Final Report Summary - TLR4-CAT (A new class of cationic lipids that activate innate immune receptors)
DiC14-amidine is a cationic lipid which induces pro-inflammatory cytokine secretion in immune cells upon interaction with the Pattern Recognition Receptor Toll-like receptor 4 (TLR4)/Myeloid Differentiation factor-2 (MD-2) membrane bound-complex, the natural sensor of bacterial lipopolysaccharides (LPS). Pro-inflammatory cytokines are important for the successful development of immunity in response to vaccines. The ability of diC14-amidine to activate pro-inflammatory signalling pathways suggests it has the potential to be developed as an adjuvant compound. The aim of our project was to study the structural basis of the interaction between diC14-amidine and TLR-4/MD-2. Most of the results have been published recently in Cell Mol Life Sci. (2015) 72(20):3971-82 (see attachment file for figures and references).
During this project, we took advantage of the species-dependent activity of TLR4/MD2 and we found that while LPS is an agonist in all species, diC14-amidine is a full agonist for human, mouse and cat receptors, but a weak agonist for horse (Fig. 1A in the publication). Using chimeric constructs made from human and horse proteins, we identified two regions in the human TLR4 that modulate the agonist activity of diC14-amidine, but do not correspond to the previously identified LPS binding domain (Fig. 3 in the publication). Specifically diC14-amidine binds to the TLR4 ectodomain at the edge of the dimerization interface. Induction of diC14-amidine signalling shows no species specificity for MD-2 (Fig. 1C in the publication).
To further characterize the interaction of diC14-amidine with human TLR4/MD2 we carried out competition assays with a known TLR4 antagonist: Rhodobacter sphaeroides LPS (RS-LPS) and we generated docking models of amidine interacting with the TLR4 ectodomain and the dimeric TLR4/MD-2 complex (Fig. 2 in the publication). Our competition assays and docking assays also confirm that diC14-amidine interacts with TLR4 via a mechanism different to that proposed previously for LPS and its derivatives. We proposed that diC14-amidine binds at the N- and C-terminal edges of the TLR4/TLR4* dimerization interface, and induces its dimerization via cross-linking of two receptors (Fig. 5 in the publication). These data allowed us to achieve the main objective of our proposal that is to understand the molecular basis of the interaction between diC14-amidine, a molecule initially designed to be used as a transfection agent in gene therapy, and TLR-4/MD-2 at the structural level. We also were able to propose a novel mechanism of TLR4 activation. The subsequent cell signalling activated by diC14-amidine is analogous to the novel mechanism proposed for TLR4 activation by nickel ions.
Characterization of the signalling activated by diC14-amidine, as a function of the co-receptors involved, showed that diC14-amidine does not require the presence of the co-receptor CD14 to activate MyD88- or TRIF-dependent TLR4/MD2 signalling pathways. CD14 nevertheless does modulate the cellular response to diC14-amidine suggesting a possible interaction of diC14-amidine liposomes with this co-receptor independent of TLR4.
Crystallization trials of the TLR4/MD2/diC14-amidine complex were, as expected, very difficult to achieve. Purification of TLR4/MD2 has not yet allowed us to generate enough material to start crystallization trials. In the meantime, however, we have generated sufficient purified CD14 recombinant proteins for biophysical analysis to demonstrate a direct binding of diC14-amidine to this co-receptor by Fourier-transform Infrared Spectroscopy and Mass Spectrometry (figures A4 and 5) which suggests that diC14-amidine might activate another CD14-dependent signalling pathway independently of TLR4 which will be the focus of future research.
For a detailed work progress summary and the list of references, please see the attached document.
During this project, we took advantage of the species-dependent activity of TLR4/MD2 and we found that while LPS is an agonist in all species, diC14-amidine is a full agonist for human, mouse and cat receptors, but a weak agonist for horse (Fig. 1A in the publication). Using chimeric constructs made from human and horse proteins, we identified two regions in the human TLR4 that modulate the agonist activity of diC14-amidine, but do not correspond to the previously identified LPS binding domain (Fig. 3 in the publication). Specifically diC14-amidine binds to the TLR4 ectodomain at the edge of the dimerization interface. Induction of diC14-amidine signalling shows no species specificity for MD-2 (Fig. 1C in the publication).
To further characterize the interaction of diC14-amidine with human TLR4/MD2 we carried out competition assays with a known TLR4 antagonist: Rhodobacter sphaeroides LPS (RS-LPS) and we generated docking models of amidine interacting with the TLR4 ectodomain and the dimeric TLR4/MD-2 complex (Fig. 2 in the publication). Our competition assays and docking assays also confirm that diC14-amidine interacts with TLR4 via a mechanism different to that proposed previously for LPS and its derivatives. We proposed that diC14-amidine binds at the N- and C-terminal edges of the TLR4/TLR4* dimerization interface, and induces its dimerization via cross-linking of two receptors (Fig. 5 in the publication). These data allowed us to achieve the main objective of our proposal that is to understand the molecular basis of the interaction between diC14-amidine, a molecule initially designed to be used as a transfection agent in gene therapy, and TLR-4/MD-2 at the structural level. We also were able to propose a novel mechanism of TLR4 activation. The subsequent cell signalling activated by diC14-amidine is analogous to the novel mechanism proposed for TLR4 activation by nickel ions.
Characterization of the signalling activated by diC14-amidine, as a function of the co-receptors involved, showed that diC14-amidine does not require the presence of the co-receptor CD14 to activate MyD88- or TRIF-dependent TLR4/MD2 signalling pathways. CD14 nevertheless does modulate the cellular response to diC14-amidine suggesting a possible interaction of diC14-amidine liposomes with this co-receptor independent of TLR4.
Crystallization trials of the TLR4/MD2/diC14-amidine complex were, as expected, very difficult to achieve. Purification of TLR4/MD2 has not yet allowed us to generate enough material to start crystallization trials. In the meantime, however, we have generated sufficient purified CD14 recombinant proteins for biophysical analysis to demonstrate a direct binding of diC14-amidine to this co-receptor by Fourier-transform Infrared Spectroscopy and Mass Spectrometry (figures A4 and 5) which suggests that diC14-amidine might activate another CD14-dependent signalling pathway independently of TLR4 which will be the focus of future research.
For a detailed work progress summary and the list of references, please see the attached document.
