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OBJECTIVE: To investigate the relationship between lymphoid neogenesis in the synovium of patients with rheumatoid arthritis (RA) and characteristics of inflammation and disease severity. METHODS: Arthroscopic synovial biopsy was performed in 103 patients with active RA (Disease Activity Score 28-joint assessment >/=3.2) who had not received treatment with biologic agents. Sections were stained and assessed by digital image analysis. Lymphocyte aggregates were counted and graded for size (1-3). Synovial lymphoid neogenesis was defined as the presence of grade 2 or 3 aggregates and subclassified based on the presence of follicular dendritic cells (FDCs). RESULTS: Lymphoid neogenesis was present in 31% of the RA synovial tissues, whereas an additional 25% contained only grade 1 aggregates. FDCs were present in 28% of the samples with lymphoid neogenesis, corresponding to 8% of the total RA cohort. Histologically, synovia with lymphoid neogenesis showed increased infiltration by T and B lymphocytes, plasma cells, and macrophages, and increased expression of tumor necrosis factor alpha and lymphotoxin beta compared with samples without lymphoid neogenesis. Patients with lymphoid neogenesis also had higher C-reactive protein levels, erythrocyte sedimentation rates, and leukocyte and thrombocyte counts, but exhibited no increase in the severity of clinical signs and symptoms. Of importance, there was no relationship between the presence of lymphoid neogenesis and IgM rheumatoid factor or anti-citrullinated protein antibodies. The presence of lymphocyte aggregates with FDCs did not define a specific clinical phenotype compared with lymphocyte aggregates without FDCs. CONCLUSION: These findings indicate that synovial lymphoid neogenesis is associated with more severe synovial and systemic inflammation, but this is not confined to a specific clinical subset of RA. [ABSTRACT FROM AUTHOR]

OBJECTIVE: In approximately 25% of synovial tissues from rheumatoid arthritis (RA) patients, infiltrates of T cells, B cells, and follicular dendritic cells (FDCs) are spatially organized into structures resembling lymph nodes with germinal centers. The remainder of the tissues lack FDCs and show either a diffuse or an aggregated T cell and B cell infiltrate. To gain more insight into this specific disease process, we sought to identify the genes expressed in RA tissues with ectopic lymphoid structures. METHODS: Gene expression profiling of RA synovial tissues was determined by complementary DNA microarray analysis and quantitative real-time polymerase chain reaction. The presence of lymphoid follicles and localization of interleukin-7 (IL-7) in synovial tissue sections was determined by immunofluorescence staining using specific antibodies. RESULTS: Findings of gene expression analysis confirmed previous reports that tissues with lymphoid structures showed elevated expression of CXCL13, CCL21, CCR7, and lymphotoxin alpha and beta messenger RNA. In addition, the tissues also showed enhanced expression of the chemokines CXCL12 and CCL19 and the associated receptors CXCR4 and CXCR5, which are important for the attraction of T cells, B cells, and dendritic cells. Pathway analysis revealed increased expression of genes involved in JAK/STAT signaling, T cell- and B cell-specific pathways, Fcepsilon receptor type I signaling in mast cells, and IL-7 signal transduction in the tissues with ectopic lymphoid follicles, accompanied by increased expression of IL-7 receptor alpha (IL-7Ralpha)/IL-2Rgamma chains and IL-7. Protein expression of IL-7 in RA tissues was localized within fibroblast-like synoviocytes, macrophages, and blood vessels and was colocalized with extracellular matrix structures around the B cell follicles. CONCLUSION: Activation of the IL-7 pathway may play an important role in lymphoid neogenesis, analogous to its role in the development of normal lymphoid tissue. [ABSTRACT FROM AUTHOR]

Background: In order to identify factors that stimulate arteriogenesis after ischemia, we followed gene expression profiles in two extreme models for collateral artery formation over 28 days after hindlimb ischemia, namely "good-responding" C57BL/6 mice and "poor-responding" BALB/c mice., Methods and Results: Although BALB/c mice show very poor blood flow recovery after ischemia, most known proarteriogenic genes were upregulated more excessively and for a longer period than in C57BL/6 mice. In clear contrast, chemokine genes Ccl19 , Ccl21a , and Ccl21c and the chemokine receptor CCR7 were upregulated in C57BL/6 mice 1 day after hindlimb ischemia, but not in BALB/C mice. CCL19 and CCL21 regulate migration and homing of T lymphocytes via CCR7. When subjecting CCR7 -/- /LDLR -/- mice to hindlimb ischemia, we observed a 20% reduction in blood flow recovery compared with that in LDLR -/- mice. Equal numbers of α-smooth muscle actin-positive collateral arteries were found in the adductor muscles of both mouse strains, but collateral diameters were smaller in the CCR7 -/- /LDLR -/- . Fluorescence-activated cell sorter analyses showed that numbers of CCR7 + T lymphocytes (both CD4 + and CD8 + ) were decreased in the spleen and increased in the blood at day 1 after hindlimb ischemia in LDLR -/- mice. At day 1 after hindlimb ischemia, however, numbers of activated CD4 + T lymphocytes were decreased in the draining lymph nodes of LDLR -/- mice compared with CCR7 -/- /LDLR -/- mice., Conclusions: These data show that CCR7-CCL19/CCL21 axis facilitates retention CD4 + T lymphocytes at the site of collateral artery remodeling, which is essential for effective arteriogenesis., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

During prolonged hypoxic conditions, endothelial cells change their gene expression to adjust to the low oxygen environment. This process is mainly regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Although endothelial cells do not form sprouts during prolonged hypoxic culturing, silencing of HIF-2α partially restores sprout formation. The present study identifies novel HIF-2α-target genes that may regulate endothelial sprouting during prolonged hypoxia. The gene expression profile of primary human microvascular endothelial cells (hMVECs) that were cultured at 20 % oxygen was compared to hMVECs that were cultured at 1 % oxygen for 14 days by using genome-wide RNA-sequencing. The differentially regulated genes in hypoxia were compared to the genes that were differentially regulated upon silencing of HIF-2α in hypoxia. Surprisingly, KEGG pathway analysis showed that metabolic pathways were enriched within genes upregulated in response to hypoxia and enriched within genes downregulated upon HIF-2α silencing. Moreover, 51 HIF-2α-regulated genes were screened for their role in endothelial sprouting in hypoxia, of which four genes ARRDC3, MME, PPARG and RALGPS2 directly influenced endothelial sprouting during prolonged hypoxic culturing. The manipulation of specific downstream targets of HIF-2α provides a new, but to be further evaluated, perspective for restoring reduced neovascularization in several pathological conditions, such as diabetic ulcers or other chronic wounds, for improvement of vascularization of implanted tissue-engineered scaffolds., Competing Interests: The authors declare no conflict of interest. S. Gibbs is co-founder and shareholder of A-Skin BV, a university spin out (SME) company.

