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We previously showed that monophosphoryl lipid A (MLA) activates TLR4 in dendritic cells (DCs) in a Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF)-biased manner: MLA produced from Salmonella minnesota Re595 induced signaling events and expression of gene products that were primarily TRIF dependent, whereas MyD88-dependent signaling was impaired. Moreover, when tested in TRIF-intact/MyD88-deficient DCs, synthetic MLA of the Escherichia coli chemotype (sMLA) showed the same activity as its diphosphoryl, inflammatory counterpart (synthetic diphosphoryl lipid A), indicating that TRIF-mediated signaling is fully induced by sMLA. Unexpectedly, we found that the transcript level of one proinflammatory cytokine was increased in sMLA-treated cells by MyD88 deficiency to the higher level induced by synthetic diphosphoryl lipid A, which suggested MyD88 may paradoxically help restrain proinflammatory signaling by TRIF-biased sMLA. In this article, we demonstrate that sMLA induces MyD88 recruitment to TLR4 and activates the anti-inflammatory lipid phosphatase SHIP1 in an MyD88-dependent manner. At the same time, MyD88-dependent signaling activity at the level of IL-1R-associated kinase 1 is markedly reduced. Increased SHIP1 activity is associated with reductions in sMLA-induced I kappa B kinase alpha/beta and IFN regulatory factor 3 activation and with restrained expression of their downstream targets, endothelin-1 and IFN-beta, respectively. Results of this study identify a pattern that is desirable in the context of vaccine adjuvant design: TRIF-biased sMLA can stimulate partial MyD88 activity, with MyD88-dependent SHIP1 helping to reduce proinflammatory signaling in DCs. The Journal of Immunology, 2011, 186: 3858-3865.

TLR 4 stimulation of innate immune cells induces a MyD88-independent signaling pathway that leads to the production of IFN-β. In this study, we demonstrate glycogen synthase kinase 3-β (GSK3-β) plays a fundamental role in this process. Suppression of GSK3-β activity by either pharmacological inhibition, small interfering RNA-mediated gene silencing, or ectopic expression of a kinase-dead GSK3-β mutant enhanced IFN-β production by TLR4-stimulated macrophages. Conversely, ectopic expression of a constitutively active GSK3-β mutant severely attenuated IFN-β production. GSK3-β was found to negatively control the cellular levels of the transcription factor c-Jun and its nuclear association with ATF-2. Small interfering RNA-mediated knockdown of c-Jun levels abrogated the ability of GSK3-β inhibition to augment IFN-β, demonstrating that the ability of GSK3 to control IFN-β production was due to its ability to regulate c-Jun levels. The ability of GSK3 inhibition to control IFN-β production was confirmed in vivo as mice treated with a GSK3 inhibitor exhibited enhanced systemic levels of IFN-β upon LPS challenge. These findings identify a novel regulatory pathway controlling IFN-β production by TLR4-stimulated innate immune cells.