Gasdermin D Promotes AIM2 Activation and Is Required for Host Protection against Francisella novicida

This information is current as Qifan Zhu, Min Zheng, Arjun Balakrishnan, Rajendra Karki of October 2, 2021. and Thirumala-Devi Kanneganti J Immunol 2018; 201:3662-3668; Prepublished online 7 November 2018; doi: 10.4049/jimmunol.1800788 http://www.jimmunol.org/content/201/12/3662 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2018/11/06/jimmunol.180078 Material 8.DCSupplemental http://www.jimmunol.org/ References This article cites 39 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/201/12/3662.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Gasdermin D Promotes AIM2 Inflammasome Activation and Is Required for Host Protection against Francisella novicida

Qifan Zhu,*,† Min Zheng,* Arjun Balakrishnan,* Rajendra Karki,* and Thirumala-Devi Kanneganti*

The DNA sensor absent in 2 (AIM2) forms an inflammasome complex with ASC and -1 in response to Francisella tularensis subspecies novicida infection, leading to maturation of IL-1b and IL-18 and . AIM2 is critical for host protection against F. novicida infection in vivo; however, the role of pyroptosis downstream of the AIM2 inflammasome is unknown. Recent studies have identified gasdermin D (GSDMD) as the molecule executing pyroptosis by forming pores on the plasma membrane following activation by inflammatory caspase-1 and -11. In this study, we report that GSDMD-deficient mice were susceptible to F. novicida infection compared with wild type mice. Interestingly, we observed that GSDMD is required for optimal caspase-1 activation and pyroptotic cell death in F. novicida–infected bone marrow–derived macrophages. Furthermore, Downloaded from caspase-1 activation was compromised in bone marrow–derived macrophages lacking GSDMD stimulated with other AIM2 inflammasome triggers, including poly(dA:dT) transfection and mouse CMV infection. Overall, our study highlights a function, to our knowledge previously unknown, for GSDMD in promoting caspase-1 activation by AIM2 inflammasome. The Journal of Immunology, 2018, 201: 3662–3668.

nflammasomes are multiprotein complexes formed by acti- cell lysis (9–13). Recent studies have further shown that pores http://www.jimmunol.org/ vation of innate immune sensors, including nucleotide- generated by GSDMD can function as channels to allow secretion I binding oligomerization domain–like receptors absent in of IL-1b and IL-18 independently of pyroptosis (14, 15). melanoma 2 (AIM2) or pyrin, in response to pathogen-associated AIM2 recognizes dsDNA originated from Gram-negative bac- molecular patterns or danger-associated molecular patterns (1). terium Francisella, DNA viruses mouse CMV (MCMV) and AIM2 is a cytoplasmic sensor for dsDNA. Upon activation, AIM2 virus, and host cells, thereby exhibiting crucial roles in assembles the inflammasome complex with adaptor ASC to me- microbial infections and autoimmune diseases (16–22). Mice diate caspase-1 activation, caspase-1–dependent pyroptosis, and lacking AIM2, ASC, or caspase-1 are highly susceptible to in- release of proinflammatory cytokines IL-1b and IL-18 (2–5). fection by Francisella novicida, demonstrating a protective role Gasdermin D (GSDMD) was recently discovered as an executor for AIM2 inflammasome during the infection (18, 22–27). IL-1b by guest on October 2, 2021 of pyroptotic cell death downstream of caspase-1 and caspase-11 and IL-18 have been further shown to mediate the beneficial ef- (6–8). GSDMD belongs to the gasdermin family of and is fects of AIM2 in response to F. tularensis live vaccine strain (28, maintained in an autoinhibitory form under steady state. Inflam- 29). However, the role of pyroptosis downstream of AIM2 matory , including caspase-1 and -11, cleave at a link inflammasome is unknown. In this study, we showed that mice region between the N- and C-terminal domains of GSDMD, re- lacking the effector molecule of pyroptotic cell death, GSDMD, leasing the N-terminal fragment from the inhibition by the are susceptible to F. novicida infection. We further found that al- C-terminal domain (6–8). The cleaved N-terminal end of GSDMD though cleavage of GSDMD is dependent on AIM2 inflammasome, binds to phosphoinositides located in the inner leaflet of the GSDMD itself is required for optimal caspase-1 activation. Inter- plasma membrane and forms pores on the membrane, leading to estingly, GSDMD is also required for caspase-1 activation trig- gered by other AIM2 inflammasome stimuli, including poly(dA:dT) *Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN transfection and MCMV infection. 38105; and †Integrated Biomedical Sciences Program, University of Tennessee Health Science Center, Memphis, TN 38163 Materials and Methods ORCIDs: 0000-0001-7174-7943 (Q. Z.); 0000-0002-0343-3368 (M.Z.); 0000-0002- Mice 6395-6443 (T.-D.K.). Received for publication June 8, 2018. Accepted for publication October 15, 2018. Wild type (WT) C57BL/6J mice were purchased from The Jackson Lab- oratory. Aim22/2 mice (22), Casp12/2 mice (30), Il1r2/2Il182/2 mice This work was supported by National Institutes of Health Grants AI101935, AI124346, 2/2 AR056296, and CA163507 and the American Lebanese Syrian Associated Charities (31), and Gsdmd mice (31) have been described previously. Six- to (to T.-D.K.). eight-week-old male and female mice were used in this study. Animal studies were conducted according to the protocols approved by the St. Jude Address correspondence and reprint requests to Dr. Thirumala-Devi Kanneganti, Institutional Animal Care and Use Committee. Department of Immunology, MS 351, Room E7057, St. Jude Children’s Research Hos- pital, 262 Danny Thomas Place, Memphis, TN 38105-3678. E-mail address: Thirumala- Bacterial culture and animal infection [email protected] The online version of this article contains supplemental material. F. novicida strain U112 was grown overnight at 37˚C in BBL Trypticase soy broth (Becton Dickinson) containing 0.2% L-cysteine and was 1:10 Abbreviations used in this article: AIM2, absent in melanoma 2; BMDM, bone mar- 3 4 row–derived macrophage; GBP, guanylate-binding ; GSDMD, gasdermin D; subcultured for 4 h. Mice were infected s.c. with 7.5 10 CFUs (survival 3 5 m iBMDM, immortalized BMDM; KO, knockout; LDH, lactate dehydrogenase; analysis) or 1.5 10 CFUs (day 3 CFU analysis) of F. novicida in 200 l MCMV, mouse CMV; MOI, multiplicity of infection; WT, wild type. PBS. For CFU analysis, homogenized liver and spleen tissues were plated onto Trypticase soy broth agar containing 0.2% L-cysteine and incubated Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 overnight. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800788 The Journal of Immunology 3663

