Identification of a novel mechanism of blood–brain communication during peripheral inflammation via choroid plexus‐derived extracellular vesicles
2016; Springer Nature; Volume: 8; Issue: 10 Linguagem: Inglês
10.15252/emmm.201606271
ISSN1757-4684
AutoresSriram Balusu, Elien Van Wonterghem, Riet De Rycke, Koen Raemdonck, Stephan Stremersch, Kris Gevaert, Marjana Brkić, Delphine Demeestere, Valerie Vanhooren, An Hendrix, Claude Libert, Roosmarijn E. Vandenbroucke,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoResearch Article5 September 2016Open Access Transparent process Identification of a novel mechanism of blood–brain communication during peripheral inflammation via choroid plexus-derived extracellular vesicles Sriram Balusu Sriram Balusu Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Department of Medical Protein Research, VIB, Ghent, Belgium Department of Biochemistry, Ghent University, Ghent, Belgium Search for more papers by this author Elien Van Wonterghem Elien Van Wonterghem Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Riet De Rycke Riet De Rycke Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Koen Raemdonck Koen Raemdonck Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium Search for more papers by this author Stephan Stremersch Stephan Stremersch Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium Search for more papers by this author Kris Gevaert Kris Gevaert Department of Medical Protein Research, VIB, Ghent, Belgium Department of Biochemistry, Ghent University, Ghent, Belgium Search for more papers by this author Marjana Brkic Marjana Brkic Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Department of Neurobiology, Institute for Biological Research, University of Belgrade, Belgrade, Republic of Serbia Search for more papers by this author Delphine Demeestere Delphine Demeestere Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Valerie Vanhooren Valerie Vanhooren Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author An Hendrix An Hendrix Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, Ghent, Belgium Search for more papers by this author Claude Libert Claude Libert Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Roosmarijn E Vandenbroucke Corresponding Author Roosmarijn E Vandenbroucke [email protected] orcid.org/0000-0002-8327-620X Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Sriram Balusu Sriram Balusu Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Department of Medical Protein Research, VIB, Ghent, Belgium Department of Biochemistry, Ghent University, Ghent, Belgium Search for more papers by this author Elien Van Wonterghem Elien Van Wonterghem Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Riet De Rycke Riet De Rycke Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Koen Raemdonck Koen Raemdonck Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium Search for more papers by this author Stephan Stremersch Stephan Stremersch Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium Search for more papers by this author Kris Gevaert Kris Gevaert Department of Medical Protein Research, VIB, Ghent, Belgium Department of Biochemistry, Ghent University, Ghent, Belgium Search for more papers by this author Marjana Brkic Marjana Brkic Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Department of Neurobiology, Institute for Biological Research, University of Belgrade, Belgrade, Republic of Serbia Search for more papers by this author Delphine Demeestere Delphine Demeestere Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Valerie Vanhooren Valerie Vanhooren Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author An Hendrix An Hendrix Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, Ghent, Belgium Search for more papers by this author Claude Libert Claude Libert Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Roosmarijn E Vandenbroucke Corresponding Author Roosmarijn E Vandenbroucke [email protected] orcid.org/0000-0002-8327-620X Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Author Information Sriram Balusu1,2,3,4, Elien Van Wonterghem1,2, Riet De Rycke1,2, Koen Raemdonck5, Stephan Stremersch5, Kris Gevaert3,4, Marjana Brkic1,2,6, Delphine Demeestere1,2, Valerie Vanhooren1,2, An Hendrix7, Claude Libert1,2,‡ and Roosmarijn E Vandenbroucke *,1,2,‡ 1Inflammation Research Center, VIB, Ghent, Belgium 2Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 3Department of Medical Protein Research, VIB, Ghent, Belgium 4Department of Biochemistry, Ghent University, Ghent, Belgium 5Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium 6Department of Neurobiology, Institute for Biological Research, University of Belgrade, Belgrade, Republic of Serbia 7Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, Ghent, Belgium ‡These authors contributed equally to this work *Corresponding author. Tel: +32 9 3313587; Fax: +32 9 3313609; E-mail: [email protected] EMBO Mol Med (2016)8:1162-1183https://doi.org/10.15252/emmm.201606271 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Here, we identified release of extracellular