Ytosolic pathogens could evade caspase-11 by a similar technique. Indeed, Francisella novicidaa Gram-negative cytosolic bacteria,

Ytosolic pathogens could evade caspase-11 by a similar technique. Indeed, Francisella novicidaa Gram-negative cytosolic bacteria, was not detected by caspase-11 (no signal in Nlrc4-/-Asc-/- BMMs; Fig. 3B). F. novicida lysates containing DNA activated caspase-1; nevertheless, right after DNase digestion the remaining LPS failed to activate caspase-11, which was not restored by temperature-dependent alterations in acyl chain length (12) (Fig. 3C). As with L. monocytogenesco-phagocytosis of F. novicida with exogenous S. minnesota LPS resulted in caspase-11 activation (Fig. 3D). Collectively, these benefits suggest that Francisella species evade caspase-11 by modifying their lipid A. Francisella species have peculiar PKCη Accession tetra-acylated lipid A unlike the hexa-acylated species of enteric bacteria (13). F. novicida initially synthesizes a penta-acylated lipid A structure with two phosphates and after that removes the 4′ phosphate and 3′ acyl chain in reactions that usually do not happen in lpxF mutants (14, 15) (Fig. 3E). Conversion to the penta-acylated structure restored caspase-11 activation, whereas other modifications that maintained the tetra-acylated structures (flmK mutant or 18 growth (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 diverse structural needs. Deacylation of lipid A is actually a common strategy employed by pathogenic bacteria. For 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 research 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). With each other, these information indicate that caspase-11 responds to distinct lipid A structures, and pathogens appear to exploit these structural needs in an effort to evade caspase-11. As well as detection of extracellular/vacuolar LPS by TLR4, our information indicate that an additional sensor of cytoplasmic LPS activates caspase-11. These two pathways intersect, having said that, since TLR4 primes the caspase-11 pathway. Having said that, Tlr4-/- BMMs responded to transfected or CTB-delivered LPS soon after poly(I:C) priming (Fig. 4A ). Consequently, caspase-11 can respond to cytoplasmic LPS independently of TLR4. In N-type calcium channel web established models of endotoxic shock, both Tlr4-/- and Casp11-/- mice are resistant to lethal challenge with 404 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 inside the generation of shock mediators. We sought to separate these two events by priming and then challenging with otherwise sublethal doses of LPS. C57BL/6 mice primed with LPS rapidly succumbed to secondary LPS challenge in 2 hours (Fig. 4D). TLR4 was expected 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 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. 4E). This was concomitant with hypothe.