Fluorescent, phosphorescent, magnetic resonance contrast and radioactive tracer labelling of extracellular vesicles.

This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.

Extracellular vesicles and microvascular pathology: Decoding the active dialogue.

Extracellular vesicles (EV) are a heterogeneous collection of membrane-surrounded structures released from all studied cells, under both physiological and pathological conditions. These nano-size vesicles carry complex cargoes including different classes of proteins, lipids and nucleic acids and are known to act as a communication and signalling vesicles in various cellular process. In addition to their role in development and progression of pathological disorders which make them potentially great biomarkers, EV have beneficial effects, as they take part in homeostasis. In this review we have analysed the evidence for the role of microvesicles and exosomes secreted from other cells on microvascular endothelium (EV uptake) as well as the role of endothelial-derived vesicles on their neighbouring and distant cells (EV release).

The Early Innate Immune Response to, and Phagocyte-Dependent Entry of, Cryptococcus neoformans Map to the Perivascular Space of Cortical Post-Capillary Venules in Neurocryptococcosis.

The innate immune system is the primary defense against cryptococcal infection, but paradoxically it promotes infection of the central nervous system. We performed a detailed longitudinal study of neurocryptococcosis in normal, chimeric, green fluorescent protein phagocyte-positive mice and phagocyte-depleted mice and interrogated the central nervous system innate immune response to Cryptococcus neoformans H99 using confocal microscopy, histology, flow cytometry, and quantification of brain cytokine/chemokines and fungal burdens. C. neoformans was present in the perivascular space (PVS) of post-capillary venules. This was associated with a massive influx of blood-derived monocytes, neutrophils, and T lymphocytes into the PVS and a predominantly proinflammatory cytokine/chemokine response. Phagocytes containing cryptococci were present only in the lumen and corresponding PVS of post-capillary venules. Free cryptococci were observed breaching the glia limitans, the protective barrier between the PVS and the cerebral parenchyma. Parenchymal cryptococcomas were typically in direct contact with post-capillary venules and lacked surrounding immune cell infiltrates. Phagocyte depletion abrogated cryptococcoma formation and PVS infiltrates. Together, these observations suggest that cryptococcomas can originate via phagocyte-dependent transport across post-capillary venular endothelium into the PVS and thence via passage of free cryptococci into the brain. In conclusion, we demonstrate for the first time that the PVS of cortical post-capillary venules is the major site of the early innate immune response to, and phagocyte-dependent entry of, C. neoformans.

Platelets activate a pathogenic response to blood-stage Plasmodium infection but not a protective immune response.

Clinical studies indicate that thrombocytopenia correlates with the development of severe falciparum malaria, suggesting that platelets either contribute to control of parasite replication, possibly as innate parasite killer cells or function in eliciting pathogenesis. Removal of platelets by anti-CD41 mAb treatment, platelet inhibition by aspirin, and adoptive transfer of wild-type (WT) platelets to CD40-KO mice, which do not control parasite replication, resulted in similar parasitemia compared with control mice. Human platelets at a physiologic ratio of 1 platelet to 9 red blood cells (RBCs) did not inhibit the in vitro development or replication of blood-stage Plasmodium falciparum The percentage of Plasmodium-infected (iRBCs) with bound platelets during the ascending parasitemia in Plasmodium chabaudi- and Plasmodium berghei-infected mice and the 48-hour in vitro cycle of P falciparum was <10%. P chabaudi and P berghei iRBCs with apoptotic parasites (TdT+) exhibited minimal platelet binding (<5%), which was similar to nonapoptotic iRBCs. These findings collectively indicate platelets do not kill bloodstage Plasmodium at physiologically relevant effector-to-target ratios. P chabaudi primary and secondary parasitemia was similar in mice depleted of platelets by mAb-injection just before infection, indicating that activation of the protective immune response does not require platelets. In contrast to the lack of an effect on parasite replication, adoptive transfer of WT platelets to CD40-KO mice, which are resistant to experimental cerebral malaria, partially restored experimental cerebral malaria mortality and symptoms in CD40-KO recipients, indicating platelets elicit pathogenesis and platelet CD40 is a key molecule.

Infrared spectroscopic characterization of monocytic microvesicles (microparticles) released upon lipopolysaccharide stimulation.

