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.

Transcriptional Profiling of Exosomes Derived from Staphylococcus aureus-Infected Bovine Mammary Epithelial Cell Line MAC-T by RNA-Seq Analysis.

Mastitis is a common disease in the dairy industry that causes huge economic losses worldwide. Exosomes (carrying proteins, miRNA, lncRNA, etc.) play a vital role in the regulation of immune response. lncRNA can play a variety of regulatory roles by combining with protein, RNA, and DNA. The expression of mRNA and lncRNA in exosomes derived from bovine mammary epithelial cells infected by S. aureus is rarely understood. To explore this issue, RNA sequencing analysis was performed on exosomes derived from S. aureus-infected and noninfected MAC-T cells. Analysis of the sequencing results showed that there were 186 differentially expressed genes, 431 differentially expressed mRNAs and 19 differentially expressed lncRNAs in the exosomes derived from S. aureus-infected and noninfected MAC-T cells. By predicting lncRNA target genes, it was found that 19 differentially expressed lncRNAs all acted on multiple mRNAs in cis and trans. GO analysis revealed that differentially expressed genes and lncRNA target genes played significant roles in such metabolism (reactive oxygen species metabolic processes), transmembrane transport, cellular response to DNA damage stimulus, and response to cytokines. KEGG enrichment indicated that lncRNA target genes gathered in the TNF pathway, Notch pathway, MAPK pathway, NF-kappa B pathway, Hippo pathway, p53 pathway, reactive oxygen species metabolic processes, and longevity regulating pathway. In summary, all data indicated that differentially expressed gene, mRNA, and lncRNA in transcriptional profiling of exosomes participated in bacterial invasion and adhesion, oxidative stress, inflammation, and apoptosis-related signaling pathway. The data obtained in this study would provide valuable resource for understanding the lncRNA information in exosomes derived from dairy cow mammary epithelial cells and conduced to the study of S. aureus infection in dairy cow mammary glands.

Enterotoxigenic Bacteroidesfragilis Promotes Intestinal Inflammation and Malignancy by Inhibiting Exosome-Packaged miR-149-3p.

BACKGROUND & AIMS: Enterotoxigenic Bacteroides fragilis (ETBF) is strongly associated with the occurrence of inflammatory bowel disease (IBD), colitis-associated colorectal cancer, and colorectal cancer (CRC). However, the mechanism of ETBF-induced intestinal inflammation and tumorigenesis remains unclear. METHODS: microRNA sequencing was used to detect the differentially expressed microRNAs in both ETBF-treated cells and exosomes derived from ETBF-inoculated cells. Cell Counting Kit 8 assays were used to evaluate the effect of ETBF and exosomes on CRC cell proliferation. The biological role and mechanism of ETBF-mediated miR-149-3p in colitis and colon carcinogenesis were determined both in vitro and in vivo. RESULTS: ETBF promoted CRC cell proliferation by down-regulating miR-149-3p both in vitro and in vivo. ETBF-down-regulated miR-149-3p depended on METTL14-mediated N6-methyladenosine methylation. As the target gene of miR-149-3p, PHF5A transactivated SOD2 through regulating KAT2A messenger RNA alternative splicing after ETBF treatment in CRC cells. miR-149-3p could be released in exosomes and mediated intercellular communication by modulating T-helper type 17 cell differentiation. The level of plasma exosomal miR-149-3p was gradually decreased from healthy control individuals to patients with IBD and CRC. miR-149-3p, existing in plasma exosomes, negatively correlated with the abundance of ETBF in patients with IBD and CRC. CONCLUSIONS: Exosomal miR-149-3p derived from ETBF-treated cells facilitated T-helper type 17 cell differentiation. ETBF-induced colorectal carcinogenesis depended on down-regulating miR-149-3p and further promoting PHF5A-mediated RNA alternative splicing of KAT2A in CRC cells. Targeting the ETBF/miR-149-3p pathway presents a promising approach to treat patients with intestinal inflammation and CRC with a high amount of ETBF.

Emerging Role of Exosomes in Tuberculosis: From Immunity Regulations to Vaccine and Immunotherapy.

