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

FAYETTEVILLE, GA, UNITED STATES, October 26, 2026 /EINPresswire.com/ — 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 for TB. This study demonstrates that the widely used pan-caspase inhibitor z‑VAD‑FMK triggers robust necroptosis in macrophages infected with Mycobacterium tuberculosis, M. kansasii, or Rhodococcus equi. Surprisingly, this effect stems from an off-target mechanism independent of caspase-8 inhibition. In Sp140-deficient mice with elevated type I interferon responses, MLKL deficiency reduced bacterial burden and pathology after high-dose infection. This work reveals unexpected bioactivity of z‑VAD‑FMK and highlights necroptosis as a pathogenic driver in certain TB contexts.

Tuberculosis (TB) remains a major global health threat, and the death of Mycobacterium tuberculosis (Mtb)-infected macrophages is a key driver of disease pathology. Identifying cell death pathways is crucial for therapeutic intervention.

z‑VAD‑FMK is a pan-caspase inhibitor broadly used in cell-death research. Conventionally, z‑VAD‑FMK-driven necroptosis is attributed to caspase-8 suppression. Here, researchers led by Li Zhang from Southern University of Science and Technology found that z‐VAD‐FMK triggers macrophage necroptosis during mycobacterial infection via a non‑canonical off‑target mechanism. Genetic and pharmacological evidence showed this cell death relied on the RIPK1‑RIPK3‑MLKL axis, TLR2‑TNF‑α, and type I interferon signaling. Strikingly, knockdown of mouse caspase-8 and knockout of human caspase-8 in macrophages did not abolish z‑VAD‑FMK-triggered necroptosis upon mycobacterial infection. Other selective caspase inhibitors failed to recapitulate this phenotype, demonstrating a caspase-8-independent off-target mechanism. Notably, RIPK3 became functionally activated without detectable phosphorylation at the canonical T231/S232 sites, suggesting alternative RIPK3 activation modes.

This off-target necroptosis could also be observed in macrophages infected with other bacteria that are evolutionarily related to Mycobacterium tuberculosis, including Mycobacterium kansasii and Rhodococcus equi.

In vivo, MLKL deletion barely altered TB outcome in wild-type C57BL/6 mice. However, in Sp140-/- mice featuring hyperactivated type I interferon responses and necrotic granulomas, MLKL deficiency alleviated weight loss, reduced bacterial burden, and mitigated pulmonary pathology after high-dose aerosol infection.

This work reveals important but underappreciated off-target activity of z-VAD-FMK. Identifying the real target(s) of z‑VAD‑FMK will advance our understanding of necrotic granuloma formation during tuberculosis and may inform future host-directed therapeutic development for TB.

References
DOI
10.1002/mlf2.70101

Original Source URL
https://doi.org/10.1002/mlf2.70101

Funding Information
This work was supported by the National NaturalScience Foundation of China (Grant No. 32470183 to L.Z.), the Guangdong Basic and Applied Basic Research Foundation(Grant No. 2025A1515010652 to L.Z.), the Shenzhen Scienceand Technology Program (Grant No. JCYJ20250604144221028to L.Z.), the National Institutes of Health (Grant No.1R01AI138940 to C.N., P30CA047904 and DP2GM146320 to Y.N.G.), and the Milstein Program in Chemical Biology andTranslational Medicine (to C.N.). The Department of Microbiologyand Immunology of Weill Cornell Medicine is supported by the William Randolph Hearst Foundation.

Lucy Wang
BioDesign Research
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