TLRs activate immune responses by sensing microbial structures such as bacterial LPS, viral RNA, and endogenous "danger" molecules released by damaged host cells. MyD88 is an adapter protein that mediates signal transduction for most TLRs and leads to activation of NF-kappaB and MAPKs and production of proinflammatory cytokines. TLR4-mediated signaling also leads to rapid activation of PI3K, one of a family of kinases involved in regulation of cell growth, apoptosis, and motility. LPS stimulates phosphorylation of Akt, a downstream target of PI3K, in wild-type (WT) mouse macrophages. LPS-induced phosphorylation of Akt serine 473 was blunted in MyD88(-/-) macrophages and was completely TLR4-dependent. MyD88 and p85 were shown previously to co-immunoprecipitate, and a YXXM motif within the Toll-IL-1 resistance (TIR) domain of MyD88 was suggested to be important for this interaction. To test this hypothesis, we compared expressed MyD88 variants with mutations within the YXXM motif or lacking the TIR domain or death domain and measured their capacities to bind PI3K p85, MyD88, and TLR4 by co-immunoprecipitation analyses. The YXXM --> YXXA mutant MyD88 bound more strongly to p85, TLR4, and WT MyD88 than the other variants, yet was significantly less active than WT MyD88, suggesting that sustained interaction of MyD88/PI3K with the TLR4 intracellular "signaling platform" negatively regulates signaling. We propose a hypothetical model in which sustained PI3K activity at the membrane limits the availability of the PI3K substrate, thereby negatively regulating signaling.

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Proteinase-activated receptor 2 (PAR 2 ), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR 2 , often measured by Ca 2؉ release. PAR 2 contributes to inflammation through activation of NF-B-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR 2 in HEK293T cells resulted in concentration-dependent, PAR 2 AP-inducible NF-B reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple G i proteins or sequester intracellular Ca 2؉ . Because previous studies described synergistic PAR 2 -and TLR4-mediated cytokine production, we hypothesized that PAR 2 and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR 2 -induced NF-B activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR 2 in HEK293T cells led to a synergistic increase in AP-induced NF-B signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR 2 and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4 ؊/؊ and PAR 2 ؊/؊ macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.

Endotoxin tolerance reprograms cell responses to LPS by repressing expression of proinflammatory cytokines, while not inhibiting production of anti-inflammatory cytokines and antimicrobial effectors. Molecular mechanisms of induction and maintenance of endotoxin tolerance are incompletely understood, particularly with regard to the impact of endotoxin tolerization on signalosome assembly, activation of adaptor-kinase modules, and expression of negative regulators of TLR signaling in human cells. In this study, we examined LPS-mediated activation of MyD88-dependent and Toll-IL-1R-containing adaptor inducing IFN-beta (TRIF)-dependent pathways emanating from TLR4 and expression of negative regulators of TLR signaling in control and endotoxin-tolerant human monocytes. Endotoxin tolerization suppressed LPS-inducible TLR4-TRIF and TRIF-TANK binding kinase (TBK)1 associations, induction of TBK1 kinase activity, activation of IFN regulatory factor (IRF)-3, and expression of RANTES and IFN-beta. Tolerance-mediated dysregulation of the TLR4-TRIF-TBK1 signaling module was accompanied by increased levels of suppressor of IkappaB kinase-epsilon (SIKE) and sterile alpha and Armadillo motif-containing molecule (SARM). LPS-tolerant cells showed increased expression of negative regulators Toll-interacting protein (Tollip), suppressor of cytokine signaling (SOCS)-1, IL-1R-associated kinase-M, and SHIP-1, which correlated with reduced p38 phosphorylation, IkappaB-alpha degradation, and inhibited expression of TNF-alpha, IL-6, and IL-8. To examine functional consequences of increased expression of Tollip in LPS-tolerized cells, we overexpressed Tollip in 293/TLR4/MD-2 transfectants and observed blunted LPS-inducible activation of NF-kappaB and RANTES, while TNF-alpha responses were not affected. These data demonstrate dysregulation of TLR4-triggered MyD88- and TRIF-dependent signaling pathways and increased expression of negative regulators of TLR signaling in endotoxin-tolerant human monocytes.

Results from experimental models, in vitro studies, and clinical data indicate that granulocyte colony-stimulating factor (G- CSF