Caroline Lonez

Cationic liposomal lipids: from gene carriers to cell signaling

Cationic lipids are positively charged amphiphilic molecules which, for most of them, form positively charged liposomes, sometimes in combination with a neutral helper lipid. Such liposomes are mainly used as efficient DNA, RNA or protein carriers for gene therapy or immunization trials. Over the past decade, significant progress has been made in the understanding of the cellular pathways and mechanisms involved in lipoplex-mediated gene transfection but the interaction of cationic lipids with cell components and the consequences of such an interaction on cell physiology remains poorly described. The data reported in the present review provide evidence that cationic lipids are not just carriers for molecular delivery into cells but do modify cellular pathways and stimulate immune or anti-inflammatory responses. Considering the wide number of cationic lipids currently available and the variety of cellular components that could be involved, it is likely that only a few cationic lipid-dependent functions have been identified so far.

Cationic lipids activate intracellular signaling pathways

Cationic liposomes are commonly used as a transfection reagent for DNA, RNA or proteins and as a co-adjuvant of antigens for vaccination trials. A high density of positive charges close to cell surface is likely to be recognized as a signal of danger by cells or contribute to trigger cascades that are classically activated by endogenous cationic compounds. The present review provides evidence that cationic liposomes activate several cellular pathways like pro-apoptotic and pro-inflammatory cascades. An improved knowledge of the relationship between the cationic lipid properties (nature of the lipid hydrophilic moieties, hydrocarbon tail, mode of organization) and the activation of these pathways opens the way to the use and design of cationic tailored for a specific application (e.g. for gene transport or as adjuvants).

DiC14-amidine cationic liposomes stimulate myeloid dendritic cells through Toll-like receptor 4.

DiC14‐amidine cationic liposomes were recently shown to promote Th1 responses when mixed with allergen. To further define the mode of action of diC14‐amidine as potential vaccine adjuvant, we characterized its effects on mouse and human myeloid dendritic cells (DC). First, we observed that, as compared with two other cationic liposomes, only diC14‐amidine liposomes induced the production of IL‐12p40 and TNF‐α by mouse bone marrow‐derived DC. DiC14‐amidine liposomes also activated human DC, as shown by synthesis of IL‐12p40 and TNF‐α, accumulation of IL‐6, IFN‐β and CXCL10 mRNA, and up‐regulation of membrane expression of CD80 and CD86. DC stimulation by diC14‐amidine liposomes was associated with activation of NF‐κB, ERK1/2, JNK and p38 MAP kinases. Finally, we demonstrated in mouse and human cells that diC14‐amidine liposomes use Toll‐like receptor 4 to elicit both MyD88‐dependent and Toll/IL‐1R‐containing adaptor inducing interferon IFN‐β (TRIF)‐dependent responses.

Lipid mixing between lipoplexes and plasma lipoproteins is a major barrier for intravenous transfection mediated by cationic lipids.

It has been previously shown that transfection activity of cationic liposome/DNA lipoplexes delivered systemically is drastically inhibited by lipoproteins (Tandia, B. M., Vandenbranden, M., Wattiez, R., Lakhdar, Z., Ruysschaert, J. M., and Elouahabi, A. (2003) Mol Ther. 8, 264–273). In this work, we have compared the binding/uptake and transfection activities of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) and diC14-amidine (3-tetradecylamino-N-tert-butyl-N′-tetra-decylpropionamidine)-containing lipoplexes in the presence or absence of purified low density lipoproteins and high density lipoprotein. Binding/uptake of both lipoplexes by the mouse lung endothelial cell line was inhibited to a similar extent in the presence of lipoproteins. In contrast, transfection activity of diC14-amidine-containing lipoplexes was almost completely inhibited (approximately by 95%), whereas ∼40% transfection activity of DOTAP-containing lipoplexes was preserved in the presence of lipoproteins. Interestingly, the ability of lipoproteins to inhibit the transfection efficiency of lipoplexes was well correlated with their ability to undergo lipid mixing with the cationic lipid bilayer as revealed by fluorescence resonance energy transfer assay. Incubation of lipoplexes with increased doses of lipoproteins resulted in enhanced lipid mixing and reduced transfection activity of the lipoplexes in mouse lung endothelial cells. The role of lipid mixing in transfection was further demonstrated using lipid-mixing inhibitor, lyso-phosphatidylcholine, or activator (dioleoylphosphatidylethanolamine). Incorporation of Lyso-PC into diC14-amidine-containing lipoplexes completely abolished their capacity to undergo lipid mixing with lipoproteins and allowed them to reach a high transfection efficiency in the presence of lipoproteins. On the other hand, the incorporation of dioleoylphosphatidylethanolamine into DOTAP/DNA lipoplex activated lipid mixing with the lipoproteins and was shown to be detrimental toward the transfection activity of these lipoplexes. Taken together, these results indicate that fusion of lipoplexes with lipoproteins is a limiting factor for in vivo transfection.

Activation of innate immunity by lysozyme fibrils is critically dependent on cross-β sheet structure.

Inflammation occurs in many amyloidoses, but its underlying mechanisms remain enigmatic. Here we show that amyloid fibrils of human lysozyme, which are associated with severe systemic amyloidoses, induce the secretion of pro-inflammatory cytokines through activation of the NLRP3 (NLR, pyrin domain containing 3) inflammasome and the Toll-like receptor 2, two innate immune receptors that may be involved in immune responses associated to amyloidoses. More importantly, our data clearly suggest that the induction of inflammatory responses by amyloid fibrils is linked to their intrinsic structure, because the monomeric form and a non-fibrillar type of lysozyme aggregates are both unable to trigger cytokine secretion. These lysozyme species lack the so-called cross-β structure, a characteristic structural motif common to all amyloid fibrils irrespective of their origin. Since fibrils of other bacterial and endogenous proteins have been shown to trigger immunological responses, our observations suggest that the cross-β structural signature might be recognized as a generic danger signal by the immune system.