Cytokine analysis Flow cytometry Cytokine levels were measured by performing multiplex ELISA MAbs CD11b (M1/70) from eBioscience and F4/80 (BM8) from BioLegend (MilliporeSigma) or IL-18 ELISA (MBL International) according to the were used for flow cytometry analysis. Flow cytometry data were acquired manufacturer’s instructions. on FACSCalibur (Becton Dickinson) and were analyzed with FlowJo software (FlowJo and Illumina). Cell culture and stimulation Statistical analysis Bone marrow–derived macrophages (BMDMs) were cultured for 6 d in macrophage culture medium (i.e., DMEM supplemented with 10% FBS, GraphPad Prism 7.0 software was used for data analysis. Data are shown as 30% L929 conditioned medium, and 1% penicillin and streptomycin) and mean 6 SEM. Statistical significance was determined by performing t tests seeded at a concentration of 1 3 106 cells per well onto 12-well plates. The (two-tailed), one-way ANOVA, or log-rank tests. A p value , 0.05 was next day, BMDMs were incubated in antibiotic-free media. F. novicida considered to be statistically significant. was added to BMDMs at an indicated multiplicity of infection (MOI) for 20 h. Fifty micrograms per milliliter gentamicin was added 4 h after the Results infection. The MCMV strain (K181) was obtained from E.S. Mocarski (32) (Emory University School of Medicine). MCMV was added at an GSDMD is required for host defense against MOI of 10 for 10 h. For poly(dA:dT) transfection, 1 mg of poly(dA:dT) F. novicida infection m (InvivoGen) was resuspended in PBS, mixed with 0.3 l of Xfect polymer Mice lacking AIM2 inflammasome components such as AIM2 in Xfect reaction buffer (Takara Bio USA), and incubated for 10 min. DNA 2/2 2/2 complexes were added to BMDMs in Opti-MEM and incubated for 3 h. (Aim2 ) or caspase-1 (Casp1 ) were previously reported to be susceptible to F. novicida infection (18, 22–27). Consistent Generation of immortalized BMDMs with previous findings, we found that Aim22/2 and Casp12/2 mice were susceptible to F. novicida infection (Fig. 1A). Fol- Bone marrow cells were harvested from mice and seeded on plates with Downloaded from supernatant of J2 Cre retrovirus and macrophage culture medium in the lowing assembly of the inflammasome, activated caspase-1 presence of polybrene. Three days later, the supernatant was spun down, cleaves pro–IL-1b and pro–IL-18 into mature forms and me- resuspended in fresh macrophage culture medium, and transferred back on plates, where fresh J2 Cre supernatant supplemented with polybrene was diates their release from the cell. We therefore tested the role of added. Macrophages were then split accordingly. L929 conditioned media IL-1b and IL-18 during F. novicida infection. Similar to 2 2 2 2 in the macrophage culture medium were decreased by half with each Aim2 / and Casp1 / mice, mice lacking both IL-1R and IL-18 passage. (Il1r2/2Il182/2) were also highly susceptible and succumbed to http://www.jimmunol.org/ Generation of Gsdmd2/2 immortalized BMDMs stably death within 3 d of the infection (Fig. 1A), highlighting a protective expressing FLAG-GSDMD role for IL-1b and IL-18 downstream of inflammasome activation during F. novicida infection. -encoding GSDMD tagged with FLAG was cloned into retroviral plasmid pMSCV-puro. To get the desired retrovirus, 293T cells were GSDMD mediates pyroptosis downstream of AIM2 inflammasome transfected with pMSCV-puro–FLAG-GSDMD, pAME, and pVSV-G with in response to DNA transfection (7). To test the role of pyroptosis a ratio of 2:2:1 using lipofectamine 2000. Supernatants were collected at during F. novicida infection, we infected WT mice and mice lacking 2/2 48 h after transfection, and the Gsdmd immortalized BMDM (iBMDM) GSDMD (Gsdmd2/2)withF. novicida andmonitoredthesurvival. cells were used for transduction. Positive cells were selected by puromycin m Whereas 70% of WT mice survived on day 14 postinfection, only at a concentration of 5 g/ml. 2/2

