Microvesicles Derived from Adult Human Bone Marrow and Tissue Specific Mesenchymal Stem Cells Shuttle Selected Pattern of miRNAs

Background

Cell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs).

Methodology/Principal Findings

MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation.

Conclusions

This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem cells may, at least in part, depend on MV-shuttled miRNAs. Data generated from this study, stimulate further functional investigations on the predicted target genes and pathways involved in the biological effect of human adult stem cells.     

Lactation-Related MicroRNA Expression Profiles of Porcine Breast Milk Exosomes

Breast milk is the primary source of nutrition for newborns, and is rich in immunological components. MicroRNAs (miRNAs) are present in various body fluids and are selectively packaged inside the exosomes, a type of membrane vesicles, secreted by most cell types. These exosomal miRNAs could be actively delivered into recipient cells, and could regulate target gene expression and recipient cell function. Here, we analyzed the lactation-related miRNA expression profiles in porcine milk exosomes across the entire lactation period (newborn to 28 days after birth) by a deep sequencing. We found that immune-related miRNAs are present and enriched in breast milk exosomes (p<10(-16), χ(2) test) and are generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs are present in higher numbers in the colostrums than in mature milk. It was higher in the serum of colostrum-only fed piglets compared with the mature milk-only fed piglets. These immune-related miRNA-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations are a prelude to in-depth investigations of the essential roles of breast milk in the development of the infant's immune system.     

Colorectal cancer cell-derived microvesicles containing microRNA-1246 promote angiogenesis by activating Smad 1/5/8 signaling elicited by PML down-regulation in endothelial cells

Emerging studies on circulating microRNAs (miRNAs) or microvesicles (MVs) have shown the potential of them to be novel biomarkers and therapeutic targets for cancer. However, the biological roles of these miRNAs and MVs have not been validated yet. To determine the biological significance of MVs, we used human colorectal cancer cells as the MV donor and endothelial cells (HUVECs) as the MV recipient and demonstrated the transfer of colorectal cancer cell-derived MVs (CRC-MVs) to HUVECs and evaluated the roles of these MVs and their cargo in tumor angiogenesis. Consequently, the incubation of HUVECs with CRC-MVs promoted the proliferation, migration, and tube formation activities of these cells. Among the cargoes shuttled by the MVs, miR-1246 and TGF-β were considered to be responsible for the pro-angiogenic function of MVs by activating Smad 1/5/8 signaling in the HUVECs. These results suggest that colorectal cancer cells secreted MVs to contribute to tumor angiogenesis.     

Dynamic flux of microvesicles modulate parasite–host cell interaction of Trypanosoma cruzi in eukaryotic cells

Extracellular vesicles released from pathogens may alter host cell functions. We previously demonstrated the involvement of host cell‐derived microvesicles (MVs) during early interaction between Trypanosoma cruzi metacyclic trypomastigote (META) stage and THP‐1 cells. Here, we aim to understand the contribution of different parasite stages and their extracellular vesicles in the interaction with host cells. First, we observed that infective host cell‐derived trypomastigote (tissue culture‐derived trypomastigote [TCT]), META, and noninfective epimastigote (EPI) stages were able to induce different levels of MV release from THP‐1 cells; however, only META and TCT could increase host cell invasion. Fluorescence resonance energy transfer microscopy revealed that THP‐1‐derived MVs can fuse with parasite‐derived MVs. Furthermore, MVs derived from the TCT–THP‐1 interaction showed a higher fusogenic capacity than those from META– or EPI–THP‐1 interaction. However, a higher presence of proteins from META (25%) than TCT (12%) or EPI (5%) was observed in MVs from parasite–THP‐1 interaction, as determined by proteomics. Finally, sera from patients with chronic Chagas disease at the indeterminate or cardiac phase differentially recognized antigens in THP‐1‐derived MVs resulting only from interaction with infective stages. The understanding of intracellular trafficking and the effect of MVs modulating the immune system may provide important clues about Chagas disease pathophysiology.     