vesicles (EVs) by the choroid plexus epithelium (CPE) as a new mechanism of blood–brain communication. Systemic inflammation induced an increase in EVs and associated pro-inflammatory miRNAs, including miR-146a and miR-155, in the CSF. Interestingly, this was associated with an increase in amount of multivesicular bodies (MVBs) and exosomes per MVB in the CPE cells. Additionally, we could mimic this using LPS-stimulated primary CPE cells and choroid plexus explants. These choroid plexus-derived EVs can enter the brain parenchyma and are taken up by astrocytes and microglia, inducing miRNA target repression and inflammatory gene up-regulation. Interestingly, this could be blocked in vivo by intracerebroventricular (icv) injection of an inhibitor of exosome production. Our data show that CPE cells sense and transmit information about the peripheral inflammatory status to the central nervous system (CNS) via the release of EVs into the CSF, which transfer this pro-inflammatory message to recipient brain cells. Additionally, we revealed that blockage of EV secretion decreases brain inflammation, which opens up new avenues to treat systemic inflammatory diseases such as sepsis. Synopsis New mechanism of blood–brain communication by the choroid plexus epithelial cells: uniquely positioned between blood and brain, choroid plexus epithelial cells release extracellular vesicles upon peripheral inflammation and transfer a pro-inflammatory message to the brain. Systemic inflammation induces the release of miRNA-containing extracellular vesicles by the choroid plexus epithelium cells into the cerebrospinal fluid. Choroid plexus epithelium-derived EVs are able to cross the ependymal cells lining the ventricles and reach the brain parenchyma, and they are taken up by astrocytes and microglia. The choroid plexus epithelium-derived EVs transfer a pro-inflammatory signal to the brain. Introduction The choroid plexus epithelium (CPE) is a unique single layer of epithelial cells situated at the interface of the blood and the cerebrospinal fluid (CSF) and forms the blood-CSF barrier (BCSFB). In recent years, the BCSFB has gained increasing attention, especially its role in inflammatory and neurodegenerative diseases (Vandenbroucke et al, 2012; Brkic et al, 2015; Demeestere et al, 2015a,b; Balusu et al, 2016; Gorlé et al, 2016). Systemic inflammatory response syndrome (SIRS) is a systemic inflammatory disease caused by, for example, trauma, burns, and infection. When infection is suspected, SIRS is called sepsis. Until now, treatment is mainly limited to antibiotics and support of vital functions, so understanding its pathology is of extreme importance for finding new therapeutic targets (Williams, 2012). We recently showed that systemic inflammatory conditions, such as SIRS and sepsis, compromise BCSFB barrier functionality in vivo, allowing leakage from the blood into the central nervous system (CNS) via the CSF (Vandenbroucke et al, 2012). The BCSFB is not only an anatomical barrier but also a dynamic tissue that expresses multiple transporters, receptors and enzymes. Indeed, the CPE plays a vital role in maintaining brain homeostasis by producing CSF, a transparent, colorless fluid composed of water, ions, and proteins. CSF is crucial for preserving the chemical microenvironment of the CNS and for providing lubrication to the brain. CPE cells are highly secretory, and they actively produce the CSF, including nutrients and neurotropic factors (Redzic & Segal, 2004; Smith et al, 2004; Emerich et al, 2005; Abbott et al, 2010; Redzic, 2011). Moreover, the choroid plexus transcriptome, proteome, and secretome are dynamic and respond to inflammatory triggers in the periphery (Marques et al, 2007, 2009; Thouvenot et al, 2012; Vandenbroucke et al, 2012). Extracellular vesicles (EVs), including all membrane-derived vesicles that are outside the cell, have evolved as important mediators of cell–cell communication (Akers et al, 2013; El Andaloussi et al, 2013; Raposo & Stoorvogel, 2013; Colombo et al, 2014; Yanez-Mo et al, 2015; Abels & Breakefield, 2016; Tkach & Thery, 2016). Based on their mode of biogenesis, EVs can be classified as exosomes, microvesicles, or apoptotic bodies. Exosomes (30–200 nm) are secreted via fusion of multivesicular bodies (MVBs) with the plasma membrane, whereas microvesicles (200–1,000 nm) originate by direct membrane budding (Colombo et al, 2014). Apoptotic bodies are much larger (0.5–3 μm) and are formed by random blebbing of the plasma membrane induced by cell death. EVs carry proteins, lipids, mRNA, miRNA, and other soluble factors to both adjacent and distant cells (Akers et al, 2013; El Andaloussi et al, 2013; Raposo & Stoorvogel, 2013; Colombo et al, 2014; Yanez-Mo et al, 2015; Abels & Breakefield, 2016; Tkach & Thery, 2016). MiRNAs present in EVs are of particular interest because they can modulate the gene expression pattern in recipient cells. MiRNAs are single-stranded RNA molecules of 19–23 nucleotides and regulate gene expression via mRNA degradation or translational repression (Filipowicz et