Reconstitution of immune cell interactions in free-standing membranes.

The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. Therapeutic control of immune responses therefore relies on thorough elucidation of the molecular processes occurring at these interfaces. However, the detailed investigation of each component's contribution to the formation and regulation of the contact is hampered by the complexities of cell composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially in the use of advanced imaging technology. One approach that circumvents these problems is to establish in vitro systems that faithfully mimic immune cell interactions, but allow complexity to be 'dialled-in' as needed. Here, we present an in vitro system that makes use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we began to explore the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and how this changes during the initiation of signalling. The GUV/cell system presented here is expected to be widely applicable.

In vivo induction of neutrophil extracellular traps by Mycobacterium tuberculosis in a guinea pig model.

In 2004, a novel mechanism of cellular death, called 'NETosis', was described in neutrophils. This mechanism, different from necrosis and apoptosis, is characterized by the release of chromatin webs admixed with microbicidal granular proteins and peptides (NETs). NETs trap and kill a variety of microorganisms. Diverse microorganisms, including Mycobacterium tuberculosis, are NET inducers in vitro. The aim of this study was to examine whether M. tuberculosis can also induce NETs in vivo and if the NETs are bactericidal to the microorganism. Guinea pigs were intradermally inoculated with M. tuberculosis H37Rv, and the production of NETs was investigated at several time points thereafter. NETs were detected as early as 30 min post-inoculation and were clearly evident by 4 h post-inoculation. NETs produced in vivo contained DNA, myeloperoxidase, elastase, histones, ROS and acid-fast bacilli. Viable and heat-killed M. tuberculosis, as well as Mycobacterium bovis BCG were efficient NET inducers, as were unilamellar liposomes prepared with lipids from M. tuberculosis. In vitro, guinea pig neutrophils also produced NETs in response to M. tuberculosis. However, neither the in vivo nor the in vitro-produced NETs were able to kill M. tuberculosis. Nevertheless, in vivo, neutrophils might propitiate recruitment and activation of more efficient microbicidal cells.

Liposomal constructs for antitumoral vaccination by the nasal route.

Mucosal surfaces are promising routes for vaccination. Among mucosa, airway mucosa provides the opportunity to develop non-invasive approaches for vaccine delivery. In the current study, nasal route was used to investigate the potency of highly versatile di-epitopic liposomal constructs of different size, structure and composition to exhibit antitumor efficiency after prophylactic vaccination in mice. Well-characterized small unilamellar (SUV), multilamellar (MLV), reverse-phase evaporation (REV) and ultraflexible small unilamellar vesicles (Uf-SUV), containing the ErbB2 T-cytotoxic epitope, the influenza-derived HA T-helper epitope and the lipopeptide adjuvant Pam2CAG, were formulated. These vaccines were administered into the nasal cavity of BALB/c mice, followed by i.v. or s.c. implantation of ErbB2-surexpressing cancer cells. Nasal vaccination with the SUV vaccine resulted in an efficient antitumor activity against lung tumors and a non-significant protection against s.c. tumors. Size, structure and flexibility of liposomes did not impact vaccine immunity and antitumoral efficiency against lung tumors, in contrast to total dose of vaccine or dose of adjuvant. These results showed an undeniable interest of liposomes as lipid-based carriers for antitumor vaccine delivery by the nasal route, opening new perspectives for cancer treatment.

Human GII.4 norovirus VLP induces membrane invaginations on giant unilamellar vesicles containing secretor gene dependent α1,2-fucosylated glycosphingolipids.

Norovirus is a non-enveloped virus causing acute gastroenteritis. For human norovirus, no simple cell culture system is available and consequently knowledge on cellular entry of the virus is limited. The virus binds to ABH histo-blood group glycans on glycoproteins and glycosphingolipids. Non-secretors, characterized by the lack of ABH histo-blood group glycans in the gastrointestinal tract, are resistant to most norovirus infections, suggesting that these glycans may be part of the viral receptor. Recent studies have shown that polyomavirus enters the cell via membrane invaginations induced by the multivalent binding of the virus to receptor glycosphingolipids. In this study, we have investigated whether norovirus has the ability to induce membrane invaginations on giant unilamellar vesicles (GUVs) containing purified glycosphingolipids. First, we characterized the glycosphingolipid binding pattern of VLPs from the Dijon strain (genogroup II.4), using thin-layer chromatography. The VLP recognized the ABH active glycosphingolipids H type 1, Lewis b, B type 1, A type 1 and A Lewis b, but not lactotetraosylceramide or Lewis a, typically found in non-secretors. The binding pattern to glycosphingolipids incorporated into GUVs was in full agreement with the thin-layer chromatography experiments. Upon binding to the vesicles, the VLPs formed highly mobile clusters on the surface of the GUVs. VLP containing tubular invaginations were seen on the GUVs containing glycosphingolipids recognized by the VLP. In conclusion, this study suggests that human norovirus has the ability to induce membrane curvature by binding to and clustering glycosphingolipids, which may reflect the first step in cellular entry of the virus.

