Microvesicles and intercellular communication in the context of parasitism

There is a rapidly growing body of evidence that production of microvesicles (MVs) is a universal feature of cellular life. MVs can incorporate microRNA (miRNA), mRNA, mtDNA, DNA and retrotransposons, camouflage viruses/viral components from immune surveillance, and transfer cargo between cells. These properties make MVs an essential player in intercellular communication. Increasing evidence supports the notion that MVs can also act as long-distance vehicles for RNA molecules and participate in metabolic synchronization and reprogramming eukaryotic cells including stem and germinal cells. MV ability to carry on DNA and their general distribution makes them attractive candidates for horizontal gene transfer, particularly between multi-cellular organisms and their parasites; this suggests important implications for the co-evolution of parasites and their hosts. In this review, we provide current understanding of the roles played by MVs in intracellular pathogens and parasitic infections. We also discuss the possible role of MVs in co-infection and host shifting

Freshwater Cyanobacterial Toxins, Cyanopeptides and Neurodegenerative Diseases.

Cyanobacteria produce a wide range of structurally diverse cyanotoxins and bioactive cyanopeptides in freshwater, marine, and terrestrial ecosystems. The health significance of these metabolites, which include genotoxic- and neurotoxic agents, is confirmed by continued associations between the occurrence of animal and human acute toxic events and, in the long term, by associations between cyanobacteria and neurodegenerative diseases. Major mechanisms related to the neurotoxicity of cyanobacteria compounds include (1) blocking of key proteins and channels; (2) inhibition of essential enzymes in mammalian cells such as protein phosphatases and phosphoprotein phosphatases as well as new molecular targets such as toll-like receptors 4 and 8. One of the widely discussed implicated mechanisms includes a misincorporation of cyanobacterial non-proteogenic amino acids. Recent research provides evidence that non-proteinogenic amino acid BMAA produced by cyanobacteria have multiple effects on translation process and bypasses the proof-reading ability of the aminoacyl-tRNA-synthetase. Aberrant proteins generated by non-canonical translation may be a factor in neuronal death and neurodegeneration. We hypothesize that the production of cyanopeptides and non-canonical amino acids is a more general mechanism, leading to mistranslation, affecting protein homeostasis, and targeting mitochondria in eukaryotic cells. It can be evolutionarily ancient and initially developed to control phytoplankton communities during algal blooms. Outcompeting gut symbiotic microorganisms may lead to dysbiosis, increased gut permeability, a shift in blood-brain-barrier functionality, and eventually, mitochondrial dysfunction in high-energy demanding neurons. A better understanding of the interaction between cyanopeptides metabolism and the nervous system will be crucial to target or to prevent neurodegenerative diseases

TO DIE OR NOT TO DIE—REGULATED CELL DEATH AND SURVIVAL IN CYANOBACTERIA

Regulated cell death (RCD) is central to the development, integrity, and functionality of multicellular organisms. In the last decade, evidence has accumulated that RCD is a universal phenomenon in all life domains. Cyanobacteria are of specific interest due to their importance in aquatic and terrestrial habitats and their role as primary producers in global nutrient cycling. Current knowledge on cyanobacterial RCD is based mainly on biochemical and morphological observations, often by methods directly transferred from vertebrate research and with limited understanding of the molecular genetic basis. However, the metabolism of different cyanobacteria groups relies on photosynthesis and nitrogen fixation, whereas mitochondria are the central executioner of cell death in vertebrates. Moreover, cyanobacteria chosen as biological models in RCD studies are mainly colonial or filamentous multicellular organisms. On the other hand, unicellular cyanobacteria have regulated programs of cellular survival (RCS) such as chlorosis and post-chlorosis resuscitation. The co-existence of different genetically regulated programs in cyanobacterial populations may have been a top engine in life diversification. Development of cyanobacteria-specific methods for identification and characterization of RCD and wider use of single-cell analysis combined with intelligent image-based cell sorting and metagenomics would shed more light on the underlying molecular mechanisms and help us to address the complex colonial interactions during these events. In this review, we focus on the functional implications of RCD in cyanobacterial communities

Myeloid Cells in Intact Human Cervical Explants Capture HIV and Can Transmit It to CD4 T Cells

