z‐VAD‐FMK triggers macrophage necroptosis during mycobacterial infection via a non‐canonical off‐target mechanism

Abstract 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 interferons (IFNs) and lysosomal membrane permeabilization contributes to cell death. Here, we report that the death of Mtb‐infected primary mouse macrophages in vitro became necroptotic in the presence of a pan‐caspase inhibitor, benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone (z‐VAD‐FMK, hereafter called z‐VAD) acting on a target other than caspase‐8. Macrophages infected with Mycobacterium kansasii or Rhodococcus equi likewise underwent z‐VAD‐dependent necroptosis. In TB‐resistant C57BL/6 mice, the deficiency of the key necroptosis executioner, mixed lineage kinase domain‐like (MLKL), did not affect bacterial burden or pulmonary pathology. In contrast, in mice lacking the IFN repressor SP140, which express high levels of type I IFNs 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 several Gram‐positive pathogens and highlights that necroptosis can exacerbate TB. Identifying the target of z‐VAD may help to delineate how necrotic lesions form in TB.