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1.  Type I and II interferon signatures in Sjogren's syndrome pathogenesis: Contributions in distinct clinical phenotypes and Sjogren's related lymphomagenesis 
Journal of autoimmunity  2015;63:47-58.
Both type I and II interferons (IFNs) have been implicated in the pathogenesis of Sjogren's syndrome (SS). We aimed to explore the contribution of type I and II IFN signatures in the generation of distinct SS clinical phenotypes including lymphoma development. Peripheral blood (PB) from SS patients (n=31), SS patients complicated by lymphoma (n=13) and healthy controls (HC, n=30) were subjected to real-time PCR for 3 interferon inducible genes (IFIGs) preferentially induced by type I IFN, 2 IFIGs preferentially induced by IFNγ as well as for IFNα and IFNγ genes. The same analysis was performed in minor salivary gland tissues (MSG) derived from 31 SS patients, 10 SS-lymphoma patients and 17 sicca controls (SC). In PB and MSG tissues, overexpression of both type I and type II IFIGs was observed in SS patients versus HC and SC, respectively, with a predominance of type I IFN signature in PB and a type II IFN signature in MSG tissues. In SS-lymphoma MSG tissues, lower IFNα, but higher IFNγ and type II IFIG transcripts compared to both SS and SC were observed. In receiver operating characteristic curve analysis, IFNγ/IFNα mRNA ratio in MSG tissues showed the best discrimination for lymphoma development. Discrete expression patterns of type I and II IFN signatures might be related to distinct SS clinical phenotypes. Additionally, IFNγ/IFNα mRNA ratio in diagnostic salivary gland biopsies is proposed as a novel histopathological biomarker for the prediction of in situ lymphoma development in the setting of SS.
doi:10.1016/j.jaut.2015.07.002
PMCID: PMC4564326  PMID: 26183766
Type I interferon; type II interferon; Sjogren's syndrome; lymphomagenesis; B cell activating factor
2.  Targeting of Type I Interferon in Systemic Autoimmune Diseases 
Increased blood levels of type I interferon (IFN-I) and expression of a broad signature of gene transcripts that reflect induction by IFN-I are observed in many patients with systemic autoimmune diseases, and that pattern is most striking in systemic lupus erythematosus (SLE). Persistent production of IFN-α, the most abundant subtype measured in these patients, is an important feature of the immunopathogenesis of lupus and has stimulated current efforts to develop and test therapeutics that either block IFNI or its receptor directly or target components of the IFN-I pathway involved in induction of or response to IFN-I. This review will describe data from animal models of chronic viral infection, examples of lupus-like syndromes associated with single-gene mutations that impact the IFN-I pathway, and longitudinal studies from lupus patients to support the rationale for therapeutic targeting of the IFN-I pathway in SLE. However, the complexity of IFN-I regulation and the diversity of its effects on immune system function suggest that the definitive demonstration of that pathway as a valid and productive therapeutic target will only come from clinical trials of agents tested in patients with systemic autoimmune disease, with lupus patients likely to be the most informative.
doi:10.1016/j.trsl.2014.10.005
PMCID: PMC4306610  PMID: 25468480
3.  Felty’s Syndrome Autoantibodies Bind to Deiminated Histones and Neutrophil Extracellular Chromatin Traps 
Arthritis and rheumatism  2011;64(4):982-992.
Objective
To test the hypothesis that autoantigen modifications by peptidylarginine deiminase type 4 (PAD-4) increase immunoreactivity.
Methods
We assembled sera from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Felty’s syndrome (FS), and antineutrophil cytoplasmic antibody–associated vasculitides (AAVs), as well as sera from control subjects without autoimmune diseases. The sera were tested for binding to activated neutrophils, deiminated histones, and neutrophil extracellular chromatin traps (NETs). IgG binding to lipopolysaccharide-activated neutrophils was assessed with confocal microscopy, and binding to in vitro–deiminated histones was measured using enzyme-linked immunosorbent assay (ELISA) and Western blotting. In addition, we quantitated histone deimination in freshly isolated neutrophils from the blood of patients and control subjects.
