Retinal microglia express more MHC class I and promote greater T-cell-driven inflammation than brain microglia.

Introduction: Macrophage function is determined by microenvironment and origin. Brain and retinal microglia are both derived from yolk sac progenitors, yet their microenvironments differ. Utilizing single-cell RNA sequencing (scRNA-seq) data from mice, we tested the hypothesis that retinal and brain microglia exhibit distinct transcriptional profiles due to their unique microenvironments. Methods: Eyes and brains from 2-4 month wildtype mice were combined (20 eyes; 3 brains) to yield one biologically diverse sample per organ. Each tissue was digested into single cell suspensions, enriched for immune cells, and sorted for scRNA-seq. Analysis was performed in Seurat v3 including clustering, integration, and differential expression. Multi-parameter flow cytometry was used for validation of scRNA-seq results. Lymphocytic choriomeningitis virus (LCMV) Clone 13, which produces a systemic, chronic, and neurotropic infection, was used to validate scRNA-seq and flow cytometry results in vivo. Results: Cluster analysis of integrated gene expression data from eye and brain identified 6 Tmem119 + P2ry12 + microglial clusters. Differential expression analysis revealed that eye microglia were enriched for more pro-inflammatory processes including antigen processing via MHC class I (14.0-fold, H2-D1 and H2-K1) and positive regulation of T-cell immunity (8.4-fold) compared to brain microglia. Multi-parameter flow cytometry confirmed that retinal microglia expressed 3.2-fold greater H2-Db and 263.3-fold more H2-Kb than brain microglia. On Day 13 and 29 after LCMV infection, CD8+ T-cell density was greater in the retina than the brain. Discussion: Our data demonstrate that the microenvironment of retina and brain differs, resulting in microglia-specific gene expression changes. Specifically, retinal microglia express greater MHC class I by scRNA-seq and multi-parameter flow cytometry, resulting in a possibly enhanced capability to stimulate CD8+ T-cell inflammation during LCMV infection. These results may explain tissue-specific differences between retina and brain during systemic viral infections and CD8+ T-cell driven autoimmune disease.

Induction of Broad Immunity against Invasive Salmonella Disease by a Quadrivalent Combination Salmonella MAPS Vaccine Targeting Salmonella Enterica Serovars Typhimurium, Enteritidis, Typhi, and Paratyphi A.

Bloodstream infections in low- and middle-income countries (LMICs) are most frequently attributed to invasive Salmonella disease caused by four primary serovars of Salmonella enterica: Typhi, Paratyphi A, Typhimurium, and Enteritidis. We showed previously that a bivalent vaccine targeting S. Typhi and S. Paratyphi A using a Multiple Antigen-Presenting System (MAPS) induced functional antibodies against S. Typhi and S. Paratyphi. In the current study, we describe the preclinical development of a first candidate quadrivalent combination Salmonella vaccine with the potential to cover all four leading invasive Salmonella serotypes. We showed that the quadrivalent Salmonella MAPS vaccine, containing Vi from S. Typhi, O-specific Polysaccharide (OSP) from S. Paratyphi A, S. Enteritidis and S. Typhimurium, combined with the Salmonella-specific protein SseB, elicits robust and functional antibody responses to each of the components of the vaccine. Our data indicates that the application of MAPS technology to the development of vaccines targeting invasive forms of Salmonella is practical and merits additional consideration.

A Bivalent MAPS Vaccine Induces Protective Antibody Responses against Salmonella Typhi and Paratyphi A.

Infections by Salmonella Typhi and Paratyphi A strain are still a major cause of morbidity and mortality in developing countries. Generation of antibodies against the Vi capsular polysaccharide of S. Typhi via either pure polysaccharide or protein-polysaccharide conjugate is a very effective way to protect against S. Typhi. To date, there is no commercially available vaccine against S. Paratyphi A. The O-specific polysaccharide (OSP) has been generally considered a good vaccine target for Paratyphi A. Here, a bivalent vaccine against Vi and OSP was generated using the Multiple Antigen Presenting System (MAPS). Three different protein constructs, including CRM197, rEPA of Pseudomonas, and a pneumococcal fusion protein SP1500-SP0785, were fused to Rhizavidin (Rhavi) and evaluated their impact on immunogenicity when incorporated as fusion proteins affinity-bound to the two polysaccharides. We compared the antibody responses, antibody avidity, and cidal activity of sera post-immunization with monovalent vs. combination vaccines. We also wished to evaluate the generation of Vi-specific memory B cells in mice. We found little interference when combination vaccine was compared to monovalent vaccines with respect to antibody concentration and cidal activity of sera. Significant affinity maturation was noted for both Vi and OSP antigens. Thus, our preclinical results with a combination Vi- and OSP-MAPS vaccine strongly support the feasibility of this approach and its application of this approach to other important salmonella and Shigella species.

