Extracellular vesicles derived from natural killer cells use multiple cytotoxic proteins and killing mechanisms to target cancer cells.

Extracellular vesicles (EVs) are secreted membrane vesicles, which play complex physiological and pathological functions in intercellular communication. Recently, we isolated natural killer (NK) cell-derived EVs (NK-EVs) from ex vivo expansion of NK cell cultures. The isolated NK-EVs contained cytotoxic proteins and several activated caspases, and they induced apoptosis in target cells. In this report, the protein levels of cytotoxic proteins from NK-EV isolates were analysed by ELISA. The mean values of perforin (PFN, 550 ng/mL), granzyme A (GzmA, 185 ng/mL), granzyme B (GzmB, 23.4 ng/mL), granulysin (GNLY, 56 ng/mL), and FasL (2.5 ng/mL) were obtained from >60 isolations using dot plots. The correlation between cytotoxicity and cytotoxic protein levels was examined by linear regression. PFN, GzmA, GzmB, GNLY all had a positive, moderate correlation with cytotoxicity, suggesting that there is not a single cytotoxic protein dominantly involved in killing and that all of these proteins may contribute to cytotoxicity. To further explore the possible killing mechanisms, cells were treated with NK-EVs, proteins extracted and lysates assessed by Western blotting. The levels of Gzm A substrates, SET and HMG2, were diminished in targeted cells, indicating that GzmA may induce a caspase-independent death pathway. Also, cytochrome C was released from mitochondria, a central hallmark of caspase-dependent death pathways. In addition, several ER-associated proteins were altered, suggesting that NK-EVs may induce ER stress resulting in cell death. Our results indicate that multiple killing mechanisms are activated by NK-derived EVs, including caspase-independent and -dependent cell death pathways, which can mediate cytotoxicity against cancer cells. Abbreviations: NK: natural killer cells; aNK: activated NK cells; EV: extracellular vesicles; ER: endoplasmic reticulum; ALL: acute lymphoblastic leukaemia; FBS: foetal bovine serum. GzmA: granzyme A; GzmB: granzyme B; GNLY: granulysin; PFN: perforin.

Natural Killer-Derived Exosomal miR-186 Inhibits Neuroblastoma Growth and Immune Escape Mechanisms.

In neuroblastoma, the interplay between immune cells of the tumor microenvironment and cancer cells contributes to immune escape mechanisms and drug resistance. In this study, we show that natural killer (NK) cell-derived exosomes carrying the tumor suppressor microRNA (miR)-186 exhibit cytotoxicity against MYCN-amplified neuroblastoma cell lines. The cytotoxic potential of these exosomes was partly dependent upon expression of miR-186. miR-186 was downregulated in high-risk neuroblastoma patients, and its low expression represented a poor prognostic factor that directly correlated with NK activation markers (i.e., NKG2D and DNAM-1). Expression of MYCN, AURKA, TGFBR1, and TGFBR2 was directly inhibited by miR-186. Targeted delivery of miR-186 to MYCN-amplified neuroblastoma or NK cells resulted in inhibition of neuroblastoma tumorigenic potential and prevented the TGFß1-dependent inhibition of NK cells. Altogether, these data support the investigation of a miR-186-containing nanoparticle formulation to prevent tumor growth and TGFß1-dependent immune escape in high-risk neuroblastoma patients as well as the inclusion of ex vivo-derived NK exosomes as a potential therapeutic option alongside NK cell-based immunotherapy.Significance: These findings highlight the therapeutic potential of NK cell-derived exosomes containing the tumor suppressor miR-186 that inhibits growth, spreading, and TGFß-dependent immune escape mechanisms in neuroblastoma.

Biological roles and potential applications of immune cell-derived extracellular vesicles.

Extracellular vesicles (EVs) deliver bioactive macromolecules (i.e. proteins, lipids and nucleic acids) for intercellular communication in multicellular organisms. EVs are secreted by all cell types including immune cells. Immune cell-derived EVs modulate diverse aspects of the immune system to either enhance or suppress immune activities. The extensive effects of immune cell-derived EVs have become the focus of great interest for various nano-biomedical applications, ranging from the medical use of nanoplatform-based diagnostic agents to the development of therapeutic interventions as well as vaccine applications, and thus may be ideal for 'immune-theranostic'. Here, we review the latest advances concerning the biological roles of immune cell-derived EVs in innate and acquired immunity. The intercellular communication amongst immune cells through their EVs is highlighted, showing that all immune cell-derived EVs have their unique function(s) in immunity through intricate interaction(s). Natural-killer (NK) cell-derived EVs, for example, contain potent cytotoxic proteins and induce apoptosis to targeted cancer cells. On the other hand, cancer cell-derived EVs bearing NK ligands may evade immune surveillance and responses. Finally, we discuss possible medical uses for the immune cell-derived EVs as a tool for immune-theranostic: as diagnostic biomarkers, for use in therapeutic interventions and for vaccination.

