Fig 1.
Mycobacterial cell wall proteins mediate apoptosis of bone marrow MØ.
A M. smegmatis strain was transformed to overexpress LpqH, a 19 kDA Mtb cell wall lipoprotein that is apoptogenic for MØs. The cell wall proteins of the transformed strain were disrupted with sonication and then separated by 15% SDS-PAGE. (A) A Coomassie blue stained gel is shown and LpqH (19 kDa) was detected by immunoblot with a mAb and HRP labeled rabbit antiserum. (B) Immunoblot failed to reveal LpqH in wild-type M.smegmatis. (C) Flow cytometry revealed that the great majority of MØs used in these assays were F4/80 positive. (D, E) Following the incubation of bone marrow-derived MØs with 50 μg cell wall protein for 24 h, high levels of apoptosis were revealed by immunofluorescence microscopy of TUNEL assays (excitation 496 nm, emission 575 nm) (original magnification, 20x) and by flow cytometry with Annexin V. A representative Annexin/ 7-AAD dot plot showed that 87.1% of MØs were apoptotic and 33.7% were necrotic. (F) Treatment of MØs with UV light also induced high levels of apoptosis.
Fig 2.
Mycobacterial antigens are translocated from the cytosol into the nuclei of apoptotic cells.
To examine the intracellular movement of proteins that induce apoptosis, MØs were incubated with CFSE-labeled MsmegLpqH cell wall proteins. (A) After 1 h, MØ nuclei were stained with DAPI and TUNEL. Confocal immunofluorescence images show cytoplasmic vacuoles containing phagocytosed mycobacterial proteins in most of the cells (original magnification, 40x). (B, D) In addition, 39.9% of the cells exhibited overlapping DAPI (excitation 359 nm, emission 461 nm) and CFSE fluorescence (excitation 493 nm, emission 525 nm) (original magnification 40x). (C, D) Thus, translocation of mycobacterial proteins from cytosolic deposits to the nuclei was observed; 45.7% of the nuclei were apoptotic as shown by TUNEL, and 52.6% of the apoptotic nuclei exhibited overlapping TUNEL and CFSE fluorescence (original magnification 40x). (D) Nuclear translocation of proteins and apoptosis were virtually eliminated when the MØs were pretreated with the pancaspase inhibitor, Z-VAD-FMK. To identify the mycobacterial proteins that translocated to the nucleus, MØ were incubated with MsmegLpqH cell wall proteins for 1 h. (E) Nuclear extracts of these cells were then subjected to immunoblotting with a rabbit anti-M smegmatis antiserum and a secondary HRP labeled anti-rabbit IgG antibody. Several antigenic bands were observed that ranged in size from 20 kDa to 37 kDa. Staining with a mAb confirmed the nuclear translocation of LpqH and bands with sizes of 37 kDa and 20 kDa corresponding to histones H4 and H2B. The results shown are representative of three independent experiments.
Fig 3.
Inmature DCs efficiently phagocytose apoptotic MØ.
DC precursors were obtained from mouse bone marrow and were cultured in the presence of GM-CSF for 6 days. Phagocytosis assays were then conducted with PKH-67-labeled DCs and apoptotic PKH-26-labeled MØs. (A) With confocal microscopy, classical DC morphology was observed (original magnification, 60x). (B) In addition, fluorescencent apoptotic bodies appeared to reside within vacuolar structures, (original magnification, 100x) (PKH-67 excitation 493 nm, emission 525 nm; PKH-26 excitation 496, emission 575 nm). (C, D) Flow cytometry of the DC/MØ cocultures at various time points showed that phagocytosis increased with time and high levels of phagocytosis were detected 24 h after coculturing. (C, D, E) Phagocytosis of UV light induced apoptotic bodies was similar. The results shown are representative of three independent experiments. MFI, Mean fluorescence index.
Fig 4.
Phagocytosis of mycobacteria-induced apoptotic MØs induces an immunogenic profile for DCs.
