Human T-cell leukemia virus-1 (HTLV-1) is a neglected and incurable retrovirus estimated to infect 5 to 10 million worldwide. Specific indigenous Australian populations report infection rates of more than 40%, suggesting a potential evolution of the virus with global implications. HTLV-1 causes adult T-cell leukemia/lymphoma (ATLL), and a neurological disease named HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Even though HTLV-1 transmission primarily occurs from cell-to-cell, there is still a gap of knowledge regarding the mechanisms of viral spread and disease progression. We have recently shown that Extracellular Vesicles (EVs) ubiquitously produced by cells may be used by HTLV-1 to transport viral proteins and RNA, and elicit adverse effects on recipient uninfected cells. The viral proteins Tax and HBZ are involved in disease progression and impairment of autophagy in infected cells. Here, we show that activation of HTLV-1 via ionizing radiation (IR) causes a significant increase of intracellular Tax, but not EV-associated Tax. Also, lower density EVs from HTLV-1-infected cells, separated by an Iodixanol density gradient, are positive for gp61+++/Tax+++/HBZ+ proteins (HTLV-1 EVs). We found that HTLV-1 EVs are not infectious when tested in multiple cell lines. However, these EVs promote cell-to-cell contact of uninfected cells, a phenotype which was enhanced with IR, potentially promoting viral spread. We treated humanized NOG mice with HTLV-1 EVs prior to infection and observed an increase in viral RNA synthesis in mice compared to control (EVs from uninfected cells). Proviral DNA levels were also quantified in blood, lung, spleen, liver, and brain post-treatment with HTLV-1 EVs, and we observed a consistent increase in viral DNA levels across all tissues, especially the brain. Finally, we show direct implications of EVs in viral spread and disease progression and suggest a two-step model of infection including the release of EVs from donor cells and recruitment of recipient cells as well as an increase in recipient cell-to-cell contact promoting viral spread.

The dengue virus (DENV), transmitted by Aedes spp. mosquitoes, is one of the most important arboviral infections in the world. Dengue begins as a febrile condition, and in certain patients, it can evolve severe clinical outcomes, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The reasons why certain patients develop DHF or DSS have not been thoroughly elucidated to date, and both patient and viral factors have been implicated. Previous work has shown that a severe immune dysfunction involving dendritic cells and T cells plays a key role in increasing the disease severity, especially in secondary heterologous infections. Extracellular vesicles (EVs) are membranous particles that are secreted by several cell types involved in homeostatic and pathological processes. Secretion of EVs by infected cells can enhance immune responses or favor viral evasion. In this study, we compare the molecular content of EVs that are secreted by human primary dendritic cells under different conditions: uninfected or infected with DENV3 strains isolated from patients with different infection phenotypes (a severe case involving DSS and a mild case). Human monocyte-derived dendritic cells (mdDCs) were infected with the dengue virus strains DENV3 5532 (severe) or DENV3 290 (mild), and the EVs were isolated. The presence of cup-shaped EVs was confirmed by electron microscopy and immunostaining with CD9, CD81, and CD83. The RNA content from the mdDC-infected cells contained several mRNAs and miRNAs related to immune responses compared to the EVs from mock-infected mdDCs. A number of these RNAs were detected exclusively during infection with DENV3 290 or DENV3 5532. This result suggests that the differential immune modulation of mdDCs by dengue strains can be achieved through the EV pathway. Additionally, we observed an association of EVs with DENV-infectious particles that seem to be protected from antibodies targeting the DENV envelope protein. We also showed that EVs derived from cells treated with IFN alpha have a protective effect against DENV infection in other cells. These results suggested that during DENV infection, the EV pathway could be exploited to favor viral viability, although immune mechanisms to counteract viral infection can also involve DC-derived EVs.

"Probiotic therapy" to regulate gut microbiota and intervene in intestinal diseases such as inflammatory bowel disease (IBD) has become a research hotspot. Bacteroides acidifaciens, as a new generation of probiotics, has shown beneficial effects on various diseases.