Exosomes are cell-derived nanovesicles carrying protein, lipid, and nucleic acid for secreting cells, and act as significant signal transport vectors for cell-cell communication and immune modulation. Immune-cell-derived exosomes have been found to contain molecules involved in immunological pathways, such as MHCII, cytokines, and pathogenic antigens. Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains one of the most fatal infectious diseases. The pathogen for tuberculosis escapes the immune defense and continues to replicate despite rigorous and complicate host cell mechanisms. The infected-cell-derived exosomes under this circumstance are found to trigger different immune responses, such as inflammation, antigen presentation, and activate subsequent pathways, highlighting the critical role of exosomes in anti-MTB immune response. Additionally, as a novel kind of delivery system, exosomes show potential in developing new vaccination and treatment of tuberculosis. We here summarize recent research progress regarding exosomes in the immune environment during MTB infection, and further discuss the potential of exosomes as delivery system for novel anti-MTB vaccines and therapies.

Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn's disease-associated adherent-invasive E. coli.

Adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of Crohn's disease (CD) patients, are able to adhere to and invade intestinal epithelial cells (IECs), survive and replicate within macrophages and induce a pro-inflammatory response. AIEC infection of IECs induces secretion of exosomes that increase AIEC replication in exosome-receiving IECs and macrophages. Here, we investigated the mechanism underlying the increased AIEC replication in cells receiving exosomes from AIEC-infected cells. Exosomes released by uninfected human intestinal epithelial T84 cells (Exo-uninfected) or by T84 cells infected with the clinical AIEC LF82 strain (Exo-LF82), the nonpathogenic E. coli K12 strain (Exo-K12) or the commensal E. coli HS strain (Exo-HS) were purified and used to stimulate T84 cells. Stimulation of T84 cells with Exo-LF82 inhibited autophagy compared with Exo-uninfected, Exo-K12 and Exo-HS. qRT-PCR analysis revealed increased levels of miR-30c and miR-130a in Exo-LF82 compared to Exo-uninfected, Exo-K12 and Exo-HS. These miRNAs were transferred via exosomes to recipient cells, in which they targeted and inhibited ATG5 and ATG16L1 expression and thereby autophagy response, thus favoring AIEC intracellular replication. Inhibition of these miRNAs in exosome-donor cells infected with AIEC LF82 abolished the increase in miR-30c and miR-130a levels in the released Exo-LF82 and in Exo-LF82-receiving cells, thus suppressing the inhibitory effect of Exo-LF82 on ATG5 and ATG16L1 expression and on autophagy-mediated AIEC clearance in Exo-LF82-receiving cells. Our study shows that upon AIEC infection, IECs secrete exosomes that can transfer specific miRNAs to recipient IECs, inhibiting autophagy-mediated clearance of intracellular AIEC.

Sputum Exosomal microRNAs Profiling Reveals Critical Pathways Modulated By Pseudomonas aeruginosa Colonization In Bronchiectasis.

Background: Pseudomonas aeruginosa (PA) colonization confers poor prognosis in bronchiectasis. However, the biomarkers and biological pathways underlying these associations are unclear. Objective: To identify the roles of PA colonization in bronchiectasis by exploring for sputum exosomal microRNA profiles. Methods: We enrolled 98 patients with clinically stable bronchiectasis and 17 healthy subjects. Sputum was split for bacterial culture and exosomal microRNA sequencing, followed by validation with quantitative polymerase chain reaction. Bronchiectasis patients were stratified into PA and non-PA colonization groups based on sputum culture findings. We applied Gene Ontology and Kyoto Encyclopedia of Genes and Genome pathway enrichment analysis to explore biological pathways corresponding to the differentially expressed microRNAs (DEMs) associated with PA colonization. Results: Eighty-two bronchiectasis patients and 9 healthy subjects yielded sufficient sputum that passed quality control. We identified 10 overlap DEMs for the comparison between bronchiectasis patients and healthy subjects, and between PA and non-PA colonization group. Both miR-92b-5p and miR-223-3p could discriminate PA colonization (C-statistic >0.60) and independently correlated with PA colonization in multiple linear regression analysis. The differential expression of miR-92b-5p was validated by quantitative polymerase chain reaction (P<0.05), whereas the differential expression of miR-223 trended towards statistical significance (P=0.06). These DEMs, whose expression levels correlated significantly with sputum inflammatory biomarkers (interleukin-1ß and interleukin-8) level, were implicated in the modulation of the nuclear factor-κB, phosphatidylinositol and longevity regulation pathways. Conclusion: Sputum exosomal microRNAs are implicated in PA colonization in bronchiectasis, highlighting candidate targets for therapeutic interventions to mitigate the adverse impacts conferred by PA colonization.

