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Whole-blood gene expression profiling in ankylosing spondylitis shows upregulation of toll-like receptor 4 and 5.

ABSTRACT: to identify differentially expressed genes in peripheral blood cells (PBC) of patients with ankylosing spondylitis (AS) relative to healthy controls and controls with systemic inflammation.we investigated PBC samples of 16 patients with AS and 14 matched controls, in addition to systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) samples utilizing Illumina Human Ref-8 BeadChips. Candidate genes were confirmed using quantitative PCR. Subsequently, these genes were also validated in a separate sample of 27 patients with AS [before and after anti-tumor necrosis factor (anti-TNF) treatment] and 27 matched controls.we identified 83 differentially expressed transcripts between AS patients and controls. This gene list was filtered through the lists of differentially expressed transcripts in SLE and SSc, which resulted in identification of 52 uniquely dysregulated transcripts in AS. Many of the differentially expressed genes belonged to Toll-like receptor (TLR) and related pathways. TLR4 and TLR5 were the only dysregulated TLR subtypes among AS patients. We confirmed the overexpression of TLR4 and TLR5 in AS patients in comparison to controls (p = 0.012 and p = 0.006, respectively) and SLE (p = 0.002, p = 0.008) using quantitative PCR in the same sample. Similarly, TLR4 (p = 0.007) and TLR5 (p = 0.012) were significantly upregulated among the AS patients before anti-TNF treatment in the confirmatory sample. TLR4 (p = 0.002) and TLR5 (p = 0.025) decreased significantly after anti-TNF treatment.PBC gene expression profiling in AS shows an upregulation of TLR4 and TLR5. This supports the importance of TLR subtypes in the pathogenesis of AS that are responsible for the immune response to Gram-negative bacteria.

SUBMITTER: Assassi S

PROVIDER: S-EPMC3014385 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Whole-blood gene expression profiling in ankylosing spondylitis shows upregulation of toll-like receptor 4 and 5.

Assassi Shervin S Reveille John D JD Arnett Frank C FC Weisman Michael H MH Ward Michael M MM Agarwal Sandeep K SK Gourh Pravitt P Bhula Jiten J Sharif Roozbeh R Sampat Keeran K Mayes Maureen D MD Tan Filemon K FK

The Journal of rheumatology 20101015 1

<h4>Objective</h4>to identify differentially expressed genes in peripheral blood cells (PBC) of patients with ankylosing spondylitis (AS) relative to healthy controls and controls with systemic inflammation.<h4>Methods</h4>we investigated PBC samples of 16 patients with AS and 14 matched controls, in addition to systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) samples utilizing Illumina Human Ref-8 BeadChips. Candidate genes were confirmed using quantitative PCR. Subsequently, the ...[more]

PMID: 20952467

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Project description:Introduction: A number of genetic-association studies have identified genes contributing to AS susceptibility but such approaches provide little information as to the gene activity changes occurring during the disease process. Transcriptional profiling generates a “snapshot” of the sampled cells activity and thus can provide insights into the molecular processes driving the disease process. We undertook a whole-genome microarray approach to identify candidate genes associated with AS and validated these gene-expression changes in a larger sample cohort. Methods: 18 active AS patients, classified according to the New York criteria. and 18 gender-and age-matched controls were profiled using Illumina HT-12 Whole-Genome Expression BeadChips which carry cDNAs for 48000 genes and transcripts. Class comparison analysis identified a number of differentially expressed candidate genes. These candidate genes were then validated in a larger cohort using qPCR-based TaqMan Low Density Arrays (TLDAs). Results: 239 probes corresponding to 221 genes were identified as being significantly different between patients and controls with a p-value <0.0005 (80% confidence level of false discovery rate). Forty seven genes were then selected for validation studies, using the TLDAs. Thirteen of these genes were validated in the second patient cohort with 12 down-regulated 1.3-2-fold and only 1 upregulated (1.6-fold). Among a number of identified genes with well-documented inflammatory roles we also validated genes that might be of great interest to the understanding of AS progression such as SPOCK2 (osteonectin) and EP300 which modulate cartilage and bone metabolism. Conclusion: We have validated a gene expression signature for AS from whole blood and identified strong candidate genes that may play roles in both the inflammatory and joint destruction aspects of the disease. RNA was extracted from whole blood using PAXGene tubes. 16 AS patients with active disease and 16 gender- and age-matched controls were analysed.

Project description:BackgroundThe precise pathogenesis of ankylosing spondylitis (AS) is still largely unknown at present. Our previous study found that toll-like receptor 4 (TLR4) downregulated and performed immunoregulatory dysfunction in mesenchymal stem cells from AS patients (AS-MSCs). The aim of this study was to explore the expression profiles of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in TLR4-primed AS-MSCs, and to clarify the potential mechanisms.MethodsThe immunoregulatory effects of MSCs were determined after TLR4 activation. Next, the differentially-expressed (DE) lncRNAs and mRNAs between AS-MSCs and TLR4-primed AS-MSCs [stimulated by lipopolysaccharide (LPS)] were identified via high-throughput sequencing followed by quantitative real-time PCR (qRT-PCR) confirmation. Finally, bioinformatics analyses were performed to identify the critical biological functions, signaling pathways, and associated functional networks involved in the TLR4-primed immunoregulatory function of AS-MSCs.ResultsA total of 147 DE lncRNAs and 698 DE mRNAs were identified between TLR4-primed AS-MSCs and unstimulated AS-MSCs. Of these, 107 lncRNAs were upregulated and 40 were downregulated (fold change ≥2, P<0.05), while 504 mRNAs were upregulated and 194 were downregulated (fold change ≥2, P<0.05). Five lncRNAs and five mRNAs with the largest fold changes were respectively verified by qRT-PCR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that the DE mRNAs and lncRNAs were highly associated with the inflammatory response, such as NOD-like receptor (NLR) signaling pathway, the TNF signaling pathway and the NF-κB signaling pathway. Cis-regulation prediction revealed eight novel lncRNAs, while trans-regulation prediction revealed 15 lncRNAs, respectively. Eight core pairs of lncRNA and target mRNA in the lncRNA-transcription factor (TF)-mRNA network were as follows: PACERR-PTGS2, LOC105378085-SOD2, LOC107986655-HIVEP2, MICB-DT-MICB, LOC105373925-SP140L, LOC107984251-IFIT5, LOC112268267-GBP2, and LOC101926887-IFIT3, respectively.ConclusionsTLR4 activation in AS can enhance the immunoregulatory ability of MSCs. Eight core pairs of lncRNA and target mRNA were observed in TLR4-primed AS-MSCs, which could contribute to understanding the potential mechanism of AS-MSC immunoregulatory dysfunction.

