Endotoxin Tolerance Dysregulates MyD88‐Dependent and TRIF‐Dependent Signaling Pathways and Increases Expression of Negative Regulators of TLR Signaling

Abstract

Endotoxin tolerance represses expression of proinflammatory cytokines and chemokines while not affecting anti‐inflammatory cytokines and anti‐microbial effectors (reprogramming). Septic shock patients often develop increased susceptibility to infections and decreased responsiveness to LPS, which closely resembles the endotoxin‐tolerant phenotype. Little is known regarding molecular mechanisms of induction and maintenance of endotoxin tolerance. In this study, we examined LPS‐mediated activation of MyD88‐dependent and TRIF‐dependent pathways, as well as expression levels of negative regulators of TLR signaling in control and endotoxin‐tolerant human monocytes. Tolerization resulted in decreased LPS‐inducible dimerization, phosphorylation and nuclear translocation of interferon regulatory factor (IRF)‐3. Consistent with the importance of IRF‐3 for activation of MyD88‐independent, TRIF‐dependent genes, real‐time PCR analyses of endotoxin‐tolerant cells revealed suppressed expression of RANTES and IFN‐β mRNA in response to LPS. Reduced LPS‐inducible IRAK‐1 and p38 phosphorylation, IκB‐α degradation, and nuclear translocation of IRF‐5 noted in LPS‐tolerant cells were accompanied by inhibited TNF‐α and IL‐8 mRNA expression. Real‐time PCR and immunoblotting analyses of negative regulators of TLR signaling documented increased expression levels of Tollip mRNA and protein, as well as accumulation of SOCS‐1, SHIP‐1, IRAK‐M, and ST‐2 mRNA in endotoxin‐tolerant monocytes. Taken together, these data demonstrate TLR4‐triggered MyD88‐dependent, TRAM/TRIF‐dependent intermediates, transcription factors, cytokines, and regulators of TLR signaling that are subject to modulation by endotoxin tolerance.This work was supported by NIH grant AI‐059524 (to A.E.M.).