE. This study demonstrates the utility of functional data in selecting rare variants for genetic association studies, allowing for fewer comparisons requiring statistical correction and for alternate lines of evidence implicating the particular variant in disease.
doi:10.1002/emmm.201000120
PMCID: PMC3395111  PMID: 21268286
cell signalling; immune response; lupus; polymorphisms; virus
12.  Association between the response to TNF-antagonists and plasma type I interferon activity and interferon-beta/alpha ratios in rheumatoid arthritis patients: a post-hoc analysis of a predominantly Hispanic Cohort 
Arthritis and rheumatism  2010;62(2):392.
Objective
Despite substantial clinical efficacy of tumor necrosis factor-α (TNF)-antagonist therapy in patients with rheumatoid arthritis (RA), some patients respond poorly to such agents. Since an interferon (IFN) signature is variably expressed among RA patients, we investigated whether plasma type I IFN activity might predict response to TNF-antagonist therapy.
Methods
RA patients (n=35), the majority Hispanic, from a single center were evaluated prior to and following initiation of TNF-antagonist therapy. As controls, 12 RA patients from the same center who were not treated with a TNF antagonist were studied. Plasma type I IFN activity was measured using a reporter cell assay and disease status assessed using the Disease Activity Score (DAS28). IL-1 receptor antagonist (IL-1ra) levels were determined in baseline samples using a commercial ELISA. The clinical response was classified according to the European League against Rheumatism (EULAR) RA improvement criteria.
Results
Plasma type I IFN activity at baseline was significantly associated with clinical response (OR=1.36, CI: 1.05-1.76, p=0.02), with high baseline IFN activity associated with a good response. Changes in DAS28 scores were greater among patients with a baseline plasma IFNβ/α ratio >0.8 (indicating elevated plasma IFNβ levels). Consistent with the capacity of IFNβ to induce IL-1ra, elevated baseline IL-1ra levels were associated with better therapeutic outcomes (OR=1.82, CI: 1.1-3.29, p=0.027).
Conclusion
Plasma type I IFN activity, IFNβ/α ratio, and IL-1ra levels were predictive of therapeutic response in TNF-antagonist-treated RA patients, indicating that those parameters might define clinically meaningful subgroups of RA patients with distinct responses to therapeutic agents.
doi:10.1002/art.27226
PMCID: PMC2821991  PMID: 20112385
13.  Type I interferon in organ-targeted autoimmune and inflammatory diseases 
Arthritis Research & Therapy  2010;12(Suppl 1):S5.
A significant role for IFNα in the pathogenesis of systemic lupus erythematosus is well supported, and clinical trials of anti-IFNα monoclonal antibodies are in progress in this disease. In other autoimmune diseases characterized by substantial inflammation and tissue destruction, the role of type I interferons is less clear. Gene expression analysis of peripheral blood cells from patients with rheumatoid arthritis and multiple sclerosis demonstrate an interferon signature similar to but less intense than that seen in patients with lupus. In both of those diseases, presence of the interferon signature has been associated with more significant clinical manifestations. At the same time, evidence supports an anti-inflammatory and beneficial role of IFNβ locally in the joints of patients with rheumatoid arthritis and in murine arthritis models, and many patients with multiple sclerosis show a clinical response to recombinant IFNβ. As can also be proposed for type I diabetes mellitus, type I interferon appears to contribute to the development of autoimmunity and disease progression in multiple autoimmune diseases, while maintaining some capacity to control established disease - particularly at local sites of inflammation. Recent studies in both rheumatoid arthritis and multiple sclerosis suggest that quantification of type I interferon activity or target gene expression might be informative in predicting responses to distinct classes of therapeutic agents.
doi:10.1186/ar2886
PMCID: PMC2991778  PMID: 21303493
14.  Exposure to nuclear antigens contributes to the induction of humoral autoimmunity during TNF alpha blockade 
Annals of the rheumatic diseases  2008;68(6):1022-1029.
Objective
type I interferons and apoptotic particles contribute to anti-nuclear autoimmunity in experimental models. We assessed if similar mechanisms contribute to break peripheral B cell tolerance in humans by studying the induction of anti-nuclear antibodies by TNF blockade in spondyloarthritis (SpA)
Methods
We studied 40 SpA patients treated with infliximab or etanercept and 20 renal cell carcinoma patients treated with sorafenib. Serum anti-nucleosome IgM and nucleosomes were measured by ELISA. Type I IFN serum activity was measured using a functional reporter cell assay. Synovial apoptosis was assessed by TUNEL assay and anti-active caspase-3 immunostaining. Complement was measured by nephelometry.