Galectins are an ancient family of β-galactoside-specific lectins and consist of 15 different types, each with a specific function. They play a role in the immune system, inflammation, wound healing and carcinogenesis. In particular the role of galectin in cancer is widely studied. Lately, the role of galectins in the development of cardiovascular disease has gained attention. Worldwide cardiovascular disease is still the leading cause of death. In ischemic heart disease, atherosclerosis limits adequate blood flow. Angiogenesis and arteriogenesis are highly important mechanisms relieving ischemia by restoring perfusion to the post-stenotic myocardial area. Galectins act ambiguous, both relieving ischemia and accelerating atherosclerosis. Atherosclerosis can ultimately lead to myocardial infarction or ischemic stroke, which are both associated with galectins. There is also a role for galectins in the development of myocarditis by their influence on inflammatory processes. Moreover, galectin acts as a biomarker for the severity of myocardial ischemia and heart failure. This review summarizes the association between galectins and the development of multiple cardiovascular diseases such as myocarditis, ischemic stroke, myocardial infarction, heart failure and atrial fibrillation. Furthermore it focuses on the association between galectin and more general mechanisms such as angiogenesis, arteriogenesis and atherosclerosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Activation of the type I interferon (IFN) response program is described for several autoimmune diseases, including systemic lupus erythematosus (SLE), multiple sclerosis (MS), myositis (IIM) and rheumatoid arthritis (RA). While IFNα contributes to SLE pathology, IFNβ therapy is often beneficial in MS, implying different immunoregulatory roles for these IFNs. This study was aimed to investigate potential diversification of IFNα-and IFNβ-mediated response programs in autoimmune diseases., Methods: Peripheral blood gene expression of 23 prototypical type I IFN response genes (IRGs) was determined in 54 healthy controls (HCs), 69 SLE (47 test, 22 validation), 149 IFNβ-treated MS (71 test, 78 validation), 160 untreated MS, 78 IIM and 76 RA patients. Patients with a type I IFN signature were selected for analysis., Results: We identified IFNα- and IFNβ-specific response programs (GC-A and GC-B, respectively) in SLE and IFNβ-treated MS patients. Concordantly, the GC-A/GC-B log-ratio was positive for all SLE patients and negative for virtually all IFNβ-treated MS patients, which was confirmed in additional cohorts. Applying this information to other autoimmune diseases, IIM patients displayed positive GC-A/GC-B log-ratios, indicating predominant IFNα activity. The GC-A/GC-B log-ratio in RA was lower and approached zero in part of the patients, implying relative importance of both clusters. Remarkably, GC-A/GC-B log-ratios appeared most heterogeneous in untreated MS; half of the patients displayed GC-A dominance, whereas others showed GC-B dominance or log-ratios near zero., Conclusions: Our findings show diversification of the type I IFN response in autoimmune diseases, suggesting different pathogenic roles of the type I IFNs.

The flow-responsive transcription factor Krüppel-like factor 2 (KLF2) maintains an anti-coagulant, anti-inflammatory endothelium with sufficient nitric oxide (NO)-bioavailability. In this study, we aimed to explore, both in vitro and in human vascular tissue, expression of the NO-transporting transmembrane pore aquaporin-1 (AQP1) and its regulation by atheroprotective KLF2 and atherogenic inflammatory stimuli. In silico analysis of gene expression profiles from studies that assessed the effects of KLF2 overexpression in vitro and atherosclerosis in vivo on endothelial cells, identifies AQP1 as KLF2 downstream gene with elevated expression in the plaque-free vessel wall. Biomechanical and pharmaceutical induction of KLF2 in vitro is accompanied by induction of AQP1. Chromosome immunoprecipitation (CHIP) confirms binding of KLF2 to the AQP1 promoter. Inflammatory stimulation of endothelial cells leads to repression of AQP1 transcription, which is restrained by KLF2 overexpression. Immunohistochemistry reveals expression of aquaporin-1 in non-activated endothelium overlying macrophage-poor intimae, irrespective whether these intimae are characterized as being plaque-free or as containing advanced plaque. We conclude that AQP1 expression is subject to KLF2-mediated positive regulation by atheroprotective shear stress and is downregulated under inflammatory conditions both in vitro and in vivo. Thus, endothelial expression of AQP1 characterizes the atheroprotected, non-inflamed vessel wall. Our data provide support for a continuous role of KLF2 in stabilizing the vessel wall via co-temporal expression of eNOS and AQP1 both preceding and during the pathogenesis of atherosclerosis.

Circulating angiogenic cells (CACs) are monocyte-derived cells with endothelial characteristics, which contribute to both angiogenesis and arteriogenesis in a paracrine way. Interferon-β (IFN-β) is known to inhibit these divergent processes in animals and patients. We hypothesized that IFN-β might act by affecting the differentiation and function of CACs. CACs were cultured from peripheral blood mononuclear cells and phenotypically characterized by surface expression of monocytic and endothelial markers. IFN-β significantly reduced the number of CACs by 18-64%. Apoptosis was not induced by IFN-β, neither in mononuclear cells during differentiation, nor after maturation to CACs. Rather, IFN-β impaired adhesion to, and spreading on, fibronectin, which was dependent on α5β1 (VLA-5)-integrin. IFN-β affected the function of VLA-5 in mature CACs, leading to rounding and detachment of cells, by induction of calpain 1 activity. Cell rounding and detachment was completely reversed by inhibition of calpain 1 activity in mature CACs. During in vitro capillary formation, CAC addition and calpain 1 inhibition enhanced sprouting of endothelial cells to a comparable extent, but were not sufficient to rescue tube formation in the presence of IFN-β. We show that the IFN-β-induced reduction of the numbers of in vitro differentiated CACs is based on activation of calpain 1, resulting in an attenuated adhesion to extracellular matrix proteins via VLA-5. In vivo, this could lead to inhibition of vessel formation due to reduction of the locally recruited CAC numbers and their paracrine angiogenic factors., (Copyright © 2015 the American Physiological Society.)

Inflammatory immune disorders such as inflammatory bowel disease and multiple sclerosis are major health problems. Currently, the intestinal whipworm Trichuris suis is being explored in clinical trials to reduce inflammation in these diseases; however, the mechanisms by which the parasite affects the host immune system are not known. Here we determined the effects of T. suis soluble products (SPs) on Toll-like receptor-4 (TLR4)-stimulated human dendritic cells (DCs) using Illumina bead chip gene arrays. Pathway analysis of lipopolysaccharide-stimulated DCs with or without T. suis treatment showed that co-stimulation with T. suis SPs resulted in a downregulation of both the myeloid differentiation primary response gene 88-dependent and the TIR-domain-containing adaptor-inducing interferon-β-dependent signalling pathways triggered by TLR4. These data were verified using quantitative real-time PCR of several key genes within these pathways and/or defining their protein levels. In addition, T. suis SPs induce Rab7b, a negative regulator of TLR4 signalling that interferes with its trafficking, which coincided with a reduced surface expression of TLR4. These data indicate that the mechanism by which T. suis SPs reduce inflammatory responses is through suppression of both TLR4 signalling and surface expression on DCs.

