Extracellular Vesicles from a Helminth Parasite Suppress Macrophage Activation and Constitute an Effective Vaccine for Protective Immunity

Recent studies have demonstrated that many parasites release extracellular vesicles (EVs), yet little is known about the specific interactions of EVs with immune cells or their functions during infection. We show that EVs secreted by the gastrointestinal nematode Heligmosomoides polygyrus are internalized by macrophages and modulate their activation. EV internalization causes downregulation of type 1 and type 2 immune-response-associated molecules (IL-6 and TNF, and Ym1 and RELMα) and inhibits expression of the IL-33 receptor subunit ST2. Co-incubation with EV antibodies abrogated suppression of alternative activation and was associated with increased co-localization of the EVs with lysosomes. Furthermore, mice vaccinated with EV-alum generated protective immunity against larval challenge, highlighting an important role in vivo. In contrast, ST2-deficient mice are highly susceptible to infection, and they are unable to clear parasites following EV vaccination. Hence, macrophage activation and the IL-33 pathway are targeted by H. polygyrus EVs, while neutralization of EV function facilitates parasite expulsion

A preliminary proteomic characterisation of extracellular vesicles released by the ovine parasitic nematode, Teladorsagia circumcincta

AbstractTeladorsagia circumcincta is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The development of high levels of anthelmintic resistance in this nematode species challenges its future control. Recent research indicates that many parasite species release extracellular vesicles into their environment, many of which have been classified as endocytic in origin, termed exosomes. These vesicles are considered to play important roles in the intercellular communication between parasites and their hosts, and thus represent potentially useful targets for novel control strategies. Here, we demonstrate that exosome-like extracellular vesicles can be isolated from excretory-secretory (ES) products released by T. circumcincta fourth stage larvae (Tci-L4ES). Furthermore, we perform a comparative proteomic analysis of vesicle-enriched and vesicle-free Tci-L4ES. Approximately 73% of the proteins identified in the vesicle-enriched fraction were unique to this fraction, whilst the remaining 27% were present in both vesicle-enriched and vesicle-free fraction. These unique proteins included structural proteins, nuclear proteins, metabolic proteins, proteolytic enzymes and activation-associated secreted proteins. Finally, we demonstrate that molecules present within the vesicles-enriched material are targets of the IgA and IgG response in T. circumcincta infected sheep, and could potentially represent useful targets for future vaccine intervention studies

Caveolin-1 Influences LFA-1 Redistribution upon TCR Stimulation in CD8 T Cells

TCR stimulation by peptide-MHC complexes on APCs requires precise reorganization of molecules into the area of cellular contact to form an immunological synapse from where T cell signaling is initiated. Caveolin (Cav) 1, a widely expressed transmembrane protein, is involved in the regulation of membrane composition, cellular polarity and trafficking, and the organization of signal transduction pathways. The presence of Cav1 protein in T cells was identified only recently, and its function in this context is not well understood. We show that Cav1-knockout CD8 T cells have a reduction in membrane cholesterol and sphingomyelin, and upon TCR triggering they exhibit altered morphology and polarity, with reduced effector function compared with Cav1 wild-type CD8 T cells. In particular, redistribution of the b2 integrin LFA-1 to the immunological synapse is compromised in Cav1-knockout T cells, as is the ability of LFA-1 to form high-avidity interactions with ICAM-1. Our results identify a role for Cav1 in membrane organization and b2 integrin function in primary CD8 T cells.Peer reviewe

Jaspine B induces nonapoptotic cell death in gastric cancer cells independently of its inhibition of ceramide synthase

Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.This work was supported by the Ministerio de Ciencia e Innovaci?n (Project CTQ2014-54743-R) and a predoctoral contract from Generalitat de Catalunya to F.C. A.H.F. is the Joseph Meyerhoff Professor of Biochemistry at the Weizmann Institute of Science. The authors thank Prof. R. Ghidoni and Dr. V. Gagliostro (University of Milan) for preliminary LC3-II data and Prof. A. Delgado for helpful discussions. The authors also thank E. Dalmau for excellent technical assistance.Peer reviewe

