EGFR kinase activity is required for TLR4 signaling and the septic shock response

Mammalian Toll-like receptors (TLR) recognize microbial products and elicit transient immune responses that protect the infected host from disease. TLR4—which signals from both plasma and endosomal membranes—is activated by bacterial lipopolysaccharides (LPS) and induces many cytokine genes, the prolonged expression of which causes septic shock in mice. We report here that the expression of some TLR4-induced genes in myeloid cells requires the protein kinase activity of the epidermal growth factor receptor (EGFR). EGFR inhibition affects TLR4-induced responses differently depending on the target gene. The induction of interferon-β (IFN-β) and IFN-inducible genes is strongly inhibited, whereas TNF-α induction is enhanced. Inhibition is specific to the IFN-regulatory factor (IRF)-driven genes because EGFR is required for IRF activation downstream of TLR—as is IRF co-activator β-catenin—through the PI3 kinase/AKT pathway. Administration of an EGFR inhibitor to mice protects them from LPS-induced septic shock and death by selectively blocking the IFN branch of TLR4 signaling. These results demonstrate a selective regulation of TLR4 signaling by EGFR and highlight the potential use of EGFR inhibitors to treat septic shock syndrome.     

A phosphorylation site in the Toll-like receptor 5 TIR domain is required for inflammatory signalling in response to flagellin

Flagellin, the major structural subunit of bacterial flagella, potently induces inflammatory responses in mammalian cells by activating Toll-like receptor (TLR) 5. Like other TLRs, TLR5 recruits signalling molecules to its intracellular TIR domain, leading to inflammatory responses. Phosphatidylinositol 3-kinase (PI3K) has been reported to play a role in early TLR signalling. We identified a putative binding site for PI3K at tyrosine 798 in the TLR5 TIR domain, at a site analogous to the PI3K recruitment domain in the interleukin-1 receptor. Mutation of this residue did not affect homodimerization, but prevented inflammatory responses to flagellin. While we did not detect direct interaction of PI3K with TLR5, we demonstrated by mass spectrometry that Y798 is phosphorylated in flagellin-treated HEK 293T cells. Together, these results suggest that phosphorylation of Y798 in TLR5 is required for signalling, but not for TLR5 dimerization.     

TLR5 risk-associated haplotype for canine inflammatory bowel disease confers hyper-responsiveness to flagellin

Single nucleotide polymorphisms (SNP) in the TLR5 gene have been associated with human inflammatory bowel disease (IBD) and animal models of this disease. We recently demonstrated a significant association between three non-synonymous SNPs in the canine TLR5 gene and IBD in German shepherd dogs (GSDs). However, so far, no direct link between these SNPs and a disturbance in TLR5 function was shown. In the present study, we determined the functional significance of the canine TLR5 SNPs by transfecting the identified risk-protective and risk-associated haplotype into human embryonic kidney cells (HEK) and assessed nuclear factor-kappa B (NF-κB) activation and CXCL8 production after stimulation. In addition, a whole blood assay for TLR5 activation was developed using blood derived from carrier dogs of either haplotype. There was a significant increase in NF-kB activity when cells transfected with the risk-associated TLR5 haplotype were stimulated with flagellin compared to the cells expressing the risk-protective TLR5 haplotype. This difference in NFkB activation correlated with CXCL8 expression in the supernatant measured by ELISA. Furthermore, whole blood taken from carrier dogs of the risk-associated TLR5 haplotype produced significantly more TNF after stimulation with flagellin compared to that taken from carriers of the risk-protective haplotype. Thus, we show for the first time a direct functional impact of the canine IBD risk-associated TLR5 haplotype, which results in hyper-responsiveness to flagellin compared to the IBD risk-protective TLR5 haplotype. Our data potentially suggest that similarly to human IBD and experimental models, TLR5 may also play a role in canine IBD. Blocking the hyper-responsive receptor found in susceptible dogs with IBD may alleviate the inappropriate inflammation seen in this disease.     

TLR signaling is required for Salmonella typhimurium virulence

Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and can drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella-containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection.     

