Abstract
Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.
Highlights
NINJ1 is essential for pyroptosis-related Plasma membrane rupture (PMR) Like Ninj1mt/mt bone marrow-derived macrophages (BMDMs), Ninj1–/– and Gsdmd–/– BMDMs released less LDH than wild-type BMDMs in response to LPS, nigericin, or infection with Salmonella Typhimurium, Escherichia coli, or C. rodentium (Fig. 2a, Extended Data Fig. 2a)
We further assessed PMR by time-lapse live cell imaging of BMDMs preloaded with large dextran dyes (DD-150 or Dextran dye (DD)-70 kDa)
Our data provide compelling genetic evidence that the release of IL-1β and IL-18 from macrophages is independent of PMR and likely occurs via the ~18 nm gasdermin D (GSDMD) pore
Summary
ENU-mutagenized mouse strains C57BL/6NCrl generation (G) 0 mice were treated with ethylnitrosourea (ENU) and the resulting mutations were bred to homozygosity in G3 mice as previously described[31]. Ninj1–/– mice were obtained by electroporation-based strategy of C57BL/6N zygotes with 25 ng/μl wild-type Cas[9] mRNA (Thermo Fisher Scientific) and 13 ng/μl in vitro-transcribed 2 single-guide RNAs into mouse zygotes[35]. Typhimurium (SL1344, MOI 10), primed BMDMs were cultured with bacteria for 1.5 h and gentamicin (Thermo Fisher Scientific) was added to cultures at 100 μg/ml, which was followed by additional incubation for a total 16 h. For preparation of supernatant + extract, 1.0 × 106 primed BMDMs were electroporated with 1.0 μg LPS in 100 μl R buffer using Neon (Thermo Fisher Scientific) 100 μl Tip with 1,720 Voltage, 10 Width, 2 Pulse settings. Cells were cultured in no FBS high-glucose DMEM medium for 4 h following LPS electroporation or nigericin stimulation. Infected mice were monitored for survival for 17 days post infection
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