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The Card19 Locus of Murine Chromosome 13 Regulates Terminal Cell Lysis Downstream of Caspase Activation and Gasdermin-D Cleavage

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The Card19 Locus of Murine Chromosome 13 Regulates Terminal Cell Lysis Downstream of Caspase Activation and Gasdermin-D Cleavage bioRxiv preprint doi: https://doi.org/10.1101/2021.03.19.436207; this version posted March 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The Card19 locus of murine chromosome 13 regulates terminal cell lysis downstream of caspase activation and Gasdermin-D cleavage Card19 locus regulates caspase-dependent cell lysis Elisabet Bjanes1,2,#, Reyna Garcia Sillas2, Rina Matsuda1,2, Benjamin Demarco3, Timothée Fettrelet3, Alexandra A. DeLaney1,2, Eric M. Rodriguez Lopez2,4, Daniel Grubaugh2, Meghan A. Wynosky-Dolfi2,&, Naomi H. Philip2,4,§, Elise Krespan2,5, Dorothy Tovar2,¶, Leonel Joannas2,6, Daniel P. Beiting2,5,7, Jorge Henao-Mejia2,6,7, Brian C. Schaefer8, Kaiwen W. Chen3,%, Petr Broz3, Igor E. Brodsky2,7* 1Cell and Molecular Biology Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America 2Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, United States of America 3Department of Biochemistry, University of Lausanne, Epalinges, Vaud, Switzerland 4Immunology Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America 5Center for Host Microbial Interactions, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, United States of America 6CRISPR/Cas9 Mouse Targeting Core, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, United States of America 7Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America 8Department of Microbiology and Immunology, Uniformed Services University, Bethesda, Maryland, United States of America #Current address: Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America &Current address: Innate Immunity Research Unit, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America §Current address: Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, United States of America ¶Current address: Department of Microbiology & Immunology, Stanford University School of Medicine, Palo Alto California, United States of America %Current address: Immunology Programme and Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore *Correspondence to: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.03.19.436207; this version posted March 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Author Contributions Conceptualization: E.B., and I.E.B. Formal Analysis: R.M. Investigation: E.B., R.G.S., R.M., B.D., T.F. A.D, E.M.R.L, D.G., M.A.W-D., N.H.P., and D.T. Supervision: L.J., J.H-M., K.W.C., P.B., and I.E.B. Resources: L.J., D.B., E.K., B.C.S., J.H-M., and P.B. Funding Acquisition: I.E.B. Writing: E.B., B.C.S., K.W.C., I.E.B. Abstract Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-/- mice whose macrophages were protected from cell death and showed reduced pore formation during apoptosis or pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as two independently-generated CRISPR/Cas9 Card19 knockout mice showed no defect in macrophage cell lysis. The original Card19-/- line was generated in a 129SvEvBrd background, and SNP analysis revealed a six megabase region of 129 origin co-segregating with the Card19 locus. Card19 is located on chromosome 13, adjacent to Ninj1, which was recently reported to regulate cell lysis downstream of GSDMD activation. Nonetheless, we could not detect major defects in NINJ1 protein expression or mutations in Ninj1 coding sequence in Card19-/- mice. Mice from the original Card19-/- line exhibited significantly increased susceptibility to Yersinia infection, demonstrating that cell lysis itself plays a key role in protection against bacterial infection. Our findings identify a locus on murine chromosome 13 that regulates the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage, and implicates additional NINJ1-independent factors that control terminal cell lysis. 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.19.436207; this version posted March 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Author Summary Programmed cell death is critical for regulating tissue homeostasis and host defense against infection. Pyroptosis is an inflammatory form of programmed cell death that couples cell lysis with release of inflammatory cytokines. Cell lysis is triggered by activation of particular intracellular pore forming proteins, but how regulation of cell lysis occurs is not well understood. We identified the Card19 locus on chromosome 13 as playing a key role in regulating terminal cell death in response to danger signals, such as bacterial infections or harmful chemicals. We found that immune cells from Card19-/- mice were resistant to multiple forms of cell death in response to a variety of harmful stimuli, including bacterial infections. Card19-/- mice were more susceptible to Yersinia infection, indicating that cell lysis contributes to control of bacterial infections. Our data provide new insight into the impact of terminal cell lysis on control of bacterial infection and indicate that additional factors exist that regulate lytic cell death. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.19.436207; this version posted March 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction Regulated cell death is an evolutionarily conserved mechanism by which multicellular organisms regulate fundamental biological processes ranging from tissue development to control of microbial infection. Apoptosis and pyroptosis represent two distinct forms of regulated cell death that are activated in response to diverse stimuli, including developmental signals (1, 2), tissue stress or injury (3), and microbial infection (4-7). While apoptosis is an immunologically quiescent or suppressive form of cell death during tissue homeostasis and development, certain stimuli, including infections and chemotherapeutic agents, induce apoptosis that is accompanied by inflammatory signals that contribute to anti-pathogen and anti-tumor immunity (8-10). Apoptosis is activated by specific initiator apoptotic caspases, following certain intrinsic or extrinsic signals, and mediates organized disassembly of the cell via a process that limits cellular permeability and enables phagocytosis of the dying cell (11, 12). Alternatively, pyroptosis is a lytic form of regulated cell death characterized by caspase-1- or -11-dependent plasma membrane disruption mediated by cleavage and activation of the pore forming protein Gasdermin D (GSDMD), and release of interleukin-1 (IL-1) family cytokines and other intracellular components (13-16). However, inhibition of the receptor-proximal signaling kinases TAK1 or IKK by chemotherapeutic drugs or during infection by bacterial pathogens such as Yersinia, can trigger a caspase-8-dependent apoptosis pathway that is also associated with GSDMD cleavage (17-19). Interestingly, a recent study demonstrated that only caspase-8 activated in the RIPK1-containing complex IIb can efficiently cleave GSDMD in macrophages (20). These findings suggest a potential point of intersection of cell death pathways previously viewed as distinct (21). During pyroptosis, secretion of IL-1 family cytokines and release of intracellular contents are temporally and genetically linked, and it has been suggested that IL-1 cytokines are released from cells when they undergo caspase-1 or -11-dependent lysis (22, 23). However, IL-1 secretion and cell lysis can be uncoupled in certain cell types or in response to specific stimuli. For example, the osmoprotectant glycine prevents release of lactate dehydrogenase (LDH), a key indicator of cell lysis, but does not affect IL-1β cytokine release (24, 25). Conversely, genetic ablation of IL-1 does not prevent cell lysis following pyroptotic stimuli. Moreover, IL-1 secretion can occur by living cells in the absence of apparent cytotoxicity (26, 27). Notably, neutrophils and monocytes display evidence of caspase-1 processing and IL-1 secretion in the absence of cell lysis (28, 29). Furthermore,
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