Yanmin Chen

Evidence for chronic, peripheral activation of neutrophils in polyarticular juvenile rheumatoid arthritis

Although strong epidemiologic evidence suggests an important role for adaptive immunity in the pathogenesis of polyarticular juvenile rheumatoid arthritis (JRA), there remain many aspects of the disease that suggest equally important contributions of the innate immune system. We used gene expression arrays and computer modeling to examine the function in neutrophils of 25 children with polyarticular JRA. Computer analysis identified 712 genes that were differentially expressed between patients and healthy controls. Computer-assisted analysis of the differentially expressed genes demonstrated functional connections linked to both interleukin (IL)-8- and interferon-γ (IFN-γ)-regulated processes. Of special note is that the gene expression fingerprint of children with active JRA remained essentially unchanged even after they had responded to therapy. This result differed markedly from our previously reported work, in which gene expression profiles in buffy coats of children with polyarticular JRA reverted to normal after disease control was achieved pharmacologically. These findings suggest that JRA neutrophils remain in an activated state even during disease quiescence. Computer modeling of array data further demonstrated disruption of gene regulatory networks in clusters of genes modulated by IFN-γ and IL-8. These cytokines have previously been shown to independently regulate the frequency (IFN-γ) and amplitude (IL-8) of the oscillations of key metabolites in neutrophils, including nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and superoxide ion. Using real-time, high-speed, single-cell photoimaging, we observed that 6/6 JRA patients displayed a characteristic defect in 12% to 23% of the neutrophils tested. Reagents known to induce only frequency fluctuations of NAD(P)H and superoxide ion induced both frequency and amplitude fluctuations in JRA neutrophils. This is a novel finding that was observed in children with both active (n = 4) and inactive (n = 2) JRA. A subpopulation of polyarticular JRA neutrophils are in a chronic, activated state, a state that persists when the disease is well controlled pharmacologically. Furthermore, polyarticular JRA neutrophils exhibit an intrinsic defect in the regulation of metabolic oscillations and superoxide ion production. Our data are consistent with the hypothesis that neutrophils play an essential role in the pathogenesis of polyarticular JRA.

Gene Expression Profiling in Neutrophils From Children With Polyarticular Juvenile Idiopathic Arthritis

OBJECTIVE We have previously reported a defect in neutrophil activation in children with polyarticular juvenile idiopathic arthritis (JIA). The current study was undertaken to determine whether gene expression abnormalities persist in JIA in remission and to use systems biology analysis to elucidate pathologic pathways in polyarticular JIA. METHODS We performed gene expression profiling on neutrophils from children with polyarticular JIA. Children were grouped according to disease status. We studied 14 children with active disease who were taking medication, 8 children with clinical remission of disease who were taking medication (CRM status), and 6 children with clinical remission of disease who were not taking medication (CR status). We also studied 13 healthy children whose age ranges overlapped those of the patients. RESULTS Neutrophil abnormalities persisted in children with polyarticular JIA even after disease remission was achieved. Children with active disease and those with CRM status showed no differences in expression of specific genes, although they could be separated on cluster analysis. A comparison of children with CR status and healthy control children revealed networks of pro- and antiinflammatory genes that suggested that remission is a state of homeostasis and balance rather than a return to normal immune function. Furthermore, gene overexpression in patients with CR status supports the hypothesis that neutrophils play a role in regulating adaptive immunity in this disease. CONCLUSION Neutrophil gene profiling in polyarticular JIA suggests important roles for neutrophils in disease pathogenesis. These findings suggest the presence of complex interactions between innate and adaptive immunity, that are not easily modeled in conventional, linear, reductionist systems.

T cell activation by soluble C1q-bearing immune complexes: implications for the pathogenesis of rheumatoid arthritis

Both innate and adaptive immune systems are thought to participate in the pathogenesis of rheumatoid arthritis in adults and children. The experiments reported here were undertaken to examine how immune complexes, potent stimulators of inflammation, may regulate cells of the adaptive immune system. Human T cells were prepared from peripheral blood by negative selection and incubated with bovine serum albumin (BSA)–anti‐BSA immune complexes that were formed in the presence or absence of human C1q. C1q‐bearing immune complexes, but not unopsonized complexes, elicited both TNF‐α and IFN‐γ secretion from human T cells. Secretion of both cytokines was time‐ and dose‐dependent. Cross‐linking C1q on the cell surface of T cells produced the same results. Cytokine secretion was not inhibited by blocking the C3b receptor (CR1, CD35) on T cells prior to incubation with immune complexes. Reverse transcriptase polymerase chain reaction (RT‐PCR) of immune complex‐stimulated cells revealed accumulation of both TNF‐α and IFN‐γ mRNA within 2 h post‐stimulation. IL‐2 was not detected in cell culture supernatants, but IL‐2 receptor α chain (CD25) was detected in low density on a small proportion of T cells activated by C1q‐bearing immune complexes. Secretion of both cytokines was inhibited partially, but not completely, by IL‐10. These experiments show that immune complexes, potent inflammatory mediators, may activate T cells through a novel mechanism. These findings have implications for chronic inflammatory diseases in humans.