Argonaute 2 in Cell-Secreted Microvesicles Guides the Function of Secreted miRNAs in Recipient Cells

MicroRNAs (miRNAs) secreted by cells into microvesicles (MVs) form a novel class of signal molecules that mediate intercellular communication. However, several fundamental aspects of secreted miRNAs remain unknown, particularly the mechanism that governs the function or fate of exogenous miRNAs in recipient cells. In the present study, we provide evidence indicating that Argonaute 2 (Ago2) plays a role in stabilizing miRNAs and facilitating the packaging of secreted miRNAs into MVs. More importantly, Ago2 in origin cell-secreted MVs (but not in recipient cells) directs the function of secreted miRNAs. First, Ago2 overexpression clearly increased the level of miR-16 in cells transfected with a miR-16 mimic by protecting the miRNAs from degradation in lysosomes. Second, Ago2 overexpression increased the level of miR-16 in cell-secreted MVs, suggesting that Ago2 may facilitate the packaging of secreted miRNAs into MVs. Third, exogenous miR-16 delivered by MVs within the origin cells significantly reduced the Bcl2 protein level in recipient cells, and miR-16 and Bcl2 mRNA were physically associated with exogenous HA-tagged Ago2 (HA-Ago2). Finally, the effect of MV-delivered miR-16 on the production of the Bcl2 protein in recipient cells was not abolished by knocking down Ago2 in the recipient cells.     

Immune-related microRNAs are abundant in breast milk exosomes

Breast milk is a complex liquid rich in immunological components that affect the development of the infant's immune system. Exosomes are membranous vesicles of endocytic origin that are found in various body fluids and that can mediate intercellular communication. MicroRNAs (miRNAs), a well-defined group of non-coding small RNAs, are packaged inside exosomes in human breast milk. Here, we identified 602 unique miRNAs originating from 452 miRNA precursors (pre-miRNAs) in human breast milk exosomes using deep sequencing technology. We found that, out of 87 well-characterized immune-related pre-miRNAs, 59 (67.82%) are presented and enriched in breast milk exosomes (P < 10(-16), χ(2) test). In addition, compared with exogenous synthetic miRNAs, these endogenous immune-related miRNAs are more resistant to relatively harsh conditions. It is, therefore, tempting to speculate that these exosomal miRNAs are transferred from the mother's milk to the infant via the digestive tract, and that they play a critical role in the development of the infant immune system.     

Milk-derived Exosomes: Novel Regulatory Factors for Intestinal Health

Exosomes are a type of nano-sized extracellular vesicles, which play essential roles in many cellular processes, such as signal transduction, immune response and antigen presentation, and exist in diverse body fluids, including serum, saliva, urine, cerebrospinal fluid and milk. As the main source of nutrition for mammalian offsprings after birth, breast milk contains various nutrients and bioactive ingredients, which are crucial for animals’ immune system and intestinal development. As active molecules of milk, milk-derived exosomes are involved in complex secretory mechanisms and contain multiple nucleic acids, such as mRNAs and non-coding RNAs. Bioinformatics predicted that these exosomal nucleic acids may participate in immunoregulatory pathways. Moreover, milk-derived exosomal proteins are abundant and may enter target cells to exert regulatory functions, while the lipid components contribute significantly to maintaining the stability and uptake of exosomes. It has been published that milk-derived exosomes could resist animal gastrointestinal digestion in the physiological state, and could be further absorbed by the intestinal tract and participate in regulating the processes of intestinal cell proliferation, intestinal inflammation and diversity of gut microbiota, which are beneficial to animal intestines and have become novel regulatory factor for intestinal health in recent years. This article summarizes the formation, composition, and intestinal fate of milk-derived exosomes, and emphasizes their special functions on intestinal health in the animal field, which may provide a theoretical basis for the study of milk-derived exosomes.     