Large-scale isolation and cytotoxicity of extracellular vesicles derived from activated human natural killer cells.

Extracellular vesicles (EVs) have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. EVs are secreted by all cell types, including immune cells such as natural killer cells (NK), and they may play important roles in the immune system. Currently, a large-scale procedure to obtain functional NK EVs is lacking, limiting their use clinically. In this report, we present a simple, robust, and cost-effective method to isolate a large quantity of NK EVs. After propagating and activating NK cells ex vivo and then incubating them in exosome-free medium for 48 h, EVs were isolated using a polymer precipitation method. The isolated vesicles contain the tetraspanin CD63, an EV marker, and associated proteins (fibronectin), but are devoid of cytochrome C, a cytoplasmic marker. Nanoparticle tracking analysis showed a size distribution between 100 and 200 nm while transmission electron microscopy imaging displayed vesicles with an oval shape and comparable sizes, fulfilling the definition of EV. Importantly, isolated EV fractions were cytotoxic against cancer cells. Furthermore, our results demonstrate for the first time that isolated activated NK (aNK) cell EVs contain the cytotoxic proteins perforin, granulysin, and granzymes A and B, incorporated from the aNK cells. Activation of caspase -3, -7 and -9 was detected in cancer cells incubated with aNK EVs, and caspase inhibitors blocked aNK EV-induced cytotoxicity, suggesting that aNK EVs activate caspase pathways in target cells. The ability to isolate functional aNK EVs on a large scale may lead to new clinical applications. Abbreviations: NK: natural killer cells; activated NK (aNK) cells; EVs: extracellular vesicles; ALL: acute lymphoblastic leukaemia; aAPC: artificial antigen-presenting cell; TEM: transmission electron microscope; PBMC: peripheral blood mononuclear cells; FBS: foetal bovine serum.

Identification of genes from the fungal pathogen Cryptococcus neoformans related to transmigration into the central nervous system.

BACKGROUND: A mouse brain transmigration assessment (MBTA) was created to investigate the central nervous system (CNS) pathogenesis of cryptococcal meningoencephalitis. METHODOLOGY/PRINCIPAL FINDINGS: Two cryptococcal mutants were identified from a pool of 109 pre-selected mutants that were signature-tagged with the nourseothricin acetyltransferase (NAT) resistance cassette. These two mutants displayed abnormal transmigration into the central nervous system. One mutant displaying decreased transmigration contains a null mutation in the putative FNX1 gene, whereas the other mutant possessing a null mutation in the putative RUB1 gene exhibited increased transmigration into the brain. Two macrophage adhesion-defective mutants in the pool, 12F1 and 3C9, showed reduced phagocytosis by macrophages, but displayed no defects in CNS entry suggesting that transit within macrophages (the "Trojan horse" model of CNS entry) is not the primary mechanism for C. neoformans migration into the CNS in this MBTA. CONCLUSIONS/SIGNIFICANCE: This research design provides a new strategy for genetic impact studies on how Cryptococcus passes through the blood-brain barrier (BBB), and the specific isolated mutants in this assay support a transcellular mechanism of CNS entry.

HIV-1 gp41 ectodomain enhances Cryptococcus neoformans binding to human brain microvascular endothelial cells via gp41 core-induced membrane activities.

Cryptococcus neoformans causes life-threatening meningoencephalitis, particularly prevalent in AIDS patients. The interrelationship between C. neoformans and HIV-1 is intriguing, as both pathogens elicit severe neuropathological complications. We have previously demonstrated that the HIV-1 gp41 ectodomain fragments gp41-I33 (amino acids 579-611) and gp41-I90 (amino acids 550-639) can enhance C. neoformans binding to HBMECs (human brain microvascular endothelial cells). Both peptides contain the loop region of gp41. In the present study, we used immunofluorescence microscopy and transmission and scanning electron microscopy to explore the underlying mechanisms. Our findings indicated that both C. neoformans and gp41-I90 up-regulated ICAM-1 (intercellular adhesion molecule 1) on the HBMECs and elicited membrane ruffling on the surface of HBMECs. The HIV-1 gp41 ectodomain could also induce CD44 and ß-actin redistribution to the membrane lipid rafts, but it could not enhance PKCα (protein kinase Cα) phosphorylation like C. neoformans. Instead, gp41-I90 was able to induce syncytium formation on HBMECs. The results of the present study suggest HIV-1 gp41-enhanced C. neoformans binding to HBMECs via gp41 core domain-induced membrane activities, revealing a potential mechanism of invasion for this pathogenic fungus into the brain tissues of HIV-1-infected patients.

Cryptococcus neoformans mediator protein Ssn8 negatively regulates diverse physiological processes and is required for virulence.

