bioRxiv2025
Macrophages are the predominant cell type infected by Mycobacterium tuberculosis (Mtb) in tuberculosis (TB). Death of Mtb-infected macrophages promotes tissue pathology and releases Mtb to infect other cells, suggesting that inhibiting the death of Mtb-infected macrophages could be an adjunctive treatment of TB. Prospects for such an intervention depend on identifying the molecular pathways leading to cell death. We previously reported that the death of Mtb-infected mouse macrophages in vitro depends on type I interferon (IFN) and that the ensuing upregulation of cis-aconitate decarboxylase (ACOD1; IRG1) contributes to cell death by exacerbating Mtb-induced lysosomal membrane permeabilization. Here we report that death of Mtb-infected primary mouse macrophages in vitro became necroptotic and die faster in the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD) acting on a target other than caspase-8. Macrophages infected with Mycobacterium kansasii and Rhodococcus equi likewise underwent z-VAD-dependent necroptosis. In C57BL/6 mice, which are relatively TB-resistant, we saw no impact of MLKL deficiency on bacterial burden or pulmonary pathology. In contrast, in Sp140-/- mice on the C57BL/6 background, which express high levels of type I IFN after Mtb infection and develop necrotic pulmonary lesions, MLKL-deficiency reduced bacterial burden and pathology after high-dose infection. This report illustrates that off-target action(s) of a caspase-8 inhibitor can switch the cell death pathway to necroptosis in macrophages infected with various Gram-positive pathogens. In turn, this opens the possibility that pathophysiologic circumstances may lead to inhibition of the same target(s) that z-VAD inhibited in our studies. That may be what allows MLKL to exacerbate tuberculosis in mice that are prone to formation of necrotic lesions. Author SummaryTuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), led to about 1.3 million deaths in 2023. The rapid emergence of drug resistance and slow pace of new drug development prompt attention to adjunctive host-directed therapies, an approach that relies on a thorough and detailed understanding of host-Mtb interactions. Mtb infection of macrophages can kill them, promoting tissue pathology and releasing of Mtb to infect other cells. Here we find that a pan-caspase inhibitor z-VAD hastened the death of Mtb-infected macrophages and converted the mode of cell death to RIPK1-RIPK3-MLKL-dependent necroptosis. However, the functional target(s) of z-VAD-FMK and the activation mechanism of RIPK3 differ from the canonical pathways. The pan-caspase inhibitor also promoted rapid, necroptotic death of macrophages infected with Mycobacterium kansasii and Rhodococcus equi. We further found that MLKL-deficiency in Sp140-/- mice on the C57BL/6 background resulted in less weight loss, lower Mtb burdens and mitigation of lung pathology after high-dose Mtb infection. Our findings suggest that necroptosis can contribute to TB pathogenesis in some circumstances. Further identification of the death pathway-switching mechanism could deepen our understanding of host-Mtb interactions and aid in the design of adjunctive host-directed therapies.