In the same study, the authors showed distinctly reduced vascular leakage in HUVECs incubated with sera from children with severe cerebral malaria pretreated with microvesicles containing anti-miR Others have reported elevated levels of miRa and miRb in exosomes secreted from LPS stimulated mouse macrophages The brain function regulating miRNAa-5p can reduce cytokine induced monocyte migration in brain endothelium in vitro Studies have shown that the M2 macrophages known to be anti-inflammatory, highly express miRa-5p compared to M1 proinflammatory macrophages 38 , This correlates with our current observation that increased miRa-5p expression improves monocyte migration but not adherence to HBMECs.
Given that miRa-5p is important for brain homeostasis by maintaining endothelial barrier function and immune cell balance, the associated decrease of both miRa-5p and miR could possibly have worse physiological outcomes 33 , These miRNAs are efficiently transferred to bystander brain endothelial cells via exosomes.
These exosomes are crucial to monocyte chemotaxis since blocking exosome release mitigates the inflammatory responses. These exosomes are taken up by the brain endothelial cells leading to damage in the form of abnormal upregulation of adhesion molecules, chemoattractants and pro-inflammatory cytokines. These miRNAs are constitutively expressed in endothelial cells left panel. Nonstimulated NS or activated monocytes were stained with calcein AM 1. The calcein AM stained monocytes present in the inserts described above, released calcein stained exosomes that were absorbed by the HBMECs cocultured in the bottom chamber.
Data were normalized using global means method Data were analyzed using the R statistical programming software 49 version 3. In the scatterplot graphs, each dot represents a data point and each horizontal bar represents the mean of the treatment group. Shading in the scatterplots indicates the range of the data and not the confidence interval.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Thery, C. Exosomes: composition, biogenesis and function. Nature reviews. Immunology 2 , —, doi: Tang, N. Monocyte exosomes induce adhesion molecules and cytokines via activation of NF-kappaB in endothelial cells.
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Original Research ARTICLE
Nature biotechnology 34 , —, doi: Lasser, C. Exosomes in diagnostic and therapeutic applications: biomarker, vaccine and RNA interference delivery vehicle. Expert opinion on biological therapy 15 , —, doi: Greene, C. Tight junction modulation of the blood brain barrier: CNS delivery of small molecules. Tissue barriers 4 , e, doi: Stamatovic, S. Current neuropharmacology 6 , —, doi: Watson, C. IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes. Clinical and experimental immunology , — Staphylococcal enterotoxin B SEB , a superantigen produced by Staphylococcus aureus , has deleterious effects in humans, such as food poisoning 1 and toxic shock 2.
Upon inhalation exposure, SEB can trigger acute inflammatory lung injury characterized by immune cell infiltration, excessive cytokine production, tissue damage, and pulmonary edema 4 , 5. Several studies have demonstrated a role for miRNA in modulating immune responses under various inflammatory conditions Similarly, studies have demonstrated that overexpression of the miR—92 cluster in T cells leads to lymphoproliferative disorders due to the repression of the proapoptotic molecule, BIM Furthermore, mice deficient in miR are resistant to developing experimental autoimmune encephalomyelitis EAE , a mouse model of multiple sclerosis 19 , while the overexpression of miR exacerbates the symptoms associated with the disease.
Taken together, these studies strongly suggest that miRNAs play a major role in modulating immune cell activation, particularly T cells, as well as promoting proinflammatory responses.
MicroRNAa: A Key Regulator of Astrocyte-Mediated Inflammatory Response
Further, our data identified miR as a major contributor to SEB-mediated lung inflammation. Our results may present an opportunity to further therapeutically target miR in the treatment of SEB-mediated acute inflammatory lung injury. Cg- Mir tm1. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council Mice were euthanized 48 h after SEB exposure.
Fluorescein isothiocyanate-conjugated anti-CD8 clone Mice were exposed to SEB as described above. The mononuclear cell layer isolated was then enumerated using the Trypan blue exclusion method. To determine the subsets of immune cells infiltrating the lung, cells were stained with fluorescent-conjugated antibodies anti-CD4, anti-CD8 and analyzed using the Beckman Coulter flow cytometer Indianapolis, IN.
