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Synthetic glycan-based TLR4 agonists targeting caspase-4/11 for the development of adjuvants and immunotherapeutics.

ABSTRACT: Gram-negative bacterial lipopolysaccharide (LPS)-induced Toll-like receptor 4 (TLR4) mediated pro-inflammatory signaling plays a key role in immunoprotection against infectious challenges and boosts adaptive immunity, whereas the activation of the cytosolic LPS receptor caspase-4/11 leads to cell death by pyroptosis and is deeply implicated in the development of sepsis. Despite tremendous advances in the understanding of the LPS-TLR4 interaction, predictably regulated TLR4 activation has not yet been achieved. The structural basis for the induction of caspase-4/11 protease activity by LPS is currently unknown. The modulation of innate and adaptive immune responses through the controlled induction of TLR4 signaling without triggering caspase-4/11 activity would open novel perspectives in the development of safe vaccine adjuvants and immunotherapeutics. We report the discovery of highly potent glycan-based immunostimulants with picomolar affinity for TLR4 which interact with caspase-4/11 and promote caspase-4/11 oligomerization while abolishing caspase-11 protease activity. The rigidity and twisted molecular shape of the ?,?-(1?1')-linked disaccharide core of synthetic LPS mimicking anionic glycolipids accounted for both species-independent and adjustable TLR4-mediated NF-?B signaling and the modulation of caspase-4/11 activation. By the use of crystal structure based design and advanced synthetic chemistry we created a set of versatile probes for studying the structural basis of caspase-4/11 activation and established a chemical strategy for controllable TLR4 mediated cytokine release which is dissociable from the induction of caspase-11 protease activity.

SUBMITTER: Adanitsch F

PROVIDER: S-EPMC5941199 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Synthetic glycan-based TLR4 agonists targeting caspase-4/11 for the development of adjuvants and immunotherapeutics.

Adanitsch Florian F Shi Jianjin J Shao Feng F Beyaert Rudi R Heine Holger H Zamyatina Alla A

Chemical science 20180315 16

Gram-negative bacterial lipopolysaccharide (LPS)-induced Toll-like receptor 4 (TLR4) mediated pro-inflammatory signaling plays a key role in immunoprotection against infectious challenges and boosts adaptive immunity, whereas the activation of the cytosolic LPS receptor caspase-4/11 leads to cell death by pyroptosis and is deeply implicated in the development of sepsis. Despite tremendous advances in the understanding of the LPS-TLR4 interaction, predictably regulated TLR4 activation has not yet ...[more]

PMID: 29780528

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Project description:Toll-like receptors (TLRs), a family of pattern recognition receptors, bind microbial and host-derived molecules, leading to intracellular signaling and activation of host innate immune responses. TLR4 is unique in that ligand-mediated activation initiates two signaling cascades: the MyD88-dependent pathway, localized to the cell membrane, elicits rapid MAP kinase and NF-κB activation, and the TRIF-dependent pathway, localized to TLR4-containing endosomes, results in IRF-3 activation. Previous studies have associated adjuvanticity with the TRIF pathway. Gram-negative LPS is a potent TLR4 agonist, and structurally related molecules also signal through TLR4 to different extents. Herein, we compared two synthetic, non-toxic lipid A analogs used previously as vaccine adjuvants, monophosphoryl lipid A (PHAD®, sMPL) and E6020, for their capacity to activate TLR4-mediated innate immune responses. In primary mouse macrophages, high dose sMPL activates the MyD88-dependent signaling equivalently to E6020. In contrast to sMPL, E6020 exhibits robust activation of the TRIF pathway, including TLR4 internalization and IRF-3-dependent transcription, at all doses examined. Eritoran, a TLR4/MD2 antagonist, competitively inhibited sMPL more strongly than E6020. Despite these differences, sMPL and E6020 adjuvants induced antibody responses to a similar level and with balanced Ig isotypes, in two separate mouse immunization models. However, E6020 induced superior non-specific protection in a model of Streptococcus pneumoniae infection, compared to sMPL, which correlates with the activation of reactive nitrogen and oxygen species. These data provide evidence that a TRIF bias is not necessarily predictive of superior adjuvanticity, but may activate stronger innate defenses from infection. TLR4 is unique among TLRs that it stimulates both the MyD88- and TRIF-dependent pathways (reviewed in Takeda K and Akira S, 2004, Semin Immunol). To compare the relative stimulatory capacities of two synthetic TLR4 agonist adjuvants, PHAD® (sMPL; Avanti Polar Lipids LLC) and E6020 (Eisai, Inc), we performed microarray analysis of primary thioglycollate-elicited mouse peritoneal macrophages that were either cultured in media alone (control) or stimulated with 100 ng/mL (57.3 uM sMPL, 61.6 uM E6020) of the TLR4 agonists for two hours. Total RNA was extracted from three biological repicates (numbered 1-3) per treatment, and the transcriptomes analyzed individually by whole transcriptome microarray (Affymetrix Clariom D). Of the differentially expressed genes (DE genes; average of three replicates greater or equal to 2-fold difference in any comparison of treatments and false discovery rate below 0.05) almost 94% were protein-coding genes (includes those listed as either protein-coding or complex in the database), on which we focused for further analysis. sMPL induced expression of 155 protein-coding genes, including those that encode IL-1beta, TNF-alpha, Cxcl1 and Cxcl2--proteins that are associated with classic macrophage activation and are essential for inducing fever and attracting neutrophils. sMPL suppressed 15 protein-coding genes. In contrast, E6020 induced expression of 413 protein-coding genes and suppressed 88 protein-coding genes, which included all but one of the sMPL-regulated protein-coding genes. The majority of the genes induced by E6020, but not sMPL, are associated with Type I IFN expression (reference: Interferome.org database). This interferon signature points to stronger activation of the TRIF/IRF3 pathway in E6020-treated macrophages and includes genes that are secondarily induced by Type I IFNs. Similar expression of MyD88 dependent genes Il1b, Il10, and Tnf, and differential expression of TRIF-dependent genes Ifnb1, Cxcl10, and Ccl5 between the two synthetic agonists was confirmed by qRT-PCR.

