Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection.

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-1ß 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.

Caspase-8 promotes c-Rel-dependent inflammatory cytokine expression and resistance against Toxoplasma gondii.

Caspase-8 is a key integrator of cell survival and cell death decisions during infection and inflammation. Following engagement of tumor necrosis factor superfamily receptors or certain Toll-like receptors (TLRs), caspase-8 initiates cell-extrinsic apoptosis while inhibiting RIPK3-dependent programmed necrosis. In addition, caspase-8 has an important, albeit less well understood, role in cell-intrinsic inflammatory gene expression. Macrophages lacking caspase-8 or the adaptor FADD have defective inflammatory cytokine expression and inflammasome priming in response to bacterial infection or TLR stimulation. How caspase-8 regulates cytokine gene expression, and whether caspase-8-mediated gene regulation has a physiological role during infection, remain poorly defined. Here we demonstrate that both caspase-8 enzymatic activity and scaffolding functions contribute to inflammatory cytokine gene expression. Caspase-8 enzymatic activity was necessary for maximal expression of Il1b and Il12b, but caspase-8 deficient cells exhibited a further decrease in expression of these genes. Furthermore, the ability of TLR stimuli to induce optimal IκB kinase phosphorylation and nuclear translocation of the nuclear factor kappa light chain enhancer of activated B cells family member c-Rel required caspase activity. Interestingly, overexpression of c-Rel was sufficient to restore expression of IL-12 and IL-1ß in caspase-8-deficient cells. Moreover, Ripk3-/-Casp8-/- mice were unable to control infection by the intracellular parasite Toxoplasma gondii, which corresponded to defects in monocyte recruitment to the peritoneal cavity, and exogenous IL-12 restored monocyte recruitment and protection of caspase-8-deficient mice during acute toxoplasmosis. These findings provide insight into how caspase-8 controls inflammatory gene expression and identify a critical role for caspase-8 in host defense against eukaryotic pathogens.

Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens.

Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia (CAP) across patient populations of all ages. We have developed a loop-mediated isothermal amplification (LAMP) assay that enables rapid, low-cost detection of M. pneumoniae from nucleic acid extracts and directly from various respiratory specimen types. The assay implements calcein to facilitate simple visual readout of positive results in approximately 1 h, making it ideal for use in primary care facilities and resource-poor settings. The analytical sensitivity of the assay was determined to be 100 fg by testing serial dilutions of target DNA ranging from 1 ng to 1 fg per reaction, and no cross-reactivity was observed against 17 other Mycoplasma species, 27 common respiratory agents, or human DNA. We demonstrated the utility of this assay by testing nucleic acid extracts (n = 252) and unextracted respiratory specimens (n = 72) collected during M. pneumoniae outbreaks and sporadic cases occurring in the United States from February 2010 to January 2014. The sensitivity of the LAMP assay was 88.5% tested on extracted nucleic acid and 82.1% evaluated on unextracted clinical specimens compared to a validated real-time PCR test. Further optimization and improvements to this method may lead to the availability of a rapid, cost-efficient laboratory test for M. pneumoniae detection that is more widely available to primary care facilities, ultimately facilitating prompt detection and appropriate responses to potential M. pneumoniae outbreaks and clusters within the community.