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Tumor Progression Locus 2-dependent Oxidative Burst Drives Phosphorylation of Extracellular Signal-regulated Kinase during TLR3 and 9 Signaling*

  • Teneema Kuriakose
    Affiliations
    Department of Infectious Diseases, The University of Georgia, College of Veterinary Medicine, Athens, Georgia 30602
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  • Balázs Rada
    Affiliations
    Department of Infectious Diseases, The University of Georgia, College of Veterinary Medicine, Athens, Georgia 30602
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  • Wendy T. Watford
    Correspondence
    To whom correspondence should be addressed: Dept. of Infectious Diseases, University of Georgia, College of Veterinary Medicine, 501 D.W. Brooks Dr., Athens, GA. Tel.: 706-542-4585; Fax: 706-542-5771
    Affiliations
    Department of Infectious Diseases, The University of Georgia, College of Veterinary Medicine, Athens, Georgia 30602
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  • Author Footnotes
    * This work was supported by startup funds (to W. T. W.) by the Office of the Vice President for Research at the University of Georgia.
Open AccessPublished:November 05, 2014DOI:https://doi.org/10.1074/jbc.M114.587121
      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.

      Results

      TLRs 2, 4, and 7 directly induce IKKβ-Tpl2-ERK signaling; TLRs 3 and 9 activate ERK indirectly via autocrine ROS signaling.

      Conclusion

      Tpl2-dependent ROS generation drives ERK phosphorylation during TLR 3 and 9 signaling.

      Significance

      The different contributions of Tpl2 to TLR signaling pathways influences early host defense mechanisms.