Role of lipid microdomains in TLR-mediated signalling

Over the last twenty years, evidence has been provided that the plasma membrane is partitioned with microdomains, laterally mobile in the bilayer, providing the necessary microenvironment to specific membrane proteins for signalling pathways to be initiated. We discuss here the importance of such microdomains for Toll-like receptors (TLR) localization and function. First, lipid microdomains favour recruitment and clustering of the TLR machinery partners, i.e. receptors and co-receptors previously identified to be required for ligand recognition and signal transmission. Further, the presence of the so-called Cholesterol Recognition Amino-Acid Consensus (CRAC) sequences in the intracellular juxtamembrane domain of several Toll-like receptors suggests a direct role of cholesterol in the activation process. This article is part of a Special Issue entitled: Lipid-protein interactions.

Cationic lipid nanocarriers activate Toll-like receptor 2 and NLRP3 inflammasome pathways

UNLABELLED We provide evidence that cationic lipids, usually considered as a safe alternative to viral vectors as nanocarriers for gene therapy or drug intracellular delivery, do not behave as inert material but do activate cellular signalling pathways implicated in inflammatory reactions. We show here that the cationic lipid RPR206252 induces NF-κB activation, and the production of TNF-α, IL-1β, IL-6 and IFN-γ by human or mouse macrophage cell lines. Further, we demonstrate that the activation of inflammatory cascades by RPR206252 is dependent on Toll-like receptor 2 (TLR2), the natural sensor of bacterial lipopeptides and NOD-like receptor protein 3 (NLRP3), the major inflammasome component. Our results suggest that cationic lipid nanocarriers because of their ability to stimulate the innate system can be used as a new class of synthetic and safe adjuvant for vaccination. FROM THE CLINICAL EDITOR Cationic lipid nanocarriers are typically considered neutral tools for gene delivery. However, as demonstrated in this study, they possess a clear ability to stimulate the innate immune system, and actually can be used as a new class of synthetic and safe adjuvant for vaccination.

The cationic lipid, diC14 amidine, extends the adjuvant properties of aluminum salts through a TLR-4- and caspase-1-independent mechanism.

Adjuvant efficiency is critical for inducing a protective and long-lasting immune response against weak immunogenic antigens. Discovered more than 70 years ago, aluminum salts remain the most widely used adjuvant in human vaccine. Prone to induce a strong humoral response, alum fails to drive a cell-mediated immunity, which is essential to fight against intracellular pathogens. Adjuvant systems that contain more than one component may represent an excellent alternative for completing the lack of T cell immunity associated with the injection of alum-based vaccine. In this work, we demonstrated that the adjuvant effects of alum strongly benefited from combining with a cationic lipid, the diC14 amidine. Indeed, we measured a significant improvement of alum-driven IL-1β release when human macrophages were co-cultured with a mixed suspension of alum and the diC14 amidine. Morphological analysis suggested that diC14 amidine improved the alum uptake by phagocytes. Furthermore, the addition of diC14 amidine to alum efficiently enhanced antigen processing and cross-presentation by antigen presenting cells. The biological relevance of these in vitro data was assessed by measuring the in vivo development of a cytotoxic activity and the enhanced synthesis of antigen-specific immunoglobulins after immunization with alum combined to diC14 amidine. Mechanistically, we demonstrated that diC14 amidine supported the alum adjuvanticity independently of the TLR-4 and caspase-1 agonist activities of the cationic lipid. Based on our findings, we conclude that diC14 amidine works synergistically with alum to achieve higher immune protection after vaccination.

Fusogenic activity of cationic lipids and lipid shape distribution

Addition of co-lipids into cationic lipid formulations is considered as promoting cell delivery of DNA by enhancing fusion processes with cell membranes. Here, by combining FRET and confocal microscopy, we demonstrate that some cationic lipids do not require a co-lipid to fuse efficiently with cells. These cationic lipids are able to self-organize into bilayers that are stable enough to form liposomes, while presenting some destabilizing properties reminiscent of the conically shaped fusogenic co-lipid, DOPE. We therefore analyzed the resident lipid structures in cationic bilayers by molecular dynamics simulations, clustering the individual lipid structures into populations of similarly shaped molecules, as opposed to the classical approach of using the static packing parameter to define the lipid shapes. Comparison of fusogenic properties with these lipid populations suggests that the ratio of cylindrical versus conical lipid populations correlates with the ability to fuse with cell membranes.

Cationic lipids activate cellular cascades. Which receptors are involved

Cationic lipids have been extensively used as carriers of biologically active molecules (nucleic acids, peptides and proteins) into cells. Recent data provided evidence that cationic lipids are not just inert transporters but do activate specific cellular cascades. This review illustrates these activating properties with a few examples. Cell activation raises the question of which receptors are involved. Some cationic lipids seem to satisfy specific structural requirements of Toll-like receptors (TLR4) as they activate TLR4-dependent pathways. However, cationic lipids display a large structural diversity and it is likely that they are also recognized by receptors with a broader specificity. Alternatives are proposed and discussed to explain this broad specificity.