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

Abstract

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 in vitro 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, in vitro, 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.

Fig 1. Classification of cellular reactivity around the beads, granuloma index.

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.

Fig 2. Effects of TNF-α blockade on in vitro granuloma formation.

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.

Fig 3. Effect of TNF-α blockade on cytokines production in PPD+ group.

Levels of cytokines after granuloma formation, in vitro, 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.

Fig 4. Effect of TNF-α blockade on cytokines production in treated Tb group.

Level of cytokines after granuloma formation, in vitro, Treated 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.

Fig 5. Effect of TNF-α blockade on cytokines production in active Tb group.

Level of cytokines after granuloma formation, in vitro, 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.