al, 2008). Disturbances in the expression pattern of miRNAs are related to various pathophysiological conditions, such as cancer, inflammation, and diabetes. The presence of miRNAs outside the cell in various biological fluids (Weber et al, 2010) and the mechanism of miRNA secretion and its function in intercellular cross talk have recently gained significant attention (Bang & Thum, 2012; Gutierrez-Vazquez et al, 2013; Record et al, 2013; Guay et al, 2015; Heusermann et al, 2016; Tkach & Thery, 2016), also in the CNS (Andras & Toborek, 2016; Budnik et al, 2016; Thompson et al, 2016; Zappulli et al, 2016). Interestingly, this cross talk can lead to either pro- or anti-inflammatory responses (Vincent-Schneider et al, 2002; Abusamra et al, 2005; Kim et al, 2005, 2006; Bhatnagar et al, 2007; Clayton et al, 2008), while in neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, it is believed that these EVs carry toxic proteins within the CNS (Vingtdeux et al, 2007; Vella et al, 2008; Guest et al, 2011; Fruhbeis et al, 2012; Rajendran et al, 2014). Given the highly secretory nature of CPE cells and their strategic position at the interface of blood and CSF, they might play an important role in the communication between the blood and the brain. In this study, we identified a novel way of blood-to-brain communication that is activated by peripheral inflammation and occurs via secretion of CPE-derived, miRNA-containing EVs into the CSF. These CPE-derived EVs carry miRNA molecules, cross the ependymal cell layer lining the ventricles, reach the recipient brain parenchymal cells, and induce target mRNA repression and inflammatory response activation. Results Systemic inflammation induces changes in extracellular vesicles (EVs) in the cerebrospinal fluid (CSF) Cerebrospinal fluid (CSF) occupies the subarachnoid space and the ventricular system around and inside the brain and spinal cord, serves as a shock absorber for the central nervous system (CNS) and circulates nutrients and chemicals filtered from the blood into the brain; thereby playing an important role in brain homeostasis. Next to plasma proteins, electrolytes, amino acids, etc., CSF also contains extracellular vesicles (EVs). EVs are membrane-derived vesicles, including exosomes, microvesicles, or apoptotic bodies. Indeed, transmission electron microscopy (TEM) analysis confirmed the presence of EVs in the CSF isolated from mice (Fig 1A). Interestingly, nanoparticle tracking analysis (NTA, NanoSight) measurements of the amount of particles in CSF isolated from control and LPS-injected mice revealed that the amount of EVs in CSF increases gradually upon systemic inflammation, and the rise is significant 2 h after LPS injection. Figure 1B and C displays the absolute numbers and size distributions of the particles, respectively, and indicates that systemic inflammation leads to an increased amount of EVs in the CSF, especially in EVs of ~110 nm in size. Figure 1. LPS injection induces changes in extracellular vesicles (EVs) and miRNAs in the cerebrospinal fluid (CSF) A. Representative transmission electron microscope (TEM) image showing the presence of EVs in the CSF in two independent experiments. B. NanoSight quantification of the amount of particles in the CSF 0, 1, 2, 4, and 6 h after i.p. LPS injection (n = 3–5). C. Size distribution of the EVs in vivo in the CSF before (black; n = 5) and 6 h after (gray; n = 3) LPS treatment determined by NanoSight analysis. D–G. Quantitative real-time polymerase chain reaction analysis of miR-1a (D), miR-9 (E), miR-146a (F), and miR-155 (G) (n = 4). RNA was isolated from pooled CSF (50 μl) from different mice (n = 3). Data information: Data in (B, D-G) are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *0.01 ≤ P < 0.05; **0.001 ≤ P < 0.01. Download figure Download PowerPoint Encapsulated in the extracellular vesicles are proteins and nucleic acids including miRNAs. To address whether the increase in EVs upon systemic inflammation was associated with an increase in miRNAs in the CSF, we pooled 50 μl CSF from different mice and isolated EVs, followed by total RNA isolation. Next, we analyzed expression levels of several miRNAs implicated in inflammation (Sheedy & O'Neill, 2008), namely miR-1a, miR-9, miR-146a, and miR-155, and all of them showed up-regulation in the CSF-derived EVs after in vivo systemic (intraperitoneal, i.p.) LPS injection (Fig 1D–G). Primary choroid plexus epithelial (CPE) cells secrete miRNA-containing EVs upon LPS treatment in vitro The choroid plexus hangs in the ventricles and contains a single layer of CPE cells surrounding a core of fenestrated capillaries and loose connective tissue. These CPE cells are uniquely positioned between blood and CSF and are responsible for most of the CSF production. Here, we hypothesized that the CPE cells sense peripheral inflammation at their basal side, resulting in EV secretion at the apical side. To address this, we extended our study to the in vitro response of primary cultures of mouse CPE cells. We cultured primary CPE cells as described (Menheniott et