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Neutrophil extracellular traps (NETs) and oxidative stress are considered to be beneficial in the innate immune defense against pathogens. However, defective clearance of NETs in the lung of acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients could lead to severe respiratory syndrome infection, the so-called coronavirus disease 2019 (COVID-19). To elucidate the pathways that are related to NETs within the pathophysiology of COVID-19, we utilized RNA sequencing (RNA-seq) as well as immunofluorescence and immunohistochemistry methods. RNA-seq analysis provided evidence for increased oxidative stress and the activation of viral-related signaling pathways in post-mortem lungs of COVID-19 patients compared to control donors. Moreover, an excess of neutrophil infiltration and NET formation were detected in the patients’ lungs, where the extracellular DNA was oxidized and co-localized with neutrophil granule protein myeloperoxidase (MPO). Interestingly, staining of the lipid peroxidation marker 4-hydroxynonenal (4-HNE) depicted high colocalization with NETs and was correlated with the neutrophil infiltration of the lung tissues, suggesting that it could serve as a suitable marker for the identification of NETs and the severity of the disease. Moreover, local inhalation therapy to reduce the excess lipid oxidation and NETs in the lungs of severely infected patients might be useful to ameliorate their clinical conditions.

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., 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 of Card19 on chromosome 13 resulted in decreased expression of the adjacent gene, Ninj1, which was recently found to regulate terminal lysis events in response to cell death-inducing stimuli. Consistently, macrophages from Card19-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 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 highlight the role of additional factors that regulate lytic cell death downstream of gasdermin cleavage.

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 two independently-generated CRISPR/Cas9 Card19 knockout mice showed no defect in macrophage cell lysis, and expression of CARD19 in Card19lxcn macrophages did not restore cell lysis. Card19 is located on chromosome 13, adjacent to Ninj1, which was recently reported to regulate cell lysis downstream of GSDMD activation. Intriguingly, RNA-seq and western blotting revealed that Card19lxcn BMDMs are hypomorphic for NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored cell lysis. Card19lxcn mice exhibited significantly increased susceptibility to Yersinia infection, demonstrating that cell lysis itself plays a key role in protection against bacterial infection. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent Ninj1 gene, thereby disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.Author SummaryProgrammed 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 of Card19 on chromosome 13 resulted in decreased expression of the adjacent gene, Ninj1 which was recently found to regulate terminal lysis events in response to cell death-inducing stimuli. We found that macrophages from Card19-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 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 highlight the role of additional factors that regulate lytic cell death downstream of gasdermin cleavage.

Eosinophils are a subset of granulocytes characterized by a high abundance of specific granules in their cytoplasm. To act as effector cells, eosinophils degranulate and form eosinophil extracellular traps (EETs), which contain double-stranded DNA (dsDNA) co-localized with granule proteins. The exact molecular mechanism of EET formation remains unknown. Although the term “EET release” has been used in scientific reports, it is unclear whether EETs are pre-formed in eosinophils and subsequently released. Moreover, although eosinophil degranulation has been extensively studied, a precise time-course of granule protein release has not been reported until now. In this study, we investigated the time-dependent release of eosinophil peroxidase (EPX) and mitochondrial DNA (mtDNA) following activation of both human and mouse eosinophils. Unexpectedly, maximal degranulation was already observed within 1 min with no further change upon complement factor 5 (C5a) stimulation of interleukin-5 (IL-5) or granulocyte/macrophage colony-stimulating factor (GM-CSF)-primed eosinophils. In contrast, bulk mtDNA release in the same eosinophil populations occurred much slower and reached maximal levels between 30 and 60 min. Although no single-cell analyses have been performed, these data suggest that the molecular pathways leading to degranulation and mtDNA release are at least partially different. Moreover, based on these data, it is likely that the association between the mtDNA scaffold and granule proteins in the process of EET formation occurs in the extracellular space.

Caspase-8–dependent GSDMD activation promotes antibacterial defense; however, this pathway can also mediate TNF shock in vivo., Gasdermin D (GSDMD) is a pore-forming protein that promotes pyroptosis and release of proinflammatory cytokines. Recent studies revealed that apoptotic caspase-8 directly cleaves GSDMD to trigger pyroptosis. However, the molecular requirements for caspase-8–dependent GSDMD cleavage and the physiological impact of this signaling axis are unresolved. Here, we report that caspase-8–dependent GSDMD cleavage confers susceptibility to tumor necrosis factor (TNF)–induced lethality independently of caspase-1 and that GSDMD activation provides host defense against Yersinia infection. We further demonstrate that GSDMD inactivation by apoptotic caspases at aspartate 88 (D88) suppresses TNF-induced lethality but promotes anti-Yersinia defense. Last, we show that caspase-8 dimerization and autoprocessing are required for GSDMD cleavage, and provide evidence that the caspase-8 autoprocessing and activity on various complexes correlate with its ability to directly cleave GSDMD. These findings reveal GSDMD as a potential therapeutic target to reduce inflammation associated with mutations in the death receptor signaling machinery.