Nano-sized extracellular vesicles (EVs), including exosomes, microvesicles, and other types of vesicles, are released by most mammalian cells and bacteria. We here ask whether feces contain EVs of mammalian and/or bacterial origin, and whether these EVs induce systemic inflammation. Fecal extracellular vesicles (fEVs) were isolated from mice and humans. The presence of EVs from Gram-negative and Gram-positive bacteria was detected by enzyme-linked immunosorbent assay using anti-lipid A and anti-lipoteichoic acid antibodies, whereas Western blot using anti-beta-actin antibody was employed to detect host-derived EVs in the fEVs. Further, fEVs were administered into mice by intraperitoneal injection, and inflammatory responses were investigated in the peritoneum, blood, and lungs. The role of TLR2 and TLR4 were studied using knockout mice. Significant quantities of EVs were present in feces from mice as well as humans, and derived from Gram-negative and Gram-positive bacteria, as well as the host. Bacteria-free fEVs introduced into the peritoneum induced local and systemic inflammation (including in the lungs), but fEVs from germ-free animals had weaker effects. This pronounced local and systemic inflammatory responses seemed to be induced by EVs from both Gram-negative and Gram-positive bacteria, and was attenuated in mice lacking TLR2 or TLR4. Our findings show that fEVs cause sepsis-like systemic inflammation, when introduced intraperitoneally, a process regulated by TLR2 and TLR4.

Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease. In the infected mice, emu-miR-4989-3p is present in sera, but its role remains unknown. Using high-throughput sequencing and qPCR, emu-miR-4989-3p was herein confirmed to be encapsulated into E. multilocularis extracellular vesicles. In the transfected macrophages, emu-miR-4989-3p was demonstrated to significantly inhibit NO production compared to the control (p < 0.05). Moreover, transfection of emu-miR-4989-3p also gave rise to the increased expression of TNF-α (p < 0.01). Furthermore, emu-miR-4989-3p induced the dysregulation of several key components in the LPS/TLR4 signaling pathway compared with the control, especially TLR4 and NF-κB that both were upregulated. Conversely, the NO production and the expression of TNF-α, TLR4 and NF-κB tended to be increased and decreased in the mimics-transfected cells upon emu-miR-4989-3p low expression, respectively. These results suggest that emu-miR-4989-3p is one of 'virulence' factors encapsulated into the extracellular vesicles, potentially playing a role in the pathogenesis of E. multilocularis.

Staphylococcus epidermidis causes infections associated with orthopedic implants due to its ability to establish persistent biofilms, making infections chronic and hard to treat. Extracellular vesicles (EVs) are part of the bacterial communication system, but the role of S. epidermidis-derived EVs in biofilm formation processes and survival is completely unknown. The aims of this study were (i) to investigate the effect of subinhibitory concentrations of antibiotics on vesiculation in S. epidermidis and evaluate the role of EVs in bacterial survival and adhesion under antimicrobial selective pressure and (ii) to evaluate whether EVs derived from a gentamicin-resistant S. epidermidis strain influence the susceptibility and adhesion of a gentamicin-susceptible strain. A gentamicin-susceptible (GEN S ) strain isolated from implant-associated osteomyelitis was cultured with EVs previously isolated from the same strain growing with subinhibitory concentrations of GEN (0, 0.03, and 0.06 μg × mL-1) or with EVs from a gentamicin-resistant (GEN R ) strain. EVs were characterized regarding their size, number and protein content. The growth of S. epidermidis cultured with increasing concentrations of GEN (<=> MIC of 0.12 μg × mL-1) was recorded, viability was determined by quantitative culturing and fluorescence staining, and biofilm biomass on polystyrene was quantified by crystal violet staining. Cells grown in subinhibitory concentrations of GEN produced a larger number of EVs of similar size but with greater protein content than cells grown in control (Ctrl) conditions (0 GEN). Under antimicrobial pressure, EVs promoted different mechanisms of antimicrobial tolerance depending on the EV and GEN concentrations. Cell adhesion to polystyrene decreased in the presence of 0 and 0.03 μg × mL-1 GEN upon EV stimulation. Compared with Ctrl cells, cells treated with EVs from a GEN R strain showed increased cell division during the exponential growth phase, faster maximal growth rate, shorter doubling time (8-33 min), and dramatically inhibited cell adhesion. These findings suggest that vesiculation in S. epidermidis is a survival response to subinhibitory concentrations of gentamicin. EVs may contribute to bacterial survival through their involvement (1) in the modulation of the growth rate, affecting cell division, and (2) in cell adhesion, decreasing cell attachment to polystyrene and glass.