Urinary exosome as a potential biomarker for urinary tract infection.

Unlike urinary tract infection (UTI), asymptomatic bacteriuria (ABU) should not be treated, with some exceptions such as pregnant women and patients who will undergo traumatic urologic interventions. However, there has been no clinically available marker for their differential diagnosis. Exosomes or small extracellular vesicles carry proteins contained in cells from which they are derived, thus having the potential as a biomarker of several diseases. On the basis of the hypothesis that the molecular signature of exosomes in urine may differ between UTI and ABU patients, we examined if urinary exosomes could serve as a marker for their differential diagnosis. Exosomes were isolated by ultracentrifugation or affinity-based method from cell culture medium of monocytic THP-1 and uroepithelial SV-HUC-1 cells and human urine. Protein expression was examined by Western blot analysis, ELISA, and CLEIA. The results showed that the levels of intracellular signalling molecules Akt and ERK and transcription factor NF-κB increased in exosomes isolated from THP-1 and SV-HUC-1 cells cocultured with Escherichia coli and/or treated with lipopolysaccharide. In urinary exosomes of UTI patients, Akt significantly diminished, and an exosomal marker CD9 showed a trend to decrease after treatment with antimicrobial agents. More importantly, Akt and CD9 levels in urinary exosomes were higher in UTI patients than in ABU patients, which was also observed after correction by urine creatinine. Collectively, these results suggest that Akt and CD9 in urinary exosomes could be useful markers for differential diagnosis of UTI and ABU.

Helicobacter pylori-induced exosomal MET educates tumour-associated macrophages to promote gastric cancer progression.

Helicobacter pylori (H. pylori) infection triggers chronic inflammation that has been associated with gastric cancer (GC). Exosomes are small extracellular vesicles that have become the key mediators of intercellular communication. In this study, we investigated exosome-mediated communication between H. pylori-infected GC cells and macrophages, focusing on the transfer of activated mesenchymal-epithelial transition factor (MET). We observed a significant decrease in MET protein expression in GC cells after infection with H. pylori, whereas MET mRNA levels remained unchanged. Intriguingly, MET expression, specifically the phosphorylated active form, was increased in exosomes released from H. pylori-infected GC cells. Confocal microscopy and Western blotting analyses showed that these exosomes containing MET were delivered to and internalized by macrophages. Indeed, in human GC tissues positive for H. pylori, we also observed that activated MET was highly expressed in tumour-infiltrating macrophages. After internalization, exosomal MET then appeared to educate the macrophages towards a pro-tumorigenesis phenotype. This included exosomal MET-mediated stimulation of proinflammatory cytokine secretion IL-1ß, which subsequently promoted tumour growth and progression in vitro and in vivo. Taken together, these data were the first to demonstrate H. pylori infection-induced upregulation of activated MET in exosomes and the pro-tumorigenic effect on tumour-associated macrophages.

Serum exosomes of chronic gastritis patients infected with Helicobacter pylori mediate IL-1α expression via IL-6 trans-signalling in gastric epithelial cells.

Emerging evidence has linked the exosomes to many immunological disorders, including infectious diseases. However, knowledge regarding the role of exosomes in Helicobacter pylori infection is limited. Here, we show that serum exosomes from chronic gastritis patients with H. pylori infection (Hp exosomes) stimulate the expression of the soluble interleukin (IL)-6 receptor (sIL-6R), which is involved in IL-6 trans-signalling in gastric epithelial cells. Interestingly, sIL-6R up-regulates expression of the proinflammatory cytokine IL-1α, and the neutralization of sIL-6R suppresses IL-1α secretion. Thus, Hp exosomes regulate IL-1α expression via sIL-6R-mediated IL-6 trans-signaling. Altogether, this study reveals a novel perspective in which exosomes play a vital role in immunological mechanisms during H. pylori infection.

Genome-wide microRNA profiling of bovine milk-derived exosomes infected with Staphylococcus aureus.