Results
Despite similar clinical improvement and reduction of synovial inflammation, anti-nucleosome IgM were induced by infliximab but not etanercept. This induction did not correlate with type I IFN activity, which was transiently downmodulated by infliximab but persistently upregulated by etanercept. In contrast, anti-nucleosome IgM levels did correlate with serum nucleosome levels, which were significantly upregulated by infliximab but not by etanercept treatment. This increase in serum nucleosome levels was not directly related to massive cell death, but rather to a decrease of complement 3 and 4 serum levels during infliximab treatment.
Conclusion
Infliximab and etanercept have a differential effect on both type I IFN activity and nucleosome levels. However, only elevated serum nucleosomes relate to the induction of anti-nucleosome antibodies after infliximab treatment.
doi:10.1136/ard.2008.093724
PMCID: PMC2681782  PMID: 18625621
Spondyloarthritis; TNF blockade; humoral autoimmunity; nucleosomes; type I interferon
15.  Developments in the clinical understanding of lupus 
Advances in genetics and new understanding of the molecular pathways that mediate innate and adaptive immune system activation, along with renewed focus on the role of the complement system as a mediator of inflammation, have stimulated elaboration of a scheme that might explain key mechanisms in the pathogenesis of systemic lupus erythematosus. Clinical observations identifying important comorbidities in patients with lupus have been a recent focus of research linking immune mechanisms with clinical manifestations of disease. While these advances have identified rational and promising targets for therapy, so far the therapeutic trials of new biologic agents have not met their potential. Nonetheless, progress in understanding the underlying immunopathogenesis of lupus and its impact on clinical disease has accelerated the pace of clinical research to improve the outcomes of patients with systemic lupus erythematosus.
doi:10.1186/ar2762
PMCID: PMC2787273  PMID: 19849817
16.  Activation of mTOR controls the loss of TCRζ in lupus T cells through HRES-1/Rab4-regulated lysosomal degradation 
Persistent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T-cell activation and death pathway selection in systemic lupus erythematosus. Treatment with rapamycin, which effectively controls disease activity, normalizes CD3/CD28-induced calcium fluxing but fails to influence MHP, suggesting that altered calcium fluxing is downstream or independent of mitochondrial dysfunction. Here, we show that activity of the mammalian target of rapamycin (mTOR), which is a sensor of the mitochondrial transmembrane potential, is increased in lupus T cells. Activation of mTOR causes the over-expression of the Rab5A and HRES-1/Rab4 small GTPases that regulate endocytic recycling of surface receptors. Pull-down studies revealed a direct interaction of HRES-1/Rab4 with the T-cell receptor/CD3ζ chain (TCRζ). Importantly, the deficiency of the TCRζ chain and Lck and compensatory upregulation of the Fcε receptor type I γ chain (FcεRIγ) and Syk, which mediate enhanced calcium fluxing in lupus T cells, was reversed in patients treated with rapamcyin in vivo. Knockdown of HRES-1/Rab4 by siRNA and inhibitors of lysosomal function augmented TCRζ protein levels. The results suggest that activation of mTOR causes the loss of TCRζ in lupus T cells through HRES-1/Rab4-dependent lysosomal degradation.
doi:10.4049/jimmunol.0803600
PMCID: PMC2676112  PMID: 19201859
17.  Cutting Edge: Autoimmune Disease Risk Variant of STAT4 Confers Increased Sensitivity to IFN-α in Lupus Patients In Vivo1 
Increased IFN-α signaling is a primary pathogenic factor in systemic lupus erythematosus (SLE). STAT4 is a transcription factor that is activated by IFN-α signaling, and genetic variation of STAT4 has been associated with risk of SLE and rheumatoid arthritis. We measured serum IFN-α activity and simultaneous IFN-α-induced gene expression in PBMC in a large SLE cohort. The risk variant of STAT4 (T allele; rs7574865) was simultaneously associated with both lower serum IFN-α activity and greater IFN-α-induced gene expression in PBMC in SLE patients in vivo. Regression analyses confirmed that the risk allele of STAT4 was associated with increased sensitivity to IFN-α signaling. The IFN regulatory factor 5 SLE risk genotype was associated with higher serum IFN-α activity; however, STAT4 showed dominant influence on the sensitivity of PBMC to serum IFN-α. These data provide biologic relevance for the risk variant of STAT4 in the IFN-α pathway in vivo.
