Effect of apoptotic cell recognition on macrophage polarization and mycobacterial persistence

Abstract

Intracellular Mycobacterium leprae infection modifies host macrophage programming, creating a protective niche for bacterial survival. The milieu regulating cellular apoptosis in the tissue plays an important role in defining susceptible and/or resistant phenotypes. A higher density of apoptotic cells has been demonstrated in paucibacillary leprosy lesions than in multibacillary ones. However, the effect of apoptotic cell removal on M. leprae-stimulated cells has yet to be fully elucidated. In this study, we investigated whether apoptotic cell removal (efferocytosis) induces different phenotypes in proinflammatory (Mϕ1) and anti-inflammatory (Mϕ2) macrophages in the presence of M. leprae. We stimulated Mϕ1 and Mϕ2 cells with M. leprae in the presence or absence of apoptotic cells and subsequently evaluated the M. leprae uptake, cell phenotype, and cytokine pattern in the supernatants. In the presence of M. leprae and apoptotic cells, Mϕ1 macrophages changed their phenotype to resemble the Mϕ2 phenotype, displaying increased CD163 and SRA-I expression as well as higher phagocytic capacity. Efferocytosis increased M. leprae survival in Mϕ1 cells, accompanied by reduced interleukin-15 (IL-15) and IL-6 levels and increased transforming growth factor beta (TGF-β) and IL-10 secretion. Mϕ1 cells primed with M. leprae in the presence of apoptotic cells induced the secretion of Th2 cytokines IL-4 and IL-13 in autologous T cells compared with cultures stimulated with M. leprae or apoptotic cells alone. Efferocytosis did not alter the Mϕ2 cell phenotype or cytokine secretion profile, except for TGF-β. Based on these data, we suggest that, in paucibacillary leprosy patients, efferocytosis contributes to mycobacterial persistence by increasing the Mϕ2 population and sustaining the infection.

FIG 1

M. leprae stimulation did not alter the phenotype of macrophages differentiated in vitro. (A) To determine whether M. leprae may induce a different phenotype in macrophages differentiated in vitro, CD14⁺ cells from healthy donors were stimulated with M-CSF (50 ng/ml) or GM-CSF (50 ng/ml) for 6 days to obtain Mϕ1 or Mϕ2 macrophages, respectively. (B and C) Cells were stimulated with irradiated M. leprae at 10 μg/ml for 24 h, after which the expression of CD163-APC (B) and that of SRA-I-PE (C) were evaluated by flow cytometry. (D and E) Concentrations of the proinflammatory cytokine IL-15 (D) and the anti-inflammatory cytokine IL-10 (E) in the supernatants were evaluated by ELISA. Experiments were performed at least twice in triplicate, and data are presented as means ± standard deviations (SD). #, P < 0.05 in relation to Mϕ1;*, P < 0.05 in relation to Mϕ1+ML.

FIG 2

Mϕ2 cells differentiated in vitro are more phagocytic than Mϕ1 cells. (A) Ultrastructural analyses were performed to evaluate whether there are differences in the phagocytic capacities of these cells differentiated in vitro. Mϕ1 or Mϕ2 cells were stimulated with irradiated M. leprae at 10 μg/ml for 2 h, and M. leprae uptake was analyzed by electron microscopy (A and B) or flow cytometry (C). Experiments were performed at least three times in triplicate, and data are presented as means ± SD. Red arrows point to M. leprae in vacuoles inside Mϕ1 and Mϕ2 cells. #, P < 0.05 in relation to Mϕ1.

FIG 3

The presence of apoptotic cells increases M. leprae uptake by Mϕ1 cells. (A and B) Ultrastructural analyses were performed to evaluate M. leprae (ML) uptake by Mϕ1 and Mϕ2 cells in the presence of apoptotic cells (ApoJ, irradiated Jurkat cells). Mϕ1 or Mϕ2 cells were stimulated with irradiated M. leprae at 10 μg/ml for 2 h in the presence or absence of apoptotic and live Jurkat cells or neutrophils (1:1). (C) The percentage of M. leprae uptake was analyzed by flow cytometry. Experiments were performed at least three times in triplicate, and data are presented as means ± SD. #, P < 0.05 in relation to Mϕ1; *, P < 0.05 in relation to ML-, live-Jurkat-cell-plus-ML (LiveJ+ML)-, or live-neutrophil-plus-ML (LiveN+ML)-stimulated Mϕ1 cells. Solid arrows indicate M. leprae inside cells, whereas the dashed arrow indicates an apoptotic Jurkat cell inside Mϕ1 macrophage. ApoN, irradiated neutrophils.