Extracellular vesicles (EVs) has been considered an alternative process for intercellular communication. EVs release by filamentous fungi and the role of vesicular secretion during fungus-host cells interaction remain unknown. Here, we identified the secretion of EVs from the pathogenic filamentous fungus, Aspergillus fumigatus. Analysis of the structure of EVs demonstrated that A. fumigatus produces round shaped bilayer structures ranging from 100 to 200 nm size, containing ergosterol and a myriad of proteins involved in REDOX, cell wall remodeling and metabolic functions of the fungus. We demonstrated that macrophages can phagocytose A. fumigatus EVs. Phagocytic cells, stimulated with EVs, increased fungal clearance after A. fumigatus conidia challenge. EVs were also able to induce the production of TNF-α and CCL2 by macrophages and a synergistic effect was observed in the production of these mediators when the cells were challenged with the conidia. In bone marrow-derived neutrophils (BMDN) treated with EVs, there was enhancement of the production of TNF-α and IL-1β in response to conidia. Together, our results demonstrate, for the first time, that A. fumigatus produces EVs containing a diverse set of proteins involved in fungal physiology and virulence. Moreover, EVs are biologically active and stimulate production of inflammatory mediators and fungal clearance.

Extracellular vesicles are small vesicles with a diameter of 30-150 nm that are actively secreted by eukaryotic cells and play important roles in intercellular communication, immune responses, and tumorigenesis. Previous studies have shown that extracellular vesicles are involved in the process of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. However, changes in the protein content of extracellular vesicles elicited by S. Typhimurium infection have not been determined. Here, we extracted the extracellular vesicles with high purity from S. Typhimurium-infected Henle-407 cells, a kind of human intestinal epithelial cells, by ultracentrifugation combined with an extracellular vesicles purification kit, and analyzed their protein composition using label-free relative quantitative proteomics. The extracted extracellular vesicles exhibited an oval vesicular structure under electron microscopy, with a mean diameter of 140.4 ± 32.4 nm. The exosomal marker proteins CD9, CD63, and HSP70 were specifically detected. Compared with the uninfected group, nearly 1,234 specifically loaded proteins were uncovered in S. Typhimurium-infected Henle-407 cells. Among them were 409 S. Typhimurium-derived specific proteins, indicating a significant alteration in protein composition of extracellular vesicles by S. Typhimurium infection. Notably, these proteins included 75 secretory proteins and over 300 non-secretory proteins of S. Typhimurium, implicating novel pathways for bacterial protein delivery, although it remains unclear if their loading into extracellular vesicles is active or passive. To investigate the roles of these extracellular proteins, we exemplified the function of SopB, a well-known T3SS effector protein, and showed that the extracellular SopB could be taken up by RAW264.7 macrophages, activating the phosphorylation of Akt. This study provides new insights into the mechanism of Salmonella infection through extracellular vesicles that transport virulence proteins to uninfected neighboring cells to facilitate further infection.

Recent evidence suggests that probiotics can restore the mucosal barrier integrity, ameliorate inflammation, and promote homeostasis required for metabolism in obesity by affecting the gut microbiota composition. In this study, we investigated the effect of Akkermansia muciniphila and its extracellular vesicles (EVs) on obesity-related genes in microarray datasets and evaluated the cell line and C57BL/6 mice by conducting RT-PCR and ELISA assays. A. muciniphila-derived EVs caused a more significant loss in body and fat weight of high-fat diet (HFD)-fed mice, compared with the bacterium itself. Moreover, treatment with A. muciniphila and EVs had significant effects on lipid metabolism and expression of inflammatory markers in adipose tissues. Both treatments improved the intestinal barrier integrity, inflammation, energy balance, and blood parameters (i.e., lipid profile and glucose level). Our findings showed that A. muciniphila-derived EVs contain various biomolecules, which can have a positive impact on obesity by affecting the involved genes. Also, our results showed that A. muciniphila and its EVs had a significant relationship with intestinal homeostasis, which highlights their positive role in obesity treatment. In conclusion, A. muciniphila-derived EVs can be used as new therapeutic strategies to ameliorate HFD-induced obesity by affecting various mechanisms.