PMCID: PMC2716754  PMID: 19109131
18.  Rheumatoid Arthritis is Independently Associated with Increased Left Ventricular Mass but not Reduced Ejection Fraction 
Arthritis and rheumatism  2009;60(1):22-29.
Background
Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with premature atherosclerosis, vascular stiffening, and heart failure. Whether RA is associated with underlying structural and functional abnormalities of the left ventricle (LV) is poorly understood.
Methods and Results
89 patients with RA without clinical cardiovascular disease and 89 healthy matched controls underwent echocardiography, carotid ultrasonography, and radial tonometry to measure arterial stiffness. RA patients and controls were similar in body size, hypertension and diabetes status, and cholesterol. LV diastolic diameter (4.92 vs. 4.64 cm, p <0.001), mass (136.9 vs. 121.7 g, p = 0.001 or 36.5 vs. 32.9 g/m 2.7, p = 0.01), ejection fraction (EF) (71% vs. 67%, p <0.001), and prevalence of LV hypertrophy (LVH) (18% vs. 6.7%, p = 0.023) were all higher among RA patients. In multivariate analysis, presence of RA (p = 0.004) was an independent correlate of LV mass. Furthermore, RA was independently associated with the presence of LVH (OR 4.14, [95% CI 1.24-13.80; p=0.021]). Among RA patients, age at diagnosis and disease duration were independently related to LV mass. RA patients with LVH were older and had higher systolic pressure, damage index score, C-reactive protein, homocysteine and arterial stiffness index compared to those without LVH.
Conclusion
RA is associated with increased LV mass. Disease duration is independently related to increased LV mass, suggesting a pathophysiological link between chronic inflammation and LVH. In contrast, LV systolic function is preserved in RA patients indicating that systolic dysfunction is not an intrinsic feature of RA.
doi:10.1002/art.24148
PMCID: PMC2626148  PMID: 19116901
19.  PTPN22 C1858T Polymorphism is Associated with Skewing of Cytokine Profiles Toward High IFN-α Activity and Low TNF-α in Lupus Patients 
Arthritis and rheumatism  2008;58(9):2818-2823.
Background
The C1858T polymorphism in PTPN22 has been associated with risk of systemic lupus erythematosus (SLE), as well as multiple other autoimmune diseases. We have previously shown that high serum interferon alpha (IFN-α) activity is a heritable risk factor for SLE, and we hypothesized that the PTPN22 risk variant may shift serum cytokine profiles to higher IFN-α activity resulting in risk of disease.
Methods
IFN-α was measured in 143 SLE patients using a functional reporter cell assay, and TNF-α was measured with ELISA. The rs2476601 SNP in PTPN22 (C1858T) was genotyped in the same patients. Patients were grouped using a clustering algorithm into four cytokine groups (IFN-α predominant, IFN-α and TNF-α correlated, TNF-α predominant, and IFN-α and TNF-α both low).
Results
SLE patients carrying the risk allele of PTPN22 had higher serum IFN-α activity than patients lacking the risk allele (p=0.027). TNF-α levels were lower in risk allele carriers (p=0.030), and the risk allele was more common in patients with an IFN-α predominant or IFN-α and TNF-α correlated cytokine profile as compared to patients with TNF-α predominance or both cytokines low (p=0.002). 25% of male patients carried the risk allele, compared to 10% of female patients (p=0.02), however cytokine skewing was similar in both sexes.
Conclusions
The autoimmune disease risk allele of PTPN22 is associated with skewing of serum cytokine profiles toward higher IFN-α activity and lower TNF-α in SLE patients in vivo. This serum cytokine pattern may be relevant in other autoimmune diseases associated with the PTPN22 risk allele.
doi:10.1002/art.23728
PMCID: PMC2621106  PMID: 18759295
20.  Age- and Sex-Related Patterns of Serum Interferon-α Activity in Lupus Families 
Arthritis and rheumatism  2008;58(7):2113-2119.
Objective
Interferon-α (IFNα) levels are elevated in many patients with systemic lupus erythematosus (SLE) and may play a primary role in its pathogenesis. The purpose of this study was to determine whether serum IFNα activity in SLE patients and their healthy first-degree relatives is highest in early adulthood, when the incidence of SLE is greatest.
Methods
Serum samples from 315 SLE patients, 359 healthy first-degree relatives, and 141 healthy unrelated donors were measured for IFNα activity using a functional reporter cell assay. IFNα activity was analyzed in relation to age, and subgroups with high levels of IFNα activity were identified within the large data sets using a Mann-Whitney sliding window segmentation algorithm. The significance of each subgrouping was ranked by Kruskal-Wallis testing.