FIG 4

Phagocytosis of apoptotic cells in the presence of M. leprae shifts Mϕ1 polarization toward a Mϕ2 phenotype. To determine whether M. leprae stimulation in the presence of apoptotic cells could modulate the cell phenotype, Mϕ1 and Mϕ2 cells were stimulated with irradiated M. leprae at 10 μg/ml for 24 h in the presence or absence of apoptotic Jurkat cells (1:1). (A and B) CD163-APC expression (A) and SRA-I–PE expression (B) were evaluated by flow cytometry, and the percentages of positive cells are shown. (C) M. leprae viability was determined by the ratio of 16S rRNA to 16S DNA in Mϕ1 cells stimulated or not with apoptotic cells following 24 h of infection. Experiments were performed at least three times in triplicate, and data are presented as means ± SD. *, P < 0.05 in relation to nonstimulated (N.S.) Mϕ1 cells and Mϕ1+ApoJ. ***, P < 0.001. #, P < 0.05 in relation to the N.S. and ML-stimulated Mϕ1 cells.

FIG 5

Apoptotic cell uptake changes the cytokine secretion pattern induced by M. leprae in Mϕ1 cells. Mϕ1 cells were stimulated with irradiated M. leprae at 10 μg/ml for 24 h in the presence or absence of apoptotic or live Jurkat cells (1:1), and the concentrations of IL-6 (A), IL-15 (B), IL-10 (C), and TGF-β (D) in cell supernatants were evaluated by ELISA. Experiments were performed at least three times in triplicate, and data are presented as means ± SD. *, P < 0.05; ***, P < 0.001.

FIG 6

The increased SRA-I expression in Mϕ1 cells stimulated with apoptotic cells and M. leprae is dependent on arginase. (A) Arginase expression in leprosy patient skin lesions (BT, n = 5; LL, n = 5) was evaluated by the use of immunoperoxidase. The images are representative of one BT patient and one LL patient. (B) Mϕ1 cells were stimulated with irradiated M. leprae at 10 μg/ml for 24 h in the presence or absence of apoptotic or live Jurkat cells (1:1), and the arginase 1 expression was evaluated by real-time PCR. #, P < 0.05 in relation to the nonstimulated (NS), bead, ML, LiveJ, and LiveJ+ML groups. (C) Mϕ1 cells were stimulated with irradiated M. leprae at 10 μg/ml for 24 h in the presence or absence of apoptotic Jurkat cells (1:1) or arginase inhibitor nor-NOHA at 10 μM. SRA-I-PE expression was evaluated by flow cytometry. #, P < 0.05 in relation to the N.S., vehicle, ML, and ApoJ groups. *, P < 0.05. (D and E) The concentrations of IL-15 (D) and IL-10 (E) in the cell supernatants were evaluated by ELISA. Experiments were performed at least three times in triplicate, and data are presented as means ± SD. #, P < 0.05 in relation to the nonstimulated (N.S), bead, ML, LiveJ, and LiveJ+ML groups. *, P < 0.05.

FIG 7

Efferocytosis leads Mϕ1 cells to induce Th2 responses to M. leprae antigens in vitro. (A) Mϕ1 cells were stimulated with irradiated M. leprae at 10 μg/ml for 24 h in the presence or absence of apoptotic Jurkat cells (1:1). Cells were incubated with CD3⁺ T cells (TLϕ) (1 Mϕ1 cell to 10 TLϕ cells) for 48 h, and cell supernatants were harvested for cytokine analysis. (B, C, and D) IFN-γ (B), IL-13 (C), and IL-4 (D) concentrations in cell supernatants were determined by ELISA. Experiments were performed at least three times in triplicate, and data are presented as means ± SD. *, P < 0.05 in relation to M. leprae-stimulated cells.