Forty-eight hours after SEB exposure, mice were euthanized, tracheae from vehicle- or SEB-treated mice were tied with a suture, and the lung was excised as an intact unit. Fluorescent intensities obtained from hybridization were log transformed and visualized in the form of a heatmap. Hierarchical clustering was carried out using Ward's method, and similarity measurement was calculated using half square Euclidean distance. These targets were further sorted based on their role in cytokine signaling, cellular growth and proliferation, and cellular immune response.
Snord96a was used as the small RNA endogenous control. In all experiments, the number of mice used was 4 or 5 per group, unless otherwise specified. Individual experiments were performed in triplicate, and each experiment was performed independently at least three times to test reproducibility of results.
In this study, we first sought to investigate the inflammatory effect of SEB exposure in the lungs. Immediately after euthanasia, the lungs were harvested and mononuclear cells were isolated from the lungs by density gradient centrifugation to determine the phenotypic characteristics of the cells. These data suggested that SEB administration via the intranasal route triggers acute inflammation in the lungs. SEB induces lung inflammation.
B Lung-infiltrating mononuclear cells obtained by density gradient centrifugation and total number of viable cells were counted using a hemocytometer. The percentage of the immune cell subsets was multiplied by the total number of cells found in the lung and divided by to yield the absolute cell numbers shown.
A heatmap was generated based on hierarchical clustering of miRNA, highlighting a stark difference between vehicle- and SEB-exposed mice Fig. Further examination of miRNA expression revealed that of the miRNAs assessed, most remained unchanged, but a few showed significant up- or downregulation, as seen in the fold change distribution plot Fig. This network was characterized by IPA as responses involving inflammation, cellular development, cellular growth, and proliferation.
Next, we validated the expression levels of these miRNAs in lung-infiltrating mononuclear cells by qRT-PCR, which corroborated the expression patterns seen using the microarray Fig. Analysis criteria consisted of a two-sided hypergeometric test with Benjamini Hochberg correction. Only results with a kappa score of 0.
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Total RNA was isolated from lung-infiltrating mononuclear cells. Because miR was highly upregulated in response to SEB, we hypothesized that it might play a crucial role in facilitating the inflammation observed during the disease. Additionally, the miRdeficient mice expressed significantly decreased total numbers of mononuclear cells within the lung upon SEB exposure compared to those of their WT counterparts.
Cells were then transfected with nM miR inhibitor Inh or inhibitor control Inh Control for another 24 h. To examine the link between miR and its potential target genes, we undertook a bioinformatics-based approach. First, IPA miRNA target filter tools were used, selecting only those miR target genes that were relevant to cytokine signaling, cellular immune response, and cellular growth and proliferation. Thirty six targets common to the filtering criteria applied were selected Fig.
Next, an IPA-generated network was used to sort the targets based on those that were highly predicted and experimentally observed Fig. Compared to the mimic control, miR mimic led to a significant decrease in luciferase activity Fig. Next, the impact of miR mimic on Socs1 levels was explored. We found that both in WT Fig. Identification of SEB-induced miR targets. A miR targets were filtered based on their role in cytokine signaling, cell growth and proliferation, and cellular immune response using IPA.
A proportional Venn diagram indicating the miR targets common to all three filtering criteria was generated. The list of targets is indicated within brackets, and Socs1 , a highly predicted target, is in red. B IPA network was generated highlighting the highly predicted yellow and experimentally observed brown miR targets, in addition to Socs1 red , the target of interest. Relative luciferase activity firefly normalized to renilla was determined following transfection. With the discovery of miRNA, a novel and exciting mechanism of gene regulation has arisen.
A single miRNA usually targets several mRNAs, acting as a fine-tuner of gene regulation rather than an on-off system In the context of inflammation, miRNAs have been found within a variety of immune cells often targeting genes involved in the regulation of inflammatory response This is consistent with our observation that miR was upregulated following SEB activation. Moreover, in a mouse model of collagen-induced arthritis CIA , the deficiency of miR led to a decrease in pathogenic T cells.
In humans, it has also been reported that soldiers undergoing a battlefield-like stress program demonstrated an increase in hsa-miR levels in leukocytes exposed to SEB ex vivo 32 , indicating that stress-related inflammation could also potentially lead to the increase in miR Although inflammation plays a valuable role in combating infection, its dysregulation often occurs in people and can cause a variety of pathologies, ranging from chronic inflammation, to autoimmunity, to cancer. In recent years, our understanding of both the cellular and molecular networks that regulate inflammation has improved dramatically.
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