Results
Age was inversely correlated with IFNα activity in female SLE patients (r = −0.20, P = 0.001) as well as their healthy female first-degree relatives (r = −0.16, P = 0.02). In male patients and their healthy male first-degree relatives, there was no significant overall correlation between age and serum IFNα activity. The segmentation algorithm revealed significantly increased IFNα activity between the ages of 12 and 22 years in female SLE patients and between the ages of 16 and 29 years in male SLE patients. Both male and female healthy first-degree relatives had significantly decreased IFNα activity after the age of 50 years.
Conclusion
Serum IFNα activity is higher in younger individuals in the SLE family cohorts, and this tendency is accentuated in affected individuals. This age-related pattern of IFNα activity may contribute to the increased incidence of SLE in early adulthood, and interestingly, males and females had similar age-related patterns of IFNα activity.
doi:10.1002/art.23619
PMCID: PMC2729701  PMID: 18576315
21.  Association of the IRF5 Risk Haplotype With High Serum Interferon-α Activity in Systemic Lupus Erythematosus Patients 
Arthritis and rheumatism  2008;58(8):2481-2487.
Objective
A haplotype of the interferon regulatory factor 5 (IRF5) gene has been associated with the risk of developing systemic lupus erythematosus (SLE), and our previous studies have demonstrated that high levels of serum interferon-α (IFNα) activity are a heritable risk factor for SLE. The aim of this study was to determine whether the IRF5 SLE risk haplotype mediates the risk of SLE by predisposing patients to the development of high levels of serum IFNα activity.
Methods
IFNα levels in 199 SLE patients of European and Hispanic ancestry were measured with a sensitive functional reporter cell assay. The rs2004640, rs3807306, rs10488631, and rs2280714 single-nucleotide polymorphisms (SNPs) in IRF5 were genotyped in these patients. Haplotypes were categorized as SLE risk, neutral, or protective based on published data.
Results
SLE patients with risk/risk and risk/neutral IRF5 genotypes had higher serum IFNα activity than did those with protective/protective and neutral/protective genotypes (P = 0.025). This differential effect of IRF5 genotype on serum IFNα levels was driven largely by SLE patients who were positive for either anti–RNA binding protein (anti-RBP) or anti–double-stranded DNA (anti-dsDNA) autoantibodies (P = 0.012 for risk/risk or risk/neutral versus protective/protective or neutral/protective). The rs3807306 genotype was independently associated with high serum IFNα in this autoantibody group. We found no difference in IFNα activity according to IRF5 genotype in patients lacking either type of autoantibody or in patients positive for both classes of autoantibody.
Conclusion
The IRF5 SLE risk haplotype is associated with higher serum IFNα activity in SLE patients, and this effect is most prominent in patients positive for either anti-RBP or anti-dsDNA autoantibodies. This study demonstrates the biologic relevance of the SLE risk haplotype of IRF5 at the protein level.
doi:10.1002/art.23613
PMCID: PMC2621107  PMID: 18668568
22.  Interferon-induced versus chemokine transcripts as lupus biomarkers 
Compelling support for a central role for interferon-alpha in lupus pathogenesis has led to a new focus on the role of innate immune system activation in the generation of pathogenic mediators. These insights have been extended in translational studies of patients with well-characterized disease activity and clinical manifestations in order to identify informative molecular biomarkers. Chemokines are among the interferon-inducible genes, and new data support an association between the expression of chemokines and both lupus disease activity and organ damage. Longitudinal studies that relate molecular biomarkers to disease activity will be needed to validate these promising data and establish a sensitive measure of change for interventional studies and patient care.
doi:10.1186/ar2559
PMCID: PMC2656240  PMID: 19183425
23.  Early Growth Response-1 Is Required for CD154 Transcription1 
CD154 (CD40 ligand) expression on CD4 T cells is normally tightly controlled, but abnormal or dysregulated expression of CD154 has been well documented in autoimmune diseases, such as systemic lupus erythematosus. Beyond regulation by NFAT proteins, little is known about the transcriptional activation of the CD154 promoter. We identified a species-conserved purine-rich sequence located adjacent to the CD154 transcriptional promoter proximal NFAT site, which binds early growth response (Egr) transcription factors. Gel shift assays and chromatin immunoprecipitation assays reveal that Egr-1, Egr-3, and NFAT1 present in primary human CD4 T cells are capable of binding this combinatorial site in vitro and in vivo, respectively. Multimerization of this NFAT/Egr sequence in the context of a reporter gene demonstrates this sequence is transcriptionally active upon T cell activation in primary human CD4 T cells. Overexpression of Egr-1, but not Egr-3, is capable of augmenting transcription of this reporter gene as well as that of an intact CD154 promoter. Conversely, overexpression of small interfering RNA specific for Egr-1 in primary human CD4 T cells inhibits CD154 expression. Similarly, upon activation, CD154 message is notably decreased in splenic CD4 T cells from Egr-1-deficient mice compared with wild-type controls. Our data demonstrate that Egr-1 is required for CD154 transcription in primary CD4 T cells. This has implications for selective targeting of Egr family members to control abnormal expression of CD154 in autoimmune diseases such as systemic lupus erythematosus.
