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 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 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

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

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

*<p>not significant.</p

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

*<p>CFU<b>×</b>10⁵ ± SD<b>.</b></p

A combination of baseline plasma immune markers can predict therapeutic response in multidrug resistant tuberculosis

<div><p>Objective</p><p>To identify plasma markers predictive of therapeutic response in patients with multidrug resistant tuberculosis (MDR-TB).</p><p>Methods</p><p>Fifty HIV-negative patients with active pulmonary MDR-TB were analysed for six soluble analytes in plasma at the time of initiating treatment (baseline) and over six months thereafter. Patients were identified as sputum culture positive or negative at baseline. Culture positive patients were further stratified by the median time to sputum culture conversion (SCC) as fast responders (< 76 days) or slow responders (≥ 76 days). Chest X-ray scores, body mass index, and sputum smear microscopy results were obtained at baseline.</p><p>Results</p><p>Unsupervised hierarchical clustering revealed that baseline plasma levels of IP-10/CXCL10, VEGF-A, SAA and CRP could distinguish sputum culture and cavitation status of patients. Among patients who were culture positive at baseline, there were significant positive correlations between plasma levels of CRP, SAA, VEGF-A, sIL-2Rα/CD40, and IP-10 and delayed SCC. Using linear discriminant analysis (LDA) and Receiver Operating Curves (ROC), we showed that a combination of MCP-1/CCL2, IP-10, sIL-2Rα, SAA, CRP and AFB smear could distinguish fast from slow responders and were predictive of delayed SCC with high sensitivity and specificity.</p><p>Conclusion</p><p>Plasma levels of specific chemokines and inflammatory markers measured before MDR-TB treatment are candidate predictive markers of delayed SCC. These findings require validation in a larger study.</p></div

Baseline clinical and microbiological characteristics of MDR-TB patients.

<p>Baseline clinical and microbiological characteristics of MDR-TB patients.</p

Baseline levels of plasma markers.

<p>(A) Two-dimensional unsupervised hierarchical clustering of baseline analyte profiles in 50 patients, characterized by sputum smear (SS) and sputum culture (SC) status and cavitary vs non-cavitary disease. Normalized and log2 transformed values of analyte levels are indicated by the color scale, where yellow and blue represent expression levels above and below the median, respectively. Three-dimensional plots of principal component analysis (PCA) of (B) SS negative (orange) and SS positive (blue); (C) SC negative (yellow) and SC positive (blue); (D) cavitary (pink) and non-cavitary disease (green). Statistical comparisons using non-parametric Mann-Whitney U test were corrected for multiple comparisons through a false discovery rate (FDR) step down procedure (*: q<0.05, **: q<0.01, ***: q<0.001).</p