N-canonical inflammasome (three). Casp1-/- mice generated from 129 background stem cells are also deficient in Casp11 resulting from a passenger mutation backcrossed from the 129 background into C57BL/6. Caspase-11 is responsible for certain phenotypes initially attributed to caspase-1, like shock following endotoxin challenge (three). The physiologic function of caspase-11 is always to discriminate cytosolic from vacuolar bacteria (4). Inside the absence of caspase-11, mice turn out to be acutely susceptible to infection by bacteria that escape the phagosome and replicate inside the cytosol (four), for instance Burkholderia pseudomallei and B. thailandensis. Caspase-11 also responds to vacuolar Gram-negative bacteria, albeit with delayed kinetics (three, 5), which may perhaps have relevance to its aberrant activation for the duration of sepsis. While these studies demonstrated both detrimental and protective roles for caspase-11, the precise nature from the caspase-11 activating signal remained unknown. Because caspase-11 specifically responds to cytosolic bacteria, we hypothesized that detection of a conserved microbial ligand inside the cytosol triggers caspase-11. To address*Correspondence to: Edward A. Miao: emiao@med.Troglitazone unc.edu.Hagar et al.Pagethis hypothesis, we generated lysates of Gram-negative and Gram-positive bacteria and transfected them into LPS primed Nlrc4-/-Asc-/-Casp11+/+ or Casp1-/-Casp11-/- bone marrow-derived macrophages (BMMs). By comparing these strains, we are able to examine caspase-11 activation in the absence of canonical inflammasome detection of flagellin and DNA (fig. S1). Although boiled Gram-negative bacterial lysates have been detected through caspase-11 upon transfection into BMMs, Gram-positive lysates were not (Fig. 1A). RNase, DNase, lysozyme, and proteinase K digestion was sufficient to dispose of canonical inflammasome agonists, but failed to remove the caspase-11 activating aspect(s) (Fig. 1B). We then treated boiled lysates with ammonium hydroxide, which is identified to deacylate lipid species (eight), and observed that the caspase-11 activating factor was degraded, whereas canonical inflammasome agonists persisted (Fig.IL-1 beta Protein, Human 1C). These results suggested lipopolysaccharide (LPS) as the caspase-11 agonist. Constant with this hypothesis, BMMs underwent caspase-11 dependent pyroptosis following transfection of ultra pure Salmonella minnesota RE595 LPS (Fig. 1D). Caspase-11 can promote IL-1 secretion by triggering the canonical NLRP3 pathway (3) (fig. S1). Regularly, IL-1 secretion and caspase-1 processing following transfection of LPS have been also caspase-11 dependent (Fig.PMID:36717102 1E to G). Moreover, caspase-11 alone promoted pyroptosis (Fig. 1H). In contrast to caspase-1, we were unable to convincingly visualize caspase-11 processing by western blot (Fig. 1F and G; fig. S2A), in spite of the vast majority of cells exhibiting pyroptotic morphology as observed by phase microscopy. Although these data don’t exclude the possibility that processing of a modest level of caspase-11 is expected for pyroptosis, they do indicate that processing isn’t a fantastic proxy measure for fast caspase-11 activation. That is constant with direct caspase-1 activation by NLRC4, that is not accompanied by processing (9). These results suggest that the presence of LPS within the cytosol is adequate to trigger caspase-11; nonetheless, we can’t rule out the formal possibility that this signaling arises from a membrane bound compartment which include the ER or golgi. Future identification on the non-canonical inflammasome will permit this.