Project description:Several previous studies have attempted to investigate the regulatory mechanisms underlying gene expression in ankylosing spondylitis (AS). However, the specific molecular pathways underlying this condition remain unclear. Previous research used next-generation RNA sequencing to identify a series of differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMCs) when compared between patients with AS and healthy controls, thus implying that these DEGs may be related to AS. Furthermore, by screening these DEGS, it may be possible to facilitate clinical diagnosis and optimize treatment strategies. In order to test this hypothesis, we recruited 15 patients with AS and 15 healthy controls. We randomly selected five subjects from each group of patients for RNA sequencing analysis. Sequence reads were generated by an Illumina HiSeq2500 platform and mapped on to the human reference genome using HISAT2. We successfully identified 973 significant DEGs (p < 0.05) in PBMCs. When compared with controls, 644 of these genes were upregulated (with a fold?change?(FC) > 2) in AS patients and 329 were downregulated (FC < 0.5). Our analysis identified numerous genes related to immune response. Gene Ontology (GO) analysis indicated that these DEGs were significantly related to the positive regulation of epidermal growth factor-activated receptor activity, the positive regulation of the ERBB (erb-b2 receptor tyrosine kinase) signaling pathway, the differentiation of trophoblast giant cells, oxygen transport, immune-related pathways, and inflammation-related pathways. The DEGs were also closely related to the TNF and NF-?B signaling pathways. Six DEGs were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Receiver operating characteristic (ROC) curve analysis indicated that IL6 may represent a useful biomarker for diagnosing AS. The development of new biomarkers may help us to elucidate the specific mechanisms involved in the development and progression of AS.

Project description:BackgroundCircular RNA (circRNA) is a type of closed circular noncoding RNA (ncRNA), mostly formed by back-splicing or alternative splicing of pre-messenger RNA (mRNA). The aim of this study was to explore the expression profile of circRNA in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS) and discover potential molecular markers of AS.MethodsThe circRNA microarray technology was used to detect the expression of circRNAs in the peripheral blood of 6 patients with AS and 6 healthy controls (HC). To screen the differentially expressed circRNAs by fold change (FC) and P value, these differentially expressed circRNAs were analyzed by bioinformatics. In 60 cases of AS and 30 cases of HC, 4 circRNAs were subjected to real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and their correlation with various clinical indicators was analyzed. Finally, the receiver operating characteristic (ROC) curve was used to analyze their potential as AS diagnostic markers.ResultsThe microarray results showed that there were 1369 significantly differently expressed (P < 0.05, FC > 1.5) circRNAs between the AS and HC groups (675 upregulated and 694 downregulated). The results of bioinformatics analysis suggested that they were mainly involved in "enzyme binding," "adenosine ribonucleotide binding," "MAPK signaling pathway", etc. The RT-qPCR results showed that the expressions of hsa_circRNA_001544 (U = 486.5, P < 0.05) and hsa_circRNA_102532 (U = 645, P < 0.05) were significantly different between the AS group and the HC group. The AS group was further divided into two subgroups: active AS (ASA) and stable AS (ASS). After analysis, it was found that compared with the HC group, hsa_circRNA_001544 was significantly increased in both ASA (U = 214, P < 0.05) and ASS groups (U = 273, P < 0.05), while hsa_circRNA_008961 (U = 250, P < 0.05) and hsa_circRNA_102532 (U = 295, P < 0.05) were only significantly increased in the ASA group. Furthermore, hsa_circRNA_012732 was significantly different between the ASA and ASS groups (U = 194, P < 0.05), and there was no statistical significance among the remaining groups. Correlation analysis results showed that hsa_circRNA_012732 was negatively correlated with Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), high-sensitivity C-reactive protein (hsCRP), and globulin (GLOB) and positively correlated with lymphocyte count (LY), mean corpusular volume, and albumin (ALB), and hsa_circRNA_008961 was negatively correlated with platelet (PLT) count. ROC curve analysis showed that hsa_circRNA_001544 (95% CI = 0.610-0.831, P < 0.05) and hsa_circRNA_102532 (95% CI = 0.521-0.762, P < 0.05) were statistically significant, and their area under curve (AUC) values were 0.720 and 0.642, respectively.ConclusionsThere are differentially expressed circRNAs in PBMCs of AS patients, and they may be involved in the occurrence and development of AS. Among these differentially expressed circRNAs, hsa_circRNA_012732 has the potential to become an indicator of disease activity, and hsa_circRNA_001544 has the potential to become a molecular marker for AS diagnosis.

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Whole-blood gene expression profiling in ankylosing spondylitis shows upregulation of toll-like receptor 4 and 5.

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Whole-blood gene expression profiling in ankylosing spondylitis shows upregulation of toll-like receptor 4 and 5.

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