10% of Gsdmd mice survived during the course of infection by guest on October 2, 2021 Western blotting (Fig.1A).Furthermore,Gsdmd2/2 mice had significantly increased For caspase-1 immunoblots, GAPDH immunoblots that are associated with bacterial burden in the liver and spleen 3 d after F. novicida infection caspase-1 immunoblots, and immunoblots in Fig. 2C, BMDMs and supernatant (Fig. 1B), further confirming a protective role for GSDMD. Mem- were lysed in radioimmunoprecipitation assay buffer supplemented with pro- brane pores formed by GSDMD have been shown to mediate se- tease inhibitor (Sigma-Aldrich) and sample loading buffer containing SDS and cretion of IL-1b and IL-18. Indeed, we observed reduced levels of 100 mM DTT. For immunoblotting of GSDMD and GAPDH in Fig. 4, cell 2/2 supernatant was removed. BMDMs were washed with PBS and further lysed in circulating IL-18 in Gsdmd mice compared with WT mice 1 d RIPA buffer supplemented with protease and phosphatase inhibitor (Roche). postinfection (Fig. 1C). Prepared samples were then separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (MilliporeSigma). Membranes were GSDMD is necessary for optimal caspase-1 activation by blocked by 5% milk and incubated overnight with primary Abs against F. novicida caspase-1 (AG-20B-0042; AdipoGen Life Sciences), GSDMD (ab209845; Abcam), FLAG (F1804; Sigma-Aldrich), or GAPDH (no. 5174; Cell Signaling Inflammasome activation is critical for host defense against Technology). Membranes were then incubated with HRP-conjugated second- F. novicida infection in vivo. To test whether inflammasome ac- ary Ab for 1 h. Proteins were further visualized using Luminata Forte Western tivation is affected in the absence of GSDMD, we generated HRP substrate (MilliporeSigma). BMDMs from WT, Gsdmd2/2,andAim22/2 mice. The population Lactate dehydrogenase release assay of CD11b+F4/80+ macrophages was comparable between WT and Gsdmd2/2 BMDMs (Supplemental Fig. 1), suggesting that there is Cell supernatants were collected 20 h after F. novicida infection, 3 h after poly(dA:dT) transfection, or 10 h after MCMV infection. Lactate dehy- no difference in the extent of macrophage differentiation. We then 2/2 2/2 drogenase (LDH) activity was measured using the Promega cytotoxicity kit infected BMDMs from WT, Gsdmd ,andAim2 mice with according to manufacturer’s protocols. F. novicida and measured cleavage of pro–caspase-1 into its active form (p20) following assembly of inflammasome. Interestingly, IncuCyte analysis Gsdmd2/2 BMDMs showed decreased activation of caspase-1 BMDMs were seeded at a concentration of 1 3 106 cells per well onto compared with WT cells after F. novicida infection (Fig. 2A), 12-well plates. The next day, BMDMs were stimulated and incubated with highlighting the requirement of GSDMD for inflammasome acti- 100 nM cell-impermeable DNA-binding fluorescent dye SYTOX Green (S7020; Life Technologies) to stain dead cells. Cell death was monitored in vation in response to F. novicida. Caspase-1 activation was reduced 2/2 real time using a two-color IncuCyte ZOOM in-incubator imaging system in Gsdmd BMDMs postinfection with multiple MOIs of (Essen BioScience). Nuclear-ID (ENZ-52406; Enzo Life Sciences) was F. novicida (Fig. 2A, Supplemental Fig. 2A, 2B). To further confirm added at the last time point to stain live and dead cells for the total cell the role of GSDMD in caspase-1 activation during F. novicida in- number (33). Resulting images were analyzed using IncuCyte S3 software. 2/2 + fection, we reconstituted GSDMD in Gsdmd iBMDMs and Cell death was presented as a ratio of SYTOX Green cells to the total cell 2/2 number. Experiments were conducted with three replicates for each ex- found that complementation of the Gsdmd iBMDMs with perimental condition and nine image fields per well. FLAG-GSDMD increased the caspase-1 activation induced by 3664 ROLE OF GASDERMIN D IN ACTIVATION OF AIM2 INFLAMMASOME Downloaded from