Microvesicles (MVs) are involved in cell-cell interactions, including disease pathogenesis. Nondestructive Fourier-transform infrared (FTIR) spectra from MVs were assessed as a technique to provide new biochemical insights into a LPS-induced monocyte model of septic shock. FTIR spectroscopy provided a quick method to investigate relative differences in biomolecular content of different MV populations that was complementary to traditional semiquantitative omics approaches, with which it is difficult to provide information on relative changes between classes (proteins, lipids, nucleic acids, carbohydrates) or protein conformations. Time-dependent changes were detected in biomolecular contents of MVs and in the monocytes from which they were released. Differences in phosphatidylcholine and phosphatidylserine contents were observed in MVs released under stimulation, and higher relative concentrations of RNA and α-helical structured proteins were present in stimulated MVs compared with MVs from resting cells. FTIR spectra of stimulated monocytes displayed changes that were consistent with those observed in the corresponding MVs they released. LPS-stimulated monocytes had reduced concentrations of nucleic acids, α-helical structured proteins, and phosphatidylcholine compared with resting monocytes but had an increase in total lipids. FTIR spectra of MV biomolecular content will be important in shedding new light on the mechanisms of MVs and the different roles they play in physiology and disease pathogenesis.-Lee, J., Wen, B., Carter, E. A., Combes, V., Grau, G. E. R., Lay, P. A. Infrared spectroscopic characterization of monocytic microvesicles (microparticles) released upon lipopolysaccharide stimulation.

Differential plasma microvesicle and brain profiles of microRNA in experimental cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is a fatal complication of Plasmodium infection, mostly affecting children under the age of five in the sub-Saharan African region. CM pathogenesis remains incompletely understood, although sequestered infected red blood cells, inflammatory cells aggregating in the cerebral blood vessels, and the microvesicles (MV) that they release in the circulation, have been implicated. Plasma MV numbers increase in CM patients and in the murine model, where blocking their release, genetically or pharmacologically, protects against brain pathology, suggesting a role of MV in CM neuropathogenesis. In this work, the microRNA (miRNA) cargo of MV is defined for the first time during experimental CM with the overarching hypothesis that this characterization could help understand CM pathogenesis. RESULTS: The change in abundance of miRNA was studied following infection of CBA mice with Plasmodium berghei ANKA strain (causing experimental CM), and Plasmodium yoelii, which causes severe malaria without cerebral complications, termed non-CM (NCM). miRNA expression was analyzed using microarrays to compare MV from healthy (NI) and CM mice, yielding several miRNA of interest. The differential expression profiles of these selected miRNA (miR-146a, miR-150, miR-193b, miR-205, miR-215, miR-467a, and miR-486) were analyzed in mouse MV, MV-free plasma, and brain tissue by quantitative reverse transcription PCR (RT-qPCR). Two miRNA-miR-146a and miR-193b-were confirmed as differentially abundant in MV from CM mice, compared with NCM and NI mice. These miRNA have been shown to play various roles in inflammation, and their dysregulation during CM may be critical for triggering the neurological syndrome via regulation of their potential downstream targets. CONCLUSIONS: These data suggest that, in the mouse model at least, miRNA may have a regulatory role in the pathogenesis of severe malaria.

Production, fate and pathogenicity of plasma microparticles in murine cerebral malaria.

In patients with cerebral malaria (CM), higher levels of cell-specific microparticles (MP) correlate with the presence of neurological symptoms. MP are submicron plasma membrane-derived vesicles that express antigens of their cell of origin and phosphatidylserine (PS) on their surface, facilitating their role in coagulation, inflammation and cell adhesion. In this study, the in vivo production, fate and pathogenicity of cell-specific MP during Plasmodium berghei infection of mice were evaluated. Using annexin V, a PS ligand, and flow cytometry, analysis of platelet-free plasma from infected mice with cerebral involvement showed a peak of MP levels at the time of the neurological onset. Phenotypic analyses showed that MP from infected mice were predominantly of platelet, endothelial and erythrocytic origins. To determine the in vivo fate of MP, we adoptively transferred fluorescently labelled MP from mice with CM into healthy or infected recipient mice. MP were quickly cleared following intravenous injection, but microscopic examination revealed arrested MP lining the endothelium of brain vessels of infected, but not healthy, recipient mice. To determine the pathogenicity of MP, we transferred MP from activated endothelial cells into healthy recipient mice and this induced CM-like brain and lung pathology. This study supports a pathogenic role for MP in the aggravation of the neurological lesion and suggests a causal relationship between MP and the development of CM.

