Genetic targeting ofCard19is linked to disruptedNinj1 expression, impaired cell lysis, and increased susceptibility toYersiniainfection
Authors: Bjanes Elisabet , Sillas Reyna Garcia , Matsuda Rina , Demarco Benjamin , Fettrelet Timothée , DeLaney Alexandra A. , Kornfeld Opher S. , Lee Bettina L. , Rodriguez Lopez Eric M. , Grubaugh Daniel , Wynosky-Dolfi Meghan A. , Philip Naomi H. , Krespan Elise , Tovar Dorothy , Joannas Leonel , Beiting Daniel P. , Henao-Mejia Jorge , Schaefer Brian C. , Chen Kaiwen W. , Broz Petr , Brodsky Igor E. 
Published: March 2021

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 duringYersiniainfection, we identified a line ofCard19-deficient mice (Card19lxcn)whose macrophages were protected from cell lysis and showed reduced apoptosis and 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/Cas9Card19knockout mice showed no defect in macrophage cell lysis, and expression of CARD19 inCard19lxcnmacrophages did not restore cell lysis.Card19is located on chromosome 13, adjacent toNinj1, which was recently reported to regulate cell lysis downstream of GSDMD activation. Intriguingly, RNA-seq and western blotting revealed thatCard19lxcnBMDMs are hypomorphic for NINJ1 expression, and reconstitution ofNinj1inCard19lxcnimmortalized BMDMs restored cell lysis.Card19lxcnmice exhibited significantly increased susceptibility toYersiniainfection, demonstrating that cell lysis itself plays a key role in protection against bacterial infection. Our findings identify genetic targeting ofCard19being responsible for off-target effects on the adjacentNinj1gene, thereby disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control duringYersiniainfection.

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. Genetic targeting ofCard19on chromosome 13 resulted in decreased expression of the adjacent gene,Ninj1which was recently found to regulate terminal lysis events in response to cell death-inducing stimuli. We found that macrophages fromCard19-deficient mice were resistant to multiple forms of cell death in response to a variety of inflammatory stimuli, including canonical and non-canonical inflammasome activation, as well as triggers of cell-extrinsic apoptosis. Notably,Card19-deficient mice were more susceptible toYersiniainfection, 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 highlight the role of additional factors that regulate lytic cell death downstream of gasdermin cleavage.