Project description:The innate immune response to lipopolysaccharide is essential for host defense against Gram-negative bacteria. In response to bacterial infection, the TLR4/MD-2 complex that is expressed on the surface of macrophages, monocytes, dendritic, and epithelial cells senses picomolar concentrations of endotoxic LPS and triggers the production of various pro-inflammatory mediators. In addition, LPS from extracellular bacteria which is either endocytosed or transfected into the cytosol of host cells or cytosolic LPS produced by intracellular bacteria is recognized by cytosolic proteases caspase-4/11 and hosts guanylate binding proteins that are involved in the assembly and activation of the NLRP3 inflammasome. All these events result in the initiation of pro-inflammatory signaling cascades directed at bacterial eradication. However, TLR4-mediated signaling and caspase-4/11-induced pyroptosis are largely involved in the pathogenesis of chronic and acute inflammation. Both extra- and intracellular LPS receptors-TLR4/MD-2 complex and caspase-4/11, respectively-are able to directly bind the lipid A motif of LPS. Whereas the structural basis of lipid A recognition by the TLR4 complex is profoundly studied and well understood, the atomic mechanism of LPS/lipid A interaction with caspase-4/11 is largely unknown. Here we describe the LPS-induced TLR4 and caspase-4/11 mediated signaling pathways and their cross-talk and scrutinize specific structural features of the lipid A motif of diverse LPS variants that have been reported to activate caspase-4/11 or to induce caspase-4/11 mediated activation of NLRP3 inflammasome (either upon transfection of LPS in vitro or upon infection of cell cultures with intracellular bacteria or by LPS as a component of the outer membrane vesicles). Generally, inflammatory caspases show rather similar structural requirements as the TLR4/MD-2 complex, so that a "basic" hexaacylated bisphosphorylated lipid A architecture is sufficient for activation. However, caspase-4/11 can sense and respond to much broader variety of lipid A variants compared to the very "narrow" specificity of TLR4/MD-2 complex as far as the number and the length of lipid chains attached at the diglucosamine backbone of lipid A is concerned. Besides, modification of the lipid A phosphate groups with positively charged appendages such as phosphoethanolamine or aminoarabinose could be essential for the interaction of lipid A/LPS with inflammatory caspases and related proteins.

Project description:Toll-like receptor (TLR) agonists are recognized as potential immune-enhancing adjuvants and are included in several licensed vaccines. Monophosphoryl lipid A (MPL®, GlaxoSmithKline) is one such TLR4 agonist that has been approved for use in human vaccines, such as Cervarix and Shingrix. Due to the heterogeneous nature of biologically derived MPL and the need for safer and more potent adjuvants, our groups have developed the novel TLR4 agonist candidates, BECC438 and BECC470 using the Bacterial Enzymatic Combinatorial Chemistry (BECC) platform. BECC438 and BECC470 have been included in studies to test their adjuvant potential and found to be effective in vaccines against both viral and bacterial disease agents. Here, we report detailed biophysical characterization of BECC438 and BECC470 purified from a biological source (BECC438b and BECC470b, respectively) and synthesized chemically (BECC438s and BECC470s, respectively). Both BECC438s and BECC470s have identical acyl chain configurations, BECC438s is bis-phosphorylated and BECC470s is mono-phosphorylated with the removal of the 4' phosphate moiety. We determined the phase transition temperatures for the acyl chains of BECC438b and BECC470b and found them to be different from those exhibited by their synthetic counterparts. Furthermore, the phosphate groups of BECC438b and BECC470b are more highly hydrated than are those of BECC438s and BECC470s. In addition to exploring the BECC molecules' biophysical features in aqueous solution, we explored potential formulation of BECC438 and BECC470 with the aluminum-based adjuvant Alhydrogel and as part of an oil-in-water emulsion (Medimmune Emulsion or ME). All of the lipid A analogues could be fully absorbed to Alhydrogel or incorporated onto ME. Surprisingly, the BECC470s molecule, unlike the others, displayed a nearly baseline signal when monitored using a Limulus amebocyte lysate (LAL) endotoxin detection system. Despite this, it was shown to behave as an agonist for human and mouse TLR4 when tested using multiple cell-based systems. This work paves the way for further formulation optimization of two chemically defined TLR4 agonists that are showing great promise as vaccine adjuvants.

Dataset Information

Synthetic glycan-based TLR4 agonists targeting caspase-4/11 for the development of adjuvants and immunotherapeutics.

Publications

Synthetic glycan-based TLR4 agonists targeting caspase-4/11 for the development of adjuvants and immunotherapeutics.

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