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 wildtype 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 insults 1-3 . 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 study 8 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 diameter 9 ) 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. An unbiased forward genetic screen identifies NINJ1To identify essential mediators of PMR, we performed a forward genetic screen using bone marrow-derived macrophages (BMDMs) from N-ethyl-N-nitrosourea (ENU)-mutagenized mice.

Cell death as part of innate immunity: Cause or consequence?

“…Interestingly, a very recent report demonstrates the involvement of nerve injury-induced protein 1 (NINJ1) in the plasma membrane rupture during necroptosis and other forms of programmed necrosis. This study shows that murine Ninj1-/-macrophages treated with different inducers of programmed necrosis fail to die by the typical necrotic process with cell lysis and associated release of highmobility group box 1 (HMGB1) and lactate dehydrogenase (LDH), two main markers of plasma membrane rupture [22]. Instead, they die with a persistent round balloon-like morphology, compared with stimulated dying cells with normal NINJ1 expression, which show the usual necrotic cell lysis.…”

Gasdermin E-dependent and -independent Subroutines Regulate the Passage of Dextrans During Apoptosis-driven Secondary Necrosis.

“…These observations suggest that other, GSDME-independent, subroutines exist that allow the release of FITC-labeled dextrans. The existence of different subroutines supporting membrane permeabilization during cell death has recently been shown by Kayagaki et al They report that the cell-surface protein nerve injury-induced protein 1 (NINJ1) contributes to plasma membrane permeabilization during apoptosis-driven secondary necrosis, pyroptosis and necroptosis next to GSDME, GSDMD and mixed lineage kinase domain-like (MLKL) [45]. Which subroutine is responsible for the e ux of FITC-labeled dextrans in our system remains elusive, but similar to the in ux of Texas Red-labeled dextrans, e ux of FITC-labeled dextrans occurs in a sizedependent manner.…”

Phagocytic clearance of apoptotic, necrotic, necroptotic and pyroptotic cells

“…Necrosis, either primary or secondary (occurring after the completion of apoptosis), is traditionally an unregulated form of cell death largely characterised by stochastic membrane lysis ( Figure 1 ) [ 61 ]. It was recently suggested that caspase cleavage of Gasdermin E (GSDME) may mediate the progression of apoptosis to secondary necrosis through inducing membrane lysis [ 62 ] however, results are conflicting [ 10 , 63 ]. Alternatively, NINJ1 may regulate necrotic-cell membrane permeabilization [ 10 ].…”

“…It was recently suggested that caspase cleavage of Gasdermin E (GSDME) may mediate the progression of apoptosis to secondary necrosis through inducing membrane lysis [ 62 ] however, results are conflicting [ 10 , 63 ]. Alternatively, NINJ1 may regulate necrotic-cell membrane permeabilization [ 10 ]. In contrast with apoptotic cells which tightly regulate the activation of PANX1 channels and release of ‘find-me’ signals such as ATP, necrotic cells may stochastically release ATP as a by-product of uncontrolled membrane permeabilization [ 61 ].…”