Whole blood expression profiling from the TREAT trial: insights for the pathogenesis of polyarticular juvenile idiopathic arthritis
© The Author(s). 2016
Received: 21 March 2016
Accepted: 22 June 2016
Published: 7 July 2016
The Trial of Early Aggressive Therapy in Juvenile Idiopathic Arthritis (TREAT trial) was accompanied by a once-in-a-generation sample collection for translational research. In this paper, we report the results of whole blood gene expression analyses and genomic data-mining designed to cast light on the immunopathogenesis of polyarticular juvenile idiopathic arthritis (JIA).
TREAT samples and samples from an independent cohort were analyzed on Affymetrix microarrays and compared to healthy controls. Data from the independent cohort were used to validate the TREAT data. Pathways analysis was used to characterize gene expression profiles. Furthermore, we correlated differential gene expression with new information about functional regulatory elements within the genome to develop models of aberrant gene expression in JIA.
There was a strong concordance in gene expression between TREAT samples and the independent cohort. In addition, rheumatoid factor (RF)-positive and RF-negative patients showed only small differences on whole blood expression profiles. Analysis of the combined samples showed 158 genes represented by 176 probes that showed differential expression between TREAT subjects at baseline and healthy controls. None of the differentially expressed genes were encoded within linkage disequilibrium blocks containing single nucleotide polymorphisms known to be associated with risk for JIA. Functional analysis of these genes showed functional associations with multiple processes associated with innate and adaptive immunity, and appeared to reflect overall suppression of STAT1–3/interferon response factor-mediated pathways.
Despite their limitations, whole blood expression profiles clearly distinguish children with polyarticular JIA from healthy controls. Whole blood expression profiles identify several immunologic pathways of biologic relevance that will need to be pursued in homogeneous cell populations in order to clarify mechanisms of pathogenesis.
ClinicalTrials.gov registry #NCT00443430, originally registered 2 March 2007 and last updated 30 May 2013.
The Trial of Early Aggressive Therapy in Juvenile Idiopathic Arthritis (TREAT; ClinicalTrials.gov registry #NCT00443430) was an National Institute of Health (NIH) funded clinical trial  that compared two therapeutic regimens for treatment of newly-diagnosed polyarticular juvenile idiopathic arthritis (JIA). One treatment arm used methotrexate (MTX) as an initial therapy, while the other used a combined regimen of MTX, the tumor necrosis factor (TNF) inhibitor etanercept (ET), and oral prednisone. As part of the TREAT trial, whole blood was collected for RNA expression studies at specific time points during the course of the first year of therapy. Using whole blood expression data from the TREAT subjects, we have previously reported on the feasibility of developing expression-based prognostic biomarkers for children with the polyarticular form of JIA . However, the whole blood gene expression data also provide a window through which we might also gain valuable insights into both the pathogenesis of JIA and the underlying biology of treatment response, both of which are currently poorly understood.
While whole blood (and buffy coat) expression data are inherently “noisy” (among other things, they reflect gene expressions in multiple cells and cell subsets), there are both technical [3, 4] and computational approaches  that can be used to improve the signal-to-noise ratio in whole blood expression data and derive meaningful mechanistic insights. Furthermore, projects like the NIH Encyclopedia of Functional DNA Elements (ENCODE) and Roadmap Epigenomics have provided investigators with a wealth of information from which to derive mechanistic insights from gene expression data. In this study, we used whole blood gene expression data derived from baseline samples from children enrolled in the TREAT study, coupled with data-mining from public resources, to identify novel pathways that contribute to JIA disease pathogenesis.
Phenotypic characteristics of patients at month 0 (baseline)
19 (63.3 %)
26 (76.5 %)
11 (36.7 %)
8 (23.5 %)
Aged 2–6 years
Aged 7–11 years
Aged 12–16 years
Age (years), mean ± SD
10.5 ± 4.5
11.4 ± 3.6
9 (30 %)
12 (35.3 %)
22 (73.3 %)
18 (52.9 %)a
Healthy control samples
Controls consisted of 8 healthy girls and 11 healthy boys between the ages of 7 and 13 years recruited from the OU Children’s Physicians General Pediatrics clinic. The protocol for obtaining these specimens was approved by the University of Oklahoma IRB (#13205). Anesthesia for the phlebotomy was provided using topical lidocaine/prilocaine solution. These samples are hereafter referred to as healthy children (HC).
Characteristics of JIA patients from the Oklahoma cohort
All research procedures were carried out strictly following the IRB-approved protocols.
