We previously reported that Mycobacterium tuberculosis infection primes human alveolar macrophages (HAM) for tumor necrosis factor alpha (TNF-␣)-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.Alveolar macrophages infected with avirulent or attenuated strains of Mycobacterium tuberculosis undergo apoptosis in a tumor necrosis factor alpha (TNF-␣)-dependent manner, in contrast to infection with virulent mycobacterial strains, which induce little or no apoptosis above background (1,8,9). Naive primary macrophages are resistant to TNF cytotoxicity but become primed for TNF death signals when infected with attenuated strains of M. tuberculosis and related mycobacteria. It is postulated that this apoptosis response represents an innate defense mechanism against intracellular infection. Alveolar macrophages constitute a critical growth niche for intracellular M. tuberculosis in the lung, as evidenced by the attenuation of disease after aerosol infection of mice whose macrophages were depleted by bisphosphonate liposome treatment (12). Host macrophage apoptosis, but not necrosis, is linked to killing of intracellular mycobacteria (4,19). This suggests that programmed cell death of the host macrophage not only eliminates a preferred growth niche for M. tuberculosis but also activates a unique microbicidal mechanism.M. tuberculosis-induced apoptosis in primary macrophages in vitro is mediated by TNF. ⌻here is evidence for the involvement of caspase 9 and caspase 3 in this process (24), but little else is known about the signaling pathways that regulate the f...

Both lymphoid and myeloid cells express two related members of the IFN regulatory factor (IRF) family of transcription factors, specifically IRF-4 and IFN consensus binding protein (ICSBP or IRF-8). We previously reported that macrophages express IRF-4 and in combination with the ETS-like protein PU.1 can synergistically activate a human IL-1β reporter gene. Here we report that this synergy is mediated by a composite PU.1/IRF element located within an upstream enhancer known to confer cytokine- and LPS-inducible expression. In macrophages, synergistic activation of IL-1β reporter gene expression was preferentially mediated by IRF-4, whereas IRF-4 and ICSBP were equally capable of synergizing with PU.1 when coexpressed in fibroblasts. Furthermore, coexpression of IRF-1 and IRF-2 dramatically increased the capacity of both PU.1/IRF-4 and PU.1/ICSBP to induce IL-1β reporter gene expression in fibroblasts. The additional synergy observed with IRF-1 and IRF-2 coexpression is mediated by a region of DNA distinct from either the IL-1β enhancer or promoter. We also assessed the capacity of these transcription factors to activate endogenous IL-1β gene when overexpressed in human embryonic kidney 293 cells. Although ectopic expression of PU.1 alone was sufficient to activate modest levels of IL-1β transcripts, endogenous IL-1β expression was markedly increased following coexpression of additional IRF proteins. Thus, maximal expression of both a human IL-1β reporter gene and the endogenous IL-1β gene was observed in cells that coexpressed PU.1, IRF-4 (or ICSBP), IRF1, and IRF2. Together, our observations suggest that these factors may function together as an enhanceosome.

The smallest unit of bacterial peptidoglycans known to be endowed with biological activities is muramyl dipeptide (MDP). A clinically acceptable synthetic derivative of MDP, namely murabutide (MB), has been found to present interesting pharmacological properties and to suppress HIV‐1 replication in monocyte‐derived macrophages (MDM). We have addressed the signaling events activated in MDM following stimulation with either MB or the potent immunostimulant LPS. We also examined whether signaling by muramyl peptides involves the use of cell surface receptors, including CD14 and Toll‐like receptor 2 (TLR2) or TLR4 that are known to be signal‐transducing receptors for other bacterial cell wall components. We demonstrate that, unlike LPS, the safe immunomodulator MB selectively activates extracellular signal‐regulated kinases (Erk) 1 / 2, in the absence of detectable Jun N‐terminal kinase (JNK) or p38 mitogen‐activated kinase activation. Furthermore, STAT1 activation but weakor no activation of STAT3 or STAT5 respectively, could be detected in MB‐stimulated MDM. Using MonoMac6 cells, we observed high C / EBPβ and AP‐1 but weaker and transient NF‐κB activation by MB.Moreover, the truncated form of C / EBPβ, known to repress HIV‐1 transcription, was detected in extracts from MB‐treated THP‐1 cells. Surprisingly, neither MB nor MDP were able to transduce signals via CD14 and TLR2 or 4. These findings present major differences in the early cell activation process between LPS and muramyl peptides, and strongly argue for the implication of co‐receptors other than TLR2 and TLR4 in mediating the signaling events induced by defined subunits of bacterial peptidoglycans.