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a rapid and acute infection of the central nervous system with a fatal outcome in >97% of cases. Due to the infrequent report of cases and diagnostic gaps that hinder the possibility of recovering clinic isolates, studies related to pathogenesis of the disease are scarce. However, the secretion of cytolytic molecules has been proposed as a factor involved in the progression of the infection. Several of these molecules could be included in extracellular vesicles (EVs), making them potential virulence factors and even modulators of the immune response in this infection. In this work, we evaluated the immunomodulatory effect of EVs secreted by two clinic isolates of Naegleria fowleri using in vitro models. For this purpose, characterization analyses between EVs produced by both isolates were first performed, for subsequent gene transcription analyses post incubation of these vesicles with primary cultures from mouse cell microglia and BV-2 cells. Analyses of morphological changes induced in primary culture microglia cells by the vesicles were also included, as well as the determination of the presence of nucleic acids of N. fowleri in the EV fractions. Results revealed increased expression of NOS, proinflammatory cytokines IL-6, TNF-α, and IL-23, and the regulatory cytokine IL-10 in primary cultures of microglia, as well as increased expression of NOS and IL-13 in BV-2 cells. Morphologic changes from homeostatic microglia, with small cellular body and long processes to a more amoeboid morphology were also observed after the incubation of these cells with EVs. Regarding the presence of nucleic acids, specific Naegleria fowleri DNA that could be amplified using both conventional and qPCR was confirmed in the EV fractions. Altogether, these results confirm the immunomodulatory effects of EVs of Naegleria fowleri over microglial cells and suggest a potential role of these vesicles as biomarkers of primary acute meningoencephalitis.

Sexual Zika virus (ZIKV) transmission from men to women occurs less frequently than the often-detected high viral loads in semen would suggest, but worries that this transmission route predisposes to fetal damage in pregnant women remain. To better understand sexual ZIKV pathogenesis, we studied the permissiveness of the human female genital tract to infection and the effect of semen on this process. ZIKV replicates in vaginal tissues and primary epithelial cells from the vagina, ectocervix, and endocervix and induces an innate immune response, but also continues to replicate without cytopathic effect. Infection of genital cells and tissues is strongly inhibited by extracellular vesicles (EV) in semen at physiological vesicle-to-virus ratios. Liposomes with the same composition as semen EVs also impair infection, indicating that the EV's lipid fraction, rather than their protein or RNA cargo, is responsible for this anti-viral effect. Thus, EVs in semen potently restrict ZIKV transmission, but the virus propagates well once infection in the recipient mucosa has been established.

Extracellular vesicle-mediated transfer of microRNAs is a novel mode of cell-to-cell genetic transmission. Extracellular vesicles produced by microbes have been shown to contain significant quantities of physiologically active molecules such as proteins, lipids, and RNA, which could be transported to host cells and play a key role in both inter-kingdom signaling and physiological responses. In this study, we identified sRNAs by sequencing small RNAs (sRNAs) from Lactobacillus plantarum-derived extracellular vesicles (LDEVs) and detected the expression levels of vesicular sRNAs using quantitative reverse transcription-polymerase chain reaction (RT-PCR), which demonstrated the presence of microRNA-sized RNAs (msRNAs) within these vesicles. We chose sRNA71, a highly expressed msRNA, for further investigation, predicted its potential target genes for the human genome, and indicated that it could be translocated into mammalian cells. The biological functions of this sRNA71 were subsequently explored through cellular proteomics, western blot, and luciferase reporter assay. According to the findings, transfection with synthetic sRNA71 mimics substantially reduced Tp53 expression in HEK293T cells and suppressed the gene expression through binding to the 3' UTR of Tp53 mRNA. In conclusion, it is hypothesized that microbial-derived extracellular vesicles serve as carriers of functional molecules such as sRNAs, which play an essential role in regulating microbial-host communication.

Extracellular vesicles (EVs) loaded with proteins, nucleic acids, membrane lipids, and other virulence factors could participate in pathogenic processes in some fungi such as Cryptococcus neoformans and Candida albicans. However, the specific characteristics of EVs derived from Talaromyces marneffei (TM) still have not been figured out yet. In the present study, it has been observed that TM-derived EVs were a heterogeneous group of nanosized membrane vesicles (30-300 nm) under nanoparticle tracking analysis and transmission electron microscopy. The DiI-labeled EVs could be taken up by RAW 264.7 macrophage cells. Incubation of EVs with macrophages would result in increased expression levels of reactive oxygen species, nitric oxide, and some inflammatory factors including interleukin-1β, interleukin-6, interleukin-10, and tumor necrosis factor. Furthermore, the expression of co-stimulatory molecules (CD80, CD86, and MHC-II) was also increased in macrophages stimulated with EVs. The level of inflammatory factors secreted by macrophages showed a significant decrease when EVs were hydrolyzed by protease, while that of DNA and RNA hydrolase treatment remained unchanged. Subsequently, some virulence factors in EVs including heat shock protein, mannoprotein 1, and peroxidase were determined by liquid chromatography-tandem mass spectrometry. Taken together, our results indicated that the TM-derived EVs could mediate inflammatory response and its protein would play a key role in regulating the function of RAW 264.7 macrophage cells.

