Darren J. Perkins

TLR4/MyD88/PI3K interactions regulate TLR4 signaling

TLRs activate immune responses by sensing microbial structures such as bacterial LPS, viral RNA, and endogenous “danger” molecules released by damaged host cells. MyD88 is an adapter protein that mediates signal transduction for most TLRs and leads to activation of NF‐κB and MAPKs and production of proinflammatory cytokines. TLR4‐mediated signaling also leads to rapid activation of PI3K, one of a family of kinases involved in regulation of cell growth, apoptosis, and motility. LPS stimulates phosphorylation of Akt, a downstream target of PI3K, in wild‐type (WT) mouse macrophages. LPS‐induced phosphorylation of Akt serine 473 was blunted in MyD88−/− macrophages and was completely TLR4‐dependent. MyD88 and p85 were shown previously to co‐immunoprecipitate, and a YXXM motif within the Toll‐IL‐1 resistance (TIR) domain of MyD88 was suggested to be important for this interaction. To test this hypothesis, we compared expressed MyD88 variants with mutations within the YXXM motif or lacking the TIR domain or death domain and measured their capacities to bind PI3K p85, MyD88, and TLR4 by co‐immunoprecipitation analyses. The YXXM → YXXA mutant MyD88 bound more strongly to p85, TLR4, and WT MyD88 than the other variants, yet was significantly less active than WT MyD88, suggesting that sustained interaction of MyD88/PI3K with the TLR4 intracellular “signaling platform” negatively regulates signaling. We propose a hypothetical model in which sustained PI3K activity at the membrane limits the availability of the PI3K substrate, thereby negatively regulating signaling.

5,6-Dimethylxanthenone-4-acetic Acid (DMXAA) Activates Stimulator of Interferon Gene (STING)-dependent Innate Immune Pathways and Is Regulated by Mitochondrial Membrane Potential

Background: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) activates intracellular signaling through uncharacterized pathways similar to those engaged by bacterial pathogens. Results: Mitochondrial targeting agents and absence of STING impair the response to DMXAA in mouse macrophages. Conclusion: Mitochondrial membrane potential is required for optimal response to DMXAA. Significance: This study illustrates that mitochondrial physiology is pivotal in the host response to DMXAA and possibly bacterial pathogens. The chemotherapeutic agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a potent inducer of type I IFNs and other cytokines. This ability is essential for its chemotherapeutic benefit in a mouse cancer model and suggests that it might also be useful as an antiviral agent. However, the mechanism underlying DMXAA-induced type I IFNs, including the host proteins involved, remains unclear. Recently, it was reported that the antioxidant N-acetylcysteine (NAC) decreased DMXAA-induced TNF-α and IL-6, suggesting that oxidative stress may play a role. The goal of this study was to identify host proteins involved in DMXAA-dependent signaling and determine how antioxidants modulate this response. We found that expression of IFN-β in response to DMXAA in mouse macrophages requires the mitochondrial and endoplasmic reticulum resident protein STING. Addition of the antioxidant diphenylene iodonium (DPI) diminished DMXAA-induced IFN-β, but this decrease was independent of both the NADPH oxidase, Nox2, and de novo generation of reactive oxygen species. Additionally, IFN-β up-regulation by DMXAA was inhibited by agents that target the mitochondrial electron transport chain and, conversely, loss of mitochondrial membrane potential correlated with diminished innate immune signaling in response to DMXAA. Up-regulation of Ifnb1 gene expression mediated by cyclic dinucleotides was also impaired by DPI, whereas up-regulation of Ifnb1 mRNA due to cytosolic double-stranded DNA was not. Although both stimuli signal through STING, cyclic dinucleotides interact directly with STING, suggesting that recognition of DMXAA by STING may also be mediated by direct interaction.

Transcriptional Regulation of Murine IL-33 by TLR and Non-TLR Agonists

IL-33, a member of the IL-1 family of cytokines, is produced by many cell types, including macrophages, yet its regulation is largely unknown. Treatment of primary murine macrophages with a panel of TLR (e.g., TLR2, TLR3, TLR4, and TLR9) agonists and non-TLR (e.g., MDA5, RIG-I) agonists revealed a pattern of gene and protein expression consistent with a role for IFN regulatory factor-3 (IRF-3) in the expression of IL-33. Accordingly, induction of IL-33 mRNA was attenuated in IRF-3−/− macrophages and TBK-1−/− mouse embryonic fibroblasts. Despite the fact that all IL-33 agonists were IRF-3 dependent, LPS-induced IL-33 mRNA was fully inducible in IFN-β−/− macrophages, indicating that IL-33 is not dependent on IFN-β as an intermediate. Epinephrine and Bordetella pertussis adenylate cyclase toxin (ACT), cAMP-activating agents, activate CREB and greatly synergize with LPS to induce IL-33 mRNA in macrophages. Both LPS-induced and ACT/LPS-enhanced expression of IL-33 mRNA was partially, but significantly, inhibited by the protein kinase A inhibitor H-89 but not by tyrosine kinase or protein kinase C inhibitors. Two IL-33 mRNA species derived from two alternative promoters encode full-length IL-33; however, the shorter “A” species is preferentially induced by all IL-33–inducing agonists except Newcastle disease virus, a RIG-I agonist that induced expression of both “A” and “B” transcripts. Together, these studies greatly extend what is currently known about the regulation of IL-33 induction in macrophages stimulated by bacterial and viral agonists that engage distinct innate immune signaling pathways.

