D A as opposed to the hexa-acylated species of enteric bacteria (13). F. novicida initially synthesizes a penta-acylated lipid A structure with two phosphates and then removes the 4′ phosphate and 3′ acyl chain in reactions that usually do not happen in lpxF mutants (14, 15) (Fig. 3E). Conversion for the penta-acylated structure restored caspase-11 activation, whereas other modifications that maintained the tetra-acylated structures (flmK mutant or 18 development (12, 16)) did not (Fig. 3F). lpxF mutant lipid A is not detected by TLR4 (14), suggesting that the TLR4 and caspase-11 pathways have distinctive structural needs. Deacylation of lipid A is a typical tactic employed by pathogenic bacteria. By way of example, Yersinia pestis removes two acyl chains from its lipid A upon transition from development at 25 to 37 (17) (Fig. 3G). Consistent with our structural studies of F. novicida lipid A, caspase-11 detected hexa-acylated lipid A from Y. pestis grown at 25 , but not tetraacylated lipid A from bacteria grown at 37 (Fig. 3H). Collectively, these information indicate that caspase-11 responds to distinct lipid A structures, and pathogens seem to exploit these structural requirements in order to evade caspase-11. In addition to detection of extracellular/vacuolar LPS by TLR4, our data indicate that an extra sensor of cytoplasmic LPS activates caspase-11. These two pathways intersect, nonetheless, since TLR4 primes the caspase-11 pathway. Having said that, Tlr4-/- BMMs responded to transfected or CTB-delivered LPS following poly(I:C) priming (Fig. 4A ). Hence, caspase-11 can respond to cytoplasmic LPS independently of TLR4. In established models of endotoxic shock, both Tlr4-/- and Casp11-/- mice are resistant to lethal challenge with 40?4 mg/kg LPS (three, 18, 19), whereas WT mice succumb in 18 to 48 hours (Fig. 4D). We hypothesized that TLR4 detects extracellular LPS and primes the caspase-11 pathway in vivo. Then, if high concentrations of LPS persist, aberrant localization of LPS within the cytoplasm could trigger caspase-11, resulting within the generation of shock mediators. We sought to separate these two events by priming after which difficult with otherwise sublethal doses of LPS. C57BL/6 mice primed with LPS swiftly succumbed to secondary LPS challenge in 2 hours (Fig. 4D). TLR4 was needed for LPS priming, as LPS primed Tlr4-/- mice survived secondary LPS challenge (Fig. 4E). ToNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptScience. Author manuscript; offered in PMC 2014 September 13.Hagar et al.Pagedetermine regardless of whether alternate priming pathways could substitute for TLR4 in vivowe primed mice with poly(I:C), and observed that each C57BL/6 and Tlr4-/- mice succumbed to secondary LPS challenge (Fig.1086423-62-2 manufacturer 4E).261522-33-2 Price This was concomitant with hypothermia (Fig.PMID:32695810 4F), seizures, peritoneal fluid accumulation, and occasionally intestinal hemorrhage. In contrast, poly(I:C) primed Casp11-/- mice have been much more resistant to secondary LPS challenge (Fig. 4G), demonstrating the consequences of aberrant caspase-11 activation. Collectively, our data indicate that activation of caspase-11 by LPS in vivo can result in rapid onset of endotoxic shock independent of TLR4. Mice challenged together with the canonical NLRC4 agonist flagellin coupled towards the cytosolic translocation domain of anthrax lethal toxin also expertise a rapid onset of shock (20). Within this model, NLRC4-dependent caspase-1 activation triggers lethal eicosanoid production through COX-1 with comparable kinetics to our prime-challe.