Microparticles in cancer: A review of recent developments and the potential for clinical application.

Once thought of as inert remnants of cellular processes, the significance of membrane vesicles is now expanding as their capacity to package and transfer bioactive molecules during intercellular communication is established. This ability to serve as vectors in the trafficking of cellular cargo is of mounting interest in the context of cancer, particularly in the dissemination of deleterious cancer traits from donor cells to recipient cells. Although microparticles (MPs) contribute to the pathogenesis of cancer, their unique characteristics can also be exploited in the context of cancer management. The detection of MPs in body fluids has the potential to provide an effective means for the diagnosis, prognosis and surveillance of cancer patients. The use of these readily accessible systemic biomarkers has the potential to circumvent the need for invasive biopsy procedures. In addition, the autologous nature of MPs may allow them to be used as novel drug delivery carriers. Consequently, the modulation of MP vesiculation to treat disease, the detection of MPs in disease monitoring, and the application of MPs as therapeutic delivery vehicles present prospective clinical interventions in the treatment of cancer.

Microparticles Mediate the Intercellular Regulation of microRNA-503 and Proline-Rich Tyrosine Kinase 2 to Alter the Migration and Invasion Capacity of Breast Cancer Cells.

The successful treatment of cancer is hampered by drug resistance and metastasis. While these two obstacles were once considered separately, recent evidence associates resistance with an enhanced metastatic capacity. However, the underlying mechanisms remain undefined. We previously described the intercellular transfer of drug resistance via submicron vesicles called microparticles (MPs). We now propose that MPs derived from drug-resistant cells are also involved in the intercellular transfer of components to enhance the migration and invasion capacity of cells. Thus, MPs may be a conduit between resistance and metastasis. We used microarray analysis to identify regulatory microRNAs (miRNAs), which contribute to the dissemination of metastatic traits. miR-503 was downregulated in recipient cells following co-culture with MPs isolated from drug-resistant cells. miR-503 was inversely associated with metastasis, as demonstrated using wound healing/scratch migration assays and Matrigel(®)-coated transwell invasion assays. Proline-rich tyrosine kinase 2 (PYK2) was upregulated in recipient cells and associated with increased migration and invasion, with these phenotypes being reversed using a pharmacological inhibitor of PYK2 phosphorylation, tyrphostin A9. However, the MP-mediated promotion of metastatic traits was not due to the presence of these effectors in the MP cargo but rather due to down stream effector molecules in these pathways. This is the first demonstration that the role of MPs in trait acquisition extends beyond the direct transfer of vesicle components and also includes transfer of intermediary regulators that induce down stream mediators following transfer to recipient cells. This implicates an expanding role of MPs in cancer pathogenesis.

Microparticle drug sequestration provides a parallel pathway in the acquisition of cancer drug resistance.

Expanding on our previous findings demonstrating that microparticles (MPs) spread cancer multidrug resistance, we now show that MPs sequester drugs, reducing the free drug concentration available to cells. MPs were isolated from drug-sensitive and drug-resistant sub-clones of a human breast adenocarcinoma cell line and from human acute lymphoblastic leukemia cells. MPs were assessed for size, mitochondria, RNA and phospholipid content, P-glycoprotein (P-gp) expression and orientation and ATPase activity relative to drug sequestration capacity. Of the drug classes examined, MPs sequestered the anthracycline class to a significant degree. The degree of sequestration was likely due to the size of MPs and thus the amount of cargo they contain, to which the anthracyclines bind. Moreover, a proportion of the P-gp present on MPs was inside-out in orientation, enabling it to influx drugs rather than its typical efflux function. This was confirmed by surface immunofluorescence and by assessment of drug-stimulated ATPase activity following MP permeabilization. Thus we determined that breast cancer MPs carried a proportion of their P-gp oriented inside-out, providing active sequestration within the microvesicular compartment. These results demonstrate a capacity for MPs to sequester chemotherapeutic drugs, which has a predominantly active sequestration component for MPs derived from drug-resistant cells and a predominantly passive component for MPs derived from drug-sensitive cells. This reduction in available drug concentration has potential to contribute to a parallel pathway and complements that of the intercellular transfer of P-gp. These findings lend further support to the role of MPs in limiting the successful management of cancer.