Results
Increased IgG reactivity with activated neutrophils, particularly binding to NETs, was paralleled by preferential binding to deiminated histones over nondeiminated histones by ELISA in a majority of sera from FS patients but only in a minority of sera from SLE and RA patients. Immunoblotting revealed autoantibody preference for deiminated histones H3, H4, and H2A in most FS patients and in a subset of SLE and RA patients. In patients with AAVs, serum IgG preferentially bound nondeiminated histones over deiminated histones. Increased levels of deiminated histones were detected in neutrophils from RA patients.
Conclusion
Circulating autoantibodies in FS are preferentially directed against PAD-4–deiminated histones and bind to activated neutrophils and NETs. Thus, increased reactivity with modified autoantigens in FS implies a direct contribution of neutrophil activation and the production of NET-associated nuclear autoantigens in the initiation or progression of FS.
doi:10.1002/art.33432
PMCID: PMC4729190  PMID: 22034172
4.  Interferon-α and angiogenic dysregulation in pregnant lupus patients destined for preeclampsia 
Objective
To investigate whether elevated IFN-α early in pregnancy is associated with poor pregnancy outcomes and examine its relationship to angiogenic imbalance.
Methods
Women were enrolled in a case-control longitudinal study of lupus pregnancies. Serum samples obtained monthly through pregnancy were assayed for IFN-α and for antiangiogenic factor, sFlt1, and proangiogenic factor, (PlGF). Each of 28 SLE patients with poor pregnancy outcome was matched to an SLE patient with an uncomplicated pregnancy and to a pregnant healthy control. The effects of IFN-α and/or sFlt1 on-human endothelial cells and endothelial-trophoblast interactions was assessed.
Results
Compared to SLE patients with uncomplicated pregnancies, patients with preeclampsia had increased IFN-α before clinical symptoms. Non-autoimmune patients destined for preeclampsia did not have increased IFN-α. In SLE patients with low IFN-α, marked angiogenic imbalance (higher sFlt1, lower PlGF and higher sFlt1/PlGF ratios) precedes maternal manifestations of preeclampsia, whereas in SLE with high IFN-α, preeclampsia occurs without evidence of systemic angiogenic imbalance. Treatment of human endothelial cells with sFlt1 induced expression of sFlt1 mRNA, and IFN-α dramatically amplified responses to sFlt1. In a model of spiral artery transformation, only IFN-α and sFlt1 together disrupted the ability of trophoblast cells to remodel endothelial tube structures.
Conclusions
Our studies identify a new mechanism by which IFN-α induces an antiangiogenic milieu, increases the sensitivity of endothelial cells to sFlt1, and suggest that elevated IFN-α may contribute to pathogenesis of preeclampsia in some SLE pregnancies.
doi:10.1002/art.39029
PMCID: PMC4380868  PMID: 25603823
5.  Advances in Understanding the Role of Type I Interferons in SLE 
Current opinion in rheumatology  2014;26(5):467-474.
Purpose of Review
Advances in understanding the genetic and molecular basis of innate immune system activation and function have supported the hypothesis that type I interferons (IFN-I), essential mediators of anti-viral host defense, are central contributors to the pathogenesis of systemic lupus erythematosus (SLE). This review addresses recent data that support the rationale for therapeutic targeting of the IFN-I pathway in SLE.
Recent Findings
New insights into mechanisms of cell-intrinsic innate immune system activation, driven by endogenous virus-like nucleic acids and potentially modified by environmental stressors, provide a model for induction of IFN-I that may precede clinically apparent autoimmunity in patients with lupus. Further amplification of IFN-α production, induced by nucleic acid-containing immune complexes that activate endosomal Toll-like receptors, augments and sustains immune system activation, autoimmunity and tissue damage.
Summary
As demonstrated in murine studies of persistent virus infection accompanied by sustained production of IFN-I, blockade of the IFN-I pathway may reverse the immune dysregulation and tissue damage that are essential features of the immunopathogenesis of SLE. Recent research progress has identified numerous therapeutic targets, and specific candidate therapeutics relevant to the IFN-I pathway are under investigation.