RNA was purified from whole blood PAXgene specimens using a PAXgene Blood RNA kit (Qiagen, Valencia, CA, USA) as recommended by the manufacturer, including a DNAse (Qiagen) step to remove genomic DNA. Globin transcripts, which reduce labeling efficiency of whole blood cell RNA and decrease signal-to-noise ratios on microarrays , were reduced using GLOBINclear-Human (Ambion, Austin, TX, USA). Final RNA preparations were suspended in RNase-free water, quantified spectrophotometrically, and analyzed for RNA integrity by capillary gel electrophoresis (Agilent 2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA, USA).
cRNA was produced from reverse transcribed cDNA using the Illumina® TotalPrep RNA Amplification Kit (Ambion, Inc., Austin, TX, USA), hybridized to Illumina WG-6 v3 or Illumina HT-12 v4 human whole genome microarrays, and stained according to the manufacturer’s directions. Array hybridizations were undertaken in three separate batches. The first batch consisted of the 19 healthy controls and 26 baseline samples hybridized on Illumina WG-6 v3 arrays. The second batch consisted of the remaining patient samples hybridized to Illumina HT-12 v4 arrays. The independent cohort of OK samples were hybridized on Illumina WG-6 v3 slides. Gene microarray data have been made available to the scientific public (GEO Accession Number GSE55319).
All statistical analyses were carried out in R (www.r-project.org). To facilitate statistical analyses relative to healthy controls, it was necessary to combine data from different array batches. Due to the difference in the arrays it was necessary to create combined datasets using only those probes that were present on both array formats. Illumina probe IDs were used to identify 39,426 common probes. Datasets were variance stabilized and normalized using robust spline normalization via the lumi package [8, 9]. Prior to statistical analysis non-responding probes were filtered out of the datasets using the detection p value provided by the Illumina quality control metrics to eliminate probes not responding at higher than background levels.
Differential gene expression analysis was performed using the limma package [10, 11]. The false discovery rate (FDR) was estimated using the method described by Benjamini and Hochberg . Statistical significance of gene expression was determined at FDR ≤0.05. Gene lists of interest were exported from R and uploaded to Ingenuity IPA (Ingenuity Systems, Inc., Redwood City, USA) for further functional analysis.
Network analyses of differentially expressed genes from whole blood expression profiles
We used the Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems®, Redwood City, CA, USA; IPA Summer Release, June, 2015) for network analysis, with differentially expressed (DE) genes as input following the default setting of a maximum 35 genes per network. IPA network generation transforms the query genes into a set of relevant networks based on its extensive, curated Ingenuity Pathway Knowledge Base database [12, 13]. IPA utilizes a multi-stage heuristic algorithm in constructing networks through an iterative process that optimizes both interconnectivity and number of query genes under the constraint of network size. Briefly, IPA constructs networks using gene connectivity with other genes under the assumption that the gene with the highest number of connections is the most important and, thus, has the most influence. These connections represent regulatory interactions that may be either direct (e.g., the protein product of gene “A” directly regulates the expression of gene “B”) or indirect (genes “A” and “B” are regulated by the same transcription factor). The last step of IPA network construction is score calculation using the Fisher Exact test on a hypergeometric distribution. The Fisher Exact test in IPA defines the null hypothesis as being a similar proportion of query genes map to a network in the same proportion as the entire reference gene set map to the network. A network score is derived from a p value (score = –log10 p value) that indicates the probability of the query genes (defined as target molecules in IPA) in a network being randomly associated within a connecting network. It is important to note that the network score does not infer network quality; it simply indicates the fitness between a reference network and the network of query genes.
Linking genetic and expression data
The recent completion of a genome-wide fine mapping study for JIA  extends the list of previously known disease-associated genetic variants  and provided us with the opportunity to determine whether there is a genetic linkage to gene expression in JIA. Using the bedtools program  we intersected DE genes identified in the comparison between JIA and HC with linkage disequilibrium (LD) blocks of JIA-associated single nucleotide polymorphisms (SNPs) extracted from the JIA fine mapping study  as well as other known regions of risk as reviewed by Hersh and Prahalad . We obtained LD block information from the SNAP database (http://www.broadinstitute.org/mpg/snap) using the 1000 genome project pilot1 and HapMap3 with a cutoff of r 2 < 0.9 and a distance limit of 500 bases.
