Evasion of the macrophage apoptotic response by virulent strains of Mycobacterium tuberculosis.

摘要

The goal of this work was to determine the virulence-associated mechanism(s) whereby Mycobacterium tuberculosis (Mtb) evades the innate immune response of the infected host. Initial studies evaluated the potential for phorbol myristate acetate-differentiated THP-1 cells, a monocytic leukemia cell line, to mimic the apoptotic response of primary macrophages to mycobacterial infection. The development of a model system was undertaken to overcome the limitations associated with infection of primary alveolar macrophages, namely low cell numbers and the prohibitive cost of bronchoscopy on human volunteers. Consistent with the behavior of alveolar macrophages, differentiated THP-1 cells underwent apoptosis in response to infection with attenuated or avirulent strains of mycobacteria in a tumor necrosis factor (TNF)-dependent manner. High levels of macrophage apoptosis correlated with decreased viability of the bacilli. Contrastingly, virulent strains of Mtb did not induce apoptosis over background levels. These results demonstrate that THP-1 cells faithfully model alveolar macrophage responses to Mtb infection.; Low-dose infection of THP-1 cells with avirulent Mtb activated caspases 8, 10, 9 and 3 while virulent mycobacteria failed to activate any of the caspases tested. This suggests a mycobacterial-induced blockade of apoptosis, both at the level of the TNF receptor (TNFR) and at the level of the mitochondria. Further experiments determined that virulent but not avirulent Mtb induced mRNA and protein expression of TNFR2, a subset of TNFRs linked to cell survival. This finding may explain the lack of early activation of receptor-associated caspases 8 and 10. Ribonuclease protection assays and western blots indicated an increase in Mcl-1 RNA and protein, a Bcl-2 family member that blocks mitochondrial apoptosis in THP-1 cells infected with virulent but not avirulent Mtb. Upregulation of this mediator may explain the blockade in activation of mitochondrial-associated caspase 9. Retroviral-induced overexpression of Mcl-1 in THP-1 cells rescued them from avirulent Mtb-induced apoptosis. Conversely, inhibiting expression of Mcl-1 by small inhibitory RNAs (siRNA) increased apoptosis in THP-1 cells infected with virulent Mtb strains. Taken together, these results suggest the virulent Mtb strains have evolved at least two mechanisms for evading host macrophage apoptosis: by upregulating surface TNFR2 and the mitochondrial protein Mcl-1.