Derivados de animoetanol sustituidos en C2 y su uso como antitumorales

La presente invención proporciona un grupo de compuestos con capacidad antitumoral a través de la inhibición de la ceramidasa ácida y la dihidroceramida desaturasa para provocar un aumento de los niveles de dihidroceramidas celulares, así como a una composición farmacéutica que comprende dichos compuestos y al uso de los mismos para la fabricación de un medicamento, y más particularmente un medicamento para el tratamiento de ciertos tipos de cáncer.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de BarcelonaA1 Solicitud de patente con informe sobre el estado de la técnic

Acid sphingomyelinase determines melanoma progression and metastatic behaviour via the microphtalmia-associated transcription factor signalling pathway

Melanoma is a rapidly growing and highly metastatic cancer with high mortality rates, for which a resolutive treatment is lacking. Identification of novel therapeutic strategies and biomarkers of tumour stage is thus of particular relevance. We report here on a novel biomarker and possible candidate therapeutic target, the sphingolipid metabolising enzyme acid sphingomyelinase (A- SMase). A- SMase expression correlates inversely with tumour stage in human melanoma biopsies. Studies in a mouse model of melanoma and on cell lines derived from mouse and human melanomas demonstrated that A- SMase levels of expression actually determine the malignant phenotype of melanoma cells in terms of pigmentation, tumour progression, invasiveness and metastatic ability. The action of A- SMase is mediated by the activation of the extracellular signal-regulated kinase, the subsequent proteasomal degradation of the Microphtalmia-associated transcription factor (Mitf) and inhibition of cyclin-dependent kinase 2, Bcl-2 and c-Met, downstream targets of Mitf involved in tumour cell proliferation, survival and metastatisation

Regulation of CC Ligand 5/RANTES by Acid Sphingomyelinase and Acid Ceramidase

Acid sphingomyelinase (aSMase) generates the bioactive lipid ceramide (Cer) from hydrolysis of sphingomyelin (SM). However, its precise roles in regulating specific sphingolipid-mediated biological processes remain ill defined. Interestingly, the aSMase gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase) via alternative trafficking of a shared protein precursor. Previously, our laboratory identified Ser508 as a crucial residue for the constitutive and regulated secretion of S-SMase in response to inflammatory cytokines, and demonstrated a role for S-SMase in formation of select cellular Cer species (Jenkins, R. W., Canals, D., Idkowiak-Baldys, J., Simbari, F., Roddy, P., Perry, D. M., Kitatani, K., Luberto, C., and Hannun, Y. A. (2010) J. Biol. Chem. 285, 35706–35718). In the present study using a chemokine/cytokine screen, we identified the chemokine CCL5 (formerly known as RANTES) as a candidate-specific downstream target for aSMase. Regulation of CCL5 by aSMase was subsequently validated using both loss-of-function and gain-of-function models indicating that aSMase is both necessary and sufficient for CCL5 production. Interestingly, cells deficient in acid ceramidase (aCDase) also exhibited defects in CCL5 induction, whereas cells deficient in sphingosine kinase-1 and -2 exhibited higher levels of CCL5, suggesting that sphingosine and not sphingosine 1-phosphate (S1P) is responsible for the positive signal to CCL5. Consistent with this, co-expression of aSMase and aCDase was sufficient to strongly induce CCL5. Taken together, these data identify a novel role for aSMase (particularly S-SMase) in chemokine elaboration by pro-inflammatory cytokines and highlight a novel and shared function for aSMase and aCDase.Special thanks to the Medical University of South Carolina Analytical Lipidomics Core (Dr. Jacek Bielawski, Barbara Rembiesa, Justin Snider, and Jason Pierce) and the Medical University of South Carolina Synthetic Lipidomics Core (Dr. Alicja Bielawska and Nalini Mayroo). We also express thanks to Dr. Leah Siskind (Medical University of South Carolina) for careful reading of this manuscript.Peer reviewe