We analyzed the transcriptional signatures of mouse bone marrow-derived macrophages at different times after infection with promastigotes of the protozoan parasite Leishmania major. Ingenuity Pathway Analysis revealed that the macrophage metabolic pathways including carbohydrate and lipid metabolisms were among the most altered pathways at later time points of infection. Indeed, L. major promastiogtes induced increased mRNA levels of the glucose transporter and almost all of the genes associated with glycolysis and lactate dehydrogenase, suggesting a shift to anaerobic glycolysis. On the other hand, L. major promastigotes enhanced the expression of scavenger receptors involved in the uptake of Low-Density Lipoprotein (LDL), inhibited the expression of genes coding for proteins regulating cholesterol efflux, and induced the synthesis of triacylglycerides. These data suggested that Leishmania infection disturbs cholesterol and triglycerides homeostasis and may lead to cholesterol accumulation and foam cell formation. Using Filipin and Bodipy staining, we showed cholesterol and triglycerides accumulation in infected macrophages. Moreover, Bodipy-positive lipid droplets accumulated in close proximity to parasitophorous vacuoles, suggesting that intracellular L. major may take advantage of these organelles as high-energy substrate sources. While the effect of infection on cholesterol accumulation and lipid droplet formation was independent on parasite development, our data indicate that anaerobic glycolysis is actively induced by L. major during the establishment of infection.

Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source.

Extracellular vesicles (EVs) emerge as essential mediators of intercellular communication. DNA vaccines encoding antigens presented on EVs efficiently induce T-cell responses and EV-based vaccines containing the Spike (S) proteins of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) are highly immunogenic in mice. Thus, EVs may serve as vaccine platforms against emerging diseases, going beyond traditional strategies, with the antigen displayed identically to the original protein embedded in the viral membrane and presented as such to the immune system. Compared to their viral and pseudotyped counterparts, EV-based vaccines overcome many safety issues including pre-existing immunity against these vectors. Here, we applied our technology in natural EV's engineering, to express the S proteins of SARS-CoV-2 embedded in the EVs, which mimic the virus with its fully native spikes. Immunizations with a two component CoVEVax vaccine, comprising DNA vector (DNAS-EV) primes, allowing in situ production of Spike harbouring EVs, and a boost using S-EVs produced in mammalian cells, trigger potent neutralizing and cellular responses in mice, in the absence of any adjuvants. CoVEVax would be the prototype of vaccines, where the sole exchange of the envelope proteins on EVs leads to the generation of new vaccine candidates against emerging viruses.

BackgroundLeishmania are obligated intracellular pathogens that replicate almost exclusively in macrophages. The outcome of infection depends largely on parasite pathogenicity and virulence but also on the activation status and genetic background of macrophages. Animal models are essential for a better understanding of pathogenesis of different microbes including Leishmania.ResultsHere we compared the transcriptional signatures of resistant (C57BL/6) and susceptible (BALB/c) mouse bone marrow-derived macrophages in response to Leishmania major (L. major) promastigotes infection.Microarray results were first analyzed for significant pathways using the Kyoto Encylopedia of Genes and Genomes (KEGG) database. The analysis revealed that a large set of the shared genes is involved in the immune response and that difference in the expression level of some chemokines and chemokine receptors could partially explain differences in resistance. We next focused on up-regulated genes unique to either BALB/c or C57BL/6 derived macrophages and identified, using KEGG database, signal transduction pathways among the most relevant pathways unique to both susceptible and resistant derived macrophages. Indeed, genes unique to C57BL/6 BMdMs were associated with target of rapamycin (mTOR) signaling pathway while a range of genes unique to BALB/c BMdMs, belong to p53 signaling pathway. We next investigated whether, in a given mice strain derived macrophages, the different up-regulated unique genes could be coordinately regulated. Using GeneMapp Cytoscape, we showed that the induced genes unique to BALB/c or C57BL/6 BMdMs are interconnected. Finally, we examined whether the induced pathways unique to BALB/c derived macrophages interfere with the ones unique to C57BL/6 derived macrophages. Protein-protein interaction analysis using String database highlights the existence of a cross-talk between p53 and mTOR signaling pathways respectively specific to susceptible and resistant BMdMs.ConclusionsTaken together our results suggest that strains specific pathogenesis may be due to a difference in the magnitude of the same pathways and/or to differentially expressed pathways in the two mouse strains derived macrophages. We identify signal transduction pathways among the most relevant pathways modulated by L. major infection, unique to BALB/c and C57BL/6 BMdM and postulate that the interplay between these potentially interconnected pathways could direct the macrophage response toward a given phenotype.

Exosomes are small membrane vesicles that are released into the extracellular compartment as a consequence of fusion of multivesicular endosomes with the plasma membrane. To unravel the molecular basis of protein sorting into exosomes, we have made a chimeric protein containing the cytosolic domain of the transmembrane subunit of the viral Env protein of BLV and the ectodomain of CD8 (CDTM-BLV-CD8). When expressed in K562 cells known to constitutively secrete exosomes, the chimera was found to be very efficiently targeted to the released vesicles. Very interestingly, the cytosolic domain of the Env protein contains peptide motifs potentially recognized by components of the ESCRT machinery that could be related to chimera sorting into the vesicles. Then, quantifying the chimera secretion, we investigated the site of exosome biogenesis in K562 cells using a pharmacological approach. We present different arguments indicating that CDTM-BLV-CD8-containing exosomes are likely formed from a recycling endosomal/TGN compartment.