RESEARCH IMMUNOLOGY and has been specifically implicated in the res- toration of plasma membrane integrity during necroptosis (12) or upon chemical- or laser-induced damage (10, 11). ESCRT-dependent membrane repair To investigate if cells repair membranes dam- aged by GSDMD pores, we imaged the disrup- negatively regulates pyroptosis tion of the electrochemical gradient (by using the Ca2+ dye Fluo-8) and loss of membrane integrity downstream of GSDMD activation [by using propidium iodide (PI)] in mouse bone marrow–derived macrophages (BMDMs). These cells were either untreated or transfected with Sebastian Rühl1,2, Kateryna Shkarina3*, Benjamin Demarco3*, Rosalie Heilig3*, lipopolysaccharide (LPS) to activate caspase-11. José Carlos Santos3*, Petr Broz1,3+ Within 2 hours, up to 50% of LPS-transfected wild-type (WT) BMDMs underwent pyroptosis, Pyroptosis is a lytic form of cell death that is induced by inflammatory caspases upon as demonstrated by a strong PI signal (PIhi)(fig. activation of the canonical or noncanonical inflammasome pathways. These caspases S1A). A marked spike of Fluo-8 signal preceded cleave gasdermin D (GSDMD) to generate an N-terminal GSDMD fragment, which the PIhi signal, indicating a loss of membrane executes pyroptosis by forming membrane pores. We found that calcium influx through integrity and Ca2+ influx (Fig. 1, A and B; fig. S1, B GSDMD pores serves as a signal for cells to initiate membrane repair by recruiting the to D; and movies S1 and S2). In contrast, Fluo-8 endosomal sorting complexes required for transport (ESCRT) machinery to damaged or PI signals did not change in Casp11-/- or membrane areas, such as the plasma membrane. Inhibition of the ESCRT-III machinery Downloaded from Gsdmd-/- BMDMs (Fig. 1B; fig. S1, C and D; and strongly enhances pyroptosis and interleukin-1b release in both human and murine movies S1 and S2). Unexpectedly, we found that cells after canonical or noncanonical inflammasome activation. These results not only Ca2+ influx–negative WT BMDMs that maintained attribute an anti-inflammatory role to membrane repair by the ESCRT-III system but the electrochemical gradient gradually acquired also provide insight into general cellular survival mechanisms during pyroptosis. low levels of PI (PIlo)(Fig.1C).ThisPIlo signal reachedonly10%ofthePIhi signal observed in asdermin D (GSDMD) is a pore-forming it forms large pores to initiate pyroptosis (4–7). the WT BMDMs that had lost membrane integ- science.sciencemag/ protein that induces pyroptosis, a necrotic Damage to the plasma membrane does not neces- rity upon LPS transfection. Furthermore, neither form of cell death that is initiated after in- sarily result in cell death, as it has been observed unstimulated WT BMDMs (Fig. 1A) nor LPS- G flammasome activation (1–3). The detec- thattheinfluxofCa2+ ions from the extracel- transfected Casp11 -/- or Gsdmd -/- cells showed tion of pathogen- or host-derived danger lular milieu triggers repair programs involving 1Focal Area Infection Biology, Biozentrum, University of signalsbyinflammasomestriggerstheactivation either the endocytosis of a damaged membrane Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland. of inflammatory caspases (caspase-1 and -11 in mice or its shedding in the form of ectosomes (8–11). 2Department of Immunology, St. Jude Children’s Research and caspase-1 and -4 in humans), which cleave This latter mechanism relies on components of Hospital, Memphis, TN 38105, USA. 3Department of GSDMD to release autoinhibition on its N-terminal endosomal sorting complexes required for trans- Biochemistry, University of Lausanne, Chemin des NT 1 2 NT Boveresses 155, 1066 Epalinges, Switzerland. domain (GSDMD )(, ). The GSDMD tar- port (ESCRT-0 and -III and associated factors *These authors contributed equally to this work. gets the plasma membrane and organelles, where that control ESCRT recruitment and disassembly) +Corresponding author. Email: petr.broz@unil.ch on December 28, 2018 6 A unstimulated C LPS transfection (3.5 ug / 1 × 10 cells) D BMDMs Calcium influx negative cells 60 *** Medium Wildtype Gsdmd—/— ] 0 1.5 BMDMs 25 1.5 25 Wildtype Gsdmd—/— Casp11—/— 0.5 ] BMDMs ] 1.5 1.5 0 0 1.5 1.0 )/F 20 20 BMDMs BMDMs BMDMs 40 0 1.0 1.0 )/F 15 15 )/F 1.0 1.0 1.0 2.0 0 0 -F t 10 10 4.0 -F 0.5 0.5 -F t 5 5 t 0.5 0.5 0.5 20 Cell death Cell death 0.0 0 0.0 0 0.0 0.0 0.0 (% LDH release) -5 -5 uM BAPTA-AM PI [(F -0.5 -0.5 PI [(F -0.5 -0.5 -0.5 0 Fluo8 [(F 0 30 60 90 120 0 30 60 90 120 1234567 89 1234567 89 1234567 89 WT Casp11—/— Time (minutes) Time (minutes) Time (minutes) Time (minutes) Time (minutes) mock LPS transfection (16.6 ug / 1 × 106 cells) LPS transfected LPS transfection B E FGHeLa (3.5 ug / 1 × 106 cells) (ug / 1 × 106 cells) Δ Medium 3.3 uM MOI sifA S. typhimurium Wildtype —/— ] Gsdmd 0 10 5 2.5 0 BAPTA-AM 0 100 75 50 ]