doi:10.1097/BOR.0000000000000087
PMCID: PMC4280994  PMID: 25010440
Systemic lupus erythematosus; type I interferons; interferon-α; Toll-like receptors; cytoplasmic sensors; autoimmunity; long interspersed nuclear elements
6.  Type I Interferon in the Pathogenesis of Lupus 
Investigations of patients with systemic lupus erythematosus (SLE) have applied insights from studies of the innate immune response to define type I interferon (IFN-I), with IFN-α the dominant mediator, as central to the pathogenesis of this prototype systemic autoimmune disease. Genetic association data identify regulators of nucleic acid degradation and components of TLR-independent, endosomal TLR-dependent, and IFN-I signaling pathways as contributors to lupus disease susceptibility. Together with a gene expression signature characterized by IFNI-induced gene transcripts in lupus blood and tissue, those data support the conclusion that many of the immunologic and pathologic features of this disease are a consequence of a persistent self-directed immune reaction driven by IFN-I and mimicking a sustained anti-virus response. This expanding knowledge of the role of IFN-I and the innate immune response suggests candidate therapeutic targets that are being tested in lupus patients.
doi:10.4049/jimmunol.1002795
PMCID: PMC4083591  PMID: 24907379
7.  Ongoing Immunoglobulin Class Switch DNA Recombination in Lupus B Cells: Analysis of Switch Regulatory Regions 
Autoimmunity  2004;37(0):431-443.
Inflammation and tissue damage in systemic lupus erythematosus (SLE) are mediated by class-switched autoantibodies reactive with nucleic acids, nucleic acid-binding proteins, phospholipids and other self-antigens. While some healthy individuals produce IgM antibodies with specificities similar to those of lupus patients, immunoglobulin class switching to mature downstream isotypes appears to be required for the generation of pathogenic autoantibodies. To characterize the cellular and molecular basis of pathogenic autoantibody production in SLE, we studied the capacity of peripheral blood B cells of naïve phenotype from patients with SLE, rheumatoid arthritis (RA) or healthy control subjects to spontaneously switch to IgG and IgA. In addition, we determined the DNA sequences of the upstream evolutionary conserved sequence (ECS)-Iγ promoter regulatory regions that control germline IH-CH transcription and class switch DNA recombination (CSR) to IgG1, IgG2 and IgG4. IgM+IgD+ B cells from patients with SLE, but not those from RA or healthy control subjects, underwent spontaneous CSR, as assessed by expression of germline Iγ1-Cγ1, Iγ2-Cγ2, Iγ3-Cγ3, Iγ4-Cγ4 and Iα1-Cα1 transcripts, mature (switched) VHDJH-Cγ1, VHDJH-Cγ2, VHDJH-Cγ3 and VHDJH-Cα1 transcripts and secreted IgG and IgA. Although polymorphic DNA sequences were identified in the ECS-Iγ1, ECS-Iγ2 and ECS-Iγ4 promoter regions, the transcription factor-binding sites that mediate germline Iγ-Cγ transcription were conserved in patients and controls. However, distinct patterns of nuclear protein binding to an ECS-Iγ promoter sequence that contains both positive and negative regulatory elements were observed in SLE patients and controls. These results support a role for exogenous signals, such as through CD40 ligation, rather than altered genomic sequence, in the increased production of class switched autoantibodies in SLE.
doi:10.1080/08916930400010611
PMCID: PMC4624307  PMID: 15621569
Immunoglobulin; DNA; Lupus B cells; SLE; Polymorphism
8.  Nilotinib (Tasigna™) in the treatment of early diffuse systemic sclerosis: an open-label, pilot clinical trial 
Introduction
Tyrosine kinase inhibitors (TKI) are medications of interest in the treatment of Systemic Sclerosis (SSc) because of their ability to inhibit pathways involved in fibrosis. In this open-label pilot trial, our objectives were to assess the safety, efficacy, and molecular change associated with treatment of patients with diffuse cutaneous (dc)SSc with the TKI nilotinib (Tasigna™).
Methods
Ten adult patients with early dcSSc were treated with nilotinib. Primary endpoints were safety and change in modified Rodnan Skin Score (MRSS) after 6 months. Lesional skin biopsies at baseline, 6 and 12 months of treatment were assessed by histopathology, immunohistochemistry, and DNA microarray.
