Influenza virus activates inflammasomes via its intracellular M2 ion channel

Influenza virus is responsible for annual epidemics that cause severe illness in ~5 million people worldwide. Evidence has shown that influenza infection engages the NLRP3 inflammasome complex in dendritic cells and macrophages1–4. NLRP3 forms a multiprotein complex with the adaptor protein ASC (pycard) and caspase-1, which leads to the catalytic cleavage of the immature (‘pro’) forms of interleukin 1β (IL-1β), IL-18 and IL-33. Inflammasome-mediated cytokine release via NLRP3 requires two signals5: signal 1 is induced by Toll-like receptor (TLR) stimulation and leads to the synthesis of pro-IL-1β, pro-IL-18 and pro-IL-33; signal 2, triggered by agents that can cause membrane perturbations, specifically potassium efflux, induces activation of caspase-1 and cleavage of pro-IL-1β, pro-IL-18 and pro-IL-33. Wellknown examples of signal 2 include pore-forming microbial toxins, maitotoxin, aerolysin and nigericin, which activate NLRP3 inflammasomes by allowing efflux of potassium from the cytosol5–7. In addition, lysosomal membrane damage caused by phagocytosis of crystals such as asbestos, silica and aluminum salt triggers NLRP3 activation8,9. The mechanism by which viral infection results in activation of the NLRP3 inflammasome is unclear. Infection with either DNA or RNA viruses results in NLRP3-dependent inflammasome activation1–4,10,11, and studies have identified AIM2 as a sensor of double-stranded DNA (dsDNA) that is able to stimulate inflammasomes12–15. AIM2 consists of a HIN-200 domain, which binds to DNA, and a pyrin domain, which associates with ASC to activate both the transcription factor NF-κB and caspase-1. However, AIM2-induced activation is NLRP3 independent12,13. In contrast to the mechanisms known for dsDNA viruses, the mechanism by which influenza virus, a negative-stranded RNA virus, triggers activation of the NLRP3 inflammasome is unknown. Here we examine the cellular mechanism by which influenza virus infection elicits the NLRP3 inflammasome. RESULTS Influenza subtypes induce potent inflammasome activation To determine whether inflammasome activation is generally induced by viral infection, we compared the ability of several viruses to trigger inflammasome activation by measuring IL-1β secretion from infected bone marrow–derived macrophages (BMMs; Fig. 1a). All influenza virus strains tested, including influenza A and B types, induced robust release of IL-1β from BMMs (Fig. 1a). The induction of IL-1β by influenza was also dependent on NLRP3, ASC and caspase-1 (Supplementary Fig. 1a,b), as demonstrated before1–4. In addition, IL-1β secretion induced by influenza infection (Supplementary Fig. 1a,b), but not that induced by dsDNA (Supplementary Fig. 1c), was NLRP3 dependent. In contrast, at the same multiplicity of infection (MOI), two other single-stranded RNA viruses, Sendai virus (paramyxovirus) and vesicular stomatitis virus (rhabdovirus; data not shown), as well as the dsDNA virus herpes simplex virus type 2 (HSV-2), activated inflammasomes to a much smaller extent, despite robust stimulation of inflammasome-independent cytokines, such as IL-6 (Fig. 1a) and tumor necrosis factor (TNF; Fig. 1b). These data indicate that influenza virus infection alone is able to activate both signal 1 and signal 2 in unprimed BMMs or dendritic cells (DCs). In addition, these findings suggest that inflammasome activation is mediated by a specific feature associated with influenza virus infection that is not common to other single-stranded RNA viruses.