FIGURE 1. GSDMD contributes to host defense against F. novicida infection. (A)SurvivalofWT,Gsdmd2/2, Aim22/2, Casp12/2,andIl1r2/2Il182/2 mice infected with F. novicida.(B) Bacterial burden in the liver and spleen of WT and Gsdmd2/2 mice 3 d postinfection with F. novicida. (C) Concentration of IL-18 in the sera of WT and Gsdmd2/2 1 d after the infection. Data are pooled from two independent experiments (A) or from one experiment (B and C). Mean 6 SEM in (B)and(C). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, log-rank test (A) or two-tailed t test (B and C). http://www.jimmunol.org/

F. novicida (Fig. 2B, 2C). In addition, we observed impaired IL-1b GSDMD is necessary for optimal caspase-1 activation in response and IL-18 release in F. novicida–infected Gsdmd2/2 BMDMs, to AIM2 inflammasome triggers, including F. novicida and potentially because of reduced caspase-1 activation along with de- MCMV infection and, to a lesser extent, poly(dA:dT) transfection, fective pore formation (Fig. 2D), whereas secretion of TNF was and that GSDMD plays a critical role in mediating pyroptosis after similar between the cells (Supplemental Fig. 3). To test the re- AIM2 inflammasome activation. quirement of GSDMD in pyroptosis triggered by F. novicida in- 2/2 AIM2 inflammasome cleaves GSDMD during fection, we first monitored the extent of cell death in WT, Gsdmd , F. novicida infection by guest on October 2, 2021 and Aim22/2 BMDMs after F. novicida infection using SYTOX Green staining and found that Gsdmd2/2 BMDMs exhibited lower Previous studies have shown that GSDMD is cleaved downstream numbers of SYTOX Green–positive cells than WT BMDMs over the of inflammasome activation. To test if AIM2 inflammasome is able course of the infection (Fig. 2E, 2G, Supplemental Fig. 2C). Con- to mediate GSDMD cleavage following F. novicida infection, we sistent with the SYTOX Green staining, LDH release was also de- infected cells with F. novicida and observed impaired cleavage of 2/2 2/2 creased in Gsdmd2/2 BMDMs 20 h postinfection, which further GSDMD in BMDMs lacking AIM2 (Aim2 ) or ASC (Asc ) confirms that pyroptosis in BMDMs during F. novicida infection is (Fig. 4A), suggesting that AIM2 inflammasome is required for dependent on GSDMD (Fig. 2F, 2G). Interestingly, GSDMD- GSDMD cleavage. Type I IFN signaling is required for activating independent cell death was also observed in Gsdmd2/2 BMDMs AIM2 inflammasome in macrophages during F. novicida infection after F. novicida infection, suggesting that other molecules may exist via induction of transcription factor IFN regulatory factor 1 to mediate F. novicida–induced pyroptosis (Fig. 2E–G, Supplemental (IRF1), an IFN-stimulated gene (16, 18, 22–25, 35, 36). IRF1, in Fig. 2C). turn, promotes expression of various cell-autonomous immunity proteins, including guanylate-binding protein (GBP) 2, GBP5, and GSDMD is necessary for optimal caspase-1 activation by other immunity-related GTPase B10 (IRGB10), which facilitate bacte- AIM2 inflammasome triggers riolysis and release of bacterial DNA (23–25). Because GBPs are In addition to F. novicida infection, AIM2 inflammasome can also critical for AIM2 inflammasome activation during F. novicida be activated by transfection of poly(dA:dT) or MCMV infection. infection (24, 25), we used BMDMs from mice lacking multiple We found that caspase-1 activation induced by transfection of GBPs (including GBP2 