TRAF6 Protein Couples Toll-like Receptor 4 Signaling to Src Family Kinase Activation and Opening of Paracellular Pathway in Human Lung Microvascular Endothelia

Background: Bacterial lipopolysaccharide (LPS) disrupts endothelial barrier integrity. Results: LPS increases association of a TRAF6 proline-rich SH3-binding motif (aa 461–469) with c-Src and Fyn, followed by their ubiquitination and activation, which in turn increases endothelial paracellular permeability. Conclusion: TRAF6 couples LPS stimulation to Src family kinase activation and loss of endothelial barrier integrity. Significance: TRAF6 offers a target for therapeutic intervention for LPS-induced pulmonary microvascular endothelial injury. Gram-negative bacteria release lipopolysaccharide (LPS) into the bloodstream. Here, it engages Toll-like receptor (TLR) 4 expressed in human lung microvascular endothelia (HMVEC-Ls) to open the paracellular pathway through Src family kinase (SFK) activation. The signaling molecules that couple TLR4 to the SFK-driven barrier disruption are unknown. In HMVEC-Ls, siRNA-induced silencing of TIRAP/Mal and overexpression of dominant-negative TIRAP/Mal each blocked LPS-induced SFK activation and increases in transendothelial [14C]albumin flux, implicating the MyD88-dependent pathway. LPS increased TRAF6 autoubiquitination and binding to IRAK1. Silencing of TRAF6, TRAF6-dominant-negative overexpression, or preincubation of HMVEC-Ls with a cell-permeable TRAF6 decoy peptide decreased both LPS-induced SFK activation and barrier disruption. LPS increased binding of both c-Src and Fyn to GST-TRAF6 but not to a GST-TRAF6 mutant in which the three prolines in the putative Src homology 3 domain-binding motif (amino acids 461–469) were substituted with alanines. A cell-permeable decoy peptide corresponding to the same proline-rich motif reduced SFK binding to WT GST-TRAF6 compared with the Pro → Ala-substituted peptide. Finally, LPS increased binding of activated Tyr(P)416-SFK to GST-TRAF6, and preincubation of HMVEC-Ls with SFK-selective tyrosine kinase inhibitors, PP2 and SU6656, diminished TRAF6 binding to c-Src and Fyn. During the TRAF6-SFK association, TRAF6 catalyzed Lys63-linked ubiquitination of c-Src and Fyn, whereas SFK activation increased tyrosine phosphorylation of TRAF6. The TRAF6 decoy peptide blocked both LPS-induced SFK ubiquitination and TRAF6 phosphorylation. Together, these data indicate that the proline-rich Src homology 3 domain-binding motif in TRAF6 interacts directly with activated SFKs to couple LPS engagement of TLR4 to SFK activation and loss of barrier integrity in HMVEC-Ls.

Invasive Salmonella Typhimurium ST313 with Naturally Attenuated Flagellin Elicits Reduced Inflammation and Replicates within Macrophages

Invasive non-typhoidal Salmonella (iNTS) are an important cause of septicemia in children under the age of five years in sub-Saharan Africa. A novel genotype of Salmonella enterica subsp. enterica serovar Typhimurium (multi-locus sequence type [ST] 313) circulating in this geographic region is genetically different to from S. Typhimurium ST19 strains that are common throughout the rest of the world. S. Typhimurium ST313 strains have acquired pseudogenes and genetic deletions and appear to be evolving to become more like the typhoidal serovars S. Typhi and S. Paratyphi A. Epidemiological and clinical data show that S. Typhimurium ST313 strains are clinically associated with invasive systemic disease (bacteremia, septicemia, meningitis) rather than with gastroenteritis. The current work summarizes investigations of the broad hypothesis that S. Typhimurium ST313 isolates from Mali, West Africa, will behave differently from ST19 isolates in various in vitro assays. Here, we show that strains of the ST313 genotype are phagocytosed more efficiently and are highly resistant to killing by macrophage cell lines and primary mouse and human macrophages compared to ST19 strains. S. Typhimurium ST313 strains survived and replicated within different macrophages. Infection of macrophages with S. Typhimurium ST19 strains resulted in increased apoptosis and higher production of proinflammatory cytokines, as measured by gene expression and protein production, compared to S. Typhimurium ST313 strains. This difference in proinflammatory cytokine production and cell death between S. Typhimurium ST19 and ST313 strains could be explained, in part, by an increased production of flagellin by ST19 strains. These observations provide further evidence that S. Typhimurium ST313 strains are phenotypically different to ST19 strains and instead share similar pathogenic characteristics with typhoidal Salmonella serovars.