Real-time imaging reveals the dynamics of leukocyte behaviour during experimental cerebral malaria pathogenesis.

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6C(hi) monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8(+) T lymphocytes. A critical number of CD8(+) T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8(+) T lymphocytes in regulating vascular pathology in this disease.

Endothelial microparticles interact with and support the proliferation of T cells.

Endothelial cells closely interact with circulating lymphocytes. Aggression or activation of the endothelium leads to an increased shedding of endothelial cell microparticles (MP). Endothelial MP (EMP) are found in high plasma levels in numerous immunoinflammatory diseases, such as atherosclerosis, sepsis, multiple sclerosis, and cerebral malaria, supporting their role as effectors and markers of vascular dysfunction. Given our recently described role for human brain microvascular endothelial cells (HBEC) in modulating immune responses, we investigated how HBEC-derived MP could interact with and support the proliferation of T cells. Like their mother cells, EMP expressed molecules important for Ag presentation and T cell costimulation, that is, ß2-microglobulin, MHC II, CD40, and ICOSL. HBEC were able to take up fluorescently labeled Ags with EMP also containing fluorescent Ags, suggestive of Ag carryover from HBEC to EMP. In cocultures, fluorescently labeled EMP from resting or cytokine-stimulated HBEC formed conjugates with both CD4(+) and CD8(+) subsets, with higher proportions of T cells binding EMP from cytokine-stimulated cells. The increased binding of EMP from cytokinestimulated HBEC to T cells was VCAM-1 and ICAM-1 dependent. Finally, in CFSE T cell proliferation assays using anti-CD3 mAb or T cell mitogens, EMP promoted the proliferation of CD4(+) T cells and that of CD8(+) T cells in the absence of exogenous stimuli and in the T cell mitogenic stimulation. Our findings provide novel evidence that EMP can enhance T cell activation and potentially ensuing Ag presentation, thereby pointing toward a novel role for MP in neuroimmunological complications of infectious diseases.

Microparticles from mycobacteria-infected macrophages promote inflammation and cellular migration.

Mycobacterium tuberculosis infection is characterized by a strong inflammatory response whereby a few infected macrophages within the granuloma induce sustained cellular accumulation. The mechanisms coordinating this response are poorly characterized. We hypothesized that microparticles (MPs), which are submicron, plasma membrane-derived vesicles released by cells under both physiological and pathological conditions, are involved in this process. Aerosol infection of mice with M. tuberculosis increased CD45(+) MPs in the blood after 4 wk of infection, and in vitro infection of human and murine macrophages with mycobacteria enhanced MP release. MPs derived from mycobacteria-infected macrophages were proinflammatory, and when injected into uninfected mice they induced significant neutrophil, macrophage, and dendritic cell recruitment to the injection site. When incubated with naive macrophages, these MPs enhanced proinflammatory cytokine and chemokine release, and they aided in the disruption of the integrity of a respiratory epithelial cell monolayer, providing a mechanism for the egress of cells to the site of M. tuberculosis infection in the lung. In addition, MPs colocalized with the endocytic recycling marker Rab11a within macrophages, and this association increased when the MPs were isolated from mycobacteria-infected cells. M. tuberculosis-derived MPs also carried mycobacterial Ag and were able to activate M. tuberculosis-specific CD4(+) T cells in vivo and in vitro in a dendritic cell-dependent manner. Collectively, these data identify an unrecognized role for MPs in host response against M. tuberculosis by promoting inflammation, intercellular communication, and cell migration.

Cooperation between ß- and γ-cytoplasmic actins in the mechanical regulation of endothelial microparticle formation.

Elevated endothelial microparticle (MP) levels are observed in numerous diseases, increasingly supporting roles as effectors and valuable markers of vascular dysfunction. While a contractile role for the actin cytoskeleton has been implicated in vesiculation, i.e., MP production, the precise interactions and mechanisms of its constituents, ß- and γ-cytoplasmic actins, is unknown. Human cerebral microvascular endothelial cells were stimulated with known agonists, and vesiculation development was monitored by scanning electron microscopy (SEM) and flow cytometry. These data in combination provide new insight into the kinetics, patterns of vesiculating cell recruitment, and degrees of response specific to stimuli. Reorganization of ß- and γ-actins, F-actin, vinculin, and talin accompanied significant MP release. ß-Actin redistribution into basal stress fibers following stimulation was associated with increased apically situated actin-rich particulate structures, which in turn directly correlated with electron-lucent membrane protrusions observed by SEM. Y-27632 Rho-kinase inhibition abolished basal ß-actin fiber formation, minimizing apically associated actin-rich structures, significantly reducing membrane protrusions and MP release to near basal levels. Cytoskeletal protein expression and distribution varied between MPs and mother cells, as determined by Western blot. These data strongly suggest that ß-actin plays an active facilitative role in agonist-induced protuberance formation, through mechanical interactions with newly described actin-rich structures.

