Pathogens molecules are sensed by the Toll-like receptors (TLRs) to activate macrophages and induce innate immune responses. In some circumstances host-derived Interferon-gamma (IFNγ) can augment the inflammatory production of tumour necrosis factor (TNF), interleukin-6 and anti-microbial nitric oxide. In murine macrophages, IFNγ has been shown to induce cell death when co-stimulated with TLR ligands, but the genetic requirements that determine this cell death pathway remain unknown. Here, we genetically delineate the molecular mechanism underpinning IFNγ and TLR-induced cell killing and identify a novel cell death pathway that might act to limit excessive innate immune responses.
Using a panel of genetic knockout mice, we demonstrate a requirement of the apoptotic caspase proteins for IFNγ and TLR-induced cell killing, while effector molecules of the highly inflammatory cell death pathways, pyroptosis and necroptosis, are dispensable. We describe novel links between the initiator and executioner caspases and identify a unique role for the inducible nitric oxide synthase (iNOS) protein. Chemical inhibition of iNOS, or its genetic deletion via CRISPR/Cas9, show iNOS is essential for caspase activation and macrophage killing. Characterising the mechanism by which iNOS triggers apoptotic caspase signalling and macrophage killing may reveal new therapeutic opportunities to limit pathological innate immune cell activation and cytokine production, or promote the death and clearance of pathogen infected cells.