Targeted Delivery of Adamantylated Peptidoglycan Immunomodulators in Lipid Nanocarriers: NMR Shows That Cargo Fragments are Available on the Surface

We present an in-depth investigation of the membrane interactions of peptidoglycan (PGN)-based immune adjuvants designed for lipid-based delivery systems using NMR spectroscopy. The derivatives contain a cargo peptidoglycan (PGN) dipeptide fragment and an adamantyl group, which serves as an anchor to the lipid bilayer. Furthermore, derivatives with a mannose group that can actively target cell surface receptors on immune cells are also studied. We showed that the targeting mannose group and the cargo PGN fragment are both available on the lipid bilayer surface, thereby enabling interactions with cognate receptors. We found that the nonmannosylated compounds are incorporated stronger into the lipid assemblies than the mannosylated ones, but the latter compounds penetrate deeper in the bilayer. This might be explained by stronger electrostatic interactions available for zwitterionic nonmannosylated derivatives as opposed to the compounds in which the charged N-terminus is capped by mannose groups. The higher incorporation efficiency of the nonmannosylated compounds correlated with a larger relative enhancement in immune stimulation activities upon lipid incorporation compared to that of the derivatives with the mannose group. The chirality of the adamantyl group also influenced the incorporation efficiency, which in turn correlated with membrane-associated conformations that affect possible intermolecular interactions with lipid molecules. These findings will help in improving the development of PGN-based immune adjuvants suitable for delivery in lipid nanoparticles

The Lipopolysaccharide Core of Brucella abortus Acts as a Shield Against Innate Immunity Recognition

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines

Biological properties of extracellular vesicles and their physiological functions

María Yáñez-Mó#, Pia R.-M. Siljander#, Zoraida Andreu, Apolonija Bedina Zavec, Francesc E. Borràs, Edit I. Buzas, Krisztina Buzas, Enriqueta Casal, Francesco Cappello, Joana Carvalho, Eva Colás, Anabela Cordeiro-da Silva, Stefano Fais, Juan M. Falcon-Perez, Irene M. Ghobrial, Bernd Giebel, Mario Gimona, Michael Graner, Ihsan Gursel, Mayda Gursel, Niels H. H. Heegaard, An Hendrix30, Peter Kierulf, Katsutoshi Kokubun, Maja Kosanovic, Veronika Kralj-Iglic, Eva-Maria Krämer-Albers, Saara Laitinen, Cecilia Lässer, Thomas Lener, Erzsébet Ligeti, Aija Linē, Georg Lipps, Alicia Llorente, Jan Lötvall, Mateja Manček-Keber, Antonio Marcilla, Maria Mittelbrunn, Irina Nazarenko, Esther N.M. Nolte-‘t Hoen, Tuula A. Nyman, Lorraine O'Driscoll, Mireia Olivan, Carla Oliveira, Éva Pállinger, Hernando A. del Portillo, Jaume Reventós, Marina Rigau, Eva Rohde, Marei Sammar, Francisco Sánchez-Madrid, N. Santarém1, Katharina Schallmoser, Marie Stampe Ostenfeld, Willem Stoorvogel, Roman Stukelj, Susanne G. Van der Grein, M. Helena Vasconcelos, Marca H. M. Wauben and Olivier De WeverIn the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells.While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.Peer reviewe

Adamantane Containing Peptidoglycan Fragments Enhance RANTES and IL-6 Production in Lipopolysaccharide-Induced Macrophages

We report the enhancement of the lipopolysaccharide-induced immune response by adamantane containing peptidoglycan fragments in vitro. The immune stimulation was detected by Il-6 (interleukine 6) and RANTES (regulated on activation, normal T cell expressed and secreted) chemokine expression using cell assays on immortalized mouse bone-marrow derived macrophages. The most active compound was a α-D-mannosyl derivative of an adamantylated tripeptide with L-chirality at the adamantyl group attachment, whereby the mannose moiety assumed to target mannose receptors expressed on macrophage cell surfaces. The immune co-stimulatory effect was also influenced by the configuration of the adamantyl center, revealing the importance of specific molecular recognition event taking place with its receptor. The immunostimulating activities of these compounds were further enhanced upon their incorporation into lipid bilayers, which is likely related to the presence of the adamantyl group that helps anchor the peptidoglycan fragment into lipid nanoparticles. We concluded that the proposed adamantane containing peptidoglycan fragments act as co-stimulatory agents and are also suitable for the preparation of lipid nanoparticle-based delivery of peptidoglycan fragments