TLR-4/microRNA-125a/NF-κB signaling modulates the immune response to Mycobacterium tuberculosis infection

Tuberculosis (TB), caused by Mycobacterium tuberculosis, could lead to kinds of clinical disorders and remains a leading global health problem, resulting in great morbidity and mortality worldwide. Previous studies have firmly demonstrated that M. tuberculosis (M.tb) has evolved to utilize different mechanisms to evade or attenuate the host immune response, such as regulation of immune-related genes by modulation of miRNAs of host or bacteria. However, the knowledge of functions of miRNAs during M.tb infection remains limited. Here, we reported that a host microRNA, miR-125a, was significantly up-regulated by M.tb infection in both RAW264.7 and THP-1cells, in a TLR4 signaling-dependent manner. Subsequently, our results demonstrated that miR-125a was a negative regulator of NF-kB pathway by directly targeting TRAF6, resulting in the suppression of cytokines, attenuation of immune response and promotion of M.tb survival. Taken together, our findings provide a novel detailed molecular mechanism in which miR-125a was enhanced to inhibit inflammatory cytokines secretion and attenuate the immune response during M.tb infection in RAW264.7 and THP-1 cells, and suggest an intrinsic a promising anti-M.tb therapeutic target.     

Deregulation of Type I IFN-Dependent Genes Correlates with Increased Susceptibility to Cytomegalovirus Acute Infection of Dicer Mutant Mice

Regulation of gene expression by microRNAs (miRNAs) is now considered as an essential mechanism for cell development and homeostasis. Indeed, numerous studies have reported that modulating their expression, maturation, or activity can affect cell survival, identity or activation. In particular, miRNAs are key players in the tight regulation of signaling cascades, and as such, they appear as perfectly suited immunomodulators. Several immune-related processes, including inflammation, have recently been demonstrated to require specific miRNAs. In addition, the discovery of herpesvirus-encoded miRNAs has reinforced this assumption. To decipher the potential roles of miRNAs in innate antiviral immune response, we developed an in vivo model based on the inoculation of mouse cytomegalovirus (MCMV) in mice. Furthermore, we exploited a mouse line carrying a hypomorphic mutation in the Dicer gene to visualize the impact of impaired miRNA biogenesis upon the anti-MCMV response. Our data indicate that miRNAs are important actors in mounting an efficient response against herpesviruses. We suggest that a rapid and transient interferon response following viral infection requires miRNA-dependent repressor release. In addition, our in vivo efforts identified several miRNA targets, thus providing a conceptual framework for future analyzes on the regulation of specific actors involved in the Type I interferon pathway.     

Immunomodulatory role of microRNAs transferred by extracellular vesicles

The immune system is composed of different cell types localised throughout the organism to sense and respond to pathological situations while maintaining homeostasis under physiological conditions. Intercellular communication between immune cells is essential to coordinate an effective immune response and involves both cell contact dependent and independent processes that ensure the transfer of information between bystander and distant cells. There is a rapidly growing body of evidence on the pivotal role of extracellular vesicles (EVs) in cell communication and these structures are emerging as important mediators for immune modulation upon delivery of their molecular cargo. In the last decade, EVs have been shown to be efficient carriers of genetic information, including microRNAs (miRNAs), that can be transferred between cells and regulate gene expression and function on the recipient cell. Here, we review the current knowledge of intercellular functional transfer of EV‐delivered miRNAs and their putative role in immune regulation.     

The Porcine MicroRNA Transcriptome Response to Transmissible Gastroenteritis Virus Infection

Transmissible gastroenteritis virus (TGEV; Coronaviridae family) causes huge economic losses to the swine industry. MicroRNAs (miRNAs) play a regulatory role in viral infection and may be involved in the mammalian immune response. Here, we report a comprehensive analysis of host miRNA expression in TGEV-infected swine testis (ST) cells. Deep sequencing generated 3,704,353 and 2,763,665 reads from uninfected ST cells and infected ST cells, respectively. The reads were aligned to known Sus scrofa pre-miRNAs in miRBase 19, identifying 284 annotated miRNAs. Certain miRNAs were differentially regulated during TGEV infection. 59 unique miRNAs displayed significant differentially expression between the normal and TGEV-infected ST cell samples: 15 miRNAs were significantly up-regulated and 44 were significantly down-regulated. Stem-loop RT-PCR was carried out to determine the expression levels of specific miRNAs in the two samples, and the results were consistent with those of sequencing. Gene ontology enrichment analysis of host target genes demonstrated that the differentially expressed miRNAs are involved in regulatory networks, including cellular process, metabolic process, immune system process. This is the first report of the identification of ST cell miRNAs and the comprehensive analysis of the miRNA regulatory mechanism during TGEV infection, which revealed the miRNA molecular regulatory mechanisms for the viral infection, expression of viral genes and the expression of immune-related genes. The results presented here will aid research on the prevention and treatment of viral diseases.     