Aims: IFN-beta (IFNβ) signalling is increased in patients with insufficient coronary collateral growth (i.e. arteriogenesis) and IFNβ hampers arteriogenesis in mice. A downside of most pro-arteriogenic agents investigated in the past has been their pro-atherosclerotic properties, rendering them unsuitable for therapeutic application. Interestingly, type I IFNs have also been identified as pro-atherosclerotic cytokines and IFNβ treatment increases plaque formation and accumulation of macrophages. We therefore hypothesized that mAb therapy to inhibit IFNβ signalling would stimulate arteriogenesis and simultaneously attenuate-rather than aggravate-atherosclerosis., Methods and Results: In a murine hindlimb ischaemia model, atherosclerotic low-density lipoprotein receptor knockout (LDLR(-/-)) mice were treated during a 4-week period with blocking MAbs specific for mouse IFN-α/β receptor subunit 1 (IFNAR1) or murine IgG isotype as a control. The arteriogenic response was quantified using laser Doppler perfusion imaging (LDPI) as well as immunohistochemistry. Effects on atherosclerosis were determined by quantification of plaque area and analysis of plaque composition. Downstream targets of IFNβ were assessed by real-time PCR (RT-PCR) in the aortic arch. Hindlimb perfusion restoration after femoral artery ligation was improved in mice treated with anti-IFNAR1 compared with controls as assessed by LDPI. This was accompanied by a decrease in CXCL10 expression in the IFNAR1 MAb-treated group. Anti-IFNAR1 treatment reduced plaque apoptosis without affecting total plaque area or other general plaque composition parameters. Results were confirmed in a short-term model and in apolipoprotein E knockout (APOE)(-/-) mice., Conclusion: Monoclonal anti-IFNAR1 therapy during a 4-week treatment period stimulates collateral artery growth in mice and did not enhance atherosclerotic burden. This is the first reported successful strategy using MAbs to stimulate arteriogenesis., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Background: Metabolic syndrome (MetS) is characterized by central obesity, insulin resistance, dysglycemia, and a pro-atherogenic plasma lipid profile. MetS creates a high risk for development of type 2 diabetes (T2DM) and cardiovascular disease (CVD), presumably by altering inflammatory responses. Presently, it is unknown how the chronic metabolic disturbances in acute hyperglycemia, MetS and T2DM affect the immune activity of peripheral blood cells., Methods: We performed genome-wide expression analysis of peripheral blood cells obtained from patients with T2DM (n = 6) and age-, sex- , BMI- and blood pressure-matched obese individuals with MetS (n = 4) and lean healthy normoglycemic controls (n = 3), both under fasting conditions and after controlled induction of acute hyperglycemia during a 70 min hyperglycemic clamp. Differential gene expression during fasting conditions was confirmed by real-time PCR, for which we included additional age-, sex-, BMI-, and blood pressure-matched obese individuals with (n = 4) or without (n = 4) MetS., Results: Pathway and Gene ontology analysis applied to baseline expression profiles of peripheral blood cells from MetS and T2DM patients revealed metabolic changes, highly similar to a reoviral infection gene signature in T2DM patients. Transcription factor binding site analysis indicated that increased HIF-1α activity, a transcription factor induced by either hypoxia or oxidative stress, is responsible for this aberrant metabolic profile in peripheral blood cells from T2DM patients. Acute hyperglycemia in healthy controls resulted in reduced expression of cytotoxicity-related genes, representing NK- and CD8(+) cells. In obese controls, MetS and especially T2DM patients, baseline expression of genes involved in cytotoxicity was already low, compared to healthy controls and did not further decrease upon acute hyperglycemia., Conclusions: The reduced activity of cytotoxic genes in T2DM is explained by chronic hyperglycemia, but its acute effects are restricted to healthy controls. Genome expression of circulating leukocytes from T2DM patients differs from MetS individuals by a specific reovirus signature. Our data thus suggest a role for suppressed anti-viral capacity in the etiology of diabetes.

Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

Recent clinical trials in patients with inflammatory diseases like multiple sclerosis (MS) or inflammatory bowel disease (IBD) have shown the beneficial effects of probiotic helminth administration, although the underlying mechanism of action remains largely unknown. Potential cellular targets may include innate immune cells that propagate inflammation in these diseases, like pro-inflammatory macrophages. We here investigated the effects of the helminth Trichuris suis soluble products (SPs) on the phenotype and function of human inflammatory (granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated) macrophages. Interestingly, we here show that T. suis SPs potently skew inflammatory macrophages into a more anti-inflammatory state in a Toll-like receptor 4 (TLR4)-dependent manner, and less effects are seen when stimulating macrophages with TLR2 or -3 ligands. Gene microarray analysis of GM-CSF-differentiated macrophages further revealed that many TLR4-induced inflammatory mediators, including interleukin (IL)-12B, CCL1 and CXCL9, are downregulated by T. suis SPs. In particular, we observed a strong reduction in the expression and function of P2RX7, a purinergic receptor involved in macrophage inflammation, leading to reduced IL-1β secretion. In conclusion, we show that T. suis SPs suppress a broad range of inflammatory pathways in GM-CSF-differentiated macrophages in a TLR4-dependent manner, thereby providing enhanced mechanistic insight into the therapeutic potential of this helminth for patients with inflammatory diseases.

Acute myocardial infarction (AMI) is accompanied by increased expression of Toll like receptors (TLR)-2 and TLR4 on circulating monocytes. In animal models, blocking TLR2/4 signaling reduces inflammatory cell influx and infarct size. The clinical consequences of TLR activation during AMI in humans are unknown, including its role in long-term cardiac functional outcome Therefore, we analyzed gene expression in whole blood samples from 28 patients with an acute ST elevation myocardial infarction (STEMI), enrolled in the EXenatide trial for AMI patients (EXAMI), both at admission and after 4-month follow-up, by whole genome expression profiling and real-time PCR. Cardiac function was determined by cardiac magnetic resonance (CMR) imaging at baseline and after 4-month follow-up. TLR pathway activation was shown by increased expression of TLR4 and its downstream genes, including IL-18R1, IL-18R2, IL-8, MMP9, HIF1A, and NFKBIA. In contrast, expression of the classical TLR-induced genes, TNF, was reduced. Bioinformatics analysis and in vitro experiments explained this noncanonical TLR response by identification of a pivotal role for HIF-1α. The extent of TLR activation and IL-18R1/2 expression in circulating cells preceded massive troponin-T release and correlated with the CMR-measured ischemic area (R=0.48, p=0.01). In conclusion, we identified a novel HIF-1-dependent noncanonical TLR activation pathway in circulating leukocytes leading to enhanced IL-18R expression which correlated with the magnitude of the ischemic area. This knowledge may contribute to our mechanistic understanding of the involvement of the innate immune system during STEMI and may yield diagnostic and prognostic value for patients with myocardial infarction., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: Therapeutic arteriogenesis, that is, expansive remodeling of preexisting collaterals, using single-action factor therapies has not been as successful as anticipated. Modulation of factors that act as a master switch for relevant gene programs may prove more effective. Transcriptional coactivator p300-CBP-associated factor (PCAF) has histone acetylating activity and promotes transcription of multiple inflammatory genes. Because arteriogenesis is an inflammation-driven process, we hypothesized that PCAF acts as multifactorial regulator of arteriogenesis., Approach and Results: After induction of hindlimb ischemia, blood flow recovery was impaired in both PCAF(-/-) mice and healthy wild-type mice treated with the pharmacological PCAF inhibitor Garcinol, demonstrating an important role for PCAF in arteriogenesis. PCAF deficiency reduced the in vitro inflammatory response in leukocytes and vascular cells involved in arteriogenesis. In vivo gene expression profiling revealed that PCAF deficiency results in differential expression of 3505 genes during arteriogenesis and, more specifically, in impaired induction of multiple proinflammatory genes. Additionally, recruitment from the bone marrow of inflammatory cells, in particular proinflammatory Ly6C(hi) monocytes, was severely impaired in PCAF(-/-) mice., Conclusions: These findings indicate that PCAF acts as master switch in the inflammatory processes required for effective arteriogenesis.

Blood-brain barrier (BBB) dysfunction is a major hallmark of many neurological diseases, including multiple sclerosis (MS). Using a genomics approach, we defined a microRNA signature that is diminished at the BBB of MS patients. In particular, miR-125a-5p is a key regulator of brain endothelial tightness and immune cell efflux. Our findings suggest that repair of a disturbed BBB through microRNAs may represent a novel avenue for effective treatment of MS.

