IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

Abstract

Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen.

Keywords: AG, antigen; ATG, autophagy-related; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GTP, guanosine triphosphate; HD, healthy donors; HR TB, high-responder tuberculosis patient; IFNG; IFNG, interferon gamma; IL, Interleukin; LC3, microtubule-associated protein 1A/1B-light chain 3; LR TB, low-responder tuberculosis patients; Mtb-Ag, Mycobacterium tuberculosis antigen; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; SLAM, signaling lymphocytic activation molecule; TB, tuberculosis; Th, T helper; autophagy; cytokines; defense; immune response; mAb, monoclonal antibody; patients; rIFNG, recombinant IFNG; tuberculosis.

Figure 1.

Levels of autophagy induced in response to Mtb-Ag in tuberculosis patients and healthy donors. (A and B) PBMC from HD, HR TB and LR TB were cultured at 3 × 10⁶ cells/ml in RPMI with 10% FBS. Cells were then cultivated for 16 h without stimulus to allow the adherence of monocytes. Afterwards, PBMC were stimulated with sonicated M. tuberculosis (Mtb-Ag) for 24 h, protein extracts were obtained and western blot was performed. Densitometry of the blots was performed and the means of the ratios of LC3-II to GAPDH were expressed as arbitrary units (AU). (A) A representative example of a HR TB patient is shown. (B) Bars represent the mean values ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Wilcoxon matched-pairs signed rank test; #P < 0.05, ##P < 0.01 Mann-Whitney Test. (C) PBMC from HD and HR TB and LR TB patients were incubated at 2 × 10⁶ cells/ml in RPMI with 10% FBS. PBMC were then stimulated with or without Mtb-Ag for 24 h and immunofluorescence for LC3 was performed. Samples were then analyzed in a confocal microscope. Representative images of a HD, a HR TB, and a LR TB patient are shown. Bars represent the mean values of the number of LC3 puncta/cell ± SEM. *P < 0.05, **P < 0.01, Wilcoxon matched-pairs signed rank test; ##P < 0.01 Mann-Whitney Test.

Figure 2.

Mtb-Ag induces autophagy in CD14⁺ cells from tuberculosis patients and healthy donors. PBMC from healthy donors (HD) and tuberculosis patients (TB) (High responders [HR TB] and low responders [LR TB]) were incubated at 3 × 10⁶ cells/ml in RPMI with 10% FBS. PBMC were stimulated with or without sonicated M. tuberculosis antigen (Mtb-Ag) for 24 h and intracellular saponin-resistant LC3 determination was performed on CD14⁺ and/or CD3⁺ cells by flow cytometry. (A) Dot plots were obtained first gating on monocytes by light scatter, then on CD14⁺ cells and finally on LC3⁺ cells. Representative dot plots from a HD and a LR TB are shown. (B) Bars represent the mean values of the percentage of CD14⁺LC3⁺ cells ± SEM. *P < 0.05, Wilcoxon matched-pairs signed rank test; #P < 0.05, ##P < 0.01, Mann-Whitney Test. (C) Intracellular LC3-II was determined by flow cytometry first gating on lymphocytes by light scatter and then gating on CD3⁺ T cells (upper panel); or first gating on monocytes by light scatter and then on CD14⁺ cells (lower panel). A representative dot plot from a HR tuberculosis patient is shown.

Figure 3.

The levels of autophagy are directly correlated with the amount of IFNG secreted in response to Mtb-Ag. PBMC from healthy donors (HD) and tuberculosis patients (TB) (High responders [HR TB] and low responders [LR TB]) were incubated at 3 × 10⁶ cells/ml in RPMI with 10% FBS. Cells were cultured for 16 h without stimulus to allow the adherence of monocytes. Then, PBMC were stimulated with sonicated M. tuberculosis (Mtb-Ag) for 24 and/or 48 h. (A) IFNG levels in Mtb-Ag-stimulated cell culture supernatant fractions assayed by ELISA after 24 h of incubation with Mtb-Ag. Bars represent the mean values ± SEM. #P < 0.05, ##P < 0.01, Mann-Whitney Test. (B) PBMC from HR and LR TB patients and HD were stimulated with Mtb-Ag for 24 and 48 h, and then, IFNG production was determined by flow cytometry, first, gating on lymphocytes by light scatter and then, gating on CD4⁺ T cells. A representative dot plot for each group is shown. Cells cultured with media (inset) are also shown. (C) After 24 h of Mtb-Ag stimulation, protein gel blot detection of LC3-II and GAPDH was performed from the proteins extracts by standard methods. Densitometry of the blots was then determined and the means of the ratio of LC3-II to GAPDH in each experimental condition were expressed as arbitrary units (AU). The results are represented as LC3-II/GAPDH in Mtb-Ag-stimulated cultures vs IFNG levels in Mtb-Ag-stimulated cell culture supernatant fractions. Correlation was analyzed by the Spearman r test.

Figure 4

(See previous page). IFNG induced in response to Mtb-Ag participate in the process of autophagy. PBMC from healthy donors (HD), high-responder TB patients (HR TB) and low-responder TB patients (LR TB) were incubated at 2 × 10⁶ cells/ml in RPMI with 10% FBS. Cells were stimulated with or without sonicated M. tuberculosis (Mtb-Ag) ± anti-IFNG (A) or recombinant IFNG (rIFNG) (B) for 24 h. Immunofluorescence and intracellular flow staining for LC3 was then performed and samples were analyzed using a confocal microscope or a FACSAria II flow cytometer, respectively. (A) Bars represent the mean values of the number of LC3 puncta/cell ± SEM. *P < 0.05, Wilcoxon matched-pairs signed rank test. Representative images and representative dot plots of cells obtained from a HD and a HR TB patient are depicted. (B) Representative images of cells obtained from a HD, and HR TB and LR TB patients are shown. Bars represent the mean values of the number of LC3 puncta/cell ± SEM. *P < 0.05, **P < 0.01, Wilcoxon matched-pairs signed rank test; #P < 0.05,##P < 0.01 Mann-Whitney Test.