Pulmonary CD14 diminishes lung inflammation by high dose S-LPS, but enhances lung inflammation by low dose S-LPS.

<p>Mice (n = 7–9) were treated intranasally with 10 µg S-LPS (left panel), 1 µg S-LPS (middle panel) or 0.1 µg S-LPS (right panel). Six hours later BALF was isolated and analysed for PMN counts (A–C), TNF levels (D–F) and LIX levels (G–I). Data are mean ± SEM. **, P<0.01; ***, P<0001 versus WT mice.</p

sCD14 exerts bimodal effects in acute lung inflammation depending on the dose of S-LPS.

<p>WT and CD14KO mice were treated intranasally with 10 µg S-LPS (left panel) or 0.1 µg S-LPS (right panel) and 10 µg sCD14 was administered simultaneously with S-LPS to groups of CD14KO mice. Six hours after LPS (and sCD14) administration, BALF was isolated and analyzed for PMN counts (A, B), TNF levels (C, D) and LIX levels (ER, F). Eight to nine mice were used per group. Data are are mean ± SEM. *, P<0.05; **, P<0.01; ***, P<0001 versus WT mice; ##, P<0.01; ###, P<0.001 versus CD14KO mice.</p

Pulmonary CD14 partially diminishes lung inflammation by high dose R-LPS, but enhances lung inflammation by low dose R-LPS.

<p>Mice (n = 6–9) were treated intranasally with 10 µg R-LPS (left panel), 1 µg R-LPS (middle panel) or 0.1 µg R-LPS (right panel). Six hours after LPS administration, BALF was isolated and analysed for PMN counts (A–C), TNF levels (D–F) and LIX levels (G–I). Data are mean ± SEM. *, P<0.05; **, P<0.01; ***, P<0001 versus WT mice.</p

S-LPS induces sCD14 release in the lung in a dose dependent manner.

<p>sCD14 was measured in BALF obtained from WT mice 6 hours after intranasal administration of different doses (10–0.1 µg) of S-LPS. Eight to nine mice were used per group. Data are mean ± SEM. Dotted line represents the mean value of sCD14 in BALF of naive mice.</p

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

The TM^pro/pro mutation has limited impact on bacterial growth and dissemination.

<p>Mice were inoculated intranasally with 750<i>B. pseudomallei</i> and sacrificed after 24, 48 and 72 hours. Bacterial loads were determined in lung homogenates (A), spleen (B) and liver homogenates (C) and in whole blood (D). Data are expressed as box and whisker plots showing the smallest observation, lower quartile, median, upper quartile and largest observation. Grey boxes represent WT mice, white boxes represent TM^pro/pro mice (<i>n</i> = 8 mice/group). *<i>P</i><0.05 for the difference between WT and TM^pro/pro mice (Mann-Whitney <i>U</i> test). BC+ number of positive blood cultures/total number of mice per group.</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