Mesenchymal stem cells-derived exosomal microRNAs contribute to wound inflammation

Clinical and experimental studies have highlighted the significance of inflammation in coordinating wound repair and regeneration.However,it remains challenging to control the inflammatory response and tolerance at systemic levels without causing toxicity to injured tissues.Mesenchymal stem cells(MSCs) possess potent immunomodulatory properties and facilitate tissue repair by releasing exosomes,which generate a suitable microenvironment for inflammatory resolution.Exosomes contain several effective bioactive molecules and act as a cell-cell communication vehicle to influence cellular activities in recipient cells.During this process,the horizontal transfer of exosomal microRNAs(miRNAs) to acceptor cells,where they regulate target gene expression,is of particular interest for understanding the basic biology of inflammation ablation,tissue homeostasis,and development of therapeutic approaches.In this review,we describe a signature of three specific miRNAs(miR-21,miR-146 a,and miR-181) present in human umbilical cord MSC-derived exosomes(MSC-EXO) identified microarray chip analysis and focus on the inflammatory regulatory functions of these immune-related miRNAs.We also discuss the potential mechanisms contributing to the resolution of wound inflammation and tissue healing.     

Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation

It has recently been established that exosomes can mediate intercellular cross-talk under normal and pathological conditions through the transfer of specific miRNAs. As muscle cells secrete exosomes, we addressed the question of whether skeletal muscle (SkM) exosomes contained specific miRNAs, and whether they could act as “endocrine signals” during myogenesis. We compared the miRNA repertoires found in exosomes released from C2C12 myoblasts and myotubes and found that 171 and 182 miRNAs were exported into exosomes from myoblasts and myotubes, respectively. Interestingly, some miRNAs were expressed at higher levels in exosomes than in their donor cells and vice versa, indicating a selectivity in the incorporation of miRNAs into exosomes. Moreover miRNAs from C2C12 exosomes were regulated during myogenesis. The predicted target genes of regulated exosomal miRNAs are mainly involved in the control of important signaling pathways for muscle cell differentiation (e.g., Wnt signaling pathway). We demonstrated that exosomes from myotubes can transfer small RNAs (C. elegans miRNAs and siRNA) into myoblasts. Moreover, we present evidence that exosome miRNAs secreted by myotubes are functionally able to silence Sirt1 in myoblasts. As Sirt1 regulates muscle gene expression and differentiation, our results show that myotube–exosome miRNAs could contribute to the commitment of myoblasts in the process of differentiation. Until now, myokines in muscle cell secretome provided a conceptual basis for communication between muscles. Here, we show that miRNA exosomal transfer would be a powerful means by which gene expression is orchestrated to regulate SkM metabolic homeostasis.     

Horizontal transfer of microRNAs: molecular mechanisms and clinical applications

A new class of RNA regulatory genes known as microRNAs (miRNAs) has been found to introduce a whole new layer of gene regulation in eukaryotes. The intensive studies of the past several years have demonstrated that miRNAs are not only found intracellularly, but are also detectable outside cells, including in various body fluids (e.g. serum, plasma, saliva, urine and milk). This phenomenon raises questions about the biological function of such extracellular miRNAs. Substantial amounts of extracellular miRNAs are enclosed in small membranous vesicles (e.g. exosomes, shedding vesicles and apoptotic bodies) or packaged with RNA-binding proteins (e.g. high-density lipoprotein, Argonaute 2 and nucleophosmin 1). These miRNAs may function as secreted signaling molecules to influence the recipient cell phenotypes. Furthermore, secreted extracellular miRNAs may reflect molecular changes in the cells from which they are derived and can therefore potentially serve as diagnostic indicators of disease. Several studies also point to the potential application of siRNA/miRNA delivery as a new therapeutic strategy for treating diseases. In this review, we summarize what is known about the mechanism of miRNA secretion. In addition, we describe the pathophysiological roles of secreted miRNAs and their clinical potential as diagnostic biomarkers and therapeutic drugs. We believe that miRNA transfer between cells will have a significant impact on biological research in the coming years.     

