Tumor Necrosis Factor-Alpha Antagonist Interferes With the Formation of Granulomatous Multinucleated Giant Cells: New Insights Into Mycobacterium tuberculosis Infection

Abstract

More than half of tuberculosis cases in the world are due to resuscitation of dormant Mycobacterium tuberculosis (Mtb) sequestered into cell-derived structures called granulomas. It is fairly admitted that cytokines and more particularly Tumor Necrosis Factor (TNF)-α is critical in the control of Mtb infections and that anti-TNF-α drugs constitute one of the main risk factors for reactivation of latent Mtb infection. The aim of this study was to evaluate the role of etanercept, a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human p75 TNF receptor linked to the Fc portion of human IgG1, in an in vitro model of human tuberculous granuloma. We showed that etanercept slightly delayed the formation of granuloma and reduced the generation of multinuclear giant cells (MGCs). In addition, etanercept exacerbated the expression of M1 polarization genes but also induced interleukin (IL)-10 release. In addition, our results indicated that etanercept inhibited cell fusion in an IL-10-dependent manner. Moreover, adalimumab, a human monoclonal anti-TNF-α IgG1 inhibited MGC formation in granuloma, without altering IL-10 secretion and induced macrophage apoptosis. Taken together, our data provides new insights into the role of TNF-α blockers in MGCs formation and the impact of such immunomodulatory drugs on tuberculous granuloma maturation.

Keywords: Mycobacterium tuberculosis; etanercept; granuloma; interleukin-10; interleukine-17; multinucleated giant cells; tumor necrosis factor.

Figure 1

Etanercept delays the formation of tuberculous granulomas. Isolated PBMCs from healthy donors were incubated with Sepharose beads coated with Mtb extracts for different periods of time in the presence or not of etanercept. (A) Representative pictures of beads, rosetta and granuloma are shown. (B) The number of granulomas was counted after 3, 6, and 9 days and shown as mean ± SEM (n = 10) ***P < 0.001.

Figure 2

Etanercept affects granuloma-associated macrophage populations and MGCs. Isolated granuloma cells cultured in the presence or not of etanercept for 9 days were characterized using flow cytometry. (A) Graphical plots of the percentage of CD3⁺/CD4⁺, CD3⁺/CD8⁺, and CD20⁺ lymphocytes and CD68⁺ and CD68⁺CD163⁺ macrophages population (n = 4) *P < 0.05. (B) Isolated granuloma cells cultured in the presence or not of etanercept for 9 days were characterized by May-Grünwald-Giemsa staining. Representative image of multinuclear (upper panel) and mononuclear (lower panel) cells are shown, quantified and represented as the mean percentage ± SEM (n = 10) ***P < 0.001.

Figure 3

Etanercept affects macrophage polarization. Isolated PBMCs from healthy donors were incubated with Mtb extract-coated beads for different periods of time in the presence or not of etanercept (A) The expression of macrophage polarization genes was investigated by quantitative RT-PCR normalized to the actin endogenous control and displayed as heat-map. (B) IFN-γ, IL-6, IL-10, and IL-17 cytokines were quantified by ELISA at day 3, 6, and 9 in supernatants from in vitro tuberculous granulomas treated or not with etanercept (n = 3) *P < 0.05, **P < 0.01.

Figure 4

Etanercept inhibits cell fusion. Adherent CD14⁺ cells from healthy donors (n = 5) were stimulated with IFN-γ and ConA in the presence or not of etanercept and cell-cell fusion was measured by the appearance multinucleated giant cells (≥2 nucleus). (A) Representative pictures of multinucleated cells stained with phalloidin (green) and DAPI (blue). (B) Mononuclear and multinuclear (≥ 2 nucleus) giant cells were quantified after 9 days and the mean percentage ± SEM is shown (n = 5) ***P < 0.001. (C) Formation of multinucleated cells induced by IFN-γ and ConA was also quantified in the presence of 50 ng/ml TNF-α (n = 5) *P < 0.05.

Figure 5

IL-10, not IL-17, inhibits cell fusion. (A) Adherent CD14⁺ cells from healthy donors (n = 5) were stimulated by IFN-γ and ConA in the presence or not of etanercept and IL-17 and IL-10 concentration were evaluated by ELISA in the supernatants after 9 days *P < 0.05, **P < 0.01. (B,C) Adherent CD14⁺ cells from healthy donors were stimulated by IFN-γ and ConA in the presence or not of IL-17 (10 ng/ml) or IL-10 (50 ng/ml) for 9 days. (B) Representative pictures of MGCs stained with phalloidin (green) and DAPI (blue) are shown. (C) Formation of MGCs was also quantified and the mean percentage ± SEM is shown (n = 4) *P < 0.05.

Figure 6

Adalimumab inhibits MGC formation and induces macrophage apoptosis in granuloma. Isolated PBMCs from healthy donors were incubated with Sepharose beads coated with Mtb extracts in the presence or not of adalimumab. (A) After 9 days, isolated granuloma cells were characterized by May-Grünwald-Giemsa staining and mononuclear and multinuclear giant cells were quantified (n = 3). **P < 0.05. (B) IL-10 levels were quantified by ELISA at day 3, 6, and 9 in supernatants from in vitro tuberculous granulomas treated or not with adalimumab. (C) Isolated PBMCs from healthy donors were incubated with Sepharose beads coated with Mtb extracts in the presence or not of adalimumab or etanercept. Macrophage apoptosis was assessed by flow cytometry after annexin V/7-AAD staining on CD64-gated cells. Results are expressed as mean percentage ± SEM (n = 3) *P < 0.05, **P < 0.01.