Search

Two cosegregating single-nucleotide polymorphisms (SNPs) in human TLR4, an A896G transition at SNP rs4986790 (D299G) and a C1196T transition at SNP rs4986791 (T399I), have been associated with LPS hyporesponsiveness and differential susceptibility to many infectious or inflammatory diseases. However, many studies failed to confirm these associations, and transfection experiments resulted in conflicting conclusions about the impact of these SNPs on TLR4 signaling. Using advanced protein modeling from crystallographic data of human and murine TLR4, we identified homologous substitutions of these SNPs in murine Tlr4, engineered a knock-in strain expressing the D298G and N397I TLR4 SNPs homozygously, and characterized in vivo and in vitro responses to TLR4 ligands and infections in which TLR4 is implicated. Our data provide new insights into cellular and molecular mechanisms by which these SNPs decrease the TLR4 signaling efficiency and offer an experimental approach to confirm or refute human data possibly confounded by variables unrelated to the direct effects of the SNPs on TLR4 functionality., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Richard et al.)

Toll-like receptors (TLRs), a family of "pattern recognition receptors," bind microbial and host-derived molecules, leading to intracellular signaling and proinflammatory gene expression. TLR4 is unique in that ligand-mediated activation requires the co-receptor myeloid differentiation 2 (MD2) to initiate two signaling cascades: the MyD88-dependent pathway is initiated at the cell membrane, and elicits rapid MAP kinase and NF-κB activation, while the TIR-domain containing adaptor inducing interferon-β (TRIF)-dependent pathway is initiated from TLR4-containing endosomes and results in IRF3 activation. Previous studies associated inflammation with the MyD88 pathway and adjuvanticity with the TRIF pathway. Gram-negative lipopolysaccharide (LPS) is a potent TLR4 agonist, and structurally related molecules signal through TLR4 to differing extents. Herein, we compared monophosphoryl lipid A (sMPL) and E6020, two synthetic, non-toxic LPS lipid A analogs used as vaccine adjuvants, for their capacities to activate TLR4-mediated innate immune responses and to enhance antibody production. In mouse macrophages, high dose sMPL activates MyD88-dependent signaling equivalently to E6020, while E6020 exhibits significantly more activation of the TRIF pathway (a "TRIF bias") than sMPL. Eritoran, a TLR4/MD2 antagonist, competitively inhibited sMPL more strongly than E6020. Despite these differences, sMPL and E6020 adjuvants enhanced antibody responses to comparable extents, with balanced immunoglobulin (Ig) isotypes in two immunization models. These data indicate that a TRIF bias is not necessarily predictive of superior adjuvanticity., Competing Interests: Declaration of Competing Interest Lynn Hawkins is a scientist with Eisai, Inc. (Cambridge, MA). The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The unique cell biology of Toll-like receptor 4 (TLR4) allows it to initiate two signal-transduction cascades: a signal dependent on the adaptors TIRAP (Mal) and MyD88 that begins at the cell surface and regulates proinflammatory cytokines, and a signal dependent on the adaptors TRAM and TRIF that begins in the endosomes and drives the production of type I interferons. Negative feedback circuits to limit TLR4 signals from both locations are necessary to balance the inflammatory response. We describe a negative feedback loop driven by autocrine-paracrine prostaglandin E 2 (PGE 2 ) and the PGE 2 receptor EP4 that restricted TRIF-dependent signals and the induction of interferon-β through the regulation of TLR4 trafficking. Inhibition of PGE 2 production or antagonism of EP4 increased the rate at which TLR4 translocated to endosomes and amplified TRIF-dependent activation of the transcription factor IRF3 and caspase-8. This PGE 2 -driven mechanism restricted TLR4-TRIF signaling in vitro after infection of macrophages by the Gram-negative pathogens Escherichia coli or Citrobacter rodentium and protected mice against mortality induced by Salmonella enteritidis serovar Typhimurium. Thus, PGE 2 restricted TLR4-TRIF signaling specifically in response to lipopolysaccharide.

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.

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 (IRAK4(KDKI)) were used to address the role of IRAK4 in response to TLR agonists or bacterial infection. IRAK4(KDKI) 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 IRAK4(KDKI) macrophages stimulated through either TLR2 or TLR4, suggesting that IRAK4 contributes to TLR signaling beyond the initial phosphorylation of MAPKs. Additionally, IRAK4(KDKI) macrophages produced minimal cytokine mRNA expression in response to the Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus compared with WT cells, and IRAK4(KDKI) 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 IRAK4(KDKI) mice, thereby providing a potential treatment strategy in both normal and immunocompromised patients.

Overwhelming inflammation triggered by systemic infection in bacterial sepsis contributes to the pathology of this condition. Toll-like receptors (TLRs) are important in early septic inflammation. As a safeguard, the innate immune system has evolved to counter excessive inflammation through the induction of "tolerance." In endotoxin tolerance, TLR signaling is inhibited and/or attenuated by multiple mechanisms that mitigate the ability of lipopolysaccharide (LPS) to activate critical kinases through TLR4. Here, we describe a novel mechanism. Protein kinase R (PKR), a kinase normally activated by a subset of TLRs, is rendered unresponsive to LPS in endotoxin-tolerized cells. In its naive state, PKR is subject to K63-linked ubiquitination (Ub), followed by K48-linked Ub, in response to LPS. In tolerance, the kinetics of this differential Ub is altered, resulting in a predominance of K48-linked chains, concomitant with a loss of PKR activation. These findings provide a novel mechanism by which a TLR-responsive kinase may be rendered inactive in tolerance.

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-kappaB 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.