and 5) encoded on 3 dsDNA ligand poly(dA:dT) was partially reduced in Gsdmd2/2 (Gbpchr3-knockout [KO]). We found reduced cleavage of GSDMD BMDMs compared with WT cells (Fig. 3A). Gsdmd2/2 BMDMs in Gbpchr3-KO BMDMs, further demonstrating the requirement of exhibited compromised release of IL-18 but not TNF (Fig. 3B, AIM2 inflammasome for GSDMD cleavage (Fig. 4B). Overall, Supplemental Fig. 3). Consistent with previous studies (7, 34), cell these data demonstrate that although caspase-1 activation induced death was completely abolished in poly(dA:dT)-transfected by AIM2 inflammasome triggers is largely dependent on Gsdmd2/2 BMDMs during the early phase of stimulation (Fig. GSDMD, GSDMD cleavage still requires AIM2 inflammasome. 3C–E). Furthermore, MCMV-induced caspase-1 activation was dampened in Gsdmd2/2 BMDMs compared with WT BMDMs Discussion (Fig. 3F). Release of IL-1b and IL-18 in MCMV-infected Pyroptosis is a lytic form of cell death that is induced by Gsdmd2/2 BMDMs was impaired (Fig. 3G), whereas TNF se- inflammasomes, leading to release of mature IL-1b and IL-18 and cretion was comparable between WT and Gsdmd2/2 BMDMs other cellular components, including high-mobility group box 1 (Supplemental Fig. 3). Altogether, these data demonstrate that (HMGB1) and IL-1a. It has not been discovered until recently that The Journal of Immunology 3665 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. GSDMD promotes casaspse-1 activation by F. novicida.(A) Immunoblot analysis of pro–caspase-1 (Pro-Casp1), the caspase-1 subunit p20 (Casp1-p20), and GAPDH in unprimed WT or mutant BMDMs left uninfected (medium alone [Med]) or assessed 20 h postinfection with F. novicida (MOI, 100). Densitometric quantification of immunoblots was performed. The protein band intensity of Casp1-p20 was normalized to GAPDH and expressed as a level relative to that of uninfected WT BMDMs, set at 1. (B) Immunoblot analysis of Pro-Casp1, Casp1-p20, and GAPDH in Gsdmd2/2 iBMDMs, Gsdmd2/2 iBMDMs transduced with a retroviral vector encoding FLAG-GSDMD, and Aim22/2 iBMDMs postinfection with (Figure legend continues) 3666 ROLE OF GASDERMIN D IN ACTIVATION OF AIM2 INFLAMMASOME

FIGURE 3. GSDMD is necessary for optimal caspase-1 activation by other AIM2 inflammasome triggers. (A) Immunoblot analysis of pro– caspase-1 (Pro-Casp1), the caspase- 1 subunit p20 (Casp1-p20), and GAPDH in unprimed WT or mutant BMDMs untreated (medium alone [Med]) or assessed 3 h after trans- fection with poly(dA:dT). Densito- metric quantification of immunoblots was performed. The protein band intensity of Casp1-p20 was normal- ized to GAPDH and expressed as a levelrelativetothatofuntreatedWT BMDMs,setat1.(B) Release of IL- 18 in unprimed BMDMs after treat- ment as in (A). (C and D) Cell death analysis by IncuCyte (C)orLDH release (D) after treatment as in (A). (E) Representative images of SYTOX Downloaded from Green staining by IncuCyte (Scale bar, 100 mm) or light microscopy (Scale bar, 25 mm) of BMDMs after transfection with poly(dA:dT). Ar- rowheads indicate representative pyroptotic cells. (F) Immunoblot analysis of Pro-Casp1, Casp1-p20, http://www.jimmunol.org/ and GAPDH in unprimed WT or mutant BMDMs left uninfected (Med) or assessed 10 h postinfection with10MOIofMCMV.Densito- metric quantification of immunoblots was performed. The protein band intensity of Casp1-p20 was normal- ized to GAPDH and expressed as a