Protein kinase D1: a new component in TLR9 signaling

Protein kinase D1 (PKD1) is expressed ubiquitously and regulates diverse cellular processes such as oxidative stress, gene expression, cell survival, and vesicle trafficking. However, the presence and function of PKD1 in monocytic cells are currently unknown. In this study, we provide evidence that PKD1 is involved in TLR9 signaling in macrophages. Class B-type CpG DNA (CpG-B DNA) induced activation of PKD1 via a pathway that is dependent on endosomal pH, TLR9, MyD88, and IL-1R-associated kinase 1 in macrophages. Upon CpG-B DNA stimulation, PKD1 interacted with the TLR9/MyD88/IL-1R-associated kinase/TNFR-associated factor 6 complex. Knockdown of PKD1 revealed that PKD1 is required for activation of NF-kappaB and MAPKs, and subsequent expression of cytokines in response to CpG-B DNA. Our findings identify PKD1 as a key signaling modulator in TLR9-mediated macrophage activation.     

Protein kinase Ctheta is required for autophagy in response to stress in the endoplasmic reticulum

Autophagy is an evolutionally conserved process for the bulk degradation of cytoplasmic proteins and organelles. Recent observations indicate that autophagy is induced in response to cellular insults that result in the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). However, the signaling mechanisms that activate autophagy under these conditions are not understood. Here, we report that ER stress-induced autophagy requires the activation of protein kinase C (PKC), a member of the novel-type PKC family. Induction of ER stress by treatment with either thapsigargin or tunicamycin activated autophagy in immortalized hepatocytes as monitored by the conversion LC3-I to LC3-II, clustering of LC3 into dot-like cytoplasmic structures, and electron microscopic detection of autophagosomes. Pharmacological inhibition of PKC or small interfering RNA-mediated knockdown of PKC prevented the autophagic response to ER stress. Treatment with ER stressors induced PKC phosphorylation within the activation loop and localization of phospho-PKC to LC3-containing dot structures in the cytoplasm. However, signaling through the known unfolded protein response sensors was not required for PKC activation. PKC activation and stress-induced autophagy were blocked by chelation of intracellular Ca(2+) with BAPTA-AM. PKC was not activated or required for autophagy in response to amino acid starvation. These observations indicate that Ca(2+)-dependent PKC activation is specifically required for autophagy in response to ER stress but not in response to amino acid starvation.     

PB1 domain-dependent signaling complex is required for extracellular signal-regulated kinase 5 activation

MEKK2, MEK5, and extracellular signal-regulated kinase 5 (ERK5) are members of a three-kinase cascade for the activation of ERK5. MEK5 is the only MAP2K to express a PB1 domain, and we have shown that it heterodimerizes with the PB1 domain of MEKK2. Here we demonstrate the MEK5 PB1 domain is a scaffold that also binds ERK5, functionally forming a MEKK2-MEK5-ERK5 complex. Reconstitution assays and CFP/YFP imaging (fluorescence resonance energy transfer [FRET]) measuring YFP-MEKK2/CFP-MEK5 and CFP-MEK5/YFP-ERK5 interactions define distinct MEK5 PB1 domain binding sites for MEKK2 and ERK5, with a C-terminal extension of the PB1 domain contributing to ERK5 binding. Stimulus-dependent CFP/YFP FRET in combination with mutational analysis was used to define MEK5 PB1 domain residues critical for the interaction of MEKK2/MEK5 and MEK5/ERK5 required for activation of the ERK5 pathway in living cells. Fusion of the MEK5 PB1 domain to the N terminus of MEK1 confers ERK5 regulation by a MAP2K normally regulating only ERK1/2. The MEK5 PB1 domain confers stringent MAP3K regulation of ERK5 relative to more promiscuous MAP3K control of ERK1/2, JNK, and p38.     

