Species-specific endotoxin stimulus determines toll-like receptor 4- And caspase 11-mediated pathway activation characteristics

The innate immune system is the body's first line of defense against pathogens and its protection against infectious diseases. On the surface of host myeloid cells, Toll-like receptor 4 (TLR4) senses lipopolysaccharide (LPS), the major outer membrane component of Gram-negative bacteria. Intracellularly, LPS is recognized by caspase 11 through the noncanonical inflammasome to induce pyroptosis-an inflammatory form of lytic cell death. While TLR4-mediated signaling perturbations result in secretion of cytokines and chemokines that help clear infection and facilitate adaptive immunity, caspase 11-mediated pyroptosis leads to the release of damage- associated molecular patterns and inflammatory mediators. Although the core signaling events and many associated proteins in the TLR4 signaling pathway are known, the complex signaling events and protein networks within the noncanonical inflammasome pathway remain obscure. Moreover, there is mounting evidence for pathogen-specific innate immune tuning. We characterized the major LPS structures from two different pathogens, modeled their binding to the surface receptors, systematically examined macrophage inflammatory responses to these LPS molecules, and surveyed the temporal differences in global protein secretion resulting from TLR4 and caspase 11 activation in macrophages using mass spectrometry (MS)-based quantitative proteomics. This integrated strategy, spanning functional activity assays, top-down structural elucidation of endotoxins, and secretome analysis of stimulated macrophages, allowed us to identify crucial differences in TLR4- and caspase 11- mediated protein secretion in response to two Gram-negative bacterial endotoxins.