      Introduction

      Toll-like receptors (TLRs)
      The abbreviations used are: TLR
      Toll-like receptor
      BMDMs
      bone marrow-derived macrophages
      BMDCs
      bone marrow-derived dendritic cells
      CHX
      cycloheximide
      DPI
      diphenyleneiodonium
      GSH
      glutathione
      IKKβ
      inhibitor of κB kinase-β
      MCSF
      macrophage colony stimulating factor
      MyD88
      myeloid differentiation primary response gene 88
      NOX
      NADPH oxidase
      PAMPs
      pathogen-associated molecular patterns
      pDCs
      plasmacytoid dendritic cells
      PMs
      peritoneal macrophages
      ROS
      reactive oxygen species
      TACE
      TNFα-converting enzyme
      Tpl2
      tumor progression locus 2
      TRIF
      TIR-domain-containing adapter-inducing interferon-β
      VSV
      vesicular stomatitis virus
      WCL
      whole cell lysates.
      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 (
      • Kawai T.
      • Akira S.
      The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors.
      ). 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 (
      • Kawai T.
      • Akira S.
      The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors.
      ). The magnitude and quality of this early response also regulates the initiation of adaptive responses (
      • Iwasaki A.
      • Medzhitov R.
      Toll-like receptor control of the adaptive immune responses.
      ). Despite extensive research, the precise molecular mechanisms that dictate specific cellular responses to TLRs are still incompletely understood.
      NFκB and MAP kinase pathways are the two major signaling cascades initiated after recognition of specific PAMPs by TLRs (
      • Kawai T.
      • Akira S.
      Signaling to NF-κB by Toll-like receptors.
      ). Engagement of all TLRs activates both of these pathways, and cross-talk between them coordinates the cellular responses to external stimuli (
      • Kawai T.
      • Akira S.
      Signaling to NF-κB by Toll-like receptors.
      ,
      • Banerjee A.
      • Gerondakis S.
      Coordinating TLR-activated signaling pathways in cells of the immune system.
      ). 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) (
      • Cho J.
      • Melnick M.
      • Solidakis G.P.
      • Tsichlis P.N.
      Tpl2 (tumor progression locus 2) phosphorylation at Thr290 is induced by lipopolysaccharide via an Iκ-B Kinase-β-dependent pathway and is required for Tpl2 activation by external signals.
      ,
      • Perkins N.D.
      Integrating cell-signalling pathways with NF-κB and IKK function.
      ).
      Tpl2 is a serine-threonine kinase originally identified as a proto-oncogene and expressed in both hematopoietic and non-hematopoietic compartments (
      • Makris A.
      • Patriotis C.
      • Bear S.E.
      • Tsichlis P.N.
      Genomic organization and expression of Tpl-2 in normal cells and Moloney murine leukemia virus-induced rat T-cell lymphomas: activation by provirus insertion.
      ). Differential translation initiation of Tpl2 mRNA gives rise to 52 and 58 kDa isoforms expressed in equimolar levels in macrophages (
      • Aoki M.
      • Hamada F.
      • Sugimoto T.
      • Sumida S.
      • Akiyama T.
      • Toyoshima K.
      The human cot proto-oncogene encodes two protein serine/threonine kinases with different transforming activities by alternative initiation of translation.
      ). In unstimulated cells, Tpl2 is constitutively associated with NFκB1p105, and this interaction is necessary for Tpl2 stability but blocks Tpl2 kinase activity (
      • Waterfield M.R.
      • Zhang M.
      • Norman L.P.
      • Sun S.C.
      NF-κB1/p105 regulates lipopolysaccharide-stimulated MAP kinase signaling by governing the stability and function of the Tpl2 kinase.
      ). Phosphorylation of p105 by IKKβ leads to Tpl2 release (
      • Beinke S.
      • Robinson M.J.
      • Hugunin M.
      • Ley S.C.
      Lipopolysaccharide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IκB kinase-induced proteolysis of NF-κB1 p105.
      ). IKKβ also mediates phosphorylation of Tpl2 at threonine 290 and serine 400, which regulates Tpl2 kinase activity (
      • Cho J.
      • Melnick M.
      • Solidakis G.P.
      • Tsichlis P.N.
      Tpl2 (tumor progression locus 2) phosphorylation at Thr290 is induced by lipopolysaccharide via an Iκ-B Kinase-β-dependent pathway and is required for Tpl2 activation by external signals.