Unravelling mysteries at the perivascular space: a new rationale for cerebral malaria pathogenesis.

Cerebral malaria (CM) is a severe neurological complication caused by Plasmodium falciparum parasites; it is characterized by the sequestration of infected red blood cells within the cerebral microvasculature. New findings, combined with a better understanding of the central nervous system (CNS) barriers, have provided greater insight into the players and events involved in CM, including site-specific T cell responses in the human brain. Here, we review the updated roles of innate and adaptive immune responses in CM, with a focus on the role of the perivascular macrophage-endothelium unit in antigen presentation, in the vascular and perivascular compartments. We suggest that these events may be pivotal in the development of CM.

Tetranuclear Polypyridylruthenium(II) Complexes as Selective Nucleic Acid Stains for Flow Cytometric Analysis of Monocytic and Epithelial Lung Carcinoma Large Extracellular Vesicles.

Selective staining of extracellular vesicles (EVs) is a major challenge for diagnostic and therapeutic applications. Herein, the EV labeling properties of a new class of tetranuclear polypyridylruthenium(II) complexes, Rubb7-TNL and Rubb7-TL, as phosphorescent stains are described. These new stains have many advantages over standard stains to detect and characterize EVs, including: high specificity for EV staining versus cell staining; high phosphorescence yields; photostability; and a lack of leaching from EVs until incorporation with target cells. As an example of their utility, large EVs released from control (basal) or lipopolysaccharide (LPS)-stimulated THP-1 monocytic leukemia cells were studied as a model of immune system EVs released during bacterial infection. Key findings from EV staining combined with flow cytometry were as follows: (i) LPS-stimulated THP-1 cells generated significantly larger and more numerous large EVs, as compared with those from unstimulated cells; (ii) EVs retained native EV physical properties after staining; and (iii) the new stains selectively differentiated intact large EVs from artificial liposomes, which are models of cell membrane fragments or other lipid-containing debris, as well as distinguished two distinct subpopulations of monocytic EVs within the same experiment, as a result of biochemical differences between unstimulated and LPS-stimulated monocytes. Comparatively, the staining patterns of A549 epithelial lung carcinoma-derived EVs closely resembled those of THP-1 cell line-derived EVs, which highlighted similarities in their selective staining despite their distinct cellular origins. This is consistent with the hypothesis that these new phosphorescent stains target RNA within the EVs.

Microparticle-associated nucleic acids mediate trait dominance in cancer.

Drug resistance is a major cause of cancer treatment failure, with multidrug resistance (MDR) being the most serious, whereby cancer cells display cross-resistance to structurally and functionally unrelated drugs. MDR is caused by overexpression of the efflux transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1). These transporters act to maintain sublethal intracellular drug concentrations within the cancer cell, making the population treatment unresponsive. Recently, we discovered a novel nongenetic basis to MDR whereby microparticles (MPs) transfer P-gp intercellularly from MDR donor cells to drug-sensitive recipient cells. MPs isolated from MDR leukemia and breast cancer cells were cocultured with their drug-sensitive counterparts. P-gp transfer was assessed by direct immunolabeling, and acquired transcripts and regulatory microRNAs by quantitative real-time PCR. We show that MDR MPs incorporate nucleic acids; MPs change recipient cells' transcriptional environment to reflect donor MDR phenotype, and distinct pathways exist among cancers of different origin that may be dependent on donor cells' ABCB1 overexpression. We demonstrate that this pathway exists for both hematological and nonhematological malignancies. By conferring MDR and "retemplating" the transcriptional landscape of recipient cells, MPs provide a novel pathway, having implications in the dissemination and acquisition of deleterious traits in clinical oncology.

Microparticles mediate MRP1 intercellular transfer and the re-templating of intrinsic resistance pathways.