Dominant-negative TLR5 polymorphism reduces adaptive immune response to flagellin and negatively associates with Crohn's disease

Crohn's disease (CD) is associated with elevated adaptive immunity to commensal microbes, with flagellin being a dominant antigen. In light of heightened awareness of the importance of innate immunity in regulating adaptive immunity and ambiguity as to the role of CD-associated immune responses in CD pathophysiology, we sought to determine whether natural acquisition of immune responses to flagellin were regulated by the innate immune flagellin receptor toll-like receptor 5 (TLR5) and determine whether persons carrying a recently defined common dominant-negative TLR5 polymorphism (TLR5-stop) might be protected from developing CD. Carriage rates of a recently defined dominant-negative TLR5 polymorphism (TLR5-stop) and levels of serum immunoreactivity to bacterial products were measured in inflammatory bowel disease patients, first-degree relatives, and unrelated controls. We observed that, in healthy subjects, persons carrying TLR5-stop had significantly lower levels of flagellin-specific IgG and IgA but had similar levels of total and LPS-specific Ig. Moreover, we observed that, among Jewish subjects, the carriage rate of TLR5-stop (in heterozygous state) was significantly less in CD patients, but not ulcerative colitis (UC) patients, compared with unaffected relatives and unrelated controls (5.4, 0.9, 6.0, and 6.5% for unaffected relatives, CD, UC, and unrelated Jewish controls, respectively, n = 296, 215, 185, and 416, respectively; P = 0.037 by likelihood calculation for CD vs. controls), indicating that TLR5-stop can protect persons of Jewish ethnicity against CD. We did not observe a significant association of TLR5-stop with CD in a non-Jewish cohort (11.1, 10.4, and 11.7% for unaffected relatives, CD, and UC, respectively; n = 841, 543, and 300 for unaffected relatives, respectively). These results demonstrate that natural acquisition of immune responses to flagellin are regulated by TLR5 and suggest that immune responses to flagellin are not merely associated with CD but rather promote the pathogenic response.     

TLR signaling mediated by MyD88 is required for a protective innate immune response by neutrophils to Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes breach of the colonic epithelial barrier, a vigorous Th1 inflammatory response, and colitis. Ultimately, an adaptive immune response leads to clearance of the bacteria. Whereas much is known about the adaptive response to C. rodentium, the role of the innate immune response remains unclear. In this study, we demonstrate for the first time that the TLR adaptor MyD88 is essential for survival and optimal immunity following infection. MyD88(-/-) mice suffer from bacteremia, gangrenous mucosal necrosis, severe colitis, and death following infection. Although an adaptive response occurs, MyD88-dependent signaling is necessary for efficient clearance of the pathogen. Based on reciprocal bone marrow transplants in conjunction with assessment of intestinal mucosal pathology, repair, and cytokine production, our findings suggest a model in which TLR signaling in hemopoietic and nonhemopoietic cells mediate three distinct processes: 1) induction of an epithelial repair response that maintains the protective barrier and limits access of bacteria to the lamina propria; 2) production of KC or other chemokines that attract neutrophils and thus facilitate killing of bacteria; and 3) efficient activation of an adaptive response that facilitates Ab-mediated clearance of the infection. Taken together, these experiments provide evidence for a protective role of innate immune signaling in infections caused by attaching and effacing pathogens.     

A plasma membrane syntaxin is phosphorylated in response to the bacterial elicitor flagellin

In vivo pulse labeling of suspension-cultured Arabidopsis cells with [32P]orthophosphate allows a systematic analysis of dynamic changes in protein phosphorylation. Here, we use this technique to investigate signal transduction events at the plant plasma membrane triggered upon perception of microbial elicitors of defense responses, using as a model elicitor flg22, a peptide corresponding to the most conserved domain of bacterial flagellin. We demonstrate that two-dimensional gel electrophoresis in conjunction with mass spectrometry is a suitable tool for the identification of intrinsic membrane proteins, and we show that among them a syntaxin, AtSyp122, is phosphorylated rapidly in response to flg22. Although incorporation of radioactive phosphate into the protein only occurs significantly after elicitation, immunoblot analysis after two-dimensional gel separation indicates that the protein is also phosphorylated prior to elicitation. These results indicate that flg22 elicits either an increase in the rate of turnover of phosphate or an additional de novo phosphorylation event. In vitro, phosphorylation of AtSyp122 is calcium-dependent. In vitro phosphorylated peptides separated by two-dimensional thin layer chromatography comigrate with two of the three in vivo phosphopeptides, indicating that this calcium-dependent phosphorylation is biologically relevant. These results indicate a regulatory link between elicitor-induced calcium fluxes and the rapid phosphorylation of a syntaxin. Because syntaxins are known to be important in membrane fusion and exocytosis, we hypothesize that one of the functions of the calcium signal is to stimulate exocytosis of defense-related proteins and compounds.     