Heightened pro-inflammatory effect of preeclamptic placental microvesicles on peripheral blood immune cells in humans

Normal pregnancy is associated with the presence of circulating placental microvesicles (MVs). Increased MV shedding and altered immune activation are seen in patients with preeclampsia, suggesting that placental MVs may play a role in the pathophysiology of this disease. Therefore, the aim of this study was to investigate the activation of peripheral blood mononuclear cells (PBMCs) by MVs shed by first-trimester, normal term, and preeclamptic term placenta. First-trimester and preeclamptic term, but not normal term, placental-derived MVs activated PBMCs, as evidenced by elevated IL1B. Significant changes were also seen with several other cytokines and chemokines, and in general when compared to normal term MVs, preeclamptic MVs induced a greater pro-inflammatory response in PBMCs. Pretreatment of PBMCs with first-trimester or normal term placental MVs resulted in a dampened IL1B response to a subsequent lipopolysaccharide (LPS) challenge. In contrast, treatment of PBMCs with preeclamptic term placental MVs exacerbated the LPS response. This was also the case for several other cytokines and chemokines. These studies suggest that placental MVs can modulate basal peripheral immune cell activation and responsiveness to LPS during normal pregnancy, and that in preeclampsia this effect is exacerbated.     

Immunomodulatory role for membrane vesicles released by THP-1 macrophages and respiratory pathogens during macrophage infection

Background

During infection, inflammation is partially driven by the release of mediators which facilitate intercellular communication. Amongst these mediators are small membrane vesicles (MVs) that can be released by both host cells and Gram-negative and -positive bacteria. Bacterial membrane vesicles are known to exert immuno-modulatory and -stimulatory actions. Moreover, it has been proposed that host cell-derived vesicles, released during infection, also have immunostimulatory properties. In this study, we assessed the release and activity of host cell-derived and bacterial MVs during the first hours following infection of THP-1 macrophages with the common respiratory pathogens non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa.

Results

Using a combination of flow cytometry, tunable resistive pulse sensing (TRPS)-based analysis and electron microscopy, we demonstrated that the release of MVs occurs by both host cells and bacteria during infection. MVs released during infection and bacterial culture were found to induce a strong pro-inflammatory response by naive THP-1 macrophages. Yet, these MVs were also found to induce tolerance of host cells to secondary immunogenic stimuli and to enhance bacterial adherence and the number of intracellular bacteria.

Conclusions

Bacterial MVs may play a dual role during infection, as they can both trigger and dampen immune responses thereby contributing to immune defence and bacterial survival.

     

Characterization of the relationship between FLI1 and immune infiltrate level in tumour immune microenvironment for breast cancer

Breast cancer is the most common cancer and the leading cause of cancer death among women in the world. Tumour-infiltrating lymphocytes were defined as the white blood cells left in the vasculature and localized in tumours. Recently, tumour-infiltrating lymphocytes were found to be associated with good prognosis and response to immunotherapy in tumours. In this study, to examine the influence of FLI1 in immune system in breast cancer, we interrogated the relationship between the FLI1 expression levels with infiltration levels of 28 immune cell types. By splitting the breast cancer samples into high and low expression FLI1 subtypes, we found that the high expression FLI1 subtype was enriched in many immune cell types, and the up-regulated differentially expressed genes between them were enriched in immune system processes, immune-related KEGG pathways and biological processes. In addition, many important immune-related features were found to be positively correlated with the FLI1 expression level. Furthermore, we found that the FLI1 was correlated with the immune-related genes. Our findings may provide useful help for recognizing the relationship between tumour immune microenvironment and FLI1, and may unravel clinical outcomes and immunotherapy utility for FLI1 in breast cancer.