In this review we compare expression studies on monocyte subsets as an example to show the integrated possibilities of molecular databases and bioinformatic analysis tools. Monocytes have been recognized as cells with great plasticity and differentiation potential that play a pivotal role in revascularization processes, i.e. angiogenesis and arteriogenesis. To gain more insight in the relevant developmental programs, we compared the full-genome mRNA expression profiles of several distinct human monocyte subpopulations previously identified based on surface marker expression. These included classical and non-classical, M1 and M2 macrophages, circulating angiogenic cells (CAC), and non-monocyte-derived endothelial colony-forming cells (ECFC). Their transcriptional profiles revealed distinct and overlapping gene expression signatures and pathways reminiscent of utilization of transcription factors driving polarization into the different monocytic phenotypes. Hierarchical cluster analysis revealed that CAC are most related to M2 macrophages and unstimulated macrophages, and to a lesser extent to classical monocytes, and are quite distinct from M1 macrophages and ECFC. Analysis of the promoter region of CAC-expressed genes suggests that in particular the ETS family of transcription factors is important in CAC development. These analyses show the power of combining multiple datasets with existing databases on biological knowledge, to interpret full genome expression data., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: In patients with obstructive coronary artery disease (CAD), the growth of collateral arteries, i.e. arteriogenesis, can preserve myocardial tissue perfusion and function. Monocytes modulate this process, supplying locally the necessary growth factors and degrading enzymes. Knowledge on factors involved in human arteriogenesis is scarce. Thus, the aim of the present study is to identify targets in monocytes that are critical for arteriogenesis in patients with CAD., Methods and Results: A total of 50 patients with a chronic total coronary occlusion were dichotomized according to their collateral flow index. From each patient, RNA was isolated from unstimulated peripheral blood monocytes, monocytes stimulated by lipopolysaccharide (LPS) or interleukin (IL)-4, and from macrophages. Increased mRNA expression of galectin-2 was found in three out of four monocytic cell types of patients with a low capacity of the collateral circulation (P= 0.03 for unstimulated monocytes; P= 0.02 for LPS-stimulated monocytes; P= 0.20 for IL-4-stimulated monocytes; P= 0.02 for macrophages). Additionally, galectin-2 mRNA expression was significantly associated with the rs7291467 polymorphism in LGALS2 encoding galectin-2 in all four monocytic cell types. Patient with the rs7291467 CC genotype displayed highest galectin-2 expression, and also tended to have a lower arteriogenic response. To evaluate the effect of galectin-2 on arteriogenesis in vivo, we used a murine hindlimb model. Treatment with galectin-2 markedly impaired the perfusion restoration at Day 7., Conclusion: Collectively, these results identify galectin-2 as a novel inhibitor of arteriogenesis. Modulation of galectin-2 may constitute a new therapeutic strategy for the stimulation of arteriogenesis in patients with CAD.

Interferon-β (IFNβ) therapy is effective in approximately half of the patients with relapsing-remitting multiple sclerosis (RRMS). Clinical non-responders were characterized by an increased expression of IFN response genes before the start of therapy, and a lack of a pharmacologically induced increase in IFN response gene activity. Because Interferon Regulatory Factor 5 (IRF5) is a master regulator of IFN-activity, we carried out a candidate gene study of IRF5 gene variants in relation to the pharmacological and clinical response upon IFNβ treatment. We found that patients with the IRF5 rs2004640-TT and rs47281420-AA genotype exerted a poor pharmacological response to IFNβ compared with patients carrying the respective G-alleles (P=0.0006 and P=0.0023, respectively). Moreover, patients with the rs2004640-TT genotype developed more magnetic resonance imaging (MRI)-based T2 lesions during IFNβ treatment (P=0.003). Accordingly, an association between MRI-based non-responder status and rs2004640-TT genotype was observed (P=0.010). For the rs4728142-AA genotype a trend of an association with more T2 lesions during IFNβ treatment and MRI-based non-responder status was observed (P=0.103 and P=0.154, respectively). The clinical relevance of the rs2004640-TT genotype was validated in an independent cohort wherein a shorter time to first relapse was found (P=0.037). These findings suggest a role for IRF5 gene variation in the pharmacological and clinical outcome of IFNβ therapy that might have relevance as biomarker to predict the response to IFNβ in multiple sclerosis.

Objective: Despite the fact that rituximab depletes B cells in all treated patients with RA, not all patients show a favourable clinical response. The goal of this study was to provide insight into pharmacological changes in peripheral blood that are associated with clinical response to rituximab., Methods: Gene expression profiling was performed on peripheral blood RNA of 13 patients with RA (test group) using Illumina HumanHT beadchip microarrays. An independent group of nine patients was used for validation using TaqMan quantitative PCR. Clinical responder status was determined after 6 months using change in 28-joint Disease Activity Score (ΔDAS28) and European League Against Rheumatism (EULAR) response criteria. Significance analysis of microarrays and ontology analysis were used for data analysis and interpretation., Results: Pharmacogenomic analyses demonstrated marked interindividual differences in the pharmacological responses at 3 and 6 months after start of treatment with rituximab. Interestingly, only differences in the regulation of type I interferon (IFN)-response genes after 3 months correlated with the ΔDAS28 response. Good responders (DAS>1.2; n=7) exhibited a selective increase in the expression of type I IFN-response genes, whereas this activity was unchanged or hardly changed in non-responders (DAS<1.2; n=6) (p=0.0040 at a cut-off of 1.1-fold induction). Similar results were obtained using EULAR response criteria. These results were validated in an independent cohort of nine patients (five non-responders and four responders, p=0.0317)., Conclusions: A good clinical response to rituximab in RA is associated with a selective drug-induced increase in type I IFN-response activity in patients with RA. This finding may provide insight in the biological mechanism underlying the therapeutic response to rituximab.

To provide insight into the pharmacological changes in the peripheral blood (PB) molecular profile induced by tumor necrosis factor (TNF)-blockade in patients with rheumatoid arthritis (RA), blood was obtained in PAXgene tubes from 33 RA patients before and 1 month after TNF-blocking therapy (infliximab). From 15 randomly chosen patients pre- and post-treatment gene expression profiles were determined. The remaining 18 RA patients served as validation cohort. A group-based paired analysis of the gene expression profiles from the post- vs pre-treatment samples revealed a signature of genes significantly regulated by TNF-blockade. Downregulated genes reflected several biological pathways such as inflammation, angiogenesis, B- and T-cell activation. Further analysis revealed that the pharmacological response signature was significantly regulated in all treated patients, irrespective of clinical response, which is indicative for the presence of an active TNF pathway in all RA patients. The data imply that all patients carried features of TNF bioactivity irrespective of clinical response. These results favor a model for the parallel presence of TNF-dependent and TNF-independent pathways in the individual RA patient. Clinical response status to TNF-blockade may be dependent on the relative contribution of TNF-independent effector pathways.

Background: Circulating CD34+ progenitor cells have the potential to differentiate into a variety of cells, including endothelial cells. Knowledge is still scarce about the transcriptional programs used by CD34+ cells from peripheral blood, and how these are affected in coronary artery disease (CAD) patients., Results: We performed a whole genome transcriptome analysis of CD34+ cells, CD4+ T cells, CD14+ monocytes, and macrophages from 12 patients with CAD and 11 matched controls. CD34+ cells, compared to other mononuclear cells from the same individuals, showed high levels of KRAB box transcription factors, known to be involved in gene silencing. This correlated with high expression levels in CD34+ cells for the progenitor markers HOXA5 and HOXA9, which are known to control expression of KRAB factor genes. The comparison of expression profiles of CD34+ cells from CAD patients and controls revealed a less naïve phenotype in patients' CD34+ cells, with increased expression of genes from the Mitogen Activated Kinase network and a lowered expression of a panel of histone genes, reaching levels comparable to that in more differentiated circulating cells. Furthermore, we observed a reduced expression of several genes involved in CXCR4-signaling and migration to SDF1/CXCL12., Conclusions: The altered gene expression profile of CD34+ cells in CAD patients was related to activation/differentiation by a retinoic acid-induced differentiation program. These results suggest that circulating CD34+ cells in CAD patients are programmed by retinoic acid, leading to a reduced capacity to migrate to ischemic tissues.

