Host Targeted Activity of Pyrazinamide in <i>Mycobacterium tuberculosis</i> Infection

<div><p>Pyrazinamide (PZA) is one of the first line antibiotics used for the treatment of tuberculosis (TB). In the present study, we have used in vitro and in vivo systems to investigate whether PZA, in addition to its known anti-mycobacterial properties, modulate the host immune response during <i>Mycobacterium tuberculosis</i> (Mtb) infection. In vitro we have examined the effect of PZA on cytokine and chemokine release by Mtb-infected or Toll-like receptor (TLR) -stimulated primary human monocytes. In vivo, we have investigated at the transcriptional levels using genome-wide microarray gene expression analysis, whether PZA treatment of Mtb-infected mice alters the host immune response to Mtb infection in the lungs. Here, we report that PZA treatment of Mtb-infected human monocytes and mice significantly reduces the release of pro-inflammatory cytokines and chemokines, including IL-1β, IL-6, TNF-α and MCP-1 at the protein and at the gene transcription levels, respectively. Data from microarray analysis also reveal that PZA treatment of Mtb-infected mice significantly alters the expression level of genes involved in the regulation of the pro-inflammatory mediators, lung inflammatory response and TLR signaling networks. Specifically, genes coding for adenylate cyclase and Peroxisome-Proliferator Activated Receptor (PPAR), molecules known for their anti-inflammatory effect, were found to be up-regulated in the lungs of PZA-treated Mtb-infected mice. Based on the microarray findings, we propose that PZA treatment modulates the host immune response to Mtb infection by reducing pro-inflammatory cytokine production, probably through PPAR- and NF-kB- dependent pathways. In addition, our results suggest that inclusion or exclusion of PZA in the TB treatment regimen could potentially affect the biomarker signature detected in the circulation of TB patients.</p></div

Expression of lung inflammatory response network genes in the untreated or PZA-treated mice.

<p>(A). Interaction of genes involved in the host inflammatory response network in the untreated Mtb-infected mouse lungs at 42 days. Red and green symbols in the networks indicate up-, and down-regulated SDEG and the gradation in the color intensity of symbols is proportional to their relative expression levels. (B). Intensity map of 28 SDEG in the untreated and PZA-treated Mtb-infected mouse lungs at 42 and 63 days. The scale bar ranges from +3 (up-regulated; red) to –3 (down-regulated; blue).</p

PZA effect on monocytes stimulated with selected TLR agonists

<p>. Human monocytes treated with PZA (10 or 50 µg/ml) were simultaneously stimulated with (A) TLR4 (LPS, 100 ng/ml) or (B) TLR2/6 (Pam2, 250 ng/ml) and TLR2/1 (Pam3, 250 ng/ml) agonists. Pro-inflammatory and down-regulatory mediators were measured in the culture supernatants at 24 hours post-stimulation. Data are from 4 – 7 independent experiments (independent donors; N =  4 – 7) performed in duplicate and presented as percentage induction relative to PZA-untreated TLR-stimulated cells ± SD. * statistically significant; <i>P</i> ≤ 0.05 compared with the PZA-untreated TLR-stimulated cells.</p

Morphometric analysis of mouse lungs infected with wild type and <i>cut3</i> Val223Ala mutant strains at 12 weeks of infection.

<p><b>(A) Representative image of mouse lung sections.</b> The image represents 4× magnification of lung sections stained with hematoxylin and eosin. Arrows indicate granulomatous lesions. Scale = 1mm. <b>(B-C) Proportion of involved lung and granuloma size.</b> The graphs show the proportion of lung parenchyma occupied by granulomas <b>(B)</b> and the size of granulomas <b>(C)</b>. Results represent the mean and standard deviation of data collected from 3 mice per infecting bacterial strain.</p

Growth curves of wild type, <i>cut3</i> Val223Ala and <i>plcA</i> His39Arg mutant strains in liquid medium, THP-1 cells and murine lung.

<p><b>(A and C) Growth curves in liquid medium</b>. Growth curves in liquid medium over 16 days of incubation for <i>cut3</i> Val223Ala mutant (<b>A</b>) and <i>plcA</i> His39Arg mutant (<b>C</b>) strains. <b>(B and D) Growth curves in THP-1 cells</b>. Growth curves of <i>cut3</i> Val223Ala mutant <b>(B)</b> and <i>plcA</i> His39Arg mutant (<b>D</b>) strains in THP-1 cells over 6 days of infection. Data are expressed as CFU increase relative to day 0. <b>(E) Growth curves in murine lung.</b> Growth curves of wild type, <i>cut3</i> Val223Ala mutant, and <i>plcA</i> His39Arg mutant strains in the murine lung up to 12 weeks post-infection. Panels A-D show the mean and standard deviation of results obtained from three replicates; panel E shows the mean and standard deviation of results obtained from 3–4 animals per time point.</p

Expression of canonical PPAR and NF-kB pathway genes in the untreated or PZA-treated infected mouse lungs.

<p>(A). Canonical PPAR and NF-kB pathway map showing interaction of genes in the untreated Mtb-infected mouse lungs at 42 days. The legends for gene symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074082#pone-0074082-g004" target="_blank">Figure 4</a>. Red and green symbols in the networks indicate up-, and down-regulation of SDEG and the gradation in the color intensity of symbols is proportional to their relative expression level. (B). Intensity map of 41 SDEG involved in the PPAR and NF-kB pathways in the untreated and PZA-treated mouse lungs at 42 and 63 days. The scale bar ranges from +3 (up-regulated; red) to –3 (down-regulated; blue).</p

PZA effect on the release of pro-inflammatory cytokines.

<p>Human monocytes treated with PZA (10 or 50 µg/ml) and simultaneously infected with Mtb strain CDC1551 or HN878. Pro-inflammatory mediators were measured in culture supernatants at 24 hours post-infection. Data are from seven independent experiments (independent donors; N = 7) performed in duplicate, and presented as percentage induction relative to PZA-untreated Mtb-infected cells ± SD. * statistically significant; <i>P</i>≤0.05 compared with PZA-untreated Mtb-infected cells.</p

Effect of PZA on the growth of CDC1551 and HN878 in human monocytes.

*<p>CFU<b>×</b>10⁵ ± SD<b>.</b></p

Parameters of the continuous intracellular culture model.

<p>THP-1 cells were infected with <i>M</i>. <i>tuberculosis</i> H37_RvSiena strain for 10 serial infection cycles. The graphs show the parameters evaluated for each cycle. <b>(A) Bacterial CFU count.</b> Bacterial CFU at day 1 and day 7 of infection. <b>(B) Intracellular generations.</b> Number of bacterial generations per cycle, calculated by using the formula (log₂ CFU day 7- log₂ CFU day 1)/log₂ 2. <b>(C) Count of infected THP-1 cells.</b> Number of total, viable adherent THP-1 cells (black bar), and dead cells (white bar) at day 7 of infection.</p

Number of mouse genes in the selected pro-inflammatory networks.

*<p>not significant.</p