Results
Patients had early and active dcSSc with median disease duration of 0.7 years (range 0.5, 1.7) and increasing MRSS in the month prior to baseline (mean +2.9, p=0.02). Seven out of ten patients completed 6 and 12 months of treatment. Seventy-one adverse events (AEs) including 2 serious AEs were observed, and 92 % of AEs were grade 1-2. Two patients discontinued the medication due to mild QTc prolongation. MRSS improved by a mean of 4.2 points (16 %) at 6 months and by 6.3 points (23 %) at 12 months in the 7 completers, p=0.02 and 0.01, respectively. Patients with a decrease in MRSS >20 % from baseline at 12 months (classified as improvers) had significantly higher expression of transforming growth factor beta receptor (TGFBR) and platelet-derived growth factor receptor beta (PDGFRB) signaling genes at baseline than non-improvers, and the expression of these genes significantly decreased in improvers post-treatment.
Conclusion
Nilotinib was well tolerated by the majority of patients in this study, with tolerability limited primarily by mild QTc-prolongation. Significant MRSS improvement was observed in these early, active patients, but is not conclusive of treatment effect given the open-label study-design and small number of patients in this pilot study. Improvers had higher levels of expression of genes associated with TGFBR and PDGFRB signaling at baseline, and a significant decrease in the expression of these genes occurred only in patients with higher MRSS improvement. The findings of this pilot study warrant more conclusive evaluation.
Trial registration
Clinicaltrials.gov NCT01166139, July 1, 2010.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0721-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s13075-015-0721-3
PMCID: PMC4538758  PMID: 26283632
10.  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
11.  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
12.  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
13.  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
14.  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
15.  Augmented Interferon-α Pathway Activation in Patients With Sjögren's Syndrome Treated With Etanercept 
Arthritis and rheumatism  2007;56(12):3995-4004.
Objective
Recent clinical trials suggest that etanercept is ineffective in controlling Sjögren's syndrome (SS). To address the hypothesis that tumor necrosis factor blockade can result in increased levels of interferon-α (IFNα) and BAFF, we quantified those mediators in plasma from etanercept- and placebo-treated SS patients.
Methods
We studied plasma samples from 20 patients with SS treated with etanercept (25 mg twice weekly) or placebo in a 12-week, randomized, double-blind clinical trial. In addition, we studied plasma samples from 29 healthy controls. IFNα activity was determined by reporter cell assay, and BAFF levels were determined by enzyme-linked immunosorbent assay.
Results
Baseline IFNα plasma activity and BAFF levels were increased in SS patients compared with healthy controls (mean ± SD IFNα plasma activity score 4.43 ± 2.60 versus 2.08 ± 0.91; P < 0.0001) (mean ± SD BAFF level 0.83 ± 0.27 ng/ml versus 0.60 ± 0.15 ng/ml; P = 0.008). A significant increase in IFNα activity was detected after 12 weeks of treatment in the etanercept group, but not in the placebo group (P = 0.04 and P = 0.58, respectively). Furthermore, a statistically significant increase in BAFF levels was noted in patients receiving etanercept, but not in those receiving placebo (P = 0.01 and P = 0.56, respectively). In vitro culture of control peripheral blood mononuclear cells with etanercept resulted in a dose-dependent increase in the expression of IFNα and the IFNα-inducible genes IFN-induced protein with tetratricopeptide repeats 1 and BAFF.
Conclusion
IFNα activity and BAFF levels are elevated in the plasma of patients with SS compared with healthy controls. Etanercept treatment exacerbates IFNα and BAFF overexpression, providing a possible explanation for the lack of efficacy of this agent in SS.
doi:10.1002/art.23062
PMCID: PMC2737264  PMID: 18050196
16.  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
17.  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
18.  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
19.  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
20.  Identification of a central role for complement in osteoarthritis 
Nature medicine  2011;17(12):1674-1679.
Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of “wear and tear”1. Although low-grade inflammation is detected in osteoarthritis, its role is unclear2–4. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in C5, C6, or CD59a, we show that complement, and specifically the membrane attack complex (MAC)-mediated arm of complement, is critical to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints of C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Furthermore, MAC co-localized with matrix metalloprotease (MMP)-13 and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints plays a critical role in the pathogenesis of osteoarthritis.
doi:10.1038/nm.2543
PMCID: PMC3257059  PMID: 22057346
21.  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
22.  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
23.  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
24.  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
25.  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

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