The importance of myeloid cells in HIV transmission in the female genital tract is uncertain. Because it is difficult to study the early events in HIV transmission in humans, most of our knowledge is based on animal models of SIV infection in Rhesus macaques and more recently HIV infection in humanized mice. However, these models may not accurately recapitulate transmission in the human genital tract. CD14+ myeloid cells are the most abundant hematopoietic cells in the human cervical mucosa, comprising 40–50% of CD45+ mononuclear cells. Most CD14+ cells are CD14+CD11c– macrophages and about a third are CD14+CD11c+ tissue dendritic cells, which express the HIV-binding receptors, DC-SIGN and CX3CR1. To examine the role of mucosal myeloid cells in HIV transmission, we infected intact healthy human cervical explants with CCR5–tropic HIV-1 ex vivo and then sorted populations of cervical immune cells 20 h later to determine whether they took up virus and could transmit it to activated CD4 T cells. Viral RNA was detected in CD14+ myeloid cells in all but one of 10 donor tissue samples, even when HIV RNA was not detected in CD4+ T cells. HIV RNA was detected predominantly in CD14+CD11c+ dendritic cells rather than in CD14+CD11c– macrophages. The reverse transcriptase inhibitor, nevirapine, reduced HIV RNA in CD4+ T cells, but not in CD14+ cells. Moreover, integrated HIV DNA were not detected above background in myeloid cells but was detected in T cells. These data suggest that although HIV replicates in T cells, myeloid cells in the female genital mucosa capture viral particles, but do not replicate the virus at early timepoints. However, sorted CD14+ myeloid cells isolated 20 h post-infection from 5 HIV-infected cervical explants tested all transmitted HIV to activated CD4+ T cells, while only 1 sample of sorted CD4+ T cells did. Thus, myeloid cells in human cervical tissue capture HIV and are an important early cellular storage site of infectious virus

Cyclic AMP Pathway Suppress Autoimmune Neuroinflammation by Inhibiting Functions of Encephalitogenic CD4 T Cells and Enhancing M2 Macrophage Polarization at the Site of Inflammation

Although it has been demonstrated that cAMP pathway affect both adaptive and innate cell functions, the role of this pathway in the regulation of T-cell-mediated central nervous system (CNS) autoimmune inflammation, such as in experimental autoimmune encephalomyelitis (EAE), remains unclear. It is also unclear how cAMP pathway affects the function of CD4 T cells in vivo at the site of inflammation. We found that adenylyl cyclase activator Forskolin besides inhibition of functions autoimmune CD4 T cells also upregulated microRNA (miR)-124 in the CNS during EAE, which is associated with M2 phenotype of microglia/macrophages. Our study further established that in addition to direct influence of cAMP pathway on CD4 T cells, stimulation of this pathway promoted macrophage polarization toward M2 leading to indirect inhibition of function of T cells in the CNS. We demonstrated that Forskolin together with IL-4 or with Forskolin together with IL-4 and IFNγ effectively stimulated M2 phenotype of macrophages indicating high potency of this pathway in reprogramming of macrophage polarization in Th2- and even in Th1/Th2-mixed inflammatory conditions such as EAE. Mechanistically, Forskolin and/or IL-4 activated ERK pathway in macrophages resulting in the upregulation of M2-associated molecules miR-124, arginase (Arg)1, and Mannose receptor C-type 1 (Mrc1), which was reversed by ERK inhibitors. Administration of Forskolin after the onset of EAE substantially upregulated M2 markers Arg1, Mrc1, Fizz1, and Ym1 and inhibited M1 markers nitric oxide synthetase 2 and CD86 in the CNS during EAE resulting in decrease in macrophage/microglia activation, lymphocyte and CD4 T cell infiltration, and the recovery from the disease. Forskolin inhibited proliferation and IFNγ production by CD4 T cells in the CNS but had rather weak direct effect on proliferation of autoimmune T cells in the periphery and in vitro, suggesting prevalence of indirect effect of Forskolin on differentiation and functions of autoimmune CD4 T cells in vivo. Thus, our data indicate that Forskolin has potency to skew balance toward M2 affecting ERK pathway in macrophages and indirectly inhibit pathogenic CD4 T cells in the CNS leading to the suppression of autoimmune inflammation. These data may have also implications for future therapeutic approaches to inhibit autoimmune Th1 cells at the site of tissue inflammation

Human cytokines activate JAK–STAT signaling pathway in porcine ocular tissue

Background: The JAK/STAT (Janus Tyrosine Kinase, Signal Transducers and Activators of Transcription) pathway is associated with cytokine or growth factor receptors and it is critical for growth control, developmental regulation and homeostasis. The use of porcine ocular cells as putative xenotransplants appears theoretically possible. The aim of this study was to investigate the response of various porcine ocular cells in vitro to human cytokines in regard to the activation of JAK-STAT signaling pathways. Methods: Porcine lens epithelial cells, pigmented iris epithelial cells and pigmented ciliary body cells were used in this study. These cells were isolated from freshly enucleated porcine eyes by enzymatic digestion. Cultured cells between passages 3–8 were used in all experiments. Electromobility shift assay (EMSA), proliferation assay, immunofluorescence staining and flow cytometry were used to evaluate the JAK-STAT signaling pathway in these cells. Results: JAK/STAT signaling pathways could be activated in porcine pigmented epithelial ciliary body cells, in pigmented iris epithelial cells and in lens epithelial cells in response to porcine and human interferons and cytokines. All cells showed very strong STAT1 activation upon stimulation with porcine interferon-gamma. Porcine ocular cells also respond to human cytokines; IFN-alpha induced strong activation of STAT1 in EMSA, flow cytometry and immunofluorescence experiments whereas activation of STAT3 was less strong in EMSA, but strong in flow cytometry and immunofluorescence. Human recombinant IL-6 activated STAT3 and human IL-4 activated STAT6. With the help of immunofluorescence assay and flow cytometry we observed nuclear localization of STAT proteins after activation of porcine ocular cells with cytokines and interferons. Human IFN-α had an inhibitory effect on porcine ocular cells in proliferation assays. Conclusion: Our study demonstrated that some types of human cytokines and interferon activate intracellular JAK-STAT signaling pathways in porcine ocular cells. We hypothesize that direct stimulation of the JAK-STAT pathway in porcine cells in response to human cytokines will lead to complications or failure, if pig-to-human ocular tissue xenotransplantation were to be carried out. For successful xenotransplantation among other obstacles there must be new approaches developed to regulate signaling pathways