The complexity of the pathogenesis of inflammatory bowel disease (ulcerative colitis and Crohn's disease) has led to the quest of empirically drug therapies, combining immunosuppressant agents, biological therapy and modulators of the microbiota. Helminth parasites have been proposed as an alternative treatment of these diseases based on the hygiene hypothesis, but ethical and medical problems arise. Recent reports have proved the utility of parasite materials, mainly excretory/secretory products as therapeutic agents. The identification of extracellular vesicles on those secreted products opens a new field of investigation, since they exert potent immunomodulating effects. To assess the effect of extracellular vesicles produced by helminth parasites to treat ulcerative colitis, we have analyzed whether extracellular vesicles produced by the parasitic helminth Fasciola hepatica can prevent colitis induced by chemical agents in a mouse model. Adult parasites were cultured in vitro and secreted extracellular vesicles were purified and used for immunizing both wild type C57BL/6 and RAG1-/- mice. Control and immunized mice groups were treated with dextran sulfate sodium 7 days after last immunization to promote experimental colitis. The severity of colitis was assessed by disease activity index and histopathological scores. Mucosal cytokine expression was evaluated by ELISA. The activation of NF-kB, COX-2, and MAPK were evaluated by immunoblotting. Administration of extracellular vesicles from F. hepatica ameliorates the pathological symptoms reducing the amount of pro-inflammatory cytokines and interfering with both MAPK and NF-kB pathways. Interestingly, the observed effects do not seem to be mediated by T-cells. Our results indicate that extracellular vesicles from parasitic helminths can modulate immune responses in dextran sulfate sodium (DSS)-induced colitis, exerting a protective effect that should be mediated by other cells distinct from B- and T-lymphocytes.

Trichomonas vaginalis is a parasitic protist that infects the human urogenital tract. During the infection, trichomonads adhere to the host mucosa, acquire nutrients from the vaginal/prostate environment, and release small extracellular vesicles (sEVs) that contribute to the trichomonad adherence and modulate the host-parasite communication. Approximately 40-70% of T. vaginalis strains harbor a double-stranded RNA virus called Trichomonasvirus (TVV). Naked TVV particles have the potential to stimulate a proinflammatory response in human cells, however, the mode of TVV release from trichomonads to the environment is not clear. In this report, we showed for the first time that TVV particles are released from T. vaginalis cells within sEVs. The sEVs loaded with TVV stimulated a higher proinflammatory response of human HaCaT cells in comparison to sEVs from TVV negative parasites. Moreover, a comparison of T. vaginalis isogenic TVV plus and TVV minus clones revealed a significant impact of TVV infection on the sEV proteome and RNA cargo. Small EVs from TVV positive trichomonads contained 12 enriched and 8 unique proteins including membrane-associated BspA adhesine, and about a 2.5-fold increase in the content of small regulatory tsRNA. As T. vaginalis isolates are frequently infected with TVV, the release of TVV via sEVs to the environment represents an important factor with the potential to enhance inflammation-related pathogenesis during trichomoniasis.

Bacterial extracellular vesicles (BEVs) produced by gut commensal bacteria have been proposed to play an important role in maintaining host homeostasis via interactions with the immune system. Details of the mediators and pathways of BEV-immune cell interactions are however incomplete. In this study, we provide evidence for the anti-inflammatory and immunomodulatory properties of extracellular vesicles produced by the prominent human gut commensal bacterium Bacteroides thetaiotaomicron (Bt BEVs) and identify the molecular mechanisms underlying their interaction with innate immune cells. Administration of Bt BEVs to mice treated with colitis-inducing dextran sodium sulfate (DSS) ameliorates the symptoms of intestinal inflammation, improving survival rate and reducing weight loss and disease activity index scores, in association with upregulation of IL-10 production in colonic tissue and in splenocytes. Pre-treatment (conditioning) of murine bone marrow derived monocytes (BMDM) with Bt BEVs resulted in higher ratio of IL-10/TNFα production after an LPS challenge when compared to LPS pre-conditioned or non-conditioned BMDM. Using the THP-1 monocytic cell line the interactions between Bt BEVs and monocytes/macrophages were shown to be mediated primarily by TLR2. Histone (H3K4me1) methylation analysis showed that Bt BEVs induced epigenetic reprogramming which persisted after infectious challenge, as revealed by increased levels of H3K4me1 in Bt BEV-conditioned LPS-challenged BMDM. Collectively, our findings highlight the important role of Bt BEVs in maintaining host immune homeostasis and raise the promising possibility of considering their use in immune therapies.

