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 TGFβ1-dependent inhibition of NK cells. Altogether, these data support the investigation of a miR-186-containing nanoparticle formulation to prevent tumor growth and TGFβ1-dependent immune escape in high-risk neuroblastoma patients as well as the inclusion of
-derived NK exosomes as a potential therapeutic option alongside NK cell-based immunotherapy.
These findings highlight the therapeutic potential of NK cell-derived exosomes containing the tumor suppressor miR-186 that inhibits growth, spreading, and TGFβ-dependent immune escape mechanisms in neuroblastoma.
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.
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
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.
How exosomic microRNAs (miRNAs) contribute to the development of drug resistance in the context of the tumor microenvironment has not been previously described in neuroblastoma (NBL).
Coculture ...experiments were performed to assess exosomic transfer of miR-21 from NBL cells to human monocytes and miR-155 from human monocytes to NBL cells. Luciferase reporter assays were performed to assess miR-155 targeting of TERF1 in NBL cells. Tumor growth was measured in NBL xenografts treated with Cisplatin and peritumoral exosomic miR-155 (n = 6 mice per group) CD163, miR-155, and TERF1 levels were assessed in 20 NBL primary tissues by Human Exon Arrays and quantitative real-time polymerase chain reaction. Student's t test was used to evaluate the differences between treatment groups. All statistical tests were two-sided.
miR-21 mean fold change (f.c.) was 12.08±0.30 (P < .001) in human monocytes treated with NBL derived exosomes for 48 hours, and miR-155 mean f.c. was 4.51±0.25 (P < .001) in NBL cells cocultured with human monocytes for 48 hours. TERF1 mean luciferase activity in miR-155 transfected NBL cells normalized to scrambled was 0.36 ± 0.05 (P <.001). Mean tumor volumes in Dotap-miR-155 compared with Dotap-scrambled were 322.80±120mm(3) and 76.00±39.3mm(3), P = .002 at day 24, respectively. Patients with high CD163 infiltrating NBLs had statistically significantly higher intratumoral levels of miR-155 (P = .04) and lower levels of TERF1 mRNA (P = .02).
These data indicate a unique role of exosomic miR-21 and miR-155 in the cross-talk between NBL cells and human monocytes in the resistance to chemotherapy, through a novel exosomic miR-21/TLR8-NF-кB/exosomic miR-155/TERF1 signaling pathway.
Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, ...we found that C. neoformans-derived microvesicles (CnMVs) can enhance the traversal of the blood-brain barrier (BBB) by C. neoformans invitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs), the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein)-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion) signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Assembly of a scaffold consisting of CARD9, BCL10, and MALT1 (CBM complex) is critical for effective signaling by multiple pattern recognition receptors (PRRs) including Dectin and RIG-I. The RUN ...domain Beclin-1-interacting cysteine-rich-containing Rubicon protein associates constitutively with the Beclin-UVRAG-Vps34 complex under normal conditions to regulate autophagy. Rubicon also interacts with the phagocytic NADPH-oxidase complex upon TLR stimulation to induce potent antimicrobial responses. Here, we show Rubicon is a physiological feedback inhibitor of CBM-mediated PRR signaling, preventing unbalanced proinflammatory responses. Upon Dectin-1- or RIG-I-mediated activation, Rubicon dynamically exchanges binding partners from 14-3-3β to CARD9 in a stimulation-specific and phosphorylation-dependent manner, disassembling the CBM signaling complex and ultimately terminating PRR-induced cytokine production. Remarkably, Rubicon's actions in the autophagy complex, phagocytosis complex, and CBM complex are functionally and genetically separable. Rubicon thus differentially targets signaling complexes, depending on environmental stimuli, and may function to coordinate various immune responses against microbial infection.
► Rubicon changes binding partners from 14-3-3β to CARD9 upon Dectin-1 stimulation ► Rubicon and CARD9 interaction disassembles the CARD9-BCL10-MALT1 signaling complex ► Rubicon is a feedback inhibitor of CARD9-mediated signal pathway ► Rubicon negatively affects CBM-mediated antifungal immunity
Despite aggressive research, central nervous system (CNS) disorders, including blood-brain barrier (BBB) injury caused by microbial infection, stroke, abused drugs e.g., methamphetamine (METH) and ...nicotine, and other pathogenic insults, remain the world's leading cause of disabilities. In our previous work, we found that dysfunction of brain microvascular endothelial cells (BMECs), which are a major component of the BBB, could be caused by nicotine, meningitic pathogens and microbial factors, including HIV-1 virulence factors gp41 and gp120. One of the most challenging issues in this area is that there are no available cell-based biomarkers in peripheral blood for BBB disorders caused by microbial and non-microbial insults. To identify such cellular biomarkers for BBB injuries, our studies have shown that mice treated with nicotine, METH and gp120 resulted in increased blood levels of CD146+(endothelial marker)/S100B+ (brain marker) circulating BMECs (cBMECs) and CD133+progenitor cell (PC) marker/CD146+ endothelial PCs (EPCs), along with enhanced Evans blue and albumin extravasation into the brain. Nicotine and gp120 were able to significantly increase the serum levels of ubiquitin C-terminal hydrolase 1 (UCHL1) (a new BBB marker) as well as S100B in mice, which are correlated with the changes in cBMECs and EPCs. Nicotine- and meningitic E. coli K1-induced enhancement of cBMEC levels, leukocyte migration across the BBB and albumin extravasation into the brain were significantly reduced in alpha7 nAChR knockout mice, suggesting that this inflammatory regulator plays an important role in CNS inflammation and BBB disorders caused by microbial and non-microbial factors. These results demonstrated that cBMECs as well as EPCs may be used as potential cell-based biomarkers for indexing of BBB injury.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A mouse brain transmigration assessment (MBTA) was created to investigate the central nervous system (CNS) pathogenesis of cryptococcal meningoencephalitis.
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.
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.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PSF is required for meningitic E. coli K1 penetration and leukocyte transmigration across the blood-brain ...barrier (BBB), which are the hallmarks of bacterial meningitis. However, it is unknown how vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB, which are required for bacteria-mediated pathogenicities.
IbeA-induced E. coli K1 invasion, polymorphonuclear leukocyte (PMN) transmigration and IKK/NF-κB activation are blocked by Caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB. IKKα/β phosphorylation is blocked by ERK inhibitors. Co-immunoprecipitation analysis shows that vimentin forms a complex with IκB, NF-κB and tubulins in the resting cells. A dissociation of this complex and a simultaneous association of PSF with NF-κB could be induced by IbeA in a time-dependent manner. The head domain of vimentin is required for the complex formation. Two cytoskeletal components, vimentin filaments and microtubules, contribute to the regulation of NF-κB. SiRNA-mediated knockdown studies demonstrate that IKKα/β phosphorylation is completely abolished in HBMECs lacking vimentin and PSF. Phosphorylation of ERK and nuclear translocation of NF-κB are entirely dependent on PSF. These findings suggest that vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB activation. PSF is essential for translocation of NF-κB and ERK to the nucleus.
These findings reveal previously unappreciated facets of the IbeA-binding proteins. Cooperative contributions of vimentin and PSF to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB may represent a new paradigm in pathogen-induced signal transduction and lead to the development of novel strategies for the prevention and treatment of bacterial meningitis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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