Identification of conserved domains in Salmonella muenchen flagellin that are essential for its ability to activate TLR5 and to induce an inflammatory response in vitro

The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-kappaB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95-108 (motif N) in the N terminus and 441-449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface.     

Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression

Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-kappaB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.     

Protein kinase C-theta is required for efficient positive selection

Protein kinase C-theta (PKCtheta) is critical for TCR-initiated signaling in mature T cells, but initial reports found no requirement for PKCtheta in thymocyte development. Thymocytes and peripheral T cells utilize many of the same signaling components and, given the significant role of PKCtheta in peripheral T cells, it was surprising that it was not involved at all in TCR signaling in thymocytes. We decided to re-evaluate the role of PKCtheta in thymocyte development using the well-characterized class II-restricted n3.L2 TCR-transgenic TCR model. Analysis of n3.L2 PKCtheta(-/-) mice revealed a defect in thymocyte-positive selection, resulting in a 50% reduction in the generation of n3.L2 CD4 single-positive thymocytes and n3.L2 CD4 mature T cells. Competition between n3.L2 WT and n3.L2 PKCtheta(-/-) thymocytes in bone marrow chimeras revealed a more dramatic defect, with a >80% reduction in generation of n3.L2 CD4 single-positive thymocytes derived from PKCtheta(-/-) mice. Inefficient positive selection of n3.L2 PKCtheta(-/-) CD4 single-positive cells resulted from "weaker" signaling through the TCR and correlated with diminished ERK activation. The defect in positive selection was not complete in the PKCtheta(-/-) mice, most likely accounted for by compensation by other PKC isoforms not evident in peripheral cells. Similar decreased positive selection of both CD4 and CD8 single-positive thymocytes was also seen in nontransgenic PKCtheta(-/-) mice. These findings now place PKCtheta as a key signaling molecule in the positive selection of thymocytes as well as in the activation of mature T cells.     

Protein kinase A is required for chromosomal DNA replication.

Passage through mitosis resets cells for a new round of chromosomal DNA replication [1]. In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle [1]. The nuclear membrane also contributes to limit genome replication to once per cell cycle [2]. Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk [3] [4]. Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase.     

Protein kinase C beta is required for human monocyte chemotaxis to MCP-1

Monocyte chemoattractant protein 1 (MCP-1) is important in attracting monocytes to sites of inflammation. Using predominantly pharmacological approaches, prior studies have indicated that serine/threonine kinases are involved in the MCP-1-induced signaling pathways. We report here that there is substantial inhibition of MCP-1-stimulated chemotaxis of human monocytes treated with inhibitors selective for the subset of serine/threonine kinases, protein kinase C (PKC). Selective inhibitors of PKC such as GF109203X and Calphostin C both caused approximately 80% inhibition of chemotaxis. Because these pharmacological inhibitors do not specifically inhibit individual PKC isoforms, we chose to use antisense oligodeoxyribonucleotides (ODN) to specifically reduce PKC isoform expression, first by inhibiting expression of the conventional PKC family, and next by using specific antisense ODN for PKCalpha and PKCbeta. Conventional PKC-antisense ODN treatment completely and significantly inhibited monocyte chemotaxis to MCP-1, whereas sense-control ODN caused no significant inhibition. PKCbeta-antisense ODN caused 89.2% inhibition of chemotaxis at its highest dose. In contrast, PKCbeta-sense ODN and PKCalpha-antisense and -sense ODN were without effect. Further studies evaluating the calcium response that is triggered upon MCP-1 interaction with its receptor, CCR2, indicate that this response is not altered by antisense or sense ODN treatment, thus supporting our hypothesis that PKCbeta is critical for post-receptor signal transduction downstream of the immediate calcium signal. These data contribute to our developing understanding of the signal transduction pathways involved in the chemotactic response of human monocytes to MCP-1 and uniquely identify the requirement for the PKCbeta isoform in this important process.     