level relative to that of uninfected by guest on October 2, 2021 WT BMDMs, set at 1. (G) Release of IL-18 (left) and IL-1b (right) in unprimed BMDMs postinfection as in (F). Data are representative of two (B, F,andG), three (C–E), or six (A) independent experiments; mean 6 SEM in (B)–(D)and(G). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, one-way ANOVA with Dun- nett multiple comparisons test (B, D, and G).

GSDMD induces pyroptosis by forming pores on plasma mem- was observed in Gsdmd2/2 BMDMs infected with F. novicida,it brane after being activated by caspase-1 or -11 (6–8). In this study, is intriguing to hypothesize that during the early phase of we demonstrated a protective role for GSDMD during F. novicida F. novicida infection, caspase-1 is activated in a GSDMD- infection. Although caspase-1 processing is intact in the absence independent manner. Activated caspase-1 may cleave GSDMD of GSDMD downstream of canonical NLRP3, NLRC4, or pyrin to allow the N terminus to target F. novicida in the , leading inflammasome (6, 7), we observed that activation of caspase-1 is to bacteriolysis and release of bacterial DNA, which potentially impaired in Gsdmd2/2 BMDMs in response to AIM2 inflamma- amplifies activation of AIM2 inflammasome and further GSDMD some stimuli. Given that a residual amount of caspase-1 activation cleavage at the later phase of F. novicida infection in a positive

F. novicida (MOI, 100). Densitometric quantification of immunoblots was performed. The protein band intensity of Casp1-p20 was normalized to GAPDH and expressed as a level relative to that of Gsdmd2/2 iBMDMs, set at 1. (C) Immunoblot analysis of GSDMD, FLAG, and GAPDH in uninfected Gsdmd2/2 iBMDMs, Gsdmd2/2 iBMDMs transduced with a retroviral vector encoding FLAG-GSDMD, and Aim22/2 iBMDMs (Med). (D) Release of IL-18 (left) and IL-1b (right) in unprimed BMDMs postinfection as in (A). (E and F) Cell death analysis by IncuCyte (E) or LDH release (F) postinfection as in (A). (G) Representative images of SYTOX Green staining by IncuCyte (Scale bar, 100 mm) or light microscopy (Scale bar, 25 mm) of BMDMs postinfection with F. novicida (MOI, 100). Arrowheads indicate representative pyroptotic cells. Data are representative of two (B–D), three (E–G), or six (A) independent experiments; mean 6 SEM in (D)–(F). **p , 0.01, ***p , 0.001, ****p , 0.0001, one-way ANOVA with Dunnett multiple comparisons test (D and F). The Journal of Immunology 3667

FIGURE 4. GSDMD cleavage is dependent on AIM2 inflammasome. (A and B) Immunoblot analysis of pro-GSDMD (GSDMD), cleaved N terminus of GSDMD (GSDMD-NT), and GAPDH in unprimed WT, Aim22/2, and Asc2/2 BMDMs (A) or WT and Gbpchr3-KO BMDMs (B) infected with F. novicida (MOI, 20). Data are representative of two in- dependent experiments (A and B). Downloaded from feedback manner. Indeed, GSDMD has been shown to be Disclosures associated with phosphoinositides and cardiolipin (9–11), the The authors have no financial conflicts of interest. latter of which is a component of F. novicida membrane as well as mitochondria (37). Moreover, incubation of the N-terminal References fragment of GSDMD with Escherichia coli or Staphylococcus http://www.jimmunol.org/ 1. Broz, P., and V. M. Dixit. 2016. Inflammasomes: mechanism of assembly, aureus in vitro leads to reduced bacterial CFU (11). Furthermore, regulation and signalling. Nat. Rev. Immunol. 16: 407–420. higher CFU of is observed in GSDMD– 2. 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