Cryptococcus neoformans is a ubiquitously distributed human pathogen. It is also a model system for studying fungal virulence, physiology and differentiation. Light is known to inhibit sexual development via the evolutionarily conserved white collar proteins in C. neoformans. To dissect molecular mechanisms regulating this process, we have identified the SSN8 gene whose mutation suppresses the light-dependent CWC1 overexpression phenotype. Characterization of sex-related phenotypes revealed that Ssn8 functions as a negative regulator in both heterothallic a-α mating and same-sex mating processes. In addition, Ssn8 is involved in the suppression of other physiological processes including invasive growth, and production of capsule and melanin. Interestingly, Ssn8 is also required for the maintenance of cell wall integrity and virulence. Our gene expression studies confirmed that deletion of SSN8 results in de-repression of genes involved in sexual development and melanization. Epistatic and yeast two hybrid studies suggest that C. neoformans Ssn8 plays critical roles downstream of the Cpk1 MAPK cascade and Ste12 and possibly resides at one of the major branches downstream of the Cwc complex in the light-mediated sexual development pathway. Taken together, our studies demonstrate that the conserved Mediator protein Ssn8 functions as a global regulator which negatively regulates diverse physiological and developmental processes and is required for virulence in C. neoformans.

HIV-1 gp41 ectodomain enhances Cryptococcus neoformans binding to HBMEC.

Cryptococcus neoformans infection has significantly increased recently, particularly in AIDS patients and immunocompromised individuals. C. neoformans has a predilection to the brain, resulting in devastating meningoencephalitis. We have previously shown the invasion of C. neoformans into the human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. Here, we demonstrated that C. neoformans invasion of HBMEC was enhanced by HIV-1 gp41 protein. Peptide mapping defined its functional domain around the disulfide-bond linkage of gp41 molecule (a.a. 579-611). Recombinant protein gp41-I90 (a.a. 550-639) can also enhance the binding activity. The enhancement of C. neoformans binding to HBMEC is a strain-independent manner, suggesting that gp41 ectodomain peptide exerts its function directly on HBMEC. Importantly, the enhancement could be observed in mouse animal model. Our results suggest that HIV-1 gp41 ectodomain and C. neoformans may follow a similar invasion mechanism, possibly actin reorganization and/or membrane activation, during pathogen infections on HBMEC.

Downregulation of human Cdc6 protein using a lentivirus RNA interference expression vector.

Eukaryotic CDC6 gene function is required for the initiation of DNA replication and is a key regulatory protein during cell cycle progression. The human CDC6 gene is not expressed in most normal tissues, in contrast with its marked expression in proliferating cancer cells. An effective way to explore the gene functions of CDC6 is to knock-down the CDC6 messenger RNA (mRNA) and examine the phenotypic consequences. In this chapter, we describe the construction of a lentivirus vector to express a CDC6 DNA segment. The transcript is able to fold by itself because the sense and antisense regions are complementary. There is a 9-nucleotide (nt) loop region allowing for the short hairpin RNA (shRNA) to form. Cellular ribonucleases process the shRNA into a functional short interfering RNA (siRNA). Down-regulation of Cdc6 protein is confirmed by Western blots.

Spatial distribution of nerve sprouting after myocardial infarction in mice.

BACKGROUND: Myocardial infarction (MI) elicits nerve sprouting. OBJECTIVES: The purpose of this study was to determine the spatial distribution of nerve sprouting and neurotrophic gene expression after MI. METHODS: We created MI in mice by coronary artery ligation. The hearts were removed 3 hours to 2 months after MI and examined for nerve fiber density and neurotrophic factor gene expression using Affymetrix microarray and mRNA analyses. RESULTS: The density of nerve fibers immunopositive for growth-associated protein (GAP)-43 was the highest 3 hours after MI both in the peri-infarct area and in the area remote to infarct, resulting in sympathetic (but not parasympathetic) hyperinnervation in the ventricles. The GAP-43-positive nerve fiber density of myocardium was greater in the outer transverse loop than in the inner vertical loop. The differences between these two myocardial loops peaked within 3 hours after MI and persisted for 2 months afterward. Gene expression of nerve growth factor, insulin-like growth factor, leukemia inhibitory factor, transforming growth factor-beta(3), and interleukin-1alpha was increased up to 2 months after MI compared with normal control. Expression of these growth factors was more pronounced and persistent in the peri-infarct area than in the remote area. CONCLUSION: MI induces sympathetic nerve sprouting in both peri-infarct and remote areas, more in the outer transverse loop. Selective up-regulation of nerve growth factor, insulin-like growth factor, leukemia inhibitory factor, transforming growth factor-beta(3), and interleukin-1alpha occurred in the peri-infarct area and, to a lesser extent, in the remote area.