Bovine milk is rich in exosomes, which contain abundant miRNAs and play important roles in the regulation of neonatal growth and development of adaptive immunity. Here, we analyzed miRNA expression profiles of bovine milk exosomes from three healthy and three mastitic cows, and then six miRNA libraries were constructed. Interestingly, we detected no scRNAs and few snRNAs in milk exosomes; this result indicated a potential preference for RNA packaging in milk exosomes. A total of 492 known and 980 novel exosomal miRNAs were detected, and the 10 most expressed miRNAs in the six samples accounted for 80-90% of total miRNA-associated reads. Expression analyses identified 18 miRNAs with significantly different expression between healthy and infected animals; the predicted target genes of differentially expressed miRNAs were significantly enriched in immune system process, response to stimulus, growth, etc. Moreover, target genes were significantly enriched in several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including inflammatory, immune, and cancer pathways. Our survey provided comprehensive information about milk exosomes and exosomal miRNAs involved in mastitis. Moreover, the differentially expressed miRNAs, especially miR-223 and miR-142-5p, could be considered as potential candidates for mastitis.

Extracellular Vesicles: New Players in Lung Immunity.

Extracellular vesicles (EVs), such as exosomes and microvesicles, play an important autocrine/paracrine role in intercellular communication. Details on the involvement of EVs in the pathogenesis of lung diseases have emerged over the past several years. Moreover, EVs package numerous DNA, proteins, mRNAs, and microRNAs that can regulate immune responses in recipient cells. Almost all respiratory cells release EVs, and these EVs can have protective or detrimental functions, depending on the type of donor cells, type of stimuli, and components. In lung cancer, tumor-derived EVs carry multiple immunoinhibitory signals, disable antitumor immune effector cells, and promote tumor escape from immune control. Furthermore, bacteria- and microbiota-derived EVs can shape the immune system and lead to the development of lung disease. These EVs are capable of maintaining airway homeostasis, inducing proinflammatory effects, and promoting antigen presentation, thus regulating lung inflammation and immune responses. From these viewpoints, we summarize recent findings on EVs in lung biology and immunity. EVs provide a new avenue for understanding the mechanism of inflammatory disease progression and for developing therapeutic approaches for lung immune responses.

Exosomes isolated from sera of mice fed Lactobacillus strains affect inflammatory cytokine production in macrophages in vitro.

Orally administered Lactobacillus strains, including L. plantarum No.14 and L. rhamnosus GG, reportedly reduce inflammatory cytokine production in mice. The present study tested our idea that circulating exosomes mediate the action of Lactobacillus strains. The lipopolysaccharide-induced production of TNF-α and IL-6 in vitro was attenuated in peritoneal exudate cells (PECs) isolated from C57BL/6N mice that had been fed L. plantarum No.14. When PECs were cultured for 24 h with exosomes isolated from the serum of mice fed L. plantarum No.14 or L. rhamnosus GG, accumulation of both TNF-α and of the corresponding mRNA was lowered. Growth in the presence of these exosomes also decreased the production of TNF-α and IL-6 by the murine macrophage cell line RAW264.7. In contrast, supplementation with exosome-depleted serum of mice fed L. plantarum No.14 or L. rhamnosus GG failed to affect the production of TNF-α and IL-6 by RAW264.7 cells. When PECs and RAW264.7 cells were cultured for 24 h with PKH67-labeled exosomes isolated from murine serum, fluorescent signal was observed inside the cells, suggesting that these cells incorporate serum exosomes. We propose that the anti-inflammatory activity of orally administered L. plantarum No.14 and L. rhamnosus GG is mediated, at least in part, by circulating exosomes.

Changes in the Membrane-Associated Proteins of Exosomes Released from Human Macrophages after Mycobacterium tuberculosis Infection.

Tuberculosis (TB) is the deadliest infectious disease worldwide. One obstacle hindering the elimination of TB is our lack of understanding of host-pathogen interactions. Exosomes, naturally loaded with microbial molecules, are circulating markers of TB. Changes in the host protein composition of exosomes from Mycobacterium tuberculosis (Mtb)-infected cells have not been described, can contribute to our understanding of the disease process, and serve as a direct source of biomarkers or as capture targets to enrich for exosomes containing microbial molecules. Here, the protein composition of exosomes from Mtb-infected and uninfected THP-1-derived macrophages was evaluated by tandem-mass-spectrometry and differences in protein abundances were assessed. Our results show that infection with Mtb leads to significant changes in the protein composition of exosomes. Specifically, 41 proteins were significantly more abundant in exosomes from Mtb-infected cells; 63% of these were predicted to be membrane associated. Thus, we used a novel biotinylation strategy to verify protein localization, and confirmed the localization of some of these proteins in the exosomal membrane. Our findings reveal another important scenario where Mtb could be influencing changes in host cells that unveil new features of the host-pathogen interaction and may also be exploited as a source of biomarkers for TB.