Objective: Increased expression of insulin-like growth factor 2 (IGF2) by fibroblast-like synoviocytes (FLS) was associated with low inflammatory synovium of patients with rheumatoid arthritis (RA). The aim of this study was to analyse whether the differential expression of IGF2, whose expression is normally restricted to one allele, is due to activation of the normally suppressed allele., Methods: IGF2 gene expression of RA FLS was quantified by quantitative real-time PCR. FLS heterozygous for a 3'-untranslated region IGF2 polymorphism were selected to measure the relative contribution of the allelic transcripts by allele-specific transcript quantification assay. Proliferation was determined by [(3)H]thymidine incorporation., Results: IGF2 was shown to contribute to RA FLS proliferation. FLS could be classified in IGF2 high and IGF2 low-expressing cell lines. Allelic IGF2 transcript quantification analysis revealed that in part of the RA FLS the normally suppressed allele was activated, resulting in biallelic expression of the IGF2 gene. Biallelic expression was associated with increased levels of IGF2 mRNA production., Conclusion: The findings indicate that the imprinting status of IGF2 might underlie the increased expression of IGF2, which may contribute to autonomous growth of RA FLS of low inflammatory synovial tissues.

Objective: To investigate the clinical relevance of synovial tissue subtypes in rheumatoid arthritis (RA) and to search for peripheral blood (PB) markers that may serve as biomarkers for tissue subtypes., Methods: Gene expression analysis using complementary DNA microarrays was applied on paired synovial tissue biopsy and PB samples obtained from 17 RA patients. Molecular tissue subtypes were correlated with histologic parameters (CD3, CD22, CD38, CD68, CD163, tumor necrosis factor alpha, intercellular adhesion molecule 1, vascular cell adhesion molecule, and E-selectin), disease characteristics, and PB markers. PANTHER classification was used for pathway analysis., Results: Genomic subtyping of high- and low-inflammation rheumatoid synovial tissues based on gene expression profiles exactly matched immunohistochemical classification. The patients with the high-inflammation tissue type had higher Disease Activity Scores in 28 joints, higher C-reactive protein levels, higher erythrocyte sedimentation rates, increased numbers of platelets, and shorter disease durations. Comparative analysis of PB gene expression profiles yielded no statistically significant differences between the 2 tissue groups at the single-gene expression level. PANTHER pathway analysis revealed a significant association of increased protein biosynthesis with high-inflammation tissue., Conclusion: High-inflammation tissue is associated with more severe disease and shorter disease duration. While pathway-level analysis revealed that coordinate differential expression of genes involved in protein synthesis in PB is associated with high-inflammation tissue types, differential tissue pathology was not reflected in the PB by differential expression of single genes.

Objective: To identify molecular features associated with the development of rheumatoid arthritis (RA), to understand the pathophysiology of preclinical development of RA, and to assign predictive biomarkers., Methods: The study group comprised 109 anti-citrullinated protein antibody (ACPA)- and/or rheumatoid factor-positive patients with arthralgia who did not have arthritis but were at risk of RA, and 25 patients with RA. The gene expression profiles of blood samples obtained from these patients were determined by DNA microarray analysis and quantitative polymerase chain reaction., Results: In 20 of the 109 patients with arthralgia who were at risk of RA, arthritis developed after a median of 7 months. Gene expression profiling of blood cells revealed heterogeneity among the at-risk patients, based on differential expression of immune-related genes. This report is the first to describe gene signatures relevant to the development of arthritis. Signatures significantly associated with arthritis development were involved in interferon (IFN)-mediated immunity, hematopoiesis, and chemokine/cytokine activity. Logistic regression analysis revealed that the odds ratio (OR) for developing arthritis within 12 months was 21.0 (95% confidence interval [95% CI] 2.8-156.1 [P = 0.003]) for the subgroup characterized by increased expression of genes involved in IFN-mediated immunity and/or cytokine/chemokine-activity. Genes involved in B cell immunology were associated with protection against progression to arthritis (OR 0.38, 95% CI 0.21-0.70 [P = 0.002]). These processes were reminiscent of those in patients with RA, implying that the preclinical phase of disease is associated with features of established disease., Conclusion: The results of this study indicate that IFN-mediated immunity, hematopoiesis, and cell trafficking specify processes relevant to the progression of arthritis independent of ACPA positivity. These findings strongly suggest that certain gene signatures have value for predicting the progression to arthritis, which will pave the way to preventive medicine.

Objective: Type I IFNs have recently been implicated in autoantibody-mediated diseases such as SLE. As half the RA patients display a type I IFN(high) signature, we investigated in a pilot study if type I IFN determines the autoantibody response in RA., Methods: Serum and peripheral blood cells were obtained from 52 RA patients, with paired samples before and after infliximab treatment in 21 patients. Additional samples were collected from 8 anti-citrullinated protein antibody (ACPA)-positive individuals without arthritis and from 10 ACPA-negative healthy controls. The type I IFN signature was determined by peripheral blood cell gene expression analysis and quantitative RT-PCR. ACPA IgG and IgM, RF IgM, anti-nucleosome IgM and anti-dsDNA were measured by ELISA., Results: The type I IFN signature was not related to the presence and titers of ACPA and RF during active disease. TNF blockade induced a similar rise of ANAs, and a similar decrease in RF titers in both groups. ACPA IgG and IgM levels appeared to be down-modulated only in the type I IFN(low) group. These changes were independent of the changes in type I IFN response gene activity after TNF blockade. Furthermore, the ACPA response in individuals without arthritis and inflammation was not related to an increase of type I IFN., Conclusions: In this explorative study, type I IFN signature does not appear to have a major impact on the humoral autoimmune response in RA. Replication of these data remains warranted.

Introduction: Cross-regulation between TNF and type I IFN has been postulated to play an important role in autoimmune diseases. Therefore, we determined the effect of TNF blockade in rheumatoid arthritis (RA) on the type I IFN response gene activity in relation to clinical response., Methods: Peripheral blood from 33 RA patients was collected in PAXgene tubes before and after the start of infliximab treatment. In a first group of 15 patients the baseline expression of type I IFN-regulated genes was determined using cDNA microarrays and compared to levels one month after treatment. The remaining 18 patients were studied as an independent group for validation using quantitative polymerase chain reaction (qPCR)., Results: Gene expression analysis revealed that anti-TNF antibody treatment induced a significant increase in type I IFN response gene activity in a subset of RA patients, whereas expression levels remained similar or were slightly decreased in others. The findings appear clinically relevant since patients with an increased IFN response gene activity after anti-TNF therapy had a poor clinical outcome. This association was confirmed and extended for an IFN response gene set consisting of OAS1, LGALS3BP, Mx2, OAS2 and SERPING1 in five EULAR good and five EULAR poor responders, by qPCR., Conclusions: Regulation of IFN response gene activity upon TNF blockade in RA is not as consistent as previously described, but varies between patients. The differential changes in IFN response gene activity appear relevant to the clinical outcome of TNF blockade in RA.

The objective of this study was to identify molecular profiles that may distinguish clinical subtypes in systemic sclerosis (SSc). Large-scale gene expression profiling was performed on peripheral blood (PB) from 12 SSc patients and 6 healthy individuals. Significance analysis of microarrays, two-way hierarchical cluster analysis and PANTHER (Protein ANalysis THrough Evolutionary Relationships) ontology classification were used to analyze the data. Quantitative PCR was applied for validation in a cohort of 43 SSc patients. The results show that the expression of genes involved in immune defense, cell cycle and signal transduction was significantly elevated in PB of SSc patients (n=12) compared with healthy individuals (n=6). SSc patients could be stratified into subgroups based on differential expression of genes induced by type I interferon (IFN) and genes involved in antimicrobial (AM) activity. Differential expression of type I IFN or AM signature genes was validated and extended in an independent cohort of 31 patients by quantitative PCR. Low expression of IFN response genes was associated with the presence of anti-centromere antibodies, whereas increased expression was associated with the appearance of digital ulcers. In conclusion, patients with SSc can be classified on the basis of differential expression of immune defense genes. Differences in the activity of the type I IFN response program stratify patients into two clinically relevant subgroups.