Correlation of TREAT samples with the Oklahoma cohort
From the TREAT study we analyzed 44 baseline samples from 28 RF-negative patients and 16 RF-positive patients, plus 19 control samples from healthy children. In addition, we analyzed independent samples from 10 RF-negative patients (the OK cohort). Oklahoma and TREAT data were first normalized without using the COMBAT algorithm that we previously applied to the TREAT data to filter out batch effects , as there is only one condition (baseline) in the OK subjects for comparison and, thus, the algorithm cannot differentiate between biological variation and technical variation (batch effects). Our assumption was that batch effects would weaken the statistical correlation relative to the true biological situation, and any correlation we might find using this approach would thus be significant.
We also note that in our biomarker paper  we were able to corroborate gene expression in the TREAT and Oklahoma cohorts using quantitative polymerase chain reaction (qPCR) . Having assured ourselves of the reproducibility of the array results, we proceeded to data analysis.
Differential gene expression
Differentially expressed genes in juvenile idiopathic arthritis between RF-positive and RF-negative patients
Mean expression RF– (Log2)
Mean expression RF+ (Log2)
Fold change (Log2)
Linking genetic and expression data
The search for overlaps between the DE genes identified in the comparison between healthy controls and baseline TREAT subjects and LD blocks containing JIA-associated SNPs yielded no DE genes within any of the observed LD blocks. We next determined the closest LD blocks to the DE genes, and identified 75 LD blocks containing JIA-associated SNPs situated near 38 DE genes, with distances ranging between 345 and 390 kilobases. Next, we interrogated the upstream regulators of the DE genes as identified on Ingenuity analysis, i.e., IRF1, IRF3, IRF5, STAT1, STAT2, and STAT3. We queried the LD blocks containing the JIA-associated SNPs using the bedtools program. We downloaded ENCODE transcription factor binding site (TFBS) data from UCSC Genome Browser ENCODE data at http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeRegTfbsClustered/and extracted TFBSs of the regulators of interest. Intersection analysis demonstrated that 53 LD blocks containing JIA-associated SNPs overlapped 232 TF binding sites for the regulators of interest. We applied Fisher’s Exact test for enrichment analysis of TF binding sites overlapping with the LD blocks containing JIA-associated SNPs using all ENCODE TF binding sites as background. This analysis showed no statistically significant evidence for enrichment of IRF1, IRF3, IRF5, STAT1, STAT2, or STAT3 binding within the LD blocks containing JIA-associated SNPs (Fisher’s Exact test p value 0.091).
In this paper, we analyzed the TREAT whole blood gene microarray data in an attempt to gather insights into disease mechanisms in polyarticular JIA. We found that the whole blood expression profiles reflect complex interactions between innate and adaptive immunity, a finding that is consistent with our previous reports that both peripheral blood mononuclear cell (PBMC)  and neutrophil  gene expression profiles are abnormal in untreated children with JIA. We were not able to answer the question of whether the innate or adaptive immune aberrations are primary. While an increasing body of data shows that neutrophils are important mediators of adaptive immune responses [26–31], it is equally plausible that the transcriptional aberrations we see in neutrophils are the result of an altered cytokine milieu generated from altered T-cell function. In support of the idea that the neutrophil defect is a primary aspect of the disease is our finding that neutrophil gene expression profiles in JIA patients remain distinctly abnormal even after PBMC profiles begin to resemble those of healthy children in response to therapy .
These findings are also significant for what they do not tell us. Among the 158 genes that showed differential expression between baseline TREAT samples and those of healthy controls, none was located within LD blocks where there is known genetic risk for JIA [14, 15], nor are the identified LD blocks enriched for binding sites for the TFs that the whole blood expression data suggest may be important regulators of the differentially expressed genes. This finding corroborates published work demonstrating that most of the genetic risk for JIA lies within the non-coding genome . We have recently demonstrated that most of the regions identified by Hinks et al.  are enriched (above genome background) for H3K4me1/H3K27ac-marked enhancers that can be identified in both neutrophils and CD4+ T cells . Thus, if polyarticular JIA, like many complex diseases, can be characterized by the presence of so-called expression quantitative trait loci (eQTL) , it seems likely that the loci that most strongly influence expression will be located in non-promoter regulatory regions (e.g., enhancers, insulators, and so forth) and reflect complex layers of transcriptional control rather than perturbed function of the protein products of specific genes.