Multidrug resistance (MDR) is a major impediment to the overall success of chemotherapy in clinical oncology. MDR has been primarily attributed by the ATP-dependent transmembrane proteins, P-glycoprotein (P-gp, ABCB1) and Multidrug Resistance-Associated Protein 1 (MRP1, ABCC1). These proteins maintain sublethal concentrations of intracellular chemotherapeutics by virtue of their drug efflux capacity. In this study, we report the acquisition and dissemination of functional MRP1 via microparticle (MP) mediated intercellular transfer. After we showed the transfer and functionality of P-gp in drug sensitive recipient cells, we report the transfer and time-dependent functionality of MRP1 in drug sensitive leukaemia cells following exposure to MPs shed by MRP1-overexpressing MDR cells. We also demonstrate a remarkable capacity for MPs shed from cells with a P-gp dominant resistance profile to re-template a pre-existing MRP1 dominant profile in recipient cells. These findings have significance in understanding the molecular basis for tumour dominant phenotypes and introduce potential new strategies and targets for the acquisition of MDR and other deleterious traits.

Cell-derived microparticles: new targets in the therapeutic management of disease.

Intercellular communication is essential to maintain vital physiological activities and to regulate the organism's phenotype. There are a number of ways in which cells communicate with one another. This can occur via autocrine signaling, endocrine signaling or by the transfer of molecular mediators across gap junctions. More recently communication via microvesicular shedding has gained important recognition as a significant pathway by which cells can coordinate the spread and dominance of selective traits within a population. Through this communication apparatus, cells can now acquire and secure a survival advantage, particularly in the context of malignant disease. This review aims to highlight some of the functions and implications of microparticles in physiology of various disease states, and present a novel therapeutic strategy through the regulation of microparticle production.

A tetranuclear polypyridylruthenium(II) complex as a selective stain for extracellular vesicle penetration through brain microvascular endothelium.

A new photoluminescent polypyridylruthenium(II) stain for extracellular vesicles (EVs) released from lipopolysaccharide-stimulated THP-1 monocytes enabled important new insights into how the bacteria-induced immune system affects the blood-brain barrier (BBB). These included previously unknown aspects of EV interactions with BBB microvascular endothelial cells and the extracellular matrix relevant to human brain diseases.

Exploring the role of extracellular vesicles and their protein cargo in lung cancer metastasis: A review.

Extracellular vesicles (EV) are membrane-enclosed structures of varying size released from all cells and contain a variety of cargo including proteins, lipids, and nucleic acids. They are postulated to play a pivotal role in cancer metastasis through delivery of oncogenic material to neighbouring and distant cells to promote development of a metastatic niche and tumour seeding. Here we reviewed protein data in relevant literature to determine whether specific proteins known to be involved in metastasis can be reliably identified in lung cancer EV, whether these proteins are important in all or specific lung cancers, and whether results from in-vitro cell studies are supported by research examining EV in human biofluids. Our analysis suggests that specific proteins may be more important for individual lung cancers, but interpretation of the literature is currently limited by a relative lack of research investigating EV proteins in some cancers and in clinical studies using biofluids.

Differential microRNA expression in experimental cerebral and noncerebral malaria.

MicroRNAs (miRNAs) are posttranscriptional regulatory molecules that have been implicated in the regulation of immune responses, but their role in the immune response to Plasmodium infection is unknown. We studied the expression of selected miRNAs following infection of CBA mice with Plasmodium berghei ANKA (PbA), which causes cerebral malaria (CM), or Plasmodium berghei K173 (PbK), which causes severe malaria but without cerebral complications, termed non-CM. The differential expression profiles of selected miRNAs (let-7i, miR-27a, miR-150, miR-126, miR-210, and miR-155) were analyzed in mouse brain and heart tissue by quantitative reverse transcription-PCR (qRT-PCR). We identified three miRNAs that were differentially expressed in the brain of PbA-infected CBA mice: let7i, miR-27a, and miR-150. In contrast, no miRNA changes were detected in the heart, an organ with no known pathology during acute malaria. To investigate the involvement of let-7i, miR-27a, and miR-150 in CM-resistant mice, we assessed the expression levels in gamma interferon knockout (IFN-γ(-/-)) mice on a C57BL/6 genetic background. The expression of let-7i, miR-27a, and miR-150 was unchanged in both wild-type (WT) and IFN-γ(-/-) mice following infection. Overexpression of these three miRNAs during PbA, but not PbK, infection in WT mice may be critical for the triggering of the neurological syndrome via regulation of their potential downstream targets. Our data suggest that in the CBA mouse at least, miRNA may have a regulatory role in the pathogenesis of severe malaria.