THP-1 cell apoptosis in response to Mycobacterial infection

Abstract

We previously reported that Mycobacterium tuberculosis infection primes human alveolar macrophages (HAM) for tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis and that macrophage apoptosis is associated with killing internalized bacilli. Virulent mycobacterial strains elicit much less apoptosis than attenuated strains, implying that apoptosis is a defense against intracellular infection. The present study evaluated the potential for phorbol myristate acetate-differentiated THP-1 cells to mimic this response of primary macrophages. Consistent with the behavior of alveolar macrophages, attenuated M. tuberculosis H37Ra and Mycobacterium bovis BCG strongly induce THP-1 apoptosis, which requires endogenous TNF. THP-1 apoptosis is associated with reduced viability of infecting BCG. In contrast, virulent wild-type M. tuberculosis H37Rv and M. bovis do not increase THP-1 apoptosis over baseline. BCG induced early activation of caspase 10 and 9, followed by caspase 3. In contrast, wild-type M. bovis infection failed to activate any caspases in THP-1 cells. BCG-induced THP-1 apoptosis is blocked by retroviral transduction with vectors expressing crmA but not bcl-2. We conclude that differentiated THP-1 cells faithfully model the apoptosis response of HAM. Analysis of the THP-1 cell response to infection with virulent mycobacteria suggests that TNF death signals are blocked proximal to initiator caspase activation, at the level of TNF receptor 1 or its associated intracytoplasmic adaptor complex. Interference with TNF death signaling may be a virulence mechanism that allows M. tuberculosis to circumvent innate defenses leading to apoptosis of infected host cells.

FIG. 1.

(a) Nuclear fragmentation ELISA from THP-1 cells 3 days after infection with avirulent BCG at an infectious dose of 5 or 10 bacilli per macrophage. Data are means plus standard errors of four experiments. ∗, statistically significant difference between groups (P < 0.05). (b) Annexin V-FITC staining of THP-1 cells uninfected (gray) or infected with BCG (black overlay). At 5 days postinfection, 72% of cells were apoptotic.

FIG. 2.

Nuclear fragmentation ELISA of THP-1 cells uninfected or infected with BCG alone, BCG plus neutralizing monoclonal anti-TNF antibodies (anti-TNF), or BCG plus the TNF inhibitor pentoxifylline (pentox). Data are means plus standard deviations.

FIG. 3.

DAPI nuclear staining of uninfected THP-1 cells (a), THP-1 cells infected with BCG for 5 days (b), and uninfected THP-1 cells treated with 100 U of rTNF/ml (c). Arrows, condensed nuclei of apoptotic cells. (d) Annexin V-FITC staining of uninfected (gray) and rTNF-treated (black overlay) THP-1 cells. (e) Nuclear fragmentation ELISA of THP-1 cells unstimulated (Un) or treated with 100 U of rTNF/ml for 3 days. Data are means plus standard errors of four experiments (P = 0.530 by Student's t test).

FIG. 4.

(a) Nucleosomal-fragmentation ELISA of THP-1 cells uninfected (Un) or infected with avirulent BCG or virulent wild-type M. bovis. Data are means plus standard errors of three experiments. ∗, statistically significant difference between groups (P < 0.05). (b) Same experiment as in panel a with attenuated H37Ra and virulent H37Rv. Data are means plus standard errors of five experiments. (c and d) DAPI nuclear staining of THP-1 cells treated with BCG (c) or M. bovis (d). The BCG-treated cells show condensed DNA consistent with apoptosis, while the M. bovis-treated cells retain large intact nuclei.

FIG. 5.

Annexin V-FITC staining of uninfected (gray) or BCG-infected (black overlay) THP-1 cells at day 1 (a), day 3 (b), and day 5 (c) postinfection shows an increase in apoptosis from 7 to 67% through the course of the infection. (d) CFU of viable BCG decrease through the course of the infection. Colonies were counted 2 weeks after plating THP-1 lysates on 7H11 agar.

FIG. 6.

Annexin V-PE staining of THP-1 cells infected with PINCO retroviral vector alone or vectors containing CrmA or Bcl-2 cDNA. The data were gated on PINCO-positive (GFP⁺) cells prior to annexin V analysis. Solid bars, uninfected cells; hatched bars, BCG-infected cells. The data are representative of three separate experiments.

FIG. 7.

Colorimetric caspase activity assay 12 h postinfection shows an increase in protease activity of caspases 8, 9, and 10 due to BCG (solid bars) but not to M. bovis (hatched bars).