Our findings here suggest potentially useful targets for therapy in JIA. For example, the broad suppression of type 1 and type 2 interferon responses in JIA may reflect overall suppression of TLR9-mediated processes. TLR9 is an intracellular pattern recognition receptor that detects highly methylated DNA, which is common in bacterial and viral genomes (and relatively rare in mammalian genomes) . In recent years, TLR9 has become an attractive therapeutic target for immune modulation in immune diseases [36, 37], as well as cancer [38–42]. Given these findings, it is hardly surprising that hydroxychloroquine, which suppresses TLR9 pathways [43, 44], has been shown to be ineffective in JIA. The TREAT whole blood expression data suggest that strategies to augment TLR9 responses might be more promising.
There are obviously limitations to these data and their interpretation. The first is the inherent “noisiness” of whole blood expression profiles. Whole blood expression profiles represent an amalgam of peripheral blood cells, including abundant leukocyte subtypes such as neutrophils and platelets, and less abundant cells such as monocytes, natural killer (NK) cells, and even circulating CD34+ cells . Furthermore, while the complete blood counts of the TREAT baseline subjects did not deviate from the range typically seen in children of the same age, it is possible that the differences in expression profiles reflect expansion of small leukocyte subsets not typically identified on complete blood counts performed in a standard clinical laboratory. The noisiness and relative insensitivity of whole blood expression profiling can be reduced by removing globin genes before the RNA labeling step [3, 46], as we did here, but this step reduces only a small portion of the complexity that limits the utility of whole blood expression data. Leukocyte subset transcriptomes show a considerable degree of specificity, reflecting the specific immunologic functions of each cell type. Thus, while there are considerable commonalities in the transcriptomes and regulatory regions of peripheral blood leukocytes [47–49], it is likely that there are critical elements of leukocyte function/dysfunction in polyarticular JIA (e.g., B cells, monocytes, Th17 cells) that simply cannot be identified on whole blood expression profiling. Thus, while whole blood or blood leukocyte expression profiling has been invaluable in allowing us to develop a mechanistic understanding of significant pathologic disturbances, such as sepsis or blunt trauma , it seems likely that complex functional genomics approaches of specific leukocyte subsets will be required to fully elucidate the pathogenesis of more subtle phenotypes where inflammation is chronic and more indolent, as is the case in polyarticular JIA .
Whole genome expression profiling of untreated children with polyarticular JIA reveals complex transcriptional differences when compared with healthy controls. Activation of leukocyte chemotaxis/extravasation pathways and neutrophil activation by CSF3 are reflected in whole blood transcription analyses. At the same time, suppression of STAT1–3/IRF pathways, as we have previously reported in JIA neutrophils , is revealed in the whole blood expression profile. None of the genes that showed differential expression between children with JIA and healthy control children is encoded within LD blocks containing known JIA-associated SNPs. These findings suggest that genetic risk loci for JIA either exert their effects before the disease phenotype emerges or involves more subtle and complex layers of transcriptional regulation (e.g., by trans-acting enhancers ) than can be discerned from whole blood expression profiles.
DE, differentially expressed; ET, etanercept; FDR, false discovery rate, HC, healthy children; IL, interleukin; IPA, Ingenuity Pathway Analysis; IRF, interferon response factor; JIA, juvenile idiopathic arthritis; LD, linkage disequilibrium; MTX, methotrexate; NIH, National Institute of Health; OK, independent Oklahoma cohort; PBMC, peripheral blood mononuclear cell; RF, rheumatoid factor; SNP, single nucleotide polymorphism; TFBS, transcription factor binding site; TGF, transforming growth factor; TLR, Toll-like receptor; TREAT, Trial of Early Aggressive Therapy in Juvenile Idiopathic Arthritis
This study was supported by NIH grants R01 AI-084200, R01 AR-060604 (JNJ) and R01-AR 049762 (CAW).
KJ and YC performed the wet lab experiments. This included sample processing, RNA purification, and ascertainment of RNA purity. LW, ADS, and KJ performed data analysis. This included differential gene expression analysis and corroboration of the OK and TREAT cohorts (ADS), querying of LD blocks for DE genes and TF binding (LW), and Ingenuity Pathway analysis (KJ). MBF was responsible for performing microarray procedure and assisted in data analysis and interpretation. This included RNA labeling and hybridization procedures as well as preliminary statistical procedures to monitor quality of hybridization reactions. CAW was the principal investigator on the TREAT study and assisted in data interpretation. JNJ designed and organized this study and assisted in data analysis and interpretation. All authors assisted in drafting and revising the manuscript, and read and approved the manuscript.
The authors declare that they have no competing interests.
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