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.

Glioma microvesicles carry selectively packaged coding and non-coding RNAs which alter gene expression in recipient cells.

Interactions between glioma cells and their local environment are critical determinants of brain tumor growth, infiltration and neovascularisation. Communication with host cells and stroma via microvesicles represents one pathway by which tumors can modify their surroundings to achieve a tumor-permissive environment. Here we have taken an unbiased approach to identifying RNAs in glioma-derived microvesicles, and explored their potential to regulate gene expression in recipient cells. We find that glioma microvesicles are predominantly of exosomal origin and contain complex populations of coding and noncoding RNAs in proportions that are distinct from those in the cells from which they are derived. Microvesicles show a relative depletion in microRNA compared with their cells of origin, and are enriched in unusual or novel noncoding RNAs, most of which have no known function. Short-term exposure of brain microvascular endothelial cells to glioma microvesicles results in many gene expression changes in the endothelial cells, most of which cannot be explained by direct delivery of transcripts. Our data suggest that the scope of potential actions of tumor-derived microvesicles is much broader and more complex than previously supposed, and highlight a number of new classes of small RNA that remain to be characterized.

Microparticle conferred microRNA profiles--implications in the transfer and dominance of cancer traits.

BACKGROUND: Microparticles (MPs) are membrane vesicles which are released from normal and malignant cells following a process of budding and detachment from donor cells. MPs contain surface antigens, proteins and genetic material and serve as vectors of intercellular communication. MPs comprise the major source of systemic RNA including microRNA (miRNA), the aberrant expression of which appears to be associated with stage, progression and spread of many cancers. Our previous study showed that MPs carry both transcripts and miRNAs associated with the acquisition of multidrug resistance in cancer. RESULTS: Herein, we expand on our previous finding and demonstrate that MPs carry the transcripts of the membrane vesiculation machinery (floppase and scramblase) as well as nucleic acids encoding the enzymes essential for microRNA biogenesis (Drosha, Dicer and Argonaute). We also demonstrate using microarray miRNA profiling analysis, the selective packaging of miRNAs (miR-1228*, miR-1246, miR-1308, miR-149*, miR-455-3p, miR-638 and miR-923) within the MP cargo upon release from the donor cells. CONCLUSIONS: These miRNAs are present in both haematological and non-haematological cancer cells and are involved in pathways implicated in cancer pathogenesis, membrane vesiculation and cascades regulated by ABC transporters. Our recent findings reinforce our earlier reports that MP transfer 're-templates' recipient cells so as to reflect donor cell traits. We now demonstrate that this process is likely to occur via a process of selective packaging of nucleic acid species, including regulatory nucleic acids upon MP vesiculation. These findings have significant implications in understanding the cellular basis governing the intercellular acquisition and dominance of deleterious traits in cancers.

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.

Time- and passage-dependent characteristics of a Calu-3 respiratory epithelial cell model.

BACKGROUND: Although standard protocols for the study of drug delivery in the upper airways using the sub-bronchial epithelial cell line Calu-3 model, particularly that of the air-liquid interface configuration, are readily available, the model remains un-validated with respect to culture conditions, barrier integrity, mucous secretion, and transporter function. With respect to the latter, the significance of functional P-glycoprotein (P-gp) activity in Calu-3 cells has recently been questioned, despite previous reports demonstrating a significant contribution by the same transporter in limiting drug uptake across the pulmonary epithelium. Therefore, the aim of this study was the standardization of this model as a tool for drug discovery. METHODS: Calu-3 cells were grown using air-interfaced condition (AIC) on polyester cell culture supports. Monolayers were evaluated for transepithelial electrical resistance (TEER), permeability to the paracellular marker fluorescein sodium (flu-Na), surface P-gp expression, and functionality. Mucous secretion was also identified by alcian blue staining. RESULTS: TEER and permeability values obtained for Calu-3 monolayers were shown to plateau between day 5 and day 21 in culture with values reaching 474 +/- 44 omega cm(2) and 2.33 +/- 0.36 x 10(-7) cm/s, respectively, irrespective of the passage number examined. 32.7 +/- 1.49% of Calu-3 cells cultured under these conditions detected positive for cell surface P-gp expression from day 7 onwards. Functional cell surface expression was established by rhodamine 123 drug extrusion assays. CONCLUSION: This study establishes a clear dependence on culture time and passage number for optimal barrier integrity, mucous secretion, and cell-surface P-gp expression and function in Calu-3 cells. Furthermore it provides initial guidelines for the optimization of this model for high throughput screening applications.