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

Background: Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Methods: Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. Results: A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Discussion Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events

Neuronal extracellular microRNAs miR-124 and miR-9 mediate cell-cell communication between neurons and microglia

In contrast to peripheral macrophages, microglia in the central nervous system (CNS) exhibit a specific deactivated phenotype; however, it is not clear how this phenotype is maintained. Two alternative hypotheses were postulated recently: (a) microglia differ from peripheral macrophages being derived from the yolk sac (YS), whereas peripheral macrophages originate from bone marrow (BM); (b) microglia acquire a specific phenotype under the influence of the CNS microenvironment. We have previously shown that microglia express miR-124, which was also induced in BM-derived macrophages co-cultured with a neurons. We here investigated the possibility of horizontal transfer of the neuron-specific microRNAs miR-124 and miR-9 from primary neurons to microglia/macrophages. We found that after incubation with neuronal conditioned media (NCM), macrophages downregulated activation markers MHC class II and CD45. Neither cultured adult microglia nor YS- and BM-derived macrophages demonstrated intrinsic levels of miR-124 expression. However, after incubation with NCM, miR-124 was induced in both YS- and BM-derived macrophages. Biochemical analysis demonstrated that the NCM contained miR-124 and miR-9 in complex with small proteins, large high-density lipoproteins (HDLs), and exosomes. MiR-124 and miR-9 were promptly released from neurons, and this process was inhibited by tetrodotoxin, indicating an important role of neuronal electric activity in secretion of these microRNAs. Incubation of macrophages with exogenous miR-124 resulted in efficient translocation of miR-124 into the cytoplasm. This study demonstrates an important role of neuronal miRNAs in communication of neurons with microglia, which favors the hypothesis that microglia acquire a specific phenotype under the influence of the CNS microenvironment

The use of HaloTag-based technology in flow and laser scanning cytometry analysis of live and fixed cells

<p>Abstract</p> <p>Background</p> <p>Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.</p> <p>Findings</p> <p>Here, we describe development of applications using protein tag technology (HaloTag (HT)-based) for flow and laser scanning cytometry (LSC). Cell lines, expressing recombinant surface β1-integrin-HT and HT-p65 fusion protein, and a CD4 T cell line (Jurkat) infected with human immunodeficiency virus type 1 (HIV-1) reporter virus expressing the unfused HT (HIV-1_Lai-Halo), were stained with different HT ligands and successfully detected by flow cytometers equipped with 488 and 561 nm lasers as well as a laser scanning cytometer (equipped with 488 and 405 nm lasers) alone or combined with cell cycle and viability markers.</p> <p>Conclusions</p> <p>Use of HT technology for cytometric applications has advantages over its use in microscopy as it allows for the statistical measurement of protein expression levels in individual cells within a heterogeneous cell population in combination with cell cycle analysis. Another advantage is the ability of the HaloTag to withstand long fixation and high concentration of fixative, which can be useful in research of infectious agents like HIV and/or mycobacteria.</p

Visualization and quantitation of the expression of microRNAs and their target genes in neuroblastoma single cells using imaging cytometry

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are regulatory molecules that play an important role in many physiological processes, including cell growth, differentiation, and apoptosis. In addition to modulating normal cellular functions, it has also been reported that miRNAs are involved in the development of many pathologies, including cardiovascular diseases, cancer, inflammation, and neurodegeneration. Methods for the sensitive detection and measurement of specific miRNAs and their cellular targets are essential for both basic research endeavours, as well as diagnostic efforts aimed at understanding the role of miRNAs in disease processes.</p> <p>Findings</p> <p>In this study, we describe a novel, imaging cytometry-based protocol that allows for simultaneous visualisation and quantification of miRNAs and their putative targets. We validated this methodology in a neuronal cell line by examining the relationship of the miRNA miR-124 and its known target, cyclin dependent kinase 6 (CDK6). We found that ectopic overexpression of miR-124 resulted in the downregulation of CDK6, decreased cellular proliferation, and induced cellular morphological changes.</p> <p>Conclusions</p> <p>This method is suitable for analysing the expression and cellular localisation of miRNAs and target proteins in small cell subsets within a heterogeneous cell suspension. We believe that our cytometry-based methodology will be easily adaptable to miRNA studies in many areas of biomedical research including neuroscience, stem cell biology, immunology, and oncology.</p