Dissociation of Endotoxin Tolerance and Differentiation of Alternatively Activated Macrophages

Endotoxin tolerance is a complex phenomenon characterized primarily by decreased production of proinflammatory cytokines, chemokines, and other inflammatory mediators, whereas the expression of other genes are induced or unchanged. Endotoxin tolerance is induced by prior exposure of murine macrophages/human monocytes, experimental animals, or people to TLR ligands. Although recent studies reported a possible relationship between endotoxin tolerance and differentiation of alternatively activated macrophages (AA-MΦs or M2), we show in this study that LPS pretreatment of IL-4Rα−/− and STAT6−/− macrophages, which fail to develop into AA-MΦs, resulted in tolerance of proinflammatory cytokines, as well as molecules and chemokines previously associated with AA-MΦs (e.g., arginase-1, mannose receptor, CCL2, CCL17, and CCL22). In contrast to LPS, wild-type (WT) MΦs pretreated with IL-4, the prototype inducer of AA-MΦs, did not induce endotoxin tolerance with respect to proinflammatory cytokines, AA-MΦ–associated chemokines, negative regulators, NF-κB binding and subunit composition, and MAPKs; conversely, IL-13−/− macrophages were tolerized equivalently to WT MΦs by LPS pretreatment. Further, IL-4Rα deficiency did not affect the reversal of endotoxin tolerance exerted by the histone deacetylase inhibitor trichostatin A. Like WT mice, 100% of LPS-tolerized IL-4Rα–deficient mice survived LPS + d-galactosamine–induced lethal toxicity and exhibited decreased serum levels of proinflammatory cytokines and AA-MΦ–associated chemokines induced by LPS challenge compared with nontolerized mice. These data indicate that the signaling pathways leading to endotoxin tolerance and differentiation of AA-MΦs are dissociable.

CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance.

Significance MyD88-dependent signaling is cluster of differentiation 14 (CD14)-dependent only at low LPS concentrations, whereas activation of the TIR-domain–containing adapter-inducing IFN-β (TRIF) pathway requires CD14 at all LPS concentrations, leading to internalization of the Toll-like receptor 4 (TLR4) complex into endosomes whereupon TRIF is recruited. Using alternative TLR4 agonists, or macrophages rendered tolerant to LPS, we dissociate TLR4 complex internalization from CD14 and TRIF-dependent signaling. In response to LPS, CD14 contributes to the formation of a TLR4/MD2 complex dimer that, in turn, promotes endocytosis and IRF3 activation. Dimerization of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) heterodimers is critical for both MyD88- and TIR-domain–containing adapter-inducing IFN-β (TRIF)-mediated signaling pathways. Recently, Zanoni et al. [(2011) Cell 147(4):868–880] reported that cluster of differentiation 14 (CD14) is required for LPS-/Escherichia coli- induced TLR4 internalization into endosomes and activation of TRIF-mediated signaling in macrophages. We confirmed their findings with LPS but report here that CD14 is not required for receptor endocytosis and downstream signaling mediated by TLR4/MD2 agonistic antibody (UT12) and synthetic small-molecule TLR4 ligands (1Z105) in murine macrophages. CD14 deficiency completely ablated the LPS-induced TBK1/IRF3 signaling axis that mediates production of IFN-β in murine macrophages without affecting MyD88-mediated signaling, including NF-κB, MAPK activation, and TNF-α and IL-6 production. However, neither the MyD88- nor TRIF-signaling pathways and their associated cytokine profiles were altered in the absence of CD14 in UT12- or 1Z105-treated murine macrophages. Eritoran (E5564), a lipid A antagonist that binds the MD2 “pocket,” completely blocked LPS- and 1Z105-driven, but not UT12-induced, TLR4 dimerization and endocytosis. Furthermore, TLR4 endocytosis is induced in macrophages tolerized by exposure to either LPS or UT12 and is independent of CD14. These data indicate that TLR4 receptor endocytosis and the TRIF-signaling pathway are dissociable and that TLR4 internalization in macrophages can be induced by UT12, 1Z105, and during endotoxin tolerance in the absence of CD14.

