NINJ1 mediates plasma membrane rupture during lytic cell death

Abstract

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules termed damage-associated molecular patterns (DAMPs) that propagate the inflammatory response. The underlying mechanism for PMR, however, is unknown. Here we show that the ill-characterized nerve injury-induced protein 1 (NINJ1) — a cell surface protein with two transmembrane regions — plays 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 failed to release numerous intracellular proteins including High Mobility Group Box 1 (HMGB1, a known DAMP) and Lactate Dehydrogenase (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 Citrobacter rodentium, suggesting a role for PMR in anti-bacterial host defense. Mechanistically, NINJ1 utilized an evolutionarily conserved extracellular α-helical domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held dogma that cell death-related PMR is a passive event. Pyroptosis is a potent inflammatory mode of lytic cell death triggered by diverse infectious and sterile insults1-3. It is driven by the pore-forming fragment of gasdermin D (GSDMD)4-7 and releases two exemplar proteins: interleukin-1β (IL-1β), a pro-inflammatory cytokine, and LDH, a standard marker of PMR and lytic cell death. An early landmark study8 predicted two sequential steps for pyroptosis: (1) initial formation of a small plasma membrane pore causing IL-1β release and non-selective ionic fluxes, and (2) subsequent PMR attributable to oncotic cell swelling. PMR releases LDH (140 kDa) and large DAMPs. While the predicted size of gasdermin pores (~18 nm inner diameter9) is large enough to release IL-1β (17 kDa, ~4.5 nm diameter), the underlying mechanism for subsequent PMR has been considered a passive osmotic lysis event.

Downstream of gasdermin D cleavage, a Ragulator-Rag-mTORC1 pathway promotes pore formation and pyroptosis

“…RagA is however necessary for functional pore formation and subsequent mitochondrial inactivation. As described earlier, Ninjurin-1 is a factor that also acts downstream of GSDMD cleavage to promote pyroptosis (Kayagaki et al, 2020). Our analysis allowed us to place Ragulator-Rag upstream of Ninjurin-1 activities, as the former is required for pore formation and Ninjurin-1 acts downstream of pore formation and mitochondrial inactivation to promote cytolysis.…”

“…Ninjurin-1 (NINJ1), a cell surface adhesion protein, was recently discovered to control pyroptosis at a late stage (Kayagaki et al, 2020). NINJ1 is required to induce plasma membrane rupture downstream of GSDMD pore formation.…”

Evolutionary loss of inflammasomes in carnivores to facilitate carriage of zoonotic infections

“…Here we show that key inflammasome lytic cell death pathways thought to be critical for gut health in mammals are either genetically and/or functionally missing from the Carnivora. PI uptake without cell lysis occurs in dog cells in response to inflammasome activation suggesting a dissociation of gasdermin D pore formation from lytic cell death similar to the phenotype seen if the NINJ1 protein is deleted from human or mouse cells (Kayagaki et al, 2020) yet all Carnivora have NINJ1. Our data suggest that inefficient inflammasome activation occurs in dog cells such that insufficient gasdermin D pores form to drive cell lysis.…”

NLRP3 inflammasome of renal tubular epithelial cells induces kidney injury in acute hemolytic transfusion reactions

“…31 However, they are larger than those of tumor cells (30 nm under a scanning electron microscope) 32 and bone marrow-derived macrophages (∼18 nm). 33 Immunofluorescence analysis revealed that the percentage of GSDMD-N (the executive molecule in pyroptosis) foci significantly increased in primary RTECs and HK-2 cells after chemical heme stimulation ( Figure 2G,H). Meanwhile, western blotting showed that cleaved GSDMD was obviously increased under heme stimulation in primary RTECs and HK-2 cells (Figure 2A,C).…”

“…Inflammasome-associated cell death includes both pyroptosis and apoptosis. 33 For AHTRs, the renal tubular epithelial cells that suffer from apoptosis or pyroptosis require further investigation. Since delayed hemolytic transfusion reactions, hyperhemolysis, and passenger lymphocyte syndrome in transplant recipients are now becoming new challenges in current clinical transfusion practice, it is worth investigating whether NLPR3 also plays a role in these HTRs.…”