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5'-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1β and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-γ and TNF-α responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products.

Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered to be a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. Among probiotic Gram-positive bacteria, some Propionibacterium freudenreichii strains exhibit anti-inflammatory activity, notably via surface proteins such as the surface-layer protein B (SlpB). We have hypothesized that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs and thus export immunomodulatory proteins to interact with the host. In order to demonstrate their production in this species, EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, and their physicochemical characterization, using transmission electron microscopy and nanoparticle tracking analysis (NTA), revealed shapes and sizes typical of EVs. Proteomic characterization showed that EVs contain a broad range of proteins, including immunomodulatory proteins such as SlpB. In silico protein-protein interaction predictions indicated that EV proteins could interact with host proteins, including the immunomodulatory transcription factor NF-κB. This potential interaction has a functional significance because EVs modulate inflammatory responses, as shown by IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells. Indeed, EVs displayed an anti-inflammatory effect by modulating the NF-κB pathway; this was dependent on their concentration and on the proinflammatory inducer (LPS-specific). Moreover, while this anti-inflammatory effect partly depended on SlpB, it was not abolished by EV surface proteolysis, suggesting possible intracellular sites of action for EVs. This is the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. This study has enhanced our understanding of the mechanisms associated with the probiotic activity of P. freudenreichii and identified opportunities to employ bacterial-derived EVs for the development of bioactive products with therapeutic effects.

Sporotrichosis is a mycosis that affects the skin, lymphatic system and other organs in humans and animals. The disease has a worldwide distribution, with endemic areas in Brazil, and is caused by a complex of species, including Sporothrix brasiliensis. Some fungi release extracellular vesicles (EVs) that can interact with the host cell and modulate the host immune response. The aim of this study was to analyze the participation of S. brasiliensis EVs in the modulation of dendritic cells (DCs) and in the control of infection in vivo. Our results showed that in vitro, the EVs isolated from S. brasiliensis induced an increase in the phagocytic index and fungal burden in DCs. In addition, we observed a significant increase in IL-12p40 and TNF-α cytokine production. Then, the EVs were inoculated into BALB/c mice before subcutaneous infection with yeast, and the lesion was analyzed after 21, 35, and 42 days. An increase in fungal burden and lesion diameter were observed after 21 days in mice inoculated with a high concentration of EVs. However, after 35 days, we observed a regression of the lesion, which persisted until 42 days after infection. Interestingly, we observed an increase in fungal burden in these mice. In addition, we observed the presence of immunogenic components and proteins that could be related with virulence in EVs. These results suggest that EVs can play an important role in virulence and modulation of the host immune system during experimental S. brasiliensis infection.

The extracellular vesicles (EVs) released by Leishmania can contribute to the establishment of infection and host immunomodulation. In this study, we characterized the shedding of EVs from Leishmania (Leishmania) amazonensis promastigotes. This species is the causative agent of cutaneous leishmaniasis, and its role during interactions with bone marrow-derived macrophages (BMDMs) and peritoneal B-1 cells was evaluated. Leishmania amazonensis promastigotes cultivated in vitro at different times and temperatures spontaneously released EVs. EVs were purified using size-exclusion chromatography (SEC) and quantitated by nanoparticle tracking analysis (NTA). NTA revealed that the average size of the EVs was approximately 180 nm, with concentrations ranging from 1.8 × 108 to 2.4 × 109 vesicles/mL. In addition, the presence of LPG and GP63 were detected in EVs obtained at different temperatures. Naïve BMDMs stimulated with EVs exhibited increased IL-10 and IL-6 expression. However, incubating B-1 cells with parasite EVs did not stimulate IL-10 expression but led to an increase in the expression of IL-6 and TNFα. After 7 weeks post-infection, animals infected with L. amazonensis promastigotes in the presence of parasite EVs had significant higher parasite load and a polarization to Th2 response, as compared to the group infected with the parasite alone. This work demonstrated that EVs isolated from L. amazonensis promastigotes were able to stimulate macrophages and B-1 cells to express different types of cytokines. Moreover, the immunomodulatory properties of EVs probably contributed to an increase in parasite burden in mice. These findings suggest that the functionality of L. amazonensis EVs on immune system favor of parasite survival and disease progression.