Monocytes and T-cells play an important role in the development of atherosclerotic coronary artery disease (CAD). Transcriptome analysis of circulating mononuclear cells from carefully matched atherosclerotic and control patients will potentially provide insights into the pathophysiology of atherosclerosis and supply biomarkers for diagnostic purposes. From patients undergoing coronary angiography because of anginal symptoms, we carefully matched 18 patients with severe triple-vessel CAD to 13 control patients without angiographic signs of CAD. All patients were on statin and aspirin treatment. Elevated soluble-ICAM levels demonstrated increased vascular inflammation in atherosclerotic patients. RNA from circulating CD4+ T-cells, CD14+ monocytes, lipopolysaccharide-stimulated monocytes, and macrophages was subjected to genome-wide expression analysis. In CD14+ monocytes, few inflammatory genes were overexpressed in control patients, while atherosclerotic patients showed overexpression of a group of Krüppel-associated box - containing transcription factors involved in negative regulation of gene expression. These differences disappeared upon LPS-stimulation or differentiation towards macrophages. No consistent changes in T cell transcriptomes were detected. Large inter-individual variability prevented the use of single differentially expressed genes as biomarkers, while monocyte gene expression signature predicted patient status with an accuracy of 84%. In this comprehensive analysis of circulating cell transcriptomes in atherosclerotic CAD, cautious patient matching revealed only small differences in transcriptional activity in different mononuclear cell types. Only an indication of a negative feedback to inflammatory gene expression was detected in atherosclerotic patients. Transcriptome differences of circulating cells possibly play less of a role than hitherto thought in the individual patient's susceptibility to atherosclerotic CAD, when appropriately matched for clinical symptoms and medication taken.

Background: Ulcerative colitis (UC) and Crohn's disease (CD) are characterized by chronic inflammation of the gastrointestinal tract, with overlapping clinical characteristics and unknown etiology. We reasoned that in intestinal inflammation the initial activation of the innate immune response fails to resolve, finally resulting in uncontrolled chronic inflammatory bowel disease., Methods: To identify the early inflammatory events in colitis that remain active in human chronic colitis, we analyzed the changes of the colonic transcriptome during acute experimental colitis and compared the outcome with previously published profiles of affected tissues from patients with UC and CD, and as a control for intestinal inflammation in general, tissues from celiac disease patients. Rheumatoid arthritis synovial tissues were included as a nonintestinal inflammatory disease. The expression profiles of each disease were analyzed separately, in which diseased tissues were compared to unaffected tissues from the same anatomical location., Results: Gene ontology analysis of significantly regulated genes revealed a marked activation of immunity and defense processes in all diseases, except celiac disease, where immune activation is less prominent. The control region of upregulated genes contained an increase in Ets2 binding sites in experimental colitis, UC, and rheumatoid arthritis, and were associated with upregulated immune activity. In contrast, upregulated genes in celiac disease harbored the transcription factor binding site GLI, which binds to the Gli family of transcription factors involved in hedgehog signaling, affecting development and morphogenesis., Conclusion: Ets2 may be an important transcription factor driving inflammation in acute as well as chronic inflammatory disease

Rheumatic diseases are a diverse group of disorders. Most of these diseases are heterogeneous in nature and show varying responsiveness to treatment. Because our understanding of the molecular complexity of rheumatic diseases is incomplete and criteria for categorization are limited, we mainly refer to them in terms of group averages. The advent of DNA microarray technology has provided a powerful tool to gain insight into the molecular complexity of these diseases; this technology facilitates open-ended survey to identify comprehensively the genes and biological pathways that are associated with clinically defined conditions. During the past decade, encouraging results have been generated in the molecular description of complex rheumatic diseases, such as rheumatoid arthritis, systemic lupus erythematosus, Sjögren syndrome and systemic sclerosis. Here, we describe developments in genomics research during the past decade that have contributed to our knowledge of pathogenesis, and to the identification of biomarkers for diagnosis, patient stratification and prognostication.

B lymphocyte autoimmunity plays a crucial role in the pathogenesis of rheumatoid arthritis. The local production of autoantibodies and the presence of ectopic lymphoid neogenesis in the rheumatoid synovium suggest that these dedicated microenvironments resembling canonical lymphoid follicles may regulate the initiation and maturation of B cell autoimmunity. In this study, we assessed experimentally the relevance of ectopic lymphoid neogenesis for B cell autoimmunity by a detailed structural, molecular, and serological analysis of seropositive and seronegative human synovitis. We demonstrate that synovial lymphoid neogenesis is a reversible process associated with inflammation which is neither restricted to nor preferentially associated with autoantibody positive rheumatic conditions. Despite the abundant expression of key chemokines and cytokines required for full differentiation toward germinal center reactions, synovial lymphoid neogenesis in rheumatoid arthritis only occasionally progresses toward fully differentiated follicles. In agreement with that observation, we could not detect Ag-driven clonal expansion and affinity maturation of B lymphocytes. Furthermore, ectopic lymphoid neogenesis is not directly associated with local production of anti-citrullinated protein Abs and rheumatoid factor in the rheumatoid joint. Therefore, we conclude that synovial lymphoid neogenesis is not a major determinant of these rheumatoid arthritis-specific autoantibody responses.

Background: Multiple sclerosis (MS) is a heterogeneous disease. In order to understand the partial responsiveness to IFNbeta in Relapsing Remitting MS (RRMS) we studied the pharmacological effects of IFNbeta therapy., Methodology: Large scale gene expression profiling was performed on peripheral blood of 16 RRMS patients at baseline and one month after the start of IFNbeta therapy. Differential gene expression was analyzed by Significance Analysis of Microarrays. Subsequent expression analyses on specific genes were performed after three and six months of treatment. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated in vitro with IFNbeta. Genes of interest were measured and validated by quantitative realtime PCR. An independent group of 30 RRMS patients was used for validation., Principal Findings: Pharmacogenomics revealed a marked variation in the pharmacological response to IFNbeta between patients. A total of 126 genes were upregulated in a subset of patients whereas in other patients these genes were downregulated or unchanged after one month of IFNbeta therapy. Most interestingly, we observed that the extent of the pharmacological response correlates negatively with the baseline expression of a specific set of 15 IFN response genes (R = -0.7208; p = 0.0016). The negative correlation was maintained after three (R = -0.7363; p = 0.0027) and six (R = -0.8154; p = 0.0004) months of treatment, as determined by gene expression levels of the most significant correlating gene. Similar results were obtained in an independent group of patients (n = 30; R = -0.4719; p = 0.0085). Moreover, the ex vivo results could be confirmed by in vitro stimulation of purified PBMCs at baseline with IFNbeta indicating that differential responsiveness to IFNbeta is an intrinsic feature of peripheral blood cells at baseline., Conclusion: These data imply that the expression levels of IFN response genes in the peripheral blood of MS patients prior to treatment could serve a role as biomarker for the differential clinical response to IFNbeta.

Objective: The response of rheumatoid arthritis (RA) patients to treatment with neutralising antibodies to tumour necrosis factor alpha (TNFalpha) is highly variable. The underlying mechanism for therapy responsiveness is currently unknown. We therefore evaluated the relationship between baseline molecular profiles of synovial tissues from RA patients and the clinical response to treatment with infliximab., Methods: Synovial biopsies were obtained by arthroscopy from 18 RA patients with active disease (28 joint count Disease Activity Score (DAS28) > or = 3.2) before initiation of treatment with infliximab. All patients were on stable methotrexate treatment. Clinical response at 16 weeks was defined as a reduction in DAS28 of > or = 1.2, non-response as reduction in DAS28 < 1.2. Large-scale gene expression profiling using microarrays was performed on synovial tissue samples. To identify biological processes in synovial biopsies that could discriminate between responders and non-responders, we performed pathway analysis on the expression profiles., Results: A total of 12 patients responded to therapy, while 6 patients failed to fulfil the response criteria. We identified several biological processes, related to inflammation, which were up-regulated in patients who responded to therapy, compared to those who did not show clinical improvement., Conclusion: These results indicate that patients with a high level of tissue inflammation are more likely to benefit from anti-TNFalpha treatment.