Immature DCs were obtained after 6 d of culturing bone marrow precursor cells and approximately 65–92% of this population was CD11c positive. These DCs were subsequently cocultured for 24 h with whole apoptotic MØ that were isolated with Annexin V-coated microbeads and were free of blebs. (A, B) Representative dot plots and histograms are shown. (C, D) In DC activated with MsmegLpqH apoptotic MØ the expression of MHC-I was upregulated (p ≤ 0.05; paired t Student’s test), while expression of MHC-II remained within basal values. (E, F, G) Expression of CD40 and CD86 were both greatly increased (p ≤ 0.05 and p ≤ 0.005, respectively; paired t Student’s test), while expression of CD80 increased to a lesser extent (p ≤ 0.05; Mann Whitney test). (C) DCs that engulfed UV apoptotic MØ exhibited upregulated MHC-I expression as well (p ≤ 0.05; Wilcoxon Signed rank test). (E, G) DCs activated with LPS increased the expression of CD40 (p ≤ 0.05; Mann Whitney test) and CD86 (p ≤ 0.002; paired t Student’s test). Results shown were obtained in five independent experiments.
Fig 5.
Phagocytosis of apoptotic MØ upregulates inflammatory and anti-inflammatory cytokines in DCs.
When DCs (5 x 105) were incubated for 24 h with MsmegLpqH-induced apoptotic MØs, (A, B, C, D) ELISAs of the coculture supernatants showed that secretion of IL-12 (p ≤ 0.0001) and TNF-α (p ≤ 0.007), as well as production of IL-10 (p ≤ 0.0001) and TGF-β (p ≤ 0.01), were increased. (D) In contrast, phagocytosis of the UV-treated MØs resulted in increased production of TGF-β (p ≤ 0.05), while the remaining cytokines detected were within basal levels. The results of four independent experiments are shown. For! L-12 and IL-10 analysis a paired t Student’s test was used. For TNF-α and TGF-β the Mann Whitney test was used.
Fig 6.
Induction of T cell proliferation by DCs activated with MsmegLpqH-induced apoptotic MØs.
To examine the capacity of the DCs that engulfed the apoptotic MØs to activate T cells, DCs were cocultured with autologous T cells isolated from the spleens of naïve mice by cell sorting with an anti-Thy mAb. DCs and isolated T cells were cocultured at a 1:10 ratio for 3 d. T cell proliferation was assessed by the CFSE dilution method. (A, B) DCs that phagocytosed the mycobacteria-induced apoptotic MØs triggered the proliferation of CD8+ T cells, and not CD4+ T cells (p ≤ 0.04, paired t Student’s test). (C, D) In the supernatants increased amounts of IFN-γ (p ≤ 0.01; Kruskall-Wallis test) and IL-12 (p ≤ 0.01 Mann Whitney test). (B, C, D) In comparison, the DCs activated with UV-induced apoptotic MØs promoted the proliferation of CD4+ T cells and the release of IL-12 (p ≤ 0.001, Mann Whitney test) and IFN-γ was within basal values. (E, F, G) Proliferation assays with CD4+ and CD8+ T cells isolated by cell sorting confirmed the ability of CDs activated with MsmegLpqH apoptotic MØs to trigger a proliferative response of CD8+ T cells (p ≤ 0.05, paired t Student’s test) but not of CD4+ T cells. To identify the pathways used by DCs to activate CD8+ T cells, the DCs were treated with the proteasome inhibitor, MG-132, and a vacuolar H+ ATPase inhibitor, bafilomycin. (H) Both inhibitors reduced the proliferation of the CD8+ T cells (p ≤ 0.01 and p ≤ 0.02, respectively, Dunn’s Multiple Comparison test). (I) In comparison the proliferation of CD4+ T cells was not inhibited. The results of four independent experiments are shown. (J, K) Also, a representative experiment shows that both inhibitors decreased the production of INF-γ by the CD8+ T cells but not by CD4+ T cells.