Cryptococcus neoformans induces alterations in the cytoskeleton of human brain microvascular endothelial cells.

The fungal pathogen Cryptococcus neoformans has a predilection for the central nervous system (CNS), resulting in devastating meningoencephalitis. At present, it is unclear how C. neoformans traverses the blood-brain barrier (BBB) and causes CNS infection. The present study has examined and characterized the interaction of C. neoformans with human brain microvascular endothelial cells (HBMEC), which constitute the BBB. Adhesion of and transcytosis of HBMEC by C. neoformans was inoculum- and time-dependent and occurred with both encapsulated and acapsulated strains. C. neoformans induced marked morphological changes in HBMEC, for example membrane ruffling, irregular nuclear morphology and swelling of the mitochondria and the ER. These findings suggest that C. neoformans induced actin cytoskeletal reorganization of the host cells. In addition, it was observed that the dephosphorylated form of cofilin was increased during cryptococcal adherence to HBMEC, concomitant with the actin rearrangement. Cryptococcal binding to HBMEC was increased in the presence of Y27632, a Rho kinase (ROCK)-specific inhibitor. Since ROCK activates LIM kinase (LIMK), which phosphorylates cofilin (inactive form), this suggests the involvement of the ROCK-->LIMK-->cofilin pathway. In contrast, the phosphatase inhibitor sodium orthovanadate decreased adherence of Cryptococcus to HBMEC, concomitant with the increase of phosphorylation of cofilin. Furthermore, the tight junction marker protein occludin became Triton-extractable, indicating alteration of tight junctions in brain endothelial cells. This is the first demonstration that C. neoformans is able to adhere to and transcytose across the HBMEC monolayer and alter the cytoskeleton morphology in HBMEC. Further characterization of the interactions between C. neoformans and HBMEC should help the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.

Binding of Candida albicans enolase to plasmin(ogen) results in enhanced invasion of human brain microvascular endothelial cells.

Infection by the human opportunistic fungal pathogen Candida albicans has been increasing over recent years. In an attempt to understand the molecular mechanism of Candida invasion across host tissues, the relationship of C. albicans enolase to human plasminogen/plasmin was investigated. C. albicans enolase is a cell-surface protein and an immunodominant antigen in infected patients' sera. Plasminogen is an abundant plasma protein. Several lines of evidence support the binding of C. albicans enolase to human plasminogen. Firstly, it was found that various Candida strains were able to bind to plasminogen and its active form, plasmin. Secondly, recombinant Candida enolase was retained in a nickel-chelating affinity column matrix that can bind (125)I-labelled plasminogen or plasmin in a dose-dependent manner. Plasmin(ogen)-specific inhibitors, such as epsilon -aminocaproic acid and aprotinin, can effectively block plasmin-binding activity. Thirdly, as with many plasminogen receptors, binding of Candida enolase to plasmin(ogen) is lysine-dependent, whereas little inhibition occurred with arginine, aspartate and glutamate. Fourthly, immobilized enolase enhanced plasminogen's affinity for streptokinase at least tenfold, as demonstrated by its activation of plasmin activity. To elucidate the biological significance of this result, it was demonstrated that the plasmin(ogen)-bound Candida cells were able to induce fibrinolysis activity in a matrix-gel assay. Furthermore, plasmin-bound Candida cells had an increased ability to cross an in vitro blood-brain barrier system. The results given here indicate that Candida enolase is a plasminogen- and plasmin-binding protein and that the interaction of C. albicans enolase with the plasminogen system may contribute to invasion of the tissue barrier.

Infectomics: genomics and proteomics of microbial infections.

The completion of genomic sequences is the greatest triumph of molecular reductionism since the discovery of the DNA double helix in 1953. However, the utility of reductionism is becoming limited and holistic approaches, including theories and techniques, are desperately needed in the postgenomic era. In the field of infectious diseases there is an urgent need for global approaches that can efficiently, precisely and integratively study structural and functional genomics and proteomics of microbial infections (infectomics). The combination of new (e.g. DNA and protein microarrays) and traditional approaches (e.g. cloning, PCR, gene knockout and knockin, and antisense) will help overcome the challenges we are facing today. We assume that the global phenotypic changes (infectomes) in microbes and their host during infections are encoded by the genomes of microbial pathogens and their hosts, expressed in certain environmental conditions devoted to specific microbe-host interactions. Global drug responses (pharmacomes) in microbes and their host can be detected by genomic and proteomic approaches. Genome-wide approaches to genotyping and phenotyping or expression profiling will eventually lead to global dissection of microbial pathogenesis, efficient and rapid diagnosis of infectious diseases, and the development of novel strategies to control infections. The key fundamental issue of infectious diseases is how to globally and integratively understand the interactions between microbial pathogens and their hosts by using infectomics. In this review, we focus on the events that are considered important in infectomics.