Cellular proportions in the BAL stratified by IGRA.

<p>Proportion of the three major cell types in the BAL fluid from study participants, stratified by A) blood and B) BAL IGRA status. Notched boxplots showing five number summaries (minimum, first quartile, median, third quartile, maximum) with outliers. The lower tables indicate the number of donors in each group for which cell counts for the respective cell type were available.</p

Induction of autophagy in AZP treated macrophages.

<p>THP-1 cells were treated with AZP for 24 h and stained with antibodies against LC3 to visualize the formation of auto-phagosomes.</p

Lymphocyte and macrophage cytokine production in blood IGRA negative and positive subjects.

<p>Correlations of cytokine levels with the proportion of lymphocytes and macrophages in the BAL stratified by blood IGRA result. Left: Estimated linear association between the proportion of lymphocytes and basal IFN-γ and IL-2 levels, with 95% confidence bands, in IGRA-negative and IGRA-positive healthy donors. Right: Correlations between proportion of macrophages and MIP-1α and TNF-α levels.</p

BAL fluid donors and their IGRA status in either peripheral blood or in BAL cells.

<p>BAL fluid donors and their IGRA status in either peripheral blood or in BAL cells.</p

Antimycobacterial activity of AZP and PN granule proteins determined by CFU and gel overlay assays.

<p>(<b>A</b>) <i>M. smegmatis</i> (4–9×10⁵/ml) was incubated with 50 µg/ml AZP fractions for 3 and 6 h, and CFU assay was performed to check the antimycobacterial effect of the protein fractions. Bacteria with buffer only served as control. Data are mean ± SD of three independent experiments. Significance was referred as ** for P<0.0001 and * for P<0.005. (<b>B</b>) Gel overlay assay of AZP and PN granular protein fractions against <i>M. smegmatis</i>. The granule proteins stained with Coomassie brilliant blue (left) and the clearing zone formed by the protein (right) shows the anti-mycobacterial activity.</p

Survival of <i>M. smegmatis</i> and <i>M. bovis</i> BCG in LPS-stimulated and unstimulated whole neutrophils.

<p>4–9×10⁵/ml <i>M. smegmatis</i> (<b>A</b>) and BCG (<b>B</b>) were incubated with LPS stimulated neutrophils (+) and unstimulated neutrophils (−) for indicated time points. Bacterial survival was determined by CFU assay. Medium containing bacteria alone was used as control. Data shown are from one representative experiment of three individual experiments. Experiments were performed in triplicates; mean ± SD are shown; Significance was referred as ** for P<0.0001.</p

Cytokine induction in BAL cell IGRA negative and positive subjects.

<p>Induction of cytokines by activated lymphocytes and macrophages upon antigen stimulation (triangles) or infection (circles), stratified by BAL IGRA status (open symbols, BAL IGRA-negative donors; closed symbols, BAL IGRA-positive donors). The plots show the effect estimates with lines representing 95% confidence intervals (solid line, antigen stimulations; broken line, <i>M</i>. <i>tuberculosis</i> infection assays). Related p-values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187882#pone.0187882.s008" target="_blank">S3 Table</a>. ESAT-6, 6kD Early Secretory Antigenic Target; CFP-10, 10kD Culture Filtrate Protein; PPD, Purified Protein Derivative; LPS, Lipopolysaccharide; PHA, Phytohaemagglutinin; H37Rv, <i>M</i>. <i>tuberculosis</i> H37Rv; Isol2, <i>M</i>. <i>tuberculosis</i> isolate 2; Isol3, <i>M</i>. <i>tuberculosis</i> isolate 3.</p

Gel overlay assay of AZP, PN and cytosol proteins.

<p>Azurophile (AZP), peroxidase-negative (PN) and cytosol proteins were run on AU-PAGE, Coomassie stained (left) and analyzed for antibacterial activity against <i>M. smegmatis</i>. Formation of clearing zone indicates the antimycobacterial activity (right).</p

Identification of AZP, PN and cytosol proteins by MALDI-TOF mass spectrometry.

*<p>These proteins were identified from total PN and cytosolic proteins (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050345#pone-0050345-g003" target="_blank">Figure 3</a>).</p

Lymphocyte and macrophage cytokine production in BAL cell IGRA negative and positive subjects.

<p>Correlations of cytokine levels with the proportion of lymphocytes and macrophages in the BAL stratified by BAL IGRA result. Left: Estimated linear association between the proportion of lymphocytes and basal IFN-γ and IL-2 levels, with 95% confidence bands, in BAL IGRA-negative and BAL IGRA-positive healthy donors. Right: Correlations between proportion of macrophages and MIP-1α and TNF-α levels.</p