Genomic characterization of remission in juvenile idiopathic arthritis.

IntroductionThe attainment of remission has become an important end point for clinical trials in juvenile idiopathic arthritis (JIA), although we do not yet have a full understanding of what remission is at the cell and molecular level.MethodsTwo independent cohorts of patients with JIA and healthy child controls were studied. RNA was prepared separately from peripheral blood mononuclear cells (PBMC) and granulocytes to identify differentially expressed genes using whole genome microarrays. Expression profiling results for selected genes were confirmed by quantitative, real-time polymerase chain reaction (RT-PCR).ResultsWe found that remission in JIA induced by either methotrexate (MTX) or MTX plus a TNF inhibitor (etanercept, Et) (MTX + Et) is characterized by numerous differences in gene expression in peripheral blood mononuclear cells and in granulocytes compared with healthy control children; that is, remission is not a restoration of immunologic normalcy. Network analysis of the differentially expressed genes demonstrated that the steroid hormone receptor superfamily member hepatocyte nuclear factor 4 alpha (HNF4α) is a hub in several of the gene networks that distinguished children with arthritis from controls. Confocal microscopy revealed that HNF4a is present in both T lymphocytes and granulocytes, suggesting a previously unsuspected role for this transcription factor in regulating leukocyte function and therapeutic response in JIA.ConclusionsThese findings provide a framework from which to understand therapeutic response in JIA and, furthermore, may be used to develop strategies to increase the frequency with which remission is achieved in adult forms of rheumatoid arthritis.

RNA sequencing from human neutrophils reveals distinct transcriptional differences associated with chronic inflammatory states

BackgroundThe transcriptional complexity of mammalian cells suggests that they have broad abilities to respond to specific environmental stimuli and physiologic contexts. These abilities were not apparent a priori from the structure of mammalian genomes, but have been identified through detailed transcriptome analyses. In this study, we examined the transcriptomes of cells of the innate immune system, human neutrophils, using RNA sequencing (RNAseq).MethodsWe sequenced poly-A RNA from nine individual samples corresponding to specific phenotypes: three children with active, untreated juvenile idiopathic arthritis (JIA)(AD), three children with the same disease whose disease was inactive on medication (CRM), and three children with cystic fibrosis (CF).ResultsWe demonstrate that transcriptomes of neutrophils, typically considered non-specific in their responses and functions, display considerable specificity in their transcriptional repertoires dependent on the pathologic context, and included genes, gene isoforms, and long non-coding RNA transcripts. Furthermore, despite the small sample numbers, these findings demonstrate the potential of RNAseq approaches to biomarker development in rheumatic diseases.ConclusionsThese data demonstrate the capacity of cells previously considered non-specific in function to adapt their transcriptomes to specific biologic contexts. These data also provide insight into previously unrecognized pathological pathways and show considerable promise for elucidating disease and disease-state specific regulatory networks.

Whole Blood Gene Expression Profiling Predicts Therapeutic Response at Six Months in Patients With Polyarticular Juvenile Idiopathic Arthritis

To determine whether gene expression profiles identified in peripheral whole blood samples could be used to determine therapeutic outcome in a cohort of children with newly diagnosed polyarticular juvenile idiopathic arthritis (JIA).

Disease-associated pathophysiologic structures in pediatric rheumatic diseases show characteristics of scale-free networks seen in physiologic systems: implications for pathogenesis and treatment.

BackgroundWhile standard reductionist approaches have provided some insights into specific gene polymorphisms and molecular pathways involved in disease pathogenesis, our understanding of complex traits such as atherosclerosis or type 2 diabetes remains incomplete. Gene expression profiling provides an unprecedented opportunity to understand complex human diseases by providing a global view of the multiple interactions across the genome that are likely to contribute to disease pathogenesis. Thus, the goal of gene expression profiling is not to generate lists of differentially expressed genes, but to identify the physiologic or pathogenic processes and structures represented in the expression profile.MethodsRNA was separately extracted from peripheral blood neutrophils and mononuclear leukocytes, labeled, and hybridized to genome level microarrays to generate expression profiles of children with polyarticular juvenile idiopathic arthritis, juvenile dermatomyositis relative to childhood controls. Statistically significantly differentially expressed genes were identified from samples of each disease relative to controls. Functional network analysis identified interactions between products of these differentially expressed genes.ResultsIn silico models of both diseases demonstrated similar features with properties of scale-free networks previously described in physiologic systems. These networks were observable in both cells of the innate immune system (neutrophils) and cells of the adaptive immune system (peripheral blood mononuclear cells).ConclusionGenome-level transcriptional profiling from childhood onset rheumatic diseases suggested complex interactions in two arms of the immune system in both diseases. The disease associated networks showed scale-free network patterns similar to those reported in normal physiology. We postulate that these features have important implications for therapy as such networks are relatively resistant to perturbation.