      ,
      • Beinke S.
      • Robinson M.J.
      • Hugunin M.
      • Ley S.C.
      Lipopolysaccharide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IκB kinase-induced proteolysis of NF-κB1 p105.
      ,
      • Roget K.
      • Ben-Addi A.
      • Mambole-Dema A.
      • Gantke T.
      • Yang H.T.
      • Janzen J.
      • Morrice N.
      • Abbott D.
      • Ley S.C.
      IκB kinase 2 regulates TPL-2 activation of extracellular signal-regulated kinases 1 and 2 by direct phosphorylation of TPL-2 serine 400.
      ,
      • Robinson M.J.
      • Beinke S.
      • Kouroumalis A.
      • Tsichlis P.N.
      • Ley S.C.
      Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide activation of extracellular signal-regulated kinase in macrophages.
      ). Once phosphorylated, Tpl2 transiently transduces signals but is unstable and undergoes rapid proteosomal degradation (
      • Waterfield M.R.
      • Zhang M.
      • Norman L.P.
      • Sun S.C.
      NF-κB1/p105 regulates lipopolysaccharide-stimulated MAP kinase signaling by governing the stability and function of the Tpl2 kinase.
      ,
      • Cho J.
      • Tsichlis P.N.
      Phosphorylation at Thr-290 regulates Tpl2 binding to NF-κB1/p105 and Tpl2 activation and degradation by lipopolysaccharide.
      ). The p58 isoform is preferentially released and degraded in LPS treated macrophages, since only this isoform undergoes IKKβ-mediated Thr290 phosphorylation (
      • Cho J.
      • Melnick M.
      • Solidakis G.P.
      • Tsichlis P.N.
      Tpl2 (tumor progression locus 2) phosphorylation at Thr290 is induced by lipopolysaccharide via an Iκ-B Kinase-β-dependent pathway and is required for Tpl2 activation by external signals.
      ,
      • Beinke S.
      • Robinson M.J.
      • Hugunin M.
      • Ley S.C.
      Lipopolysaccharide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IκB kinase-induced proteolysis of NF-κB1 p105.
      ).
      Early studies on Tpl2 signaling established the non-redundant role of Tpl2 in LPS-mediated activation of ERK1/2 (
      • Dumitru C.D.
      • Ceci J.D.
      • Tsatsanis C.
      • Kontoyiannis D.
      • Stamatakis K.
      • Lin J.H.
      • Patriotis C.
      • Jenkins N.A.
      • Copeland N.G.
      • Kollias G.
      • Tsichlis P.N.
      TNF-α induction by LPS is regulated post-transcriptionally via a Tpl2/ERK-dependent pathway.
      ). 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 (
      • Das S.
      • Cho J.
      • Lambertz I.
      • Kelliher M.A.
      • Eliopoulos A.G.
      • Du K.Y.
      • Tsichlis P.N.
      Tpl2/Cot signals activate ERK, JNK, and NF-κB in a cell-type and stimulus-specific manner.
      ,
      • Mielke L.A.
      • Elkins K.L.
      • Wei L.
      • Starr R.
      • Tsichlis P.N.
      • O'Shea J.J.
      • Watford W.T.
      Tumor Progression Locus 2 (Map3k8) Is Critical for Host Defense against Listeria monocytogenes and IL-1β Production.
      ,
      • Kaiser F.
      • Cook D.
      • Papoutsopoulou S.
      • Rajsbaum R.
      • Wu X.
      • Yang H.T.
      • Grant S.
      • Ricciardi-Castagnoli P.
      • Tsichlis P.N.
      • Ley S.C.
      • O'Garra A.
      TPL-2 negatively regulates interferon-beta production in macrophages and myeloid dendritic cells.
      ,
      • Eliopoulos A.G.
      • Dumitru C.D.
      • Wang C.C.
      • Cho J.
      • Tsichlis P.N.
      Induction of COX-2 by LPS in macrophages is regulated by Tpl2-dependent CREB activation signals.
      ). 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 (
      • Banerjee A.
      • Gugasyan R.
      • McMahon M.
      • Gerondakis S.
      Diverse Toll-like receptors utilize Tpl2 to activate extracellular signal-regulated kinase (ERK) in hemopoietic cells.
      ). Tpl2 kinase activity and Tpl2-dependent ERK phosphorylation were demonstrated in macrophages in response to ligands of TLR2, 3, 4, 7, and 9 (
      • Banerjee A.
      • Gugasyan R.
      • McMahon M.
      • Gerondakis S.
      Diverse Toll-like receptors utilize Tpl2 to activate extracellular signal-regulated kinase (ERK) in hemopoietic cells.
      ). 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 (
      • Yang H.T.
      • Papoutsopoulou S.
      • Belich M.
      • Brender C.
      • Janzen J.
      • Gantke T.
      • Handley M.
      • Ley S.C.
      Coordinate regulation of TPL-2 and NF-κB signaling in macrophages by NF-κB1 p105.
      ). 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.

