Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes

Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.     

Genetic Variation of Taenia Pisiformis Collected from Sichuan,China, Based on the Mitochondrial Cytochrome b gene

Taenia pisiformis is one of the most important parasites of canines and rabbits. T. pisiformis cysticercus (the larval stage) causes severe damage to rabbit breeding, which results in huge economic losses. In this study, the genetic variation of T. pisiformis was determined in Sichuan Province, China. Fragments of the mitochondrial cytochrome b (cytb) (922 bp) gene were amplified in 53 isolates from 8 regions of T. pisiformis. Overall, 12 haplotypes were found in these 53 cytb sequences. Molecular genetic variations showed 98.4% genetic variation derived from intra-region. F_ST and Nm values suggested that 53 isolates were not genetically differentiated and had low levels of genetic diversity. Neutrality indices of the cytb sequences showed the evolution of T. pisiformis followed a neutral mode. Phylogenetic analysis revealed no correlation between phylogeny and geographic distribution. These findings indicate that 53 isolates of T. pisiformis keep a low genetic variation, which provide useful knowledge for monitoring changes in parasite populations for future control strategies.     

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10⁴–10⁶ copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10³ copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.     

The JNK/NFκB pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from Ecklonia cava

Background

The proven immunomodulatory and immune system activating properties of Ecklonia cava (E. cava) have been attributed to its plentiful polysaccharide content. Therefore, we investigated whether the sulfated polysaccharide (SP) of E. cava specifically activates the protein kinases (MAPKs) and nuclear factor-κB (NFκB) to incite immune responses.

Methods

To assess immune responsiveness, lymphocytes were isolated from spleens of ICR mice and cultured with SP and its inhibitors. Assays included ³H-thymidine incorporation, flow cytometry, real time polymerase chain reaction (rtPCR), enzyme linked immunosorbent assay (ELISA), intracellular cytokine assay, Western blot, and electrophoretic mobility shift assay (EMSA).

Results

SP dose-dependently increased the proliferation of lymphocytes without cytotoxicity. In particular, SP markedly enhanced the proliferation and differentiation of CD3⁺ mature T cells and CD45R/B220⁺ pan B cells. Additionally, SP increased the expression and/or production of IL-2, IgG_1a, and IgG_2b compared to that in untreated cells. The subsequent application of JNK (SP600125), NFκB (PDTC), and serine protease (TPCK) inhibitors significantly inhibited the proliferation and IL-2 production of SP-treated lymphocytes as well as the phosphorylation of JNK and IκB, the activation of nuclear NFκB p65, and binding of NFκB p65 DNA. Moreover, co-application of both JNK and NFκB inhibitors completely blocked the proliferation of lymphocytes even in the presence of SP.

Conclusion

These results suggest that SP induced T and B cell responses via both JNK and NFκB pathways.

General significance

The effect of SP on splenic lymphocyte activation was assayed here for the first time and indicated the underlying functional mechanism.     

Trichomonas vaginalis Exosomes Deliver Cargo to Host Cells and Mediate Host∶Parasite Interactions

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host∶parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite∶parasite communication and host cell colonization.     

A Highly Conserved Toxo1 Haplotype Directs Resistance to Toxoplasmosis and Its Associated Caspase-1 Dependent Killing of Parasite and Host Macrophage

Natural immunity or resistance to pathogens most often relies on the genetic make-up of the host. In a LEW rat model of refractoriness to toxoplasmosis, we previously identified on chromosome 10 the Toxo1 locus that directs toxoplasmosis outcome and controls parasite spreading by a macrophage-dependent mechanism. Now, we narrowed down Toxo1 to a 891 kb interval containing 29 genes syntenic to human 17p13 region. Strikingly, Toxo1 is included in a haplotype block strictly conserved among all refractory rat strains. The sequencing of Toxo1 in nine rat strains (5 refractory and 4 susceptible) revealed resistant-restricted conserved polymorphisms displaying a distribution gradient that peaks at the bottom border of Toxo1, and highlighting the NOD-like receptor, Nlrp1a, as a major candidate. The Nlrp1 inflammasome is known to trigger, upon pathogen intracellular sensing, pyroptosis programmed-cell death involving caspase-1 activation and cleavage of IL-1β. Functional studies demonstrated that the Toxo1-dependent refractoriness in vivo correlated with both the ability of macrophages to restrict T. gondii growth and a T. gondii-induced death of intracellular parasites and its host macrophages. The parasite-induced cell death of infected macrophages bearing the LEW-Toxo1 alleles was found to exhibit pyroptosis-like features with ROS production, the activation of caspase-1 and IL1-β secretion. The pharmacological inactivation of caspase-1 using YVAD and Z-VAD inhibitors prevented the death of both intravacuolar parasites and host non-permissive macrophages but failed to restore parasite proliferation. These findings demonstrated that the Toxo1-dependent response of rat macrophages to T. gondii infection may trigger two pathways leading to the control of parasite proliferation and the death of parasites and host macrophages. The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death. These data represent new insights towards the identification of a major pathway of innate resistance to toxoplasmosis and the prediction of individual resistance.     