Rheumatoid arthritis (RA) is a heterogeneous disease with unknown etiology. Here we aimed to distinguish RA subtypes based on peripheral blood (PB) gene expression profiles in comparison with a pathogen-response transcriptional program. PB was obtained from 35 RA patients and 15 healthy individuals. For expression profiling we used DNA microarrays. A combined cluster analysis of RA and control samples together with samples from a viral infection model revealed that the gene expression profile of a subgroup of RA patients (RA(A)) was reminiscent to that of poxvirus-infected macaques. Statistical analysis, followed by Gene Ontology analysis of the RA(A) patients confirmed that these patients form a distinct group, with activation of several host defense mechanisms that resemble a common host-pathogen response. Analysis of the promoter region of genes that were overexpressed in the RA(A) patients, revealed an enrichment of transcription factor binding sites for NF kappaB and interferon-activated transcription factors. Moreover, this subgroup of RA patients expressed significantly increased titers of anti-cyclic citrullinated peptide antibodies. We conclude that activation of a host-pathogen response defines a subgroup of RA patients characterized by increased autoreactivity against citrullinated proteins.

Background: Rheumatoid arthritis (RA) is a heterogeneous disease with unknown cause., Aim: To identify peripheral blood (PB) gene expression profiles that may distinguish RA subtypes., Methods: Large-scale expression profiling by cDNA microarrays was performed on PB from 35 patients and 15 healthy individuals. Differential gene expression was analysed by significance analysis of microarrays (SAM), followed by gene ontology analysis of the significant genes. Gene set enrichment analysis was applied to identify pathways relevant to disease., Results: A substantially raised expression of a spectrum of genes involved in immune defence was found in the PB of patients with RA compared with healthy individuals. SAM analysis revealed a highly significant elevated expression of interferon (IFN) type I regulated genes in patients with RA compared with healthy individuals, which was confirmed by gene ontology and pathway analysis, suggesting that this pathway was activated systemically in RA. A quantitative analysis revealed that increased expression of IFN-response genes was characteristic of approximately half of the patients (IFN(high) patients). Application of pathway analysis revealed that the IFN(high) group was largely different from the controls, with evidence for upregulated pathways involved in coagulation and complement cascades, and fatty acid metabolism, while the IFN(low) group was similar to the controls., Conclusion: The IFN type I signature defines a subgroup of patients with RA, with a distinct biomolecular phenotype, characterised by increased activity of the innate defence system, coagulation and complement cascades, and fatty acid metabolism.

In the last decade, the analysis of gene expression in tissues and cells has evolved from the analysis of a selected set of genes to an efficient high throughput whole-genome screening approach of potentially all genes expressed. Development of sophisticated methodologies such as microarray technology allows an open-ended survey to identify comprehensively the fraction of genes that are differentially expressed between samples and that define the samples' unique biology. By a global analysis of the genes that are expressed in cells and tissues of an individual under different conditions and during disease, we can build up "gene expression profiles (signatures)" which characterize the dynamic functioning of the genome under pathophysiological conditions. This strategy also provides the means to subdivide patients that suffer from a complex heterogeneous disease into more homogeneous subgroups. Such discovery-based research identifies biological processes that may include new genes with unknown function or genes not previously known to be involved in this process. The latter category may hold surprises that sometimes urge us to redirect our thinking. We have used microarrays to disclose the heterogeneity of rheumatoid arthritis (RA) patients at the level of gene expression of the affected synovial tissues. Analysis of the expression profiles of synovial tissues from different patients with RA revealed considerable variability, resulting in the identification of at least two molecularly distinct forms of RA tissues. One is characterized by genes that indicate an active inflammatory infiltrate with high immunoglobulin production, whereas the other type shows little immune activation and instead shows a higher stromal cell activity. These results confirm the heterogeneous nature of RA and suggest the existence of distinct pathogenic mechanisms that contribute to RA. The differences in expression profiles provide opportunities to stratify patients for intervention therapies based on molecular criteria.

Cytokine secretion profiles of activated T cells are critical for maintaining the immunologic balance between protection and tolerance. In mice, several cytokines have been reported to exhibit monoallelic expression. Previously, we found that the human interleukin-1 alpha (IL1A) gene exhibits a stable allele-specific expression pattern in CD4+ T-cell clones. We investigated whether DNA methylation is involved in the allele-specific expression of IL-1alpha. Here, we show that differential methylation of CpGs in the proximal promoter region is associated with allele-specific expression of IL-1alpha in CD4+ T cells. The differential methylation pattern is already observed in naive T cells. In keratinocytes, which constitutively produce IL-1alpha, the proximal promoter is hypomethylated. CpGs located further upstream and in intron 4 were almost all methylated, irrespective of expression. Treatment of nonexpressing cells and of T-cell clones with 5-aza-2'deoxycytidine induced IL-1alpha expression in the nonexpressing cells and induced expression of the formerly silent allele in T-cell clones. In addition, electrophoretic mobility shift assays showed that methylation of CpGs in the proximal promoter resulted in direct inhibition of binding of nuclear factor(s). Taken together, these results suggest that allele-specific expression of IL-1alpha in CD4+ cells is achieved, at least in part, by differential methylation of the promoter.

Given the heterogeneous nature of multiple sclerosis (MS), we applied DNA microarray technology to determine whether variability is reflected in peripheral blood (PB) cells. In this study, we studied whole-blood gene expression profiles of 29 patients with relapsing-remitting MS (RRMS) and 25 age- and sex-matched healthy controls. We used microarrays with a complexity of 43K cDNAs. The data were analyzed using sophisticated pathway-level analysis in order to provide insight into the deregulated peripheral immune response programs in MS. We found a remarkable elevated expression of a spectrum of genes known to be involved in immune defense in the PB of MS patients compared to healthy individuals. Cluster analysis revealed that the increased expression of these genes was characteristic for approximately half of the patients. In addition, the gene signature in this group of patients was comparable with a virus response program. We conclude that the transcriptional signature of the PB cells reflects the heterogeneity of MS and defines a sub-population of RRMS patients, who exhibit an activated immune defense program that resembles a virus response program, which is supportive for a link between viruses and MS.

Objective: Given the heterogeneity of gene expression patterns and cellular distribution between rheumatoid arthritis (RA) synovial tissues, we sought to determine whether this variability was also reflected at the level of the fibroblast-like synoviocyte (FLS) cultured from RA synovial tissues., Methods: Gene expression profiles in FLS cultured from synovial tissues obtained from 19 RA patients were analyzed using complementary DNA microarrays and hierarchical cluster analysis. To validate the subclassification, we performed prediction analysis and principal components analysis. Genes that differed significantly in their expression between FLS cultures were selected using Statistical Analysis of Microarrays software. Real-time quantitative polymerase chain reaction was performed to validate the microarray data. Immunocytochemistry was applied to study the expression of the genes of interest in FLS and synovial tissues., Results: Hierarchical clustering identified 2 main groups of FLS characterized by distinctive gene expression profiles. FLS from high-inflammation synovial tissues revealed increased expression of a transforming growth factor beta/activin A-inducible gene profile that is characteristic of myofibroblasts, a cell type considered to be involved in wound healing, whereas increased production of growth factor (insulin-like growth factor 2/insulin-like growth factor binding protein 5) appeared to constitute a characteristic feature of FLS derived from low-inflammation synovial tissues. The molecular feature that defines the myofibroblast-like phenotype was reflected as an increased proportion of myofibroblast-like cells in the heterogeneous FLS population. Myofibroblast-like cells were also found upon immunohistochemical analysis of synovial tissue., Conclusion: Our findings support the notion that heterogeneity between synovial tissues is reflected in FLS as a stable trait, and provide evidence of a possible link between the behavior of FLS and the inflammation status of RA synovium.

