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-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 an independently-generated CRISPR/Cas9
Card19 knockout mouse line (
Card19Null) showed no defect in macrophage cell lysis. Notably,
Card19 is located on chromosome 13, immediately adjacent to
Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that
Card19lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of
Ninj1 in
Card19lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli.
Card19lxcn mice exhibited increased susceptibility to
Yersinia infection, whereas independently-generated
Card19Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of
Card19lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of
Card19 being responsible for off-target effects on the adjacent gene
Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during
Yersinia infection.
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