      DISCUSSION

      Activation of NFκB and MAP kinases are key features of all TLR signaling pathways initiating a proinflammatory response (
      • Kawai T.
      • Akira S.
      Signaling to NF-κB by Toll-like receptors.
      ). 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.
      Figure thumbnail gr7
      FIGURE 7Model of Tpl2-ERK activation during TLR signaling. Stimulation of TLR2, 4 and 7 immediately activates the IKKβ-Tpl2-ERK inflammatory pathway. In addition to a TLR adaptor protein, activation of this pathway by TLR4 requires the TLR4 co-receptor CD14 and the tyrosine kinase Syk. Active IKKβ phosphorylates both NFκB1 and Tpl2-p58 and leads to release of active Tpl2, which in turn induces MEK-dependent ERK activation prior to Tpl2-p58 proteosomal degradation. ERK signaling facilitates processing and secretion of TNFα. TLR3 and 9 do not cause Tpl2-p58 phosphorylation-induced mobility shift, degradation, or early ERK activation. Instead, TLR3 and 9 induce delayed ERK phosphorylation via autocrine signaling by ROS, which is generated in a Tpl2-dependent manner. Consequently, TLR3 and 9 induce delayed secretion of innate TNFα compared with other TLRs.
      Activation of ERK in response to diverse TLR ligands and the critical role of Tpl2 in transducing ERK activation signals are well documented (
      • Banerjee A.
      • Gugasyan R.
      • McMahon M.
      • Gerondakis S.
      Diverse Toll-like receptors utilize Tpl2 to activate extracellular signal-regulated kinase (ERK) in hemopoietic cells.
      ). 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 (
      • Kaiser F.
      • Cook D.
      • Papoutsopoulou S.
      • Rajsbaum R.
      • Wu X.
      • Yang H.T.
      • Grant S.
      • Ricciardi-Castagnoli P.
      • Tsichlis P.N.
      • Ley S.C.
      • O'Garra A.
      TPL-2 negatively regulates interferon-beta production in macrophages and myeloid dendritic cells.
      ). We further linked this reduced ERK activation to lack of Tpl2 phosphorylation and degradation.
      Conflicting reports regarding CpG-induced ERK phosphorylation in different cell types exist (
      • Kaiser F.
      • Cook D.
      • Papoutsopoulou S.
      • Rajsbaum R.
      • Wu X.
      • Yang H.T.
      • Grant S.
      • Ricciardi-Castagnoli P.
      • Tsichlis P.N.
      • Ley S.C.
      • O'Garra A.
      TPL-2 negatively regulates interferon-beta production in macrophages and myeloid dendritic cells.
      ,
      • Yi A.K.
      • Krieg A.M.
      Rapid induction of mitogen-activated protein kinases by immune stimulatory CpG DNA.
      ,
      • Hacker H.
      • Mischak H.
      • Hacker G.
      • Eser S.
      • Prenzel N.
      • Ullrich A.
      • Wagner H.
      Cell type-specific activation of mitogen-activated protein kinases by CpG-DNA controls interleukin-12 release from antigen-presenting 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 (
      • Ray P.D.
      • Huang B.W.
      • Tsuji Y.
      Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling.
      ). For example, ROS-dependent activation of MAP3K5/ASK-1 and MAP kinase p38 was shown to be necessary for TLR4 mediated innate responses (
      • Matsuzawa A.
      • Saegusa K.
      • Noguchi T.
      • Sadamitsu C.
      • Nishitoh H.
      • Nagai S.
      • Koyasu S.
      • Matsumoto K.
      • Takeda K.
      • Ichijo H.
      ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity.
      ). 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 (
      • Hsu H.Y.
      • Wen M.H.
      Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression.
      ). 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 (
      • Robinson M.J.
      • Beinke S.
      • Kouroumalis A.
      • Tsichlis P.N.
      • Ley S.C.
      Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide activation of extracellular signal-regulated kinase in 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 (
      • Das S.
      • Cho J.
      • Lambertz I.
      • Kelliher M.A.
      • Eliopoulos A.G.
      • Du K.Y.
      • Tsichlis P.N.
      Tpl2/Cot signals activate ERK, JNK, and NF-κB in a cell-type and stimulus-specific manner.
      ,
      • Mielke L.A.
      • Elkins K.L.
      • Wei L.
      • Starr R.
      • Tsichlis P.N.
      • O'Shea J.J.
      • Watford W.T.
      Tumor Progression Locus 2 (Map3k8) Is Critical for Host Defense against Listeria monocytogenes and IL-1β Production.
      ). 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 (
      • Mielke L.A.
      • Elkins K.L.
      • Wei L.
      • Starr R.
      • Tsichlis P.N.
      • O'Shea J.J.
      • Watford W.T.
      Tumor Progression Locus 2 (Map3k8) Is Critical for Host Defense against Listeria monocytogenes and IL-1β Production.
      ). Notably, cell type-specific differences between BMDMs and BMDCs in the requirement for CD14 during TLR4 signaling have been reported (
      • Zanoni I.
      • Ostuni R.
      • Marek L.R.
      • Barresi S.
      • Barbalat R.
      • Barton G.M.
      • Granucci F.
      • Kagan J.C.
      CD14 controls the LPS-induced endocytosis of Toll-like receptor 4.
      ). Unlike macrophages and BMDCs, both isoforms of Tpl2 were completely degraded in pDCs early after stimulation, further supporting the uniqueness of signaling pathways in pDCs (
      • Kaiser F.
      • Cook D.
      • Papoutsopoulou S.
      • Rajsbaum R.
      • Wu X.
      • Yang H.T.
      • Grant S.
      • Ricciardi-Castagnoli P.
      • Tsichlis P.N.
      • Ley S.C.
      • O'Garra A.
      TPL-2 negatively regulates interferon-beta production in macrophages and myeloid dendritic cells.
      ).
      CD14 is a GPI-anchored protein without intrinsic signaling potential (
      • Wright S.D.
