Gene and metabolite time-course response to cigarette smoking in mouse lung and plasma

<div><p>Prolonged cigarette smoking (CS) causes chronic obstructive pulmonary disease (COPD), a prevalent serious condition that may persist or progress after smoking cessation. To provide insight into how CS triggers COPD, we investigated temporal patterns of lung transcriptome expression and systemic metabolome changes induced by chronic CS exposure and smoking cessation. Whole lung RNA-seq data was analyzed at transcript and exon levels from C57Bl/6 mice exposed to CS for 1- or 7 days, for 3-, 6-, or 9 months, or for 6 months followed by 3 months of cessation using age-matched littermate controls. We identified previously unreported dysregulation of pyrimidine metabolism and phosphatidylinositol signaling pathways and confirmed alterations in glutathione metabolism and circadian gene pathways. Almost all dysregulated pathways demonstrated reversibility upon smoking cessation, except the lysosome pathway. Chronic CS exposure was significantly linked with alterations in pathways encoding for energy, phagocytosis, and DNA repair and triggered differential expression of genes or exons previously unreported to associate with CS or COPD, including <i>Lox</i>, involved in matrix remodeling, <i>Gp2</i>, linked to goblet cells, and <i>Slc22a12</i> and <i>Agpat3</i>, involved in purine and glycerolipid metabolism, respectively. CS-induced lung metabolic pathways changes were validated using metabolomic profiles of matched plasma samples, indicating that dynamic metabolic gene regulation caused by CS is reflected in the plasma metabolome. Using advanced technologies, our study uncovered novel pathways and genes altered by chronic CS exposure, including those involved in pyrimidine metabolism, phosphatidylinositol signaling and lysosome function, highlighting their potential importance in the pathogenesis or diagnosis of CS-associated conditions.</p></div

Pathways overlapping between transcriptomic and metabolomic data.

<p>Checkmarks signify significant pathway perturbation in metabolomic data (<i>p</i><0.05), pink shading denotes enrichment for upregulated genes in that pathway in CS-exposed mice; blue shading indicates enrichment for downregulation in CS-exposed mice (FDR≤0.10). Perturbations indicated by shaded fields under the “Cessation” column persisted following cessation when compared to AC mice.</p

KEGG pathways displaying unique enrichment patterns at 9 months of CS (FDR≤ 0.10).

<p>The pathways were either exclusively differentially enriched or exhibited an opposite direction of enrichment after 9 months CS exposure compared to other time points. *Denotes pathways enriched for upregulated genes after 9 months of CS exposure, but was not significantly enriched at any other time point. †Denotes pathways enriched for upregulated genes after 9 months of CS exposure, but initially enriched for downregulated genes after 7 days; ‡ denotes pathways enriched for downregulated genes after 9 months of CS exposure, but initially enriched for upregulated genes after 7 days of CS exposure. No pathways were significantly enriched for downregulated genes after 9 months that were not significantly enriched at any other time point.</p

Heatmap of the relative change in gene expression.

<p>Log2 FC in CS-exposed mice compared to AC mice of the 9 genes found to statistically significantly differentially expressed at four of five time points (FDR≤0.10). Positive fold change (pink) indicates upregulation in CS-exposed mice; negative fold change (blue) indicates downregulation in CS-exposed mice.</p

Mean static lung compliance (Cst).

<p>Cst measured by the flexiVent^™ instrument for each experimental group at each time point. C57Bl/6 mice were exposed to ambient air (AC, grey bars) or chronic cigarette smoke (CS, black bars) for the indicated duration of time measured in days (d) or months (mo). In one group of mice that underwent smoking cessation (SS; stop smoking, dark grey bar), mice were allowed to recover at ambient air (AC) for 3 months following a 6 months CS exposure. N = 10 mice /group. Horizontal bars with asterisk indicate significant differences in mean static lung compliance (<i>p</i><0.05). Bars represent the standard error of the mean.</p

Number of significantly differentially expressed genes, perturbed pathways and differentially used exons identified between CS- and AC-exposed mice, and between pairwise comparisons with SS (cessation group, for stop smoking) mice (FDR≤0.10, |FC| ≥ 1.5).

<p>Number of significantly differentially expressed genes, perturbed pathways and differentially used exons identified between CS- and AC-exposed mice, and between pairwise comparisons with SS (cessation group, for stop smoking) mice (FDR≤0.10, |FC| ≥ 1.5).</p

Inhibition of Nuclear Factor-Kappa B Activation Decreases Survival of <i>Mycobacterium tuberculosis</i> in Human Macrophages

<div><p></p><p>Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to <i>Mycobacterium tuberculosis</i> (<i>MTB</i>). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to <i>MTB</i>. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular <i>MTB</i>. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular <i>MTB</i> in human macrophages via induction of apoptosis and autophagy.</p></div

TNFα and IFNγ levels in MDM and AM infected with <i>MTB</i> H37Rv with or without NFκB inhibition.

<p>Primary human (<b>A</b>) MDM or (<b>B</b>) AM were infected with <i>MTB</i> H37Rv and after 1 hr, 24 hrs, 4 days, or 8 days of infection, supernatants were assayed for TNFα by ELISA and IFNγ by electrochemiluminescence. Data shown are estimated means with standard error bars from linear mixed model fits, based on seven independent experiments (MDM) or nine independent experiments (AM). *p<0.05 and **p<0.01 for cytokine expression in macrophages infected with <i>MTB</i> alone (closed circles) vs. <i>MTB</i>+BAY (closed squares). Control = (open circles) and BAY = (open squares).</p

<i>MTB</i> H37Rv induces NFκB binding to its <i>cis</i>-regulatory element.

<p>(<b>A</b>) THP-1 cells were pre-treated with 0.1% (v/v) DMSO or 5 µM BAY for 1 hr, followed by infection with <i>MTB</i> H37Rv at the indicated times. An EMSA was performed with an oligonucleotide that corresponds to the consensus binding sequence for NFκB. Data shown are representative of three independent experiments. (<b>B</b>) Primary human MDM or (<b>C</b>) AM were pre-incubated with 0.1% DMSO or 5 µM BAY for 1 hr, then infected with <i>MTB</i> for 3 hrs, and followed by an EMSA to assay for NFκB binding. Data shown are representative of two independent experiments for MDM and AM. NS = non-specific band. SS = supershift band.</p

Inhibition of NFκB activation using a dominant-negative to IκBα.

<p>(<b>A</b>) THP-1 cells were transduced with or without AdV-GFP or AdV-S32/36A-IκBα at a MOI of 30∶1 for 5 hrs and then infected with <i>MTB</i> H37Rv. After 4 days of infection, the cells were lysed and cell-associated <i>MTB</i> quantified. (<b>B</b>) Wildtype THP-1 cells with or without pre-treatment with 5 µM BAY were infected with <i>MTB</i> H37Rv. Other THP-1 cells were transduced with AdV-GFP or AdV-S32/36A-IκBα at a MOI of 30∶1 for 5 hrs and then infected with <i>MTB</i> H37Rv. After 24 hrs of infection, the supernatants were measured for IL-8 by ELISA. Data are means ± SEM of two independent experiments performed in duplicates. *p<0.05, ** p<0.01.</p