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Gasdermin D Promotes AIM2 Inflammasome Activation and Is Required for Host Protection Against Francisella Novicida

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Gasdermin D Promotes AIM2 Inflammasome Activation and Is Required for Host Protection Against Francisella Novicida Gasdermin D Promotes AIM2 Inflammasome Activation and Is Required for Host Protection against Francisella novicida This information is current as Qifan Zhu, Min Zheng, Arjun Balakrishnan, Rajendra Karki of September 26, 2021. and Thirumala-Devi Kanneganti J Immunol published online 7 November 2018 http://www.jimmunol.org/content/early/2018/11/06/jimmun ol.1800788 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/11/06/jimmunol.180078 Material 8.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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. Published November 7, 2018, doi:10.4049/jimmunol.1800788 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 melanoma 2 (AIM2) forms an inflammasome complex with ASC and caspase-1 in response to Francisella tularensis subspecies novicida infection, leading to maturation of IL-1b and IL-18 and pyroptosis. 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: 000–000. http://www.jimmunol.org/ nflammasomes are multiprotein complexes formed by acti- cell lysis (9–13). Recent studies have further shown that pores 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 vaccinia 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 by guest on September 26, 2021 Gasdermin D (GSDMD) was recently discovered as an executor for AIM2 inflammasome during the infection (18, 22–27). IL-1b 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 proteins 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 caspases, 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 protein; 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 2 ROLE OF GASDERMIN D IN ACTIVATION OF AIM2 INFLAMMASOME 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 Bone marrow cells were harvested from mice and seeded on plates with mice were susceptible to F. novicida infection (Fig. 1A). Fol- 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.
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