Toll-like receptor 2 promiscuity is responsible for the immunostimulatory activity of nucleic acid nanocarriers

Abstract

Lipopolyamines (LPAs) are cationic lipids; they interact spontaneously with nucleic acids to form lipoplexes used for gene delivery. The main hurdle to using lipoplexes in gene therapy lies in their immunostimulatory properties, so far attributed to the nucleic acid cargo, while cationic lipids were considered as inert to the immune system. Here we demonstrate for the first time that di-C18 LPAs trigger pro-inflammatory responses through Toll-like receptor 2 (TLR2) activation, and this whether they are bound to nucleic acids or not. Molecular docking experiments suggest potential TLR2 binding modes reminiscent of bacterial lipopeptide sensing. The di-C18 LPAs share the ability of burying their lipid chains in the hydrophobic cavity of TLR2 and, in some cases, TLR1, at the vicinity of the dimerization interface; the cationic headgroups form multiple hydrogen bonds, thus crosslinking TLRs into functional complexes. Unravelling the molecular basis of TLR1 and TLR6-driven heterodimerization upon LPA binding underlines the highly collaborative and promiscuous ligand binding mechanism. The prevalence of non-specific main chain-mediated interactions demonstrates that potentially any saturated LPA currently used or proposed as transfection agent is likely to activate TLR2 during transfection. Hence our study emphasizes the urgent need to test the inflammatory properties of transfection agents and proposes the use of docking analysis as a preliminary screening tool for the synthesis of new non-immunostimulatory nanocarriers.

Cationic lipids as one-component vaccine adjuvants: A promising alternative to alum

“…The cationic lipids 209,204, 122,766 and 122,767 have similar or identical polar head group but shorter alkyl chains (12 or 14 carbon atoms) compared to the C18-lipopolyamine 120,525, 128,506 and 120,535 (Table 1). Differently from these C18-lipopolyamines, which activates only TLR2 [33], 209,204, 122,766 and 122,767 are able to activate both TLR2 and TLR4 HEK-transfected cells (Fig. 1B).…”

“…1A) in a dose-dependent manner with an induction factor ranging from 2 to 4 compared to untreated cells. We previously showed that lipids 128,506, 120,535, 120,525 and 206,252 are activators of TLR2 ( [32,33] and Table 1). In order to identify the receptor involved in the immunostimulatory properties of lipids 122,766, 122,767 and 209,204, we investigated their ability to activate NF-κB in HEK293 cells transfected with an empty plasmid (w/o TLR) or encoding TLR2 or TLR4.…”

“…Cells were washed with PBS and then lysed with passive lysis buffer (Promega). Luciferase and Renilla activity on cell lysates were quantified on a BioTek Synergy HT microplate reader using home-made luciferase reagent with luciferin (Biosynth) or coelenterazine (Biosynth) as previously described [33]. Firefly and Renilla luciferase activity on cell lysates were normalized and data were expressed as fold induction as compared to unstimulated conditions.…”

“…We have previously shown that cationic lipids lipopolyamines have TLR2 and NLRP3-dependent pro-inflammatory properties [32,33]. While such inflammatory properties are deleterious in gene therapy, they can be exploited to develop new effective one-component vaccine adjuvants.…”

Large supramolecular structures of 33-mer gliadin peptide activate toll-like receptors in macrophages

“…56 The implication of contaminants or non-canonical pathways in those activations is a matter of controversy. 57 However, TLR4 and TLR2 are activated by sterile components such as Nickel ions 58 and synthetic cationic lipids, 59 or by HMGB1 protein that activates TLR2 or TLR4 depending on cellular type, 60 which excludes the involvement of contaminations and demonstrates the promiscuity of these TLRs. Here, the synthetic nature of 33-mer gliadin peptide and the use of Polymyxin B, rule out bacterial contamination and support our findings.…”