      • Ramos R.A.
      • Tobias P.S.
      • Ulevitch R.J.
      • Mathison J.C.
      CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.
      ), however CD14 functions are necessary for Myd88-independent signaling by TLR4 (
      • Jiang Z.
      • Georgel P.
      • Du X.
      • Shamel L.
      • Sovath S.
      • Mudd S.
      • Huber M.
      • Kalis C.
      • Keck S.
      • Galanos C.
      • Freudenberg M.
      • Beutler B.
      CD14 is required for MyD88-independent LPS signaling.
      ). In their elegant study demonstrating the role of CD14 in TLR4 endocytosis, Zanoni et al. commented that all LPS responses actually initiate with CD14 (
      • Zanoni I.
      • Ostuni R.
      • Marek L.R.
      • Barresi S.
      • Barbalat R.
      • Barton G.M.
      • Granucci F.
      • Kagan J.C.
      CD14 controls the LPS-induced endocytosis of Toll-like receptor 4.
      ). 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 (
      • Zanoni I.
      • Ostuni R.
      • Marek L.R.
      • Barresi S.
      • Barbalat R.
      • Barton G.M.
      • Granucci F.
      • Kagan J.C.
      CD14 controls the LPS-induced endocytosis of Toll-like receptor 4.
      ). Interestingly, a recent study reported Tpl2-mediated ERK activation during FcγR signaling (
      • Kyrmizi I.
      • Ioannou M.
      • Hatziapostolou M.
      • Tsichlis P.N.
      • Boumpas D.T.
      • Tassiulas I.
      Tpl2 kinase regulates FcγR signaling and immune thrombocytopenia in mice.
      ). Thus, regulation of IKKβ-Tpl2-ERK signaling by CD14 and Syk supports the existence of this proposed inflammatory pathway.
      Although differences in biological responses upon TLR7 and 9 stimulation have been reported (
      • Christensen S.R.
      • Shupe J.
      • Nickerson K.
      • Kashgarian M.
      • Flavell R.A.
      • Shlomchik M.J.
      Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of lupus.
      ,
      • Fukui R.
      • Saitoh S.
      • Kanno A.
      • Onji M.
      • Shibata T.
      • Ito A.
      • Onji M.
      • Matsumoto M.
      • Akira S.
      • Yoshida N.
      • Miyake K.
      Unc93B1 restricts systemic lethal inflammation by orchestrating Toll-like receptor 7 and 9 trafficking.
      ), 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 (
      • Kawai T.
      • Akira S.
      TLR signaling.
      ). However, a recent study did report differences in UNC93B1-mediated trafficking of TLR7 and 9 (
      • Lee B.L.
      • Moon J.E.
      • Shu J.H.
      • Yuan L.
      • Newman Z.R.
      • Schekman R.
      • Barton G.M.
      UNC93B1 mediates differential trafficking of endosomal TLRs.
      ). 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 (
      • Qi R.
      • Hoose S.
      • Schreiter J.
      • Sawant K.V.
      • Lamb R.
      • Ranjith-Kumar C.T.
      • Mills J.
      • San Mateo L.
      • Jordan J.L.
      • Kao C.C.
      Secretion of the human Toll-like receptor 3 ectodomain is affected by single nucleotide polymorphisms and regulated by Unc93b1.
      ,
      • Pohar J.
      • Pirher N.
      • Benčina M.
      • Manček-Keber M.
      • Jerala R.
      The role of UNC93B1 protein in surface localization of TLR3 receptor and in cell priming to nucleic acid agonists.
      ), 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 (
      • Mielke L.A.
      • Elkins K.L.
      • Wei L.
      • Starr R.
      • Tsichlis P.N.
      • O'Shea J.J.
      • Watford W.T.
      Tumor Progression Locus 2 (Map3k8) Is Critical for Host Defense against Listeria monocytogenes and IL-1β Production.
      ,
      • McNab F.W.
      • Ewbank J.
      • Rajsbaum R.
      • Stavropoulos E.
      • Martirosyan A.
      • Redford P.S.
      • Wu X.
      • Graham C.M.
      • Saraiva M.
      • Tsichlis P.
      • Chaussabel D.
      • Ley S.C.
      • O'Garra A.
      TPL-2-ERK1/2 signaling promotes host resistance against intracellular bacterial infection by negative regulation of type I IFN production.
      ). Defective ROS production in tpl2−/− mice may contribute to decreased bacterial clearance, increased susceptibility to infection or altered redox-sensitive signaling (
      • Ray P.D.
      • Huang B.W.
      • Tsuji Y.
      Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling.
      ,
      • Corcoran A.
      • Cotter T.G.
      Redox regulation of protein kinases.
      ). In addition to its bactericidal functions, ROS is also required for RIG-I-mediated antiviral responses (
      • Soucy-Faulkner A.
      • Mukawera E.
      • Fink K.
      • Martel A.
      • Jouan L.
      • Nzengue Y.
      • Lamarre D.
      • Vande Velde C.
      • Grandvaux N.
      Requirement of NOX2 and reactive oxygen species for efficient RIG-I-mediated antiviral response through regulation of MAVS expression.
      ). 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 (
      • Schmid S.
      • Sachs D.
      • Tenoever B.R.
      Mitogen-activated Protein Kinase-mediated Licensing of Interferon Regulatory Factor 3/7 Reinforces the Cell Response to Virus.
      ). This is especially interesting because, in addition to TLR7, VSV is known to signal via the CD14-TLR4 axis, and increased replication of VSV was also reported in macrophages from CD14 mutant mice (
      • Jiang Z.
      • Georgel P.
      • Du X.
      • Shamel L.
      • Sovath S.
      • Mudd S.
      • Huber M.
      • Kalis C.
      • Keck S.
      • Galanos C.
      • Freudenberg M.
      • Beutler B.
      CD14 is required for MyD88-independent LPS signaling.
      ). 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.

      Acknowledgments

      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.

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