Abstract
Signal transduction via NFκB and MAP kinase cascades is a universal response initiated upon pathogen recognition by Toll-like receptors (TLRs). How activation of these divergent signaling pathways is integrated to dictate distinct immune responses to diverse pathogens is still incompletely understood. Herein, contrary to current perception, we demonstrate that a signaling pathway defined by the inhibitor of κB kinase β (IKKβ), MAP3 kinase tumor progression locus 2 (Tpl2/MAP3K8), and MAP kinase ERK is differentially activated by TLRs. TLRs 2, 4, and 7 directly activate this inflammatory axis, inducing immediate ERK phosphorylation and early TNFα secretion. In addition to TLR adaptor proteins, IKKβ-Tpl2-ERK activation by TLR4 is regulated by the TLR4 co-receptor CD14 and the tyrosine kinase Syk. Signals from TLRs 3 and 9 do not initiate early activation of IKKβ-Tpl2-ERK pathway but instead induce delayed, NADPH-oxidase-dependent ERK phosphorylation and TNFα secretion via autocrine reactive oxygen species signaling. Unexpectedly, Tpl2 is an essential regulator of ROS production during TLR signaling. Overall, our study reveals distinct mechanisms activating a common inflammatory signaling cascade and delineates differences in MyD88-dependent signaling between endosomal TLRs 7 and 9. These findings further confirm the importance of Tpl2 in innate host defense mechanisms and also enhance our understanding of how the immune system tailors pathogen-specific gene expression patterns.
Highlights
Tumor progression locus 2 (Tpl2) kinase plays an essential, non-redundant role in activating ERK during Tolllike receptors (TLRs) signaling
Induction of Proinflammatory Gene Expression and Secretion of TNF␣ and IL-10 Are Restricted to a Subset of TLRs—To better understand how Tpl2 regulates early innate responses induced by diverse TLR ligands, we measured the expression of various proinflammatory genes including il12b, il6, tnf␣, ccl2, and ccl5 as well as anti-inflammatory il10 in WT and tpl2Ϫ/Ϫ Bone marrowderived macrophages (BMDMs) stimulated with Pam3CSK4 (TLR2), poly I:C (TLR3), LPS (TLR4), R848 (TLR7), and CpG (TLR9) for 1 h
Since early TNF␣ secretion was abolished in WT BMDMs in response to TLR3 and 9 ligands despite transcriptional induction, we investigated whether the Tpl2-ERK TNF-processing pathway was differentially activated by these TLRs
Summary
Tpl kinase plays an essential, non-redundant role in activating ERK during TLR signaling. We demonstrate that the signaling pathway defined by IKK, Tpl, and ERK, which helps to initiate and influence the nature of the innate immune response, is differentially regulated by TLRs. Among the MyD88-coupled TLRs, TLR4 uniquely requires CD14 and the tyrosine kinase Syk for Tpl2-ERK activation. TLRs 3 and 9 do not induce Tpl2-p58 phosphorylation or early ERK activation; instead they induce delayed ERK activation that is dependent upon autocrine signaling by reactive oxygen species (ROS) generated in a Tpl2-dependent manner These findings demonstrate a differential mechanism of ERK activation by diverse TLRs and identify divergent signaling pathways emanating from the MyD88-dependent endosomal TLRs 7 and 9. Our study provides a better understanding of signaling pathways utilized by major TLRs and demonstrate a major role for Tpl in eliciting host protective immune responses, including the generation of antimicrobial reactive oxygen species
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