Interleukin-12 (IL-12) is a potent inducer of interferon-gamma production by T cells and is a major factor for the development of T-helper 1 (Th1) cells. It exerts its biological effects through binding to the IL-12 receptor (IL-12R), a heterodimer composed of a 1 and a beta2 subunits. The signaling beta2 chain is expressed on Th1 cells and to a lesser extent on Th0 cells, but not on Th2 cells, rendering these latter cells unresponsive to IL-12. Polymorphisms in the coding region of the IL-12Rbeta2 gene were shown to be associated with atopic disease. Here, we analyzed the 5'-regulatory region of the human IL-12Rbeta2 gene by denaturing high-performance liquid chromatography (Transgenomic WAVE system, San Jose, CA). We found five novel single-nucleotide polymorphisms (SNPs) in the proximal 1.2 kb IL-12Rbeta2 promoter region, i.e. -237C/T, -465A/G, -1023A/G, -1033T/C, and -1035A/G. SNP -465A/G is of particular interest as it determines the integrity of a GATA consensus site. By functional comparison of both -465 alleles in transient transfection assays, we show that promoter activity is increased in case of the -465G allele, disrupting the intact GATA site. Comparison of the prevalence of -465A/G SNP alleles in small cohorts of allergic asthmatic and healthy control individuals provided no evidence for an altered distribution in the asthmatic population. In conclusion, we have identified a novel polymorphic GATA site that may affect transciptional activity of the human IL-12Rbeta2 gene under GATA3-mediated, Th2-polarizing conditions., (Copyright 2004 Blackwell Munksgaard)

The analysis of gene expression in tissues, cells, and biologic systems has evolved in the last decade from the analysis of a selected set of genes to an efficient high throughput whole-genome screening approach of potentially all genes expressed in a tissue or cell sample. Development of sophisticated methodologies such as microarray technology allows an open-ended survey to identify comprehensively the fraction of genes that are differentially expressed between samples and define the samples' unique biology. This discovery-based research provides the opportunity to characterize either new genes with unknown function or genes not previously known to be involved in a biologic process. The latter category may hold surprises that sometimes urge us to redirect our thinking. Here, we review the impact of large-scale gene expression profiling by DNA-microarray technology on basic and clinical aspects of immunology.

Background: Expression of signal transducer and activator of transcription 1 (STAT1), the mediator of interferon (IFN) signalling, is raised in synovial tissue (ST) from patients with rheumatoid arthritis (RA)., Objectives: To determine the extent to which this pathway is activated by phosphorylation in RA synovium. Additionally, to investigate the cellular basis of STAT1 activation in RA ST., Methods: ST specimens from 12 patients with RA and 14 disease controls (patients with osteoarthritis and reactive arthritis) were analysed by immunohistochemistry, using antibodies to STAT1, tyrosine phosphorylated STAT1, and serine phosphorylated STAT1. Lysates of cultured fibroblast-like synoviocytes stimulated with IFNbeta were analysed by western blotting. Phenotypic characterisation of cells expressing STAT1 in RA ST was performed by double immunolabelling for STAT1 and CD3, CD22, CD55, or CD68., Results: Raised levels of total STAT1 protein and both its activated tyrosine and serine phosphorylated forms were seen in RA synovium as compared with controls. STAT1 was predominantly abundant in T and B lymphocytes in focal inflammatory infiltrates and in fibroblast-like synoviocytes in the intimal lining layer. Raised levels of STAT1 are sustained in cultured RA compared with OA fibroblast-like synoviocytes, and STAT1 serine and tyrosine phosphorylation is rapidly induced upon stimulation with IFNbeta., Conclusion: These results demonstrate activation of the STAT1 pathway in RA synovium by raised STAT1 protein expression and concomitantly increased tyrosine (701) and serine (727) phosphorylation. High expression of STAT1 is intrinsic to RA fibroblast-like synoviocytes in the intimal lining layer, whereas activation of the pathway by phosphorylation is an active process.

Introduction: Recent studies have indicated that the cytokines produced by CD4(+) T helper type 1 (T(h)1) and type 2 (T(h)2) cells are critically important in antitumour immunity and perhaps clinical outcome. From this perspective, we investigated the immunocompetence of patients with previously untreated cancer of the oesophagus or oesophagogastric junction (OGJ) in relation to stage of disease and postoperative survival., Methods: Blood samples were taken prior to surgery from 32 patients with adenocarcinoma of the oesophagus or OGJ. Ten healthy volunteers served as normal controls. T-cell and monocyte subpopulations were determined using flow cytometry. Monocyte as well as T(h)1- and T(h)2-lymphocyte cytokine levels were assessed in stimulated whole blood cultures., Results: Absolute T-cell and monocyte (subset) counts as well as monocyte cytokine levels were similar among patients and controls. Production of T(h)1-type cytokines was higher in patients than in controls (IFN-gamma, p=0.01; IL-2, p=0.05), whereas T(h)2-type cytokine levels were comparable (IL-4, p=0.5; IL-13, p=0.3). T-cell CD4(+)/CD8(+) ratios decreased as pTNM stage worsened (stage I/II vs stage III/IV, p=0.009). Of all measured immunological parameters, only IL-2 production significantly affected both overall survival ( p=0.015) and disease-free survival ( p=0.0062). High IL-2 levels corresponded with a favourable prognosis., Conclusions: Patients awaiting surgery for adenocarcinoma of the oesophagus or oesophagogastric junction demonstrated a shift in the T(h)1/T(h)2 balance-in favour of T(h)1-compared with healthy volunteers. The ability of T cells to produce IL-2 was related to survival indicating a crucial role of T(h)1-type cells in antitumour immunosurveillance.

A number of reports have described the monoallelic expression of murine cytokine genes. Here we describe the monoallelic expression of the human IL-1alpha gene in CD4+ T cells. Analysis of peripheral blood T cell clones derived from healthy individuals revealed that the IL-1alpha gene shows predominantly monoallelic expression. Monoallelic expression was observed in Th0, Th1, and Th2 cell clones. In addition, we demonstrate monoallelic expression in T cell clones from rheumatoid arthritis patients derived from synovial fluid of the knee joint, suggesting that the occurrence of this phenomenon is not different from that in clones derived from healthy individuals. The finding of monoallelic expression of a cytokine gene in human CD4+ T cell clones provides evidence for allele-specific silencing/activation as another layer of regulation of IL-1alpha gene expression.

Objective: To generate a molecular description of synovial tissue from rheumatoid arthritis (RA) patients that would allow us to unravel novel aspects of pathogenesis and to identify different forms of disease., Methods: We applied complementary DNA microarray analysis to profile gene expression, with a focus on immune-related genes, in affected joint tissues from RA patients and in tissues from osteoarthritis (OA) patients as a control. To validate microarray data, real-time polymerase chain reaction was performed on genes of interest., Results: The gene expression signatures of synovial tissues from RA patients showed considerable variability, resulting in the identification of at least two molecularly distinct forms of RA tissues. One class of tissues revealed abundant expression of clusters of genes indicative of an involvement of the adaptive immune response. Detailed analysis of the expression profile provided evidence for a prominent role of an activated signal transducer and activator of transcription 1 pathway in these tissues. The expression profiles of another group of RA tissues revealed an increased tissue remodeling activity and a low inflammatory gene expression signature. The gene expression pattern in the latter tissues was reminiscent of that observed in the majority of OA tissues., Conclusion: The differences in the gene expression profiles provide a unique perspective for distinguishing different pathogenetic RA subsets based on molecular criteria. These data reflect important aspects of molecular variation that are relevant for understanding the biologic dysregulation underlying these subsets of RA. This approach may also help to define homogeneous groups for clinical studies and evaluation of targeted therapies.