The protein kinase PKR is required for p38 MAPK activation and the innate immune response to bacterial endotoxin

Protein kinase RNA-regulated (PKR) is an established component of innate antiviral immunity. Recently, PKR has been shown to be essential for signal transduction in other situations of cellular stress. The relationship between PKR and the stress-activated protein kinases (SAPKs), such as p38 mitogen-activated protein kinase (MAPK), is not clear. Using embryonic fibroblasts from PKR wild-type and null mice, we established a requirement for PKR in the activation of SAPKs by double-stranded RNA, lipopolysaccharide (LPS) and proinflammatory cytokines. This does not reflect a global failure to activate SAPKs in the PKR-null background as these kinases are activated normally by anisomycin and other physicochemical stress. Activation of p38 MAPK was restored in immortalized PKR-null cells by reconstitution with human PKR. We also show that LPS induction of interleukin-6 and interleukin-12 mRNA is defective in PKR-null cells, and that production of these cytokines is impaired in PKR-null mice challenged with LPS. Our findings indicate, for the first time, that PKR is required for p38 MAPK signaling and plays a potentially important role in the innate response against bacterial endotoxin.     

Protein phosphatase 5 is required for ATR-mediated checkpoint activation

In response to DNA damage or replication stress, the protein kinase ATR is activated and subsequently transduces genotoxic signals to cell cycle control and DNA repair machinery through phosphorylation of a number of downstream substrates. Very little is known about the molecular mechanism by which ATR is activated in response to genotoxic insults. In this report, we demonstrate that protein phosphatase 5 (PP5) is required for the ATR-mediated checkpoint activation. PP5 forms a complex with ATR in a genotoxic stress-inducible manner. Interference with the expression or the activity of PP5 leads to impairment of the ATR-mediated phosphorylation of hRad17 and Chk1 after UV or hydroxyurea treatment. Similar results are obtained in ATM-deficient cells, suggesting that the observed defect in checkpoint signaling is the consequence of impaired functional interaction between ATR and PP5. In cells exposed to UV irradiation, PP5 is required to elicit an appropriate S-phase checkpoint response. In addition, loss of PP5 leads to premature mitosis after hydroxyurea treatment. Interestingly, reduced PP5 activity exerts differential effects on the formation of intranuclear foci by ATR and replication protein A, implicating a functional role for PP5 in a specific stage of the checkpoint signaling pathway. Taken together, our results suggest that PP5 plays a critical role in the ATR-mediated checkpoint activation.     

Cutting Edge: TLR2 is required for the innate response to Porphyromonas gingivalis: activation leads to bacterial persistence and TLR2 deficiency attenuates induced alveolar bone resorption

Periodontitis is a chronic inflammatory disease that leads to destruction of the attachment apparatus of the teeth. The presence of particular oral bacteria and the host inflammatory response contribute to disease progression. Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a major periodontal pathogen. Isolated Ags from P. gingivalis activate innate immune cells through TLR2 or TLR4. We challenged TLR2- and TLR4-deficient mice with live P. gingivalis and studied the inflammatory response and bacterial survival. Wild-type and TLR4-deficient mice produced high levels of cytokines in response to P. gingivalis challenge, whereas cytokine levels were nearly absent or delayed in TLR2-deficient mice. Surprisingly, P. gingivalis was cleared far more rapidly in TLR2-deficient mice. In addition, TLR2-deficient mice resisted bone loss following oral infection with P. gingivalis.