Combination of Antimicrobial and Endotoxin-Neutralizing Activities of Novel Oleoylamines

A combination of antimicrobial and endotoxin-neutralizing activities is desired in order to prevent progression from infection to sepsis due to the release of lipopolysaccharide from dying gram-negative bacteria. Lipopolyamines have emerged as a new type of endotoxin-neutralizing compound, but their antimicrobial activity has not been investigated. We synthesized a series of 10 oleoylamines differing in the polyamino head group, particularly in the number and separation between nitrogen atoms and the position of the oleoyl moiety. Compounds showed activity against both gram-negative and gram-positive bacteria in the micromolar range. Compounds were able to provide penetration of ethidium bromide into bacteria, indicating effects on the bacterial membrane. Oleoylamines neutralized endotoxin in Limulus amoebocyte lysate assays and by neutralization of tumor necrosis factor alpha release in human blood. Comparison of biological activities of compounds identified structural properties responsible for antimicrobial activity, and quantitative structure-property relationship analysis provided a quantitative model for prediction of activity of oleoylamines

Vanadate from air pollutant inhibits hrs-dependent endosome fusion and augments responsiveness to toll-like receptors.

There is a well-established association between exposure to air pollutants and pulmonary injuries. For example, metals found in ROFA (residual oil fly ash) increase susceptibility of mice as well as humans to microbial infections. In our research, we have found that vanadate substantially increased the response of several Toll-like receptors (TLRs) to stimulation with their ligands. Although vanadate caused generation of reactive oxygen species (ROS), the addition of ROS scavenger N-acetyl cysteine (NAC) had no effect on augmented lipopolysaccharide (LPS) stimulation. We further showed that vanadate inhibits endosome fusion. This effect was determined by measuring the size of endosomes, NF-κB activity and TLR4 degradation in Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) overexpressed cells. Moreover, we identified the role of Hrs phosphorylation in these processes. Based on our findings, we can conclude that vanadate potentiates TLR4 activity by increasing Hrs phosphorylation status, reducing the size of Hrs/TLR4-positive endosomes and impacting TLR4 degradation, thus contributing to the detrimental effects of air pollutants on human health

Vanadate reduced the size of endosomes and compensated for the inhibitory effect of Hrs on LPS stimulation.

<p><b>A.</b> HEK293 expressing TLR4/MD-2/CD14 were prestimulated for 10 min with Na₃VO₄ (500 µM) or bafilomycin (0.15 µM) and/or stimulated for 16 h with LPS (100 ng/ml). <b>B.</b> HEK293 expressing TLR4/MD-2/CD14 and/or Hrs were prestimulated for 10 min with Na₃VO₄ (500 µM) and/or stimulated for 16 h with LPS (100 ng/ml). A dual luciferase test for NF-κB activity was performed. Representative examples of two (A) and three (B) independent experiments are shown.</p

Vanadate increased kinase phosphorylation, inhibited TLR4 degradation after LPS stimulation and Hrs Y329/334F mutations may explain the process.

<p>RAW264.7 cells were stimulated in a timeframe between 0 to 120 minutes with Na₃VO₄ (2 mM) (<b>A.</b>), LPS (100 ng/ml) (<b>B.</b>) or both (<b>C.</b>). Cells were lysed, and the amount of phosphorylated p44/42 (P-p44/42) was detected. Anti-tubulin Abs were used as a control for equal protein loading. <b>D.</b> HEK293 cells stably expressing TLR4-CFP/MD-2 and transfected with HrsY329/334F-EGFP were prestimulated for 10 min with Na₃VO₄ (500 µM) and/or stimulated for 1 h with LPS-Cy5 (2 µg/ml). Cells were fixed and confocal imaging was performed. Hrs-positive endosome sizes were measured. The differences were not significant. <b>E.</b> HEK293 cells stably expressing TLR4-CFP/MD-2 and transfected with Hrs were pretreated with cycloheximide (80 µg/ml) for 30 minutes and prestimulated for 10 min with Na₃VO₄ (500 µM) and stimulated with LPS (100 ng/ml). After 3 h cells were lysed, 20 µg of proteins were loaded, and TLR4-CFP was detected on WB using anti-GFP Abs; the intensities of bands were normalized using anti-tubulin Abs. Representative examples of three (A–C and E) and one (D) independent experiments are shown.</p