Synthetic Toll-Like Receptor 4 Agonists Stimulate Innate Resistance to Infectious Challenge

Abstract

A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.The toll-like receptors (TLRs) comprise an evolutionarily conserved receptor family that is capable of detecting and responding to microbial challenge (1). The TLRs recognize a variety of pathogen-specific components, including lipopolysaccharide (LPS), CpG DNA, and microbial membrane and cell wall components (20). Toll-like receptor 4 (TLR4) is critical for the recognition of LPS (30, 31), and considerable progress has recently been made in understanding the interaction of TLR4 with critical accessory molecules implicated in LPS recognition (8,9,17,21,22, 32,40,41). In this regard, it appears that LPS binding protein (LBP) promotes the binding of LPS to CD14, which in turn facilitates the association of the lipid A component of LPS with MD-2 to form a soluble complex that serves as an activating ligand for TLR4 on the cell surface. Binding of the MD-2/LPS complex to TLR4 results in the aggregation of TLR4 into lipid rafts and the activation of several distinct intracellular signaling pathways that results in increased transcription of many genes encoding cytokines, defensins, chemokines, and alpha/beta interferons (28, 38). These effector molecules determine a wide range of biological activities, including further production of cytokines, enhancement of microbicidal activity of phagocytic cells, and migration/maturation of dendritic cells (5,23,36).Before the discovery that LPS interacts with TLR4, evidence demonstrating the importance of innate immune activation in controlling infection with gram-neg...

The toll-like receptor-4 (TLR-4) pathway and its possible role in the pathogenesis ofEscherichia colimastitis in dairy cattle

“…Monophosphoryl-A (MLA), a non-toxic variant of the lipid A portion of LPS that lacks the (R)-3-hydroxytetradecanoyl group and 1-phosphate, has been developed [90]. LPS and MLA induce similar cytokine profiles, but MLA is at least 100-fold less toxic [36]. Recently, aminoalkyl glucosaminide phosphates (AGP) have been designed and synthesized.…”

“…Recently, aminoalkyl glucosaminide phosphates (AGP) have been designed and synthesized. They belong to a family of lipid A mimetics, and have been shown to induce innate immune responses in a TLR-4 dependent manner: intranasal challenge with influenza virus resulted in a 70% survival rate in wild type mice that were pretreated with AGP, but not in the C3H/HeJ TLR-4 deficient mice [36]. Accordingly, microarray results showed that the TLR-4 agonists clearly enhanced gene expression of cytokines like IL-6 and TNF-α, and transcription of NF-κB in human macrophages that were stimulated for 6 h with AGP [144].…”

No Longer an Innocent Bystander: Epithelial Toll-Like Receptor Signaling in the Development of Mucosal Inflammation

“…Considerable attention has been placed on developing agents that are capable of modulation of the TLR4-mediated response, in particular through manipulation of the lipid A moiety of LPS. Such lipid A mimetics, termed aminoalkyl glucosaminide phosphates (AGPs), have been demonstrated to reduce inflammation in experimental models of systemic sepsis induced by intravenous injection of Listeria monocytogenes (116), pulmonary infection after intranasal administration of influenza virus (116), murine models of colitis including DSS-induced colitis (117), and spontaneous colitis in multidrug resistance gene 1a-deficient mice (117). In parallel studies, soluble TLRs may reduce TLR signaling by binding to circulating ligands, rendering them unable to initiate pro-inflammatory signaling.…”

Induction of innate immunity by lipid A mimetics increases survival from pneumonic plague

“…The mechanism of AGP-mediated protection is well documented and involves TLR4 stimulation of chemokine and cytokine expression, leading to a classic Th1 immune response. In murine studies, various AGPs delivered intranasally provided protection against organisms such as Listeria monocytogenes or influenza virus (Cluff et al, 2005) . One of these compounds, CRX-524, and a newer AGP (CRX-527), elicited the highest cytokine responses.…”

“…One of these compounds, CRX-524, and a newer AGP (CRX-527), elicited the highest cytokine responses. These compounds have 10-carbon acyl side chains; CRX-524 has a hydrogen on the aglycon moiety while CRX-527 has a carboxyl group at this position (Cluff et al, 2005).…”