Molecular organization of the non-bilayer phospholipid arrangements that induce an autoimmune disease resembling human lupus in mice.

Non-bilayer phospholipid arrangements are three-dimensional structures that can form when anionic phospholipids with an intermediate form of the tubular hexagonal phase II (H(II)), such as phosphatidic acid, phosphatidylserine or cardiolipin, are present in a bilayer of lipids. The drugs chlorpromazine and procainamide, which trigger a lupus-like disease in humans, can induce the formation of non-bilayer phospholipid arrangements, and we have previously shown that liposomes with non-bilayer arrangements induced by these drugs cause an autoimmune disease resembling human lupus in mice. Here we show that liposomes with non-bilayer phospholipid arrangements induced by Mn²âº cause a similar disease in mice. We extensively characterize the physical properties and immunological reactivity of liposomes made of the zwitterionic lipid phosphatidylcholine and a H(II)-preferring lipid, in the absence or presence of Mn²âº, chlorpromazine or procainamide. We use an hapten inhibition assay to define the epitope recognized by sera of mice with the disease, and by a monoclonal antibody that binds specifically to non-bilayer phospholipid arrangements, and we report that phosphorylcholine and glycerolphosphorylcholine, which form part of the polar region of phosphatidylcholine, are the only haptens that block the binding of the tested antibodies to non-bilayer arrangements. We propose a model in which the negatively charged H(II)-preferring lipids form an inverted micelle by electrostatic interactions with the positive charge of Mn²âº, chlorpromazine or procainamide; the inverted micelle is inserted into the bilayer of phosphatidylcholine, whose polar regions are exposed and become targets for antibody production. This model may be relevant in the pathogenesis of human lupus.

Antibody binding to a tethered vesicle assembly using QCM-D.

The bilayer-tethered vesicle assembly has recently been proposed as a biomimetic model membrane platform for the analysis of integral membrane proteins. Here, we explore the binding of antibodies to membrane components of the vesicle assembly through the use of quartz crystal microbalance with dissipation monitoring (QCM-D). The technique provides a quantitative, label-free avenue to study binding processes at membrane surfaces. However, converting the signal generated upon binding to the actual amount of antibody bound has been a challenge for a viscoelastic system such as the tethered vesicle assembly. In this work, we first established an empirical relationship between the amount of bound antibody and the corresponding QCM-D response. Then, the results were examined in the context of an existing model describing the QCM-D response under a variety of theoretical loading conditions. As a model system, we investigated the binding of monoclonal antidinitrophenyl (DNP) IgG(1) to tethered vesicles displaying DNP hapten groups. The measured frequency and dissipation responses upon binding were compared to an independent measure of the amount of bound antibody obtained through the use of an in situ ELISA assay. At saturation, the surface mass density of bound antibody was approximately 900 ng/cm(2). Further, through the application of QCM-D models that describe the response of the quartz when loaded by either a single homogeneous viscoelastic film or by a two-layered viscoelastic film, we found that a homogeneous, one-layer model accurately predicts the amount of antibody bound to the tethered vesicles near antibody surface saturation, but a two-layer model must be invoked to accurately describe the kinetic response of the dissipation factor, which suggests that the binding of the antibody results in a stiffening of the top layer of the film.

[Experimental verification of a model of complement-dependent immune lysis of liposomes].

The quantitative dependences of the immune complement-dependent lysis of a monodisperse suspension of 200-nm liposomes whose membrane was sensitized by monovalent hapten (2,4-DNP-epsilon-caproyl-DPPE) or a polyvalent antigen (LPS from F. tularensis) on the initial concentration of specific antibodies (IgG) to hapten and the antigen have been investigated. The quantity of antibodies binding sites on the surface of liposome was evaluated. The difference between the complement-dependent immune lysis of poly- and monodisperse suspensions of liposomes was shown. The experimental results are well described by the direct binding model.

Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles.

Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion.

Electrostatic interactions and complement activation on the surface of phospholipid vesicle containing acidic lipids: effect of the structure of acidic groups.

Anionic vesicles containing acidic phospholipids are known complement activators. To clarify which negative physicochemical electrostatic charges on vesicles and structural specificities of acidic lipids are critical to complement activation, the electrostatic properties and activity to complement of two anionic vesicles modified with a carboxylic acid derivative or a conventional acidic phospholipid were compared. Electrophoretic mobility measurements indicated that the negative zeta potential and the electrostatic interactivity of these two anionic vesicles were equal at pH 7.4. However, the infusion of vesicles containing acidic phospholipid induced significant complement activation, while vesicles containing the carboxylic acid derivative failed to activate complement. These results indicate that the negative charge on the surface of vesicles is not critical for the activation complement, suggesting that complement activation is specific to the structure of acidic groups. This finding is likely to be important to the design of anionic biointerfaces and may support the promising medical applications of this anionic vesicle modified with a carboxylic acid derivative.