Exosomes released by Brucella-infected macrophages inhibit the intracellular survival of Brucella by promoting the polarization of M1 macrophages

Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella-invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti-Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo-M5)-infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo-M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor-α and interferon-γ) through NF-κB signalling pathways and inhibited the secretion of M2 cytokines (IL-10), thereby inhibiting the intracellular survival of Brucella. Exo-M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo-M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates.     

Antigen-encapsulating host extracellular vesicles derived from Salmonella-infected cells stimulate pathogen-specific Th1-type responses in vivo

Salmonella Typhimurium is a causative agent of nontyphoidal salmonellosis, for which there is a lack of a clinically approved vaccine in humans. As an intracellular pathogen, Salmonella impacts many cellular pathways. However, the intercellular communication mechanism facilitated by host-derived small extracellular vesicles (EVs), such as exosomes, is an overlooked aspect of the host responses to this infection. We used a comprehensive proteome-based network analysis of exosomes derived from Salmonella-infected macrophages to identify host molecules that are trafficked via these EVs. This analysis predicted that the host-derived small EVs generated during macrophage infection stimulate macrophages and promote activation of T helper 1 (Th1) cells. We identified that exosomes generated during infection contain Salmonella proteins, including unique antigens previously shown to stimulate protective immune responses against Salmonella in murine studies. Furthermore, we showed that host EVs formed upon infection stimulate a mucosal immune response against Salmonella infection when delivered intranasally to BALB/c mice, a route of antigen administration known to initiate mucosal immunity. Specifically, the administration of these vesicles to animals stimulated the production of anti-Salmonella IgG antibodies, such as anti-OmpA antibodies. Exosomes also stimulated antigen-specific cell-mediated immunity. In particular, splenic mononuclear cells isolated from mice administered with exosomes derived from Salmonella-infected antigen-presenting cells increased CD4+ T cells secreting Th1-type cytokines in response to Salmonella antigens. These results demonstrate that small EVs, formed during infection, contribute to Th1 cell bias in the anti-Salmonella responses. Collectively, this study helps to unravel the role of host-derived small EVs as vehicles transmitting antigens to induce Th1-type immunity against Gram-negative bacteria. Understanding the EV-mediated defense mechanisms will allow the development of future approaches to combat bacterial infections.     

Docosahexaenoic acid attenuates macrophage-induced inflammation and improves insulin sensitivity in adipocytes-specific differential effects between LC n-3 PUFA

ObjectiveAdipose tissue inflammation with immune cell recruitment plays a key role in obesity-induced insulin resistance (IR). Long-chain (LC) n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have anti-inflammatory potential; however, their individual effects on adipose IR are ill defined. We hypothesized that EPA and DHA may differentially affect macrophage-induced IR in adipocytes.MethodsJ774.2 macrophages pretreated with EPA or DHA (50 μM for 5 days) were stimulated with lipopolysaccharide (LPS, 100 ng/ml for 30 min–48 h). Cytokine secretion profiles and activation status of macrophages were assessed by enzyme-linked immunosorbent assay and flow cytometry. Pretreated macrophages were seeded onto transwell inserts and placed over 3T3-L1 adipocytes for 24–72 h; effects on adipocyte–macrophage cytokine cross-talk and insulin-stimulated ³H-glucose transport into adipocytes were monitored.ResultsDHA had more potent anti-inflammatory effects relative to EPA, with marked attenuation of LPS-induced nuclear factor (NF)κB activation and tumor necrosis factor (TNF)α secretion in macrophages. DHA specifically enhanced anti-inflammatory interleukin (IL)-10 secretion and reduced the expression of proinflammatory M1 (F4/80⁺/CD11⁺) macrophages. Co-culture of DHA-enriched macrophages with adipocytes attenuated IL-6 and TNFα secretion while enhancing IL-10 secretion. Conditioned media (CM) from DHA-enriched macrophages attenuated adipocyte NFκB activation. Adipocytes co-cultured with DHA-enriched macrophages maintained insulin sensitivity with enhanced insulin-stimulated ³H-glucose transport, GLUT4 translocation and preservation of insulin-receptor substrate-1 expression compared to co-culture with untreated macrophages. We confirmed that IL-10 expressed by DHA-enriched macrophages attenuates the CM-induced proinflammatory IR phenotype in adipocytes.ConclusionsWe demonstrate an attenuated proinflammatory phenotype of DHA-pretreated macrophages, which when co-cultured with adipocytes partially preserved insulin sensitivity.     

Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis‐infected and culture filtrate protein‐treated macrophages

Exosomes are small 30–100 nm membrane vesicles released from hematopoietic and nonhematopoietic cells and function to promote intercellular communication. They are generated through fusion of multivesicular bodies with the plasma membrane and release of interluminal vesicles. Previous studies from our laboratory demonstrated that macrophages infected with Mycobacterium release exosomes that promote activation of both innate and acquired immune responses; however, the components present in exosomes inducing these host responses were not defined. This study used LC‐MS/MS to identify 41 mycobacterial proteins present in exosomes released from M. tuberculosis‐infected J774 cells. Many of these proteins have been characterized as highly immunogenic. Further, since most of the mycobacterial proteins identified are actively secreted, we hypothesized that macrophages treated with M. tuberculosis culture filtrate proteins (CFPs) would release exosomes containing mycobacterial proteins. We found 29 M. tuberculosis proteins in exosomes released from CFP‐treated J774 cells, the majority of which were also present in exosomes isolated from M. tuberculosis‐infected cells. The exosomes from CFP‐treated J774 cells could promote macrophage and dendritic cell activation as well as activation of naïve T cells in vivo. These results suggest that exosomes containing M. tuberculosis antigens may be alternative approach to developing a tuberculosis vaccine.     

Exosome-derived uterine microRNAs isolated from cows with endometritis impede blastocyst development

As a common disease of cows occurring during their perinatal period, endometritis is known to affect fertility. At present, the studies on endometritis mainly focus on preventing microbial invasion. However, the mechanism that uterine inflammation affects embryo activity and implantation is unclear. Mainly containing lipids, proteins, mRNAs, and microRNAs, exosomes widely exist in various tissues and body fluids. Exosome extractions were used by commercial kits and confirmed through morphological examinations and Western blot. After exosomes’ mRNA profiles were generated using RNA sequencing, it was investigated how uterine cavity fluid exosomes affect the developmental competence of in vitro fertilization (IVF) embryos in case of endometritis. In this study, the isolated exosomes were spherical particles with a diameter of 30–150 nm according to the transmission electron microscopy. Identified with Western blotting, positive CD63 and CD9 expressions showed that the isolated exosomes could be used for the subsequent tests. We found 118 differentially expressed miRNAs in the exosomes of the uterine cavity fluid in healthy cows and those with endometritis, among which, 52 miRNAs were down regulated and 66 up regulated. Furthermore, the qRT-PCR results confirmed the up-regulation of three miRNAs and down-regulation of six miRNAs, which were consistent with the deep sequencing results. IVF embryos co-incubated with the endometritis exosomes significantly decreased the blastocyst formation rate in comparison with those co-incubated with the healthy exosomes (21.84+3.17 vs. 32.37+2.69). Therefore, exosome miRNAs may be a cause of infertility in cows with endometritis.     

Exosomal Hsp70 Induces a Pro-Inflammatory Response to Foreign Particles Including Mycobacteria

Background

Exosomes are endosome-derived vesicles that are released when multi-vesicular bodies (MVBs) fuse with the plasma membrane. Exosomes released from mycobacteria-infected cells have recently been shown to be pro-inflammatory. A prominent host molecule that is found within these exosomes is Hsp70, a member of the heat-shock family of proteins.

Methodology/Principal Findings

We first characterized the exosomes purified from control and mycobacteria-infected cells. We found that relative to uninfected cells, macrophages infected with M. smegmatis and M. avium release more exosomes and the exosomes they released had more Hsp70 on their surface. Both exosomes and exogenous Hsp70 treatment of macrophages led to NF-κB activation and TNFα release in uninfected macrophages; Hsp70 levels were elevated in mycobacteria-infected cells. Macrophage treatment with Hsp70 also led to increase in the phagocytosis and maturation of latex-bead phagosomes. Finally, Hsp70 pre-incubation of M. smegmatis- and M. avium-infected cells led to increased phago-lysosome fusion, as well as more killing of mycobacteria within macrophages.

Conclusions/Significance

Our results fit into an emerging concept whereby exosomes-containing Hsp70 are effective inducers of inflammation, also in response to mycobacterial infection.     

Detection of Mycobacterium tuberculosis Peptides in the Exosomes of Patients with Active and Latent M. tuberculosis Infection Using MRM-MS

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.