A dynamic model of gene expression in monocytes reveals differences in immediate/early response genes between adult and neonatal cells

Neonatal monocytes display immaturity of numerous functions compared with adult cells. Gene expression arrays provide a promising tool for elucidating mechanisms underlying neonatal immune function. We used a well-established microarray to analyze differences between LPS-stimulated human cord blood and adult monocytes to create dynamic models for interactions to elucidate observed deficiencies in neonatal immune responses.We identified 168 genes that were differentially expressed between adult and cord monocytes after 45 min incubation with LPS. Of these genes, 95% (159 of 167) were over-expressed in adult relative to cord monocytes. Differentially expressed genes could be sorted into nine groups according to their kinetics of activation. Functional modelling suggested differences between adult and cord blood in the regulation of apoptosis, a finding confirmed using annexin binding assays. We conclude that kinetic studies of gene expression reveal potentially important differences in gene expression dynamics that may provide insight into neonatal innate immunity.

Ablation of Stat3 by siRNA Alters Gene Expression Profiles in JEG-3 Cells: A Systems Biology Approach

Control of inflammation at the maternal-fetal interface is a critical element in mammalian pregnancy. Previous work from our laboratory has shown that Stat3 may be a placental mediator involved in maintaining immunologic homeostasis at the maternal-fetal interface. The aim of the current study is to further elucidate the role of Stat3 in response to inflammation. As ablation of Stat3 in mice results in embryonic lethality, we evaluated the role of Stat3 in vitro using an siRNA approach. Trophoblast-like JEG-3 cells were transfected with an siRNA construct specific to Stat3. Experimental and control cells were exposed to conditioned medium from PHA-activated peripheral blood mononuclear cells and incubated for 45 min. Cells were then collected and RNA isolated for transcriptional profiling using human Affymetrix U133 plus 2.0 GeneChips. Differences in gene expression between control and Stat3-ablated cells were evaluated using conventional statistical methods. Fifty-two genes were detected as up-regulated in conditioned medium in both mock transfected and in Stat3 siRNA transfected JEG-3 cells. Two genes (EPAS1 and RASGEF1B) were up-regulated only in cells transfected with negative control siRNA, while 36 genes were up-regulated only in cells transfected with Stat3 siRNA. Sixty genes were differentially expressed between Stat3 siRNA transfected cells relative to mock transfected cells both in basal and conditioned medium. These included 31 genes up-regulated with Stat3 siRNA transfected cells and 29 genes down-regulated with Stat3 siRNA. Eleven genes were differentially expressed only in basal medium. Seven of these were up-regulated in the presence of Stat3 siRNA and four were down-regulated. Nine genes were differentially expressed only in conditioned medium. Six of these were up-regulated and three down-regulated in the presence of Stat3 siRNA. Off-target effects were excluded in a second set of experiments in which Stat3 mRNA was targeted at a different site and quantitative real-time PCR performed on selected genes derived from the microarray analysis. While some of the genes that showed differential expression between Stat3-ablated cells and mock transfected controls were genes typically associated with immune response (e.g., CCR7 and IRAK1), in silico modeling of the microarray data also revealed complex networks of signaling molecules and molecules associated with cellular metabolism previously seen in transcription factor ablation in model organisms. We conclude thus: Stat3 controls a specific gene set in trophoblast-like JEG-3 cells. While some differentially expressed genes and in silico models of their functions are consistent with the hypothesis that Stat3 plays a role in regulating inflammation, Stat3-mediated response to inflammation appears to also involve complex homeostatic adaptations of a non-immunologic nature.

Disease-Associated Single-Nucleotide Polymorphisms From Noncoding Regions in Juvenile Idiopathic Arthritis Are Located Within or Adjacent to Functional Genomic Elements of Human Neutrophils and CD4+ T Cells

Juvenile idiopathic arthritis (JIA) is considered a complex disease in which the environment interacts with inherited genes to produce a phenotype that shows broad interindividual variability. Twenty‐four regions of genetic risk for JIA were identified in a recent genome‐wide association study (GWAS); however, as is typical of the results of GWAS, most of the regions of genetic risk (22 of 24) were in noncoding regions of the genome. This study was undertaken to identify functional elements (other than genes) that might be located within the regions of genetic risk.