Bordetella pertussis adenylate cyclase toxin (ACT) induces cyclooxygenase‐2 (COX‐2) in murine macrophages and is facilitated by ACT interaction with CD11b/CD18 (Mac‐1)

Bordetella pertussis causes a profound inflammatory response in lungs of infected individuals. The adenylate cyclase toxin (ACT) of B. pertussis is a potent enzyme that converts cytosolic ATP into cAMP, and is required for virulence in vivo. During infection, secreted ACT binds to macrophages utilizing the β2 integrin, Mac‐1 (CR3, CD11b/CD18), and subsequent intoxication by ACT inhibits essential antibacterial activities of macrophages. Additionally, Mac‐1 has been reported to be a co‐receptor for TLR4 required for the full induction of some LPS‐responsive genes, including pro‐inflammatory cyclooxygenase 2 (COX‐2). We have examined the effect of ACT on COX‐2 expression in HEK293T cells expressing Mac‐1 and in murine macrophages. We report that ACT induces COX‐2 in a manner that absolutely requires the catalytic activity of this enzyme and Mac‐1 expression dramatically enhanced the sensitivity of cells to ACT‐dependent COX‐2 induction. The mechanism of COX‐2 induction by ACT utilizes the cAMP‐PKA‐CREB‐dependent pathway. Finally, ACT and TLR2 or TLR4 act synergistically to increase COX‐2 expression. These data suggest that ACT contributes significantly to the inflammatory response induced by B. pertussis infection by augmenting COX‐2 expression and provides evidence against the concept that ACT functions exclusively via its inhibitory effects on phagocytic leucocytes.

Reprogramming of murine macrophages through TLR2 confers viral resistance via TRAF3-mediated, enhanced interferon production.

The cell surface/endosomal Toll-like Receptors (TLRs) are instrumental in initiating immune responses to both bacteria and viruses. With the exception of TLR2, all TLRs and cytosolic RIG-I-like receptors (RLRs) with known virus-derived ligands induce type I interferons (IFNs) in macrophages or dendritic cells. Herein, we report that prior ligation of TLR2, an event previously shown to induce “homo” or “hetero” tolerance, strongly “primes” macrophages for increased Type I IFN production in response to subsequent TLR/RLR signaling. This occurs by increasing activation of the transcription factor, IFN Regulatory Factor-3 (IRF-3) that, in turn, leads to enhanced induction of IFN-β, while expression of other pro-inflammatory genes are suppressed (tolerized). In vitro or in vivo “priming” of murine macrophages with TLR2 ligands increase virus-mediated IFN induction and resistance to infection. This priming effect of TLR2 is mediated by the selective upregulation of the K63 ubiquitin ligase, TRAF3. Thus, we provide a mechanistic explanation for the observed antiviral actions of MyD88-dependent TLR2 and further define the role of TRAF3 in viral innate immunity.

Complete Dependence on IRAK4 Kinase Activity in TLR2, but Not TLR4, Signaling Pathways Underlies Decreased Cytokine Production and Increased Susceptibility to Streptococcus pneumoniae Infection in IRAK4 Kinase–Inactive Mice

IRAK4 is critical for MyD88-dependent TLR signaling, and patients with Irak4 mutations are extremely susceptible to recurrent bacterial infections. In these studies, mice homozygous for a mutant IRAK4 that lacks kinase activity (IRAK4KDKI) were used to address the role of IRAK4 in response to TLR agonists or bacterial infection. IRAK4KDKI macrophages exhibited diminished responsiveness to the TLR4 agonist LPS and little to no response to the TLR2 agonist Pam3Cys compared with wild-type macrophages as measured by cytokine mRNA, cytokine protein expression, and MAPK activation. Importantly, we identified two kinases downstream of the MAPKs, MNK1 and MSK1, whose phosphorylation is deficient in IRAK4KDKI macrophages stimulated through either TLR2 or TLR4, suggesting that IRAK4 contributes to TLR signaling beyond the initial phosphorylation of MAPKs. Additionally, IRAK4KDKI macrophages produced minimal cytokine mRNA expression in response to the Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus compared with WT cells, and IRAK4KDKI mice exhibited increased susceptibility and decreased cytokine production in vivo upon S. pneumoniae infection. Treatment of infected mice with a complex of polyinosinic-polycytidylic acid with poly-L-lysine and carboxymethyl cellulose (Hiltonol), a potent TLR3 agonist, significantly improved survival of both WT and IRAK4KDKI mice, thereby providing a potential treatment strategy in both normal and immunocompromised patients.