IL-36γ is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components.

Interleukin-36 is a family of novel interleukin-1-like proinflammatory cytokines that are highly expressed in epithelial tissues and several myeloid-derived cell types. Like those of classic interleukin-1 cytokines, the secretion mechanisms of interleukin-36 are not well understood. Interleukin-36γ secretion in dermal epithelial cells requires adenosine 5'-triphosphate, which suggests a nonclassical mechanism of secretion. In this study, murine pulmonary macrophages and human alveolar macrophages were treated with recombinant pathogen-associated molecular patterns (intact bacteria: Klebsiella pneumoniae or Streptococcus pneumoniae). Cell lysates were analyzed for messenger ribonucleic acid by quantitative real-time polymerase chain reaction, and conditioned medium was analyzed for interleukin-36γ by enzyme-linked immunosorbent assay, with or without sonication. In addition, conditioned medium was ultracentrifuged at 25,000 g and 100,000 g, to isolate microparticles and exosomes, respectively, and interleukin-36γ protein was assessed in each fraction by Western blot analysis. Interleukin-36γ mRNA was induced in both murine and human lung macrophages by a variety of pathogen-associated molecular patterns, as well as heat-killed and live Klebsiella pneumoniae and Streptococcus pneumoniae, and induction occurred in a myeloid differentiation response gene 88-dependent manner. Secretion of interleukin-36γ protein was enhanced by adenosine 5'-triphosphate. Furthermore, extracellular interleukin-36γ protein detection was markedly enhanced by sonication to disrupt membrane-bound structures. Interleukin-36γ protein was detected by Western blot in microparticles and exosome fractions isolated by ultracentrifugation. Interleukin-36γ was induced and secreted from lung macrophages in response to Gram-negative and -positive bacterial stimulation. The results suggest that interleukin-36γ is secreted in a non-Golgi-dependent manner by lung macrophages in response to Gram-positive and -negative bacterial challenge.

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes.

Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.

Exosomes Released from Cells Infected with Crohn's Disease-associated Adherent-Invasive Escherichia coli Activate Host Innate Immune Responses and Enhance Bacterial Intracellular Replication.

BACKGROUND: Crohn's disease is a chronic inflammatory bowel disease, of which the etiology involves environmental, genetic, and microbial factors. A high prevalence of adherent-invasive Escherichia coli, named AIEC, has been reported in the intestinal mucosa of patients with Crohn's disease. Exosomes are extracellular vesicles that function in intercellular communication and have been implicated in host responses to intracellular pathogens. We investigated the potential involvement of exosomes in host response to AIEC infection. METHODS: Human intestinal epithelial T84 cells, THP-1 macrophages, and CEABAC10 transgenic mice were infected with the AIEC reference strain LF82 or the nonpathogenic E. coli K-12 MG1655 strain. Exosomes were purified using the ExoQuick reagent. RESULTS: LF82 infection induced the release of exosomes by T84 and THP-1 cells. Compared with exosomes released from the uninfected or MG1655-infected T84 cells, those released from LF82-infected cells activated nuclear factor-kappa B, mitogen-activated protein kinases p38, and c-Jun N-terminal kinase and increased the secretion of proinflammatory cytokines in naive THP-1 macrophages. LF82 infection of THP-1 macrophages also induced the release of exosomes that triggered a proinflammatory response in recipient THP-1 cells. Importantly, stimulation of T84 or THP-1 cells with exosomes released from LF82-infected cells increased LF82 intracellular replication compared with stimulation with exosomes secreted by uninfected cells. Exosomes purified from intestinal lumen of CEABAC10 transgenic mice infected with LF82 increased proinflammatory responses in murine RAW 264.7 macrophages compared with those from uninfected or MG1655-infected mice. CONCLUSIONS: Exosomes are new mediators of host-AIEC interaction with their capacity to activate innate immune responses and subvert the control of AIEC replication.

Evaluation of the inflammatory response in macrophages stimulated with exosomes secreted by Mycobacterium avium-infected macrophages.

