Cytokine plasma levels in active and clinically cured tuberculosis.

<p>Plasma levels of TNF-α, IFN-γ, IL-10, IL-4, IL-13, IL-6, IL-17, TGF-β and IL-22 in Healthy Donors, patients with active tuberculosis and clinically cured patients (<b>A–G</b>). Horizontal lines represent the median, bars represent 25–75 percentiles, and vertical lines represent 10–90 percentiles. *p<0.0167: Kruskal-Wallis test (followed by post-hoc Bonferroni/Dunn test for multiple comparisons).</p

T cell activation in active and clinical cured tuberculosis.

<p>Variation in the activation of helper and cytolytic T cells in Healthy Donors, patients with active tuberculosis and clinically cured patients. <b>A</b>. Schematic representation of the gating strategy and determination of Δ activation of CD69+ cells in stimulated (4 µg/mL <i>M. bovis</i> antigen) and unstimulated (medium only) cultures. From left to right, T cells were separated based on FSC and SSC patterns. These cells were divided into CD4+ and CD8+ and the expression of CD69 was evaluated. Dot plots representative of each studied group are shown. Blue dots represent the staining for CD69 in unstimulated cultures, and red dots represent the stimulated cultures. The percentage of specific antigen activation was calculated by simple subtraction of the percentage of CD69+ cells in unstimulated cultures from the percentage observed in stimulated cultures and the Δ activation for each depicted dot plot is shown. The superior panel of dot plots represents the activation in CD4+, and the inferior panel represents the activation in CD8+. Dot plots representative of a single participant are shown. <b>B, C</b>. Comparison between Healthy Donors, Active-TB patients and TB-treated patients. <b>D, E</b>. Comparison between Healthy Donors, Active-TB and different times after clinical cure (TB-treated <1 year and TB-treated >1 year). <b>F</b>. Comparisons between Healthy Donors, Active-TB patients and TB-treated patients after culture in the presence of polyclonal stimulation– 2 ug/mL PHA (Polyclonal activation was calculated by simple subtraction of the percentage of CD69+ cells in unstimulated cultures from the percentage observed in PHA-stimulated cultures). Bars represent the mean and vertical lines the standard error. <b>B, C, F</b>. *p<0.0167: ANOVA test (followed by post-hoc Bonferroni test for multiple comparisons). <b>D, E</b>. *p<0.0083: ANOVA test (followed by post-hoc Bonferroni test for multiple comparisons).</p

Th2, Th17 and Th22 cytokines in active and clinical cured tuberculosis.

<p>Production of IL-4, IL-13, IL-17, IL-6 and IL-22 by PBMCs from Healthy Donors, patients with active tuberculosis and clinically cured patients in unstimulated (medium only) and stimulated (4 µg/mL <i>M. bovis</i> antigen) cultures. <b>A, B, C, E, F</b>. Comparison between patients with active tuberculosis and clinically cured patients (TB-treated). <b>D</b>. Comparison between active tuberculosis and different times after clinical cure (TB-treated<1 year and TB-treated>1 year). Horizontal lines represent the median, bars represent 25–75 percentiles, and vertical lines represent 10–90 percentiles. #p<0.05 in unstimulated versus stimulated cultures (4 µg/mL <i>M. bovis</i> antigen), Wilcoxon test.</p

Correlation between IL-10 and Th1 cytokines in active and clinical cured tuberculosis.

<p>Correlation between IL-10 and IFN-γ or TNF-α levels produced by PBMCs from Healthy Donors (<b>A</b>), patients with active tuberculosis (<b>B</b>) and clinically cured patients (<b>C</b>) in stimulated cultures (4 µg/mL <i>M. bovis</i> antigen). Values of <i>p</i> and <i>r</i> determined using Spearman's correlation test.</p

TNF-α blockade impairs <i>in vitro</i> tuberculous granuloma formation and down modulate Th1, Th17 and Treg cytokines

<div><p>Tuberculosis (TB) is a granulomatous disease that has affected humanity for thousands of years. The production of cytokines, such as IFN-γ and TNF-α, is fundamental in the formation and maintenance of granulomas and in the control of the disease. Recently, the introduction of TNF-α-blocking monoclonal antibodies, such as Infliximab, has brought improvements in the treatment of patients with chronic inflammatory diseases, but this treatment also increases the risk of reactivation of latent tuberculosis. Our objective was to analyze, in an <i>in vitro</i> model, the influence of Infliximab on the granulomatous reactions and on the production of antigen-specific cytokines (TNF-α, IFN-γ, IL-12p40, IL-10 and IL-17) from beads sensitized with soluble Bacillus Calmette-Guérin (BCG) antigens cultured in the presence of peripheral blood mononuclear cells (PBMC) from TB patients. We evaluated 76 individuals, with tuberculosis active, treated and subjects with positive PPD. Granuloma formation was induced in the presence or absence of Infliximab for up to 10 days. The use of Infliximab in cultures significantly blocked TNF-α production (p <0.05), and led to significant changes in granuloma structure, <i>in vitro</i>, only in the treated TB group. On the other hand, there was a significant reduction in the levels of IFN-γ, IL-12p40, IL-10 and IL-17 after TNF-α blockade in the three experimental groups (p <0.05). Taken together, our results demonstrate that TNF-α blockade by Infliximab directly influenced the structure of granuloma only in the treated TB group, but negatively modulated the production of Th1, Th17 and regulatory T cytokines in the three groups analyzed.</p></div

Effect of TNF-α blockade on cytokines production in active Tb group.

<p>Level of cytokines after granuloma formation, <i>in vitro</i>, Active Tb group (A-F) under different culture conditions—Medium or Antigen Medium and Bead or BCG-Bead, in the presence or absence of Infliximab. *Statistical differences between the conditions tested after the addition of Infliximab, in the different cytokines. Wilcoxon and Kruskal-Wallis tests, p-value <0.05. The horizontal lines represent the medians, the bars represent the 25–75% percentiles and the vertical lines represent the 10–90% percentiles.</p

Effect of TNF-α blockade on cytokines production in PPD+ group.

<p>Levels of cytokines after granuloma formation, <i>in vitro</i>, PPD+ Control group (A-F) under different culture conditions—Medium or Antigen Medium and Bead or BCG-Bead, in the presence or absence of Infliximab. *Statistical differences between the conditions tested after the addition of Infliximab, in the different cytokines. Wilcoxon and Kruskal-Wallis tests, p-value <0.05. The horizontal lines represent the medians, the bars represent the 25–75% percentiles and the vertical lines represent the 10–90% percentiles.</p

Effects of TNF-α blockade on in vitro granuloma formation.

<p>Granuloma index under different culture conditions–bead or BCG-bead—on days 5 and 10, in the presence or absence of infliximab. *significant difference between groups on the same day and in the same culture condition. Δ significant difference in the same group and same day of culture, after addition of Infliximab. Wilcoxon or Kruskal-Wallis tests followed by Dunn's post-test. Significant difference was p-value <0.05. The horizontal lines represent the medians, the bars represent the 25–75% percentiles and the vertical lines represent the 10–90% percentiles.</p

Classification of cellular reactivity around the beads, granuloma index.

<p>Representative image of cell reactivity index, in which A (No bead-bound cell), B (up to 5 bead-bound cells), C (5 or more bead-bound cells without cell migration or blast transformation), D (5 or more bead-bound cells, with cell migration and blast transformation), E (monolayer of cells covering the whole bead, cell migration and presence of blastic cells), and F (Multiple layers of bead-bound cells, cell migration and blast transformation). Representative image of an Treated TB patient at 10^th day without (G) or with Infliximabe.</p