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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.Tpl2 kinase plays an essential, non-redundant role in activating ERK during TLR signaling.
TLRs 2, 4, and 7 directly induce IKKβ-Tpl2-ERK signaling; TLRs 3 and 9 activate ERK indirectly via autocrine ROS signaling.
Tpl2-dependent ROS generation drives ERK phosphorylation during TLR 3 and 9 signaling.
The different contributions of Tpl2 to TLR signaling pathways influences early host defense mechanisms.
are a major class of pattern recognition receptors that specifically detect conserved pathogen-associated molecular patterns (PAMPs) and alarm the host of an infection. TLRs are expressed either on the cell surface or within specific intracellular compartments. Cell surface TLRs (TLR1, 2, 4, 5, and 6) detect outer membrane components of microbes, whereas endosomal TLRs (TLR3, 7, 8, and 9) sense microbial nucleic acids (
). Signals emanating from TLRs activate various intracellular signaling cascades including NFκB, mitogen-activated protein (MAP) kinases, and interferon regulatory factors that collectively induce the secretion of host protective proinflammatory cytokines and interferons (
). One of the key regulatory molecules known to coordinate the activation of both NFκB and MAP kinase pathways is the inhibitor of κB kinase β (IKKβ). IKKβ is activated in response to proinflammatory stimuli, including TLRs and cytokines, and it regulates activation of NFκB and MAP kinases by phosphorylating IκBα, NFκB1p105, and the MAP3 kinase, Tumor progression locus 2 (Tpl2) (
). Tpl2−/− mice are resistant to endotoxin-induced shock due to defective ERK-dependent TNFα secretion. Further studies demonstrated a cell type- and stimulus-specific role for Tpl2 in transducing signals leading to the production of a variety of immune mediators, including IL-1β, IL-10, IL-12, and COX-2 (
). Because of its role in regulating expression, secretion and signaling of proinflammatory cytokines like TNFα and IL-1β, Tpl2 is considered an attractive target for immunotherapy of inflammatory conditions. Several studies have examined Tpl2 regulation of signal transduction and cellular responses to diverse TLR ligands (
). Moreover, ERK phosphorylation in response to LPS, TNFα, CpG, Pam3CSK, poly I:C, flagellin, and R848 were blocked in nfkb1SSAA macrophages which express a p105 mutant that cannot be phosphorylated by IKKβ (
). From these studies, it has been concluded that all TLRs similarly activate the Tpl2-ERK signaling pathway.
To better understand the molecular mechanisms utilized by different TLRs to distinguish their cellular responses, we examined the induction of proinflammatory genes and signal transduction events by diverse TLR ligands, focusing on Tpl2 signaling. Contrary to prevailing thought, we demonstrate that the signaling pathway defined by IKKβ, Tpl2, 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 also identify divergent signaling pathways emanating from the MyD88-dependent endosomal TLRs 7 and 9. Overall, our study provides a better understanding of signaling pathways utilized by major TLRs and also demonstrate a major role for Tpl2 in eliciting host protective immune responses, including the generation of antimicrobial reactive oxygen species.
Activation of NFκB and MAP kinases are key features of all TLR signaling pathways initiating a proinflammatory response (
). In this study, we made several important discoveries regarding differential mechanisms activating a common inflammatory signaling cascade during TLR signaling as summarized in Fig. 7. First, we demonstrated an indirect, delayed mechanism of ERK activation by a subset of TLRs that limits early innate responses, including early TNFα and IL-10 secretion, to TLR3 and TLR9 ligands. This pathway is distinguished by the lack of Tpl2-p58 phosphorylation and degradation despite evidence of NFκB activation, including IκBα phosphorylation and degradation, and despite Tpl2-dependent biological responses to these ligands. Second, we delineated an inflammatory pathway controlled by CD14 and the tyrosine kinase Syk in the activation of the IKKβ-Tpl2-ERK axis during TLR4 signaling. Third, we identified a ROS-dependent autocrine loop responsible for the delayed, indirect ERK phosphorylation during TLR3 and 9 signaling. Finally, we demonstrated the critical role of Tpl2 in ROS generation during TLR signaling.