Exosomes secreted from Mycobacterium avium-infected macrophages contain numerous antigens of both M. avium and the host cell and are involved in the induction and expression of the inflammatory responses in macrophages. The interaction between exosomes secreted from M. avium-infected macrophages and macrophage phagocytosis, cytokine secretion, immunostimulation, and apoptosis was analyzed. Upon stimulation with exosomes secreted from M. avium-infected macrophages, the phagocytosis of dextran by treated macrophages was increased. Furthermore, the expression of CD40, CD80, CD81, CD86, HLA-DR, and most notably CD195 was enhanced. Additionally, the secretion of IL-6, IL-8, IL-10, IFN-γ, and TNF-α was increased by stimulated macrophages. Exosome stimulation did not induce macrophage apoptosis when compared with macrophages infected with M. avium. Caspase expression, including that of caspases 3, 6, and 8, was also not altered in exosome stimulated macrophages. Thus exosomes trigger the inflammatory response in macrophages owing to the presence of bacterial antigens but have no effect on macrophage viability.

Blood/plasma secretome and microvesicles.

A major but hitherto overseen component of the blood/plasma secretome is that of extracellular vesicles (EVs) which are shed from all blood cell types. These EVs are made up of microvesicles (MVs) and exosomes. MVs, 100nm-1µm in diameter, are released from the cell surface, and are a rich source of non-conventionally secreted proteins lacking a conventional signal peptide, and thus not secreted by the classical secretory pathways. Exosomes are smaller vesicles (≤100nm) having an endocytic origin and released upon multivesicular body fusion with the plasma membrane. Both vesicle types play major roles in intercellular cross talk and constitute an important component of the secretome especially in the area of biomarkers for cancer. The release of EVs, which are found in all the bodily fluids, is enhanced in cancer and a major focus of cancer proteomics is therefore targeted at EVs. The blood/plasma secretome is also a source of EVs, potentially diagnostic of infectious disease, whether from EVs released from infected cells or from the pathogens themselves. Despite the great excitement in this field, as is stated here and in other parts of this Special issue entitled: An Updated Secretome, much of the EV research, whether proteomic or functional in nature, urgently needs standardisation both in terms of nomenclature and isolation protocols. This article is part of a Special Issue entitled: An Updated Secretome.

The role of exosomes in infectious diseases.

An exosome is a nano vesicle that buds from the endosomal compartment; it is produced and released by all kinds of mammalian cells. This vesicle contains a variety of proteins, lipids, mRNAs and miRNAs. These components are specific to the origin of the exosomes and contribute to cell-cell communications. Recently, it has been reported that a few single cell eukaryotic pathogens such as Cryptoccoccus neoformance and Leishmania major and donovanican secrete an exosome and influence the host immune system. In addition, it has been observed that cells infected by intracellular pathogens are capable of secreting an exosome which is involved in the fate of the infection. Furthermore, retroviruses recruit the host`s endosomal compartments in order to generate viral vesicles which are similar to the exosome. Most of the exosomes involved in infectious biology can either spread or limit an infection based on the type of pathogen and its target cells. Hence, an exosome may be an appropriate candidate for a vaccine therapy in prophylaxis and treatment.

[Changes of cytokines and protein expression of macrophages stimulated with exosome from macrophages by Mycobacterium avium infection].

OBJECTIVE: To explore the mechanism underlying the molecular immune response of macrophages stimulated with exosome [(+)exosome] from macrophages after Mycobacterium avium (M. avium) infection and analyze the differential protein component of the exosome. METHODS: The culture supernatants of M.avium-infected macrophages and uninfected ones were collected and exosome was harvested from the supernatants by frozen ultra centrifugation. The concentrations of IFN-γ, TNF-α in supernatants were detected by enzyme-linked immunosorbent assay (ELISA), and CD80, CD86 expressions on macrophages were analyzed by flow cytometry after macrophages were stimulated with exosome. Meanwhile, 2-DE MALDI TOF/TOF MS was used to identify differentially expressed proteins of exosome between M. avium infected group and uninfected group. RESULTS: IFN-γ, TNF-α concentrations were increased in the supernatant after stimulated with (+)exosome and CD80, CD86 were raised on macrophage surface by stimulation with (+)exosome. With 2-DE MALDI TOF/TOF MS analysis, we obtained 18 differentially expressed proteins and 12 proteins were identified successfully. CONCLUSION: (+)exosome induces TNF-α, IFN-γ secretion from macrophages and result in the promotion of inflammatory response. In addition, (+)exosome enhances CD80 and CD86 protein expressions. The function of the differentially expressed proteins we identified is closely related to cytoskeleton, protein synthesis and processing, inflammatory response.