PMCID: PMC1424665  PMID: 16393964
24.  Microarray analysis of gene expression in lupus 
Arthritis Research & Therapy  2003;5(6):279-287.
Recent advances in the study of global patterns of gene expression with the use of microarray technology, coupled with data analysis using sophisticated statistical algorithms, have provided new insights into pathogenic mechanisms of disease. Complementary and reproducible data from multiple laboratories have documented the feasibility of analysis of heterogeneous populations of peripheral blood mononuclear cells from patients with rheumatic diseases through use of this powerful technology. Although some patterns of gene expression, including increased expression of immune system cell surface activation molecules, confirm previous data obtained with other techniques, some novel genes that are differentially expressed have been identified. Most interesting is the dominant pattern of interferon-induced gene expression detected among blood mononuclear cells from patients with systemic lupus erythematosus and juvenile dermatomyositis. These data are consistent with longstanding observations indicating increased circulating interferon-α in the blood of patients with active lupus, but draw attention to the dominance of the interferon pathway in the hierarchy of gene expression pathways implicated in systemic autoimmunity.
doi:10.1186/ar1015
PMCID: PMC333417  PMID: 14680503
gene expression; interferon; microarray; statistical algorithms; systemic lupus erythematosus
25.  IRF5 haplotypes demonstrate diverse serological associations which predict serum interferon alpha activity and explain the majority of the genetic association with systemic lupus erythematosus 
Annals of the Rheumatic Diseases  2011;71(3):463-468.
Objective
High serum interferon α (IFNα) activity is a heritable risk factor for systemic lupus erythematosus (SLE). Auto-antibodies found in SLE form immune complexes which can stimulate IFNα production by activating endosomal Toll-like receptors and interferon regulatory factors (IRFs), including IRF5. Genetic variation in IRF5 is associated with SLE susceptibility; however, it is unclear how IRF5 functional genetic elements contribute to human disease.
Methods
1034 patients with SLE and 989 controls of European ancestry, 555 patients with SLE and 679 controls of African–American ancestry, and 73 patients with SLE of South African ancestry were genotyped at IRF5 polymorphisms, which define major haplotypes. Serum IFNα activity was measured using a functional assay.
Results
In European ancestry subjects, anti-double-stranded DNA (dsDNA) and anti-Ro antibodies were each associated with different haplotypes characterised by a different combination of functional genetic elements (OR > 2.56, p >003C; 1.9×10−14 for both). These IRF5 haplotype-auto-antibody associations strongly predicted higher serum IFNα in patients with SLE and explained > 70% of the genetic risk of SLE due to IRF5. In African–American patients with SLE a similar relationship between serology and IFNα was observed, although the previously described European ancestry-risk haplotype was present at admixture proportions in African–American subjects and absent in African patients with SLE.
Conclusions
The authors define a novel risk haplotype of IRF5 that is associated with anti-dsDNA antibodies and show that risk of SLE due to IRF5 genotype is largely dependent upon particular auto-antibodies. This suggests that auto-antibodies are directly pathogenic in human SLE, resulting in increased IFNα in cooperation with particular combinations of IRF5 functional genetic elements.
SLE is a systemic autoimmune disorder affecting multiple organ systems including the skin, musculoskeletal, renal and haematopoietic systems. Humoral autoimmunity is a hallmark of SLE, and patients frequently have circulating auto-antibodies directed against dsDNA, as well as RNA binding proteins (RBP). Anti-RBP autoantibodies include antibodies which recognize Ro, La, Smith (anti-Sm), and ribonucleoprotein (anti-nRNP), collectively referred to as anti-retinol-binding protein). Anti-retinol-binding protein and anti-dsDNA auto-antibodies are rare in the healthy population.1 These auto-antibodies can be present in sera for years preceding the onset of clinical SLE illness2 and are likely pathogenic in SLE.34
doi:10.1136/annrheumdis-2011-200463
PMCID: PMC3307526  PMID: 22088620

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