Activation of ERK in response to diverse TLR ligands and the critical role of Tpl2 in transducing ERK activation signals are well documented (
). Our results are in agreement with previous studies demonstrating that stimulation of all major TLRs induce Tpl2-dependent ERK activation in BMDMs. Consistent with the data reported by Kaiser et al., we observed delayed ERK phosphorylation in both CpG- and poly I:C-treated cells (
). Our findings clarify a controversy and demonstrate that TLR3 and 9 signaling do not directly couple to ERK activation. Instead, the observed ERK phosphorylation by TLR3 and 9 is due to NADPH oxidase-dependent autocrine ROS signaling. The significance of ROS as second messengers during innate immune responses and in regulating the production of various inflammatory mediators is well appreciated (
). In addition to ROS-mediated ERK phosphorylation during TLR3 and 9 signaling, our study identified Tpl2 as a critical regulator of ROS production during TLR signaling. The requirement of Tpl2 in ROS production may contribute in part to the defective induction of IL-1β in tpl2−/− macrophages, since ROS is important for IL-1β expression in response to LPS (
). The signaling events linking Tpl2 to NOX enzymes are currently unknown. Therefore, further studies are needed to determine the precise mechanisms by which Tpl2 regulates ROS production.
Tpl2-p58 mobility shift and degradation, while excellent predictors of Tpl2-dependent MEK/ERK activation, are poor indicators of overall Tpl2 biological activity. For example, Tpl2 is required for TNF processing and secretion in response to both poly I:C and CpG (Fig. 1C), both of which fail to induce Tpl2-p58 phosphorylation-induced mobility shift, degradation or early ERK activation. Tpl2 is also required for normal IFNβ production in response to poly I:C (Fig. 3D). Similarly, IL-1β also utilizes Tpl2 to transduce signals, but fails to induce Tpl2-p58 degradation (data not shown). These findings raise the possibility of phosphorylation-independent functions for Tpl2-p58 or Tpl2-p52 isoforms in cell signaling. Thr290 phosphorylation occurs only on the Tpl2 p58 isoform, whereas both p52 and p58 isoforms undergo phosphorylation on Ser400 in LPS-treated macrophages (
). Despite the fact that no functional differences between Tpl2-p58 and p52 have been reported so far, it is tempting to speculate that Tpl2-p52 transduces signals from receptors that do not induce IKKβ-mediated Thr290 phosphorylation and p58 degradation, such as poly I:C, CpG, and IL-1β.
Cell type-specific requirements for Tpl2 in transducing TLR signals have been demonstrated previously (
). However cell type-specific differences in Tpl2 phosphorylation is a novel finding. While LPS induced Tpl2-p58 Thr290 phosphorylation and mobility shift in macrophages, a decrease in Tpl2-p58 mobility was not observed in BMDCs. This difference in Tpl2 activation could account for the partial requirement of Tpl2 for TNFα secretion in BMDCs compared with BMDMs (
). Our data confirming the necessity of CD14 in IKKβ-Tpl2-ERK signaling support their placement of CD14 as the “king of all LPS responses” although either one of the TLR adaptor proteins is also necessary for this response. An inflammatory endocytosis pathway regulated by Syk was proposed for endocytosed receptors like TLR4, Dectin-1 and FcγRI (
), the molecular basis for these differences has remained enigmatic. Herein, we demonstrate the direct coupling of TLR7, but not TLR9, to the IKKβ-Tpl2-ERK signaling pathway. To our knowledge, differences between TLR7 and 9 signaling per se have not been demonstrated. This finding was surprising, as both of these endosomal TLRs transduce signals via the same MyD88 adaptor (
). Identification of discrete trafficking pathways suggests the possibility of distinct signaling compartments for TLR7 and 9 that may correlate with their activation of distinct signaling cascades and cellular responses. Since cell surface expression of TLR3 has been reported (
), a trafficking route similar to that of TLR9 was proposed for this receptor. Hence, the differential activation of the IKKβ-Tpl2-ERK pathway could correlate with the involvement of distinct signaling compartments for these endosomal TLRs.
In addition to the new insights into TLR signaling pathways, our findings have many implications regarding the role of Tpl2 in innate immune responses during infections. We and others have previously demonstrated the critical role of Tpl2 in host defense against intracellular bacteria like Listeria monocytogenes and Mycobacterium tuberculosis (
). Moreover, direct and immediate activation of Tpl2 and ERK during TLR7 signaling suggests that Tpl2 is likely to play a preferential role in host defense against RNA viruses that trigger TLR7. In this regard, a recent study reported increased replication of vesicular stomatitis virus (VSV) in Tpl2-deficient mouse embryonic fibroblasts (
). These findings suggest a role for Tpl2 in controlling virus replication and warrant further studies to assess the contribution of Tpl2 in antiviral host responses. Overall, our study provides a better understanding about key events that distinguish signal transduction by diverse TLRs and further underscores the significance of Tpl2 in eliciting host protective immune responses against diverse pathogens.
We thank Rebecca Kirkland for excellent technical assistance. We also thank Julie Nelson and the Center for Tropical and Emerging Global Diseases Flow Cytometry Core Facility for cell sorting and Dr. Barbara Reaves and the CVM Cytometry Core Facility for confocal microscopy. We would also like to acknowledge UGA's Veterinary Medicine Central Animal Facility for animal care.
The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors.