Role of Tumor Necrosis Factor-α in the Human Systemic Endotoxin-Induced Transcriptome

<div><p>TNFα has been implicated in the pathogenesis of various inflammatory diseases. Different strategies to inhibit TNFα in patients with sepsis and chronic inflammatory conditions have shown contrasting outcomes. Although TNFα inhibitors are widely used in clinical practice, the impact of TNFα antagonism on white blood cell gene expression profiles during acute inflammation in humans <i>in vivo</i> has not been assessed. We here leveraged the established model of human endotoxemia to examine the effect of the TNFα antagonist, etanercept, on the genome-wide transcriptional responses in circulating leukocytes induced by intravenous LPS administration in male subjects. Etanercept pre-treatment resulted in a markedly dampened transcriptional response to LPS. Gene co-expression network analysis revealed this LPS-induced transcriptome can be categorized as TNFα responsive and non-responsive modules. Highly significant TNFα responsive modules include NF-kB signaling, antiviral responses and T-cell mediated responses. Within these TNFα responsive modules we delineate fundamental genes involved in epigenetic modifications, transcriptional initiation and elongation. Thus, we provide comprehensive information about molecular pathways that might be targeted by therapeutic interventions that seek to inhibit TNFα activity during human inflammatory diseases.</p></div

TNFα responsive module hub (driver) genes and co-expression network visualization.

<p>Genes within transcriptional modules can be categorized as peripheral or hubs on the basis of how correlated a gene is with all other genes in the network, defined as the genes' connectivity measure, <b>k</b>. High intramodulr connectivities denote highly important module genes oftentimes possessing transcriptional factor activity. <b>A</b>. Unsupervised hierarchical clustering heatmap plot of the TNFα responsive module hub genes. Red denotes high expression; blue denotes low expression. The relative importance of each module within the co-expression network can be highlighted by unsupervised visualizations of each genes' weighted correlation coefficient. This was implemented in the Cytoscape® platform <b>B</b>. TNFα responsive co-expression modules were visualized by an organic layout considering weighted correlation coefficients >0.1 (equivalent to correlation coefficient >0.9).</p

Genomic analysis of the systemic LPS-induced transcriptional response and impact of TNFα inhibition.

<p><b>A</b>. Volcano plot analysis (integrating p-values and log2 foldchanges) for the LPS-induced response in subjects treated with placebo. <b>B</b>. Volcano plot analysis of the LPS-induced response in subjects treated with the TNFα antagonist etanercept. Red dots in panels A and B indicate probes that showed a fold-change ≥1.5 or ≤1.5. <b>C</b>. Unsupervised hierarchical clustering heatmap of the 4077 LPS-induced transcripts that were influenced by etanercept treatment as identified by ANOVA (q-value <0.05). Columns represent subject samples and rows represent transcripts. Red indicates increased gene expression, and blue indicates decreased gene expression.</p

Functional annotation and hub genes for the LPS-induced co-expression modules.

<p>LPS-induced transcriptome is organized into 38 co-expression network modules. Each module was analyzed for enrichment of biological pathways by IPA (Ingenuity® systems, <a href="http://www.ingenuity.com" target="_blank">www.ingenuity.com</a>).</p

LPS-induced TNFα responsive module genes linked to transcriptional initiation, elongation and epigenetic regulation.

<p>Genes within LPS-induced TNFα responsive co-expression modules possessing epigenetic regulation, transcriptional initiation and elongation properties. kTotal, total connectivity, k. kWithin, intra-modular connectivity. kOut, extra-modular connectivity. <i>log2</i> FC LPS, log2 transformed foldchange for the placebo-treated pre- and post-LPS challenged samples. <i>log2</i> FC LPS+Etan, log2 transformed foldchange for the etanercept-treated pre- and post-LPS challenged samples. Gene names marked in bold type denote module genes identified as top module hub genes.</p

Genotype effects at rs13477506.

<p>(A) <i>Tnni3k</i> expression (Illumina probe ILMN_3023962) as a function of genotype at rs13477506 in F2 mice; homozygous 129P2: AA, green; 129P2-FVBN/J heterozygous: AB, turquoise; homozygous FVBN/J: BB, blue; the darker shades represent the independent validation of the <i>Tnni3k</i> transcription levels by Q-PCR (right y-axis). (B) PR interval as a function of genotype at rs13477506 in F2 mice. Error bars indicate standard errors.</p

Overview of the locations of the linkage regions on chromosome 3.

<p>(A) Entire mouse chromosome 3 with refseq genes indicated in dark blue, black bar: 1.5 LOD drop of the PR-QTL; dark red bar 20 Mb DBA.AKR congenic region; purple bar 1.5 LOD drop of the Tnni3k eQTL; grey bar minimal region of overlap. (B) 1 Mb close up of the minimal region of overlap; the positions of rs49812611 (associated with nonsense mediated decay in DBA/2J) and rs13477506 (QTLs) are indicated. (C) Haplotypes of a panel of 9 inbred mouse strains as determined by the mouse phylogeny viewer (<a href="http://msub.csbio.unc.edu/" target="_blank">http://msub.csbio.unc.edu/</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003113#pgen.1003113-Yang1" target="_blank">[19]</a>.</p

LOD plot of PR interval (black, left y-axis) and <i>Tnni3k (purple</i> right y-axis) on chromosome 3.

<p>LOD plot of PR interval (black, left y-axis) and <i>Tnni3k (purple</i> right y-axis) on chromosome 3.</p

Tnni3k protein levels.

<p>Immunoblot of atrial (A) and ventricular (V) protein lysates of AKR/J and DBA/2J mice using α-mTnni3k and α-ILK as loading control.</p

<i>In silico</i> and <i>in vivo</i> correlation of <i>Tnni3k</i> levels and PR interval duration.

<p>(A) <i>In silico</i> analysis: mouse inbred lines are colored based on the haplotypes in the minimal region of overlap; PR interval duration obtained from the mouse phenome database (<a href="http://phenome.jax.org/" target="_blank">http://phenome.jax.org/</a>) FVBN/J (blue) (low expression) and green (high <i>Tnni3k</i> expression), inbred lines with the reddish colors carry rs49812611 (associated with nonsense mediated decay). (B) <i>In vivo</i> analysis of <i>Tnni3k</i> expression levels (y-axis) and PR interval duration (x-axis) in six inbred mouse lines, colors again denote the haplotype (gray is unknown haplotype).</p