al, 2010) and plated them onto transwells to mimic the in vivo situation (Fig EV1A). We thoroughly characterized the primary CPE cells by the expression of transthyretin (data not shown) and the presence and functionality of tight junctions. The primary CPE cells were strongly positive for zona occludens (ZO1, red), E-cadherin (ECDH, green), and claudin-1 (CLDN1, red) (Fig EV1B–D). Additionally, transepithelial electrical resistance (TEER) measurements confirmed the formation of a tight barrier (Fig EV1E). Click here to expand this figure. Figure EV1. Characterization of the primary CPE cell culture A. Schematic representation of the procedures of primary CPE cell isolation and culture in the transwell system. B, D. Representative confocal images showing the expression of zona occludens (ZO1, red, B), E-cadherin (ECDH, green, C), and claudin-1 (CLDN1, red, D) in the primary CPE culture. Scale bars, 30 μm. E. TEER values of the primary CPE cells grown in the transwell system. Significance was calculated compared with day 0 (n = 6). Data are presented as means ± SEM. Data were analyzed by Student's t-test. Significance levels are indicated on the graphs: ***0.0001 ≤ P < 0.001; ****P < 0.0001. Download figure Download PowerPoint The primary CPE cells were stimulated with LPS from the basal side, after which the supernatant was analyzed. Figure 2A and B displays the number and size distribution of the particles in the supernatant determined by NTA analysis (NanoSight). This revealed that LPS stimulation of primary CPE cells from the basal side results in increased secretion of EVs into the supernatant. Next, EVs were isolated, followed by RNA isolation and miRNA expression analysis. Analysis of the EV-associated miRNAs (Fig 2C–E) showed LPS-dependent miR-9, miR-146a, and miR-155 up-regulation, while miR-1a expression level was below detection limit. In parallel, we also analyzed miRNA expression of the CPE cells. qPCR analysis revealed that miR-1a/-9 were down-regulated and miR-146a/-155 were up-regulated in LPS-stimulated primary CPE cells (Fig 2F–I). This might indicate that miR-1a and miR-9 are directly secreted into the CSF without new synthesis of the miRNAs (resulting in up-regulation in supernatant and down-regulation in the CPE cells), while miR-146a and miR-155 are secreted but also their transcription is highly increased (resulting in up-regulation both in supernatant and CPE cells). Figure 2. Primary choroid plexus epithelial (CPE) cells secrete miRNA-containing EVs upon LPS incubation in vitro A, B. In vitro quantification (A) and size distribution (B) of EVs isolated from conditioned medium of primary CPE cells grown in a transwell system after 12 h in the absence (black) or presence (gray) of LPS (n = 5) determined by NanoSight analysis. C–E. TaqMan qPCR assay for the quantification of miR-9 (C), miR-146a (D), and miR-155 (E) in the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS (n = 3). F–I. Quantification of the miRNAs miR-1a (F), miR-9 (G), miR-146a (H), and miR-155 (I) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation (n = 3). Data information: Data in (A, C–I) are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *0.01 ≤ P < 0.05; **0.001 ≤ P < 0.01; ***0.0001 ≤ P < 0.001. Download figure Download PowerPoint Exosomes are EVs of 30–200 nm that are secreted by exocytosis from multivesicular bodies (MVBs) and that are characterized by the presence of specific marker proteins (Mathivanan & Simpson, 2009). In agreement with the observed increase in EV secretion by the CPE cells upon LPS stimulation, we also observed increased MVB production in vitro. Indeed, CD63 immunostaining (red) of primary CPE cells showed large intracellular vesicles in the presence of LPS (Fig EV2B; white arrow heads), which were absent in untreated primary CPE cells (Fig EV2A). Additionally, this increase was also detected by RiboGreen staining (Ganguly et al, 2009; Chiba et al, 2012) (green) of unstimulated (Fig EV2C) and LPS-stimulated primary CPE cells (Fig EV2D). Click here to expand this figure. Figure EV2. Analysis of the exosomal machinery in the CPE cells upon systemic inflammation A–D. Representative CD63 (red, A, B) and RiboGreen (green, C, D) staining of primary CPE cells after 12 h in the absence (A, C) or presence (B, D) of LPS in vitro from two independent experiments with n = 3. The white arrows indicate CD63 (A, B) or RiboGreen (C, D) positive vesicles. Scale bars, 70 μm. Download figure Download PowerPoint Inhibiting the exosome-mediated miRNA secretion by addition of the neutral sphingomyelinase inhibitor GW4869, a validated inhibitor of exosome production (Trajkovic et al, 2008), to primary CPE cells further suggested that the observed LPS effects were exosome dependent. NanoSight analysis showed that GW4869 treatment resulted in a reduction of LPS-induced EV secretion (Fig 3A). Interestingly, this decrease in EV secretion was correlated with a reduction in miR-9, miR-146a, and miR-155 expression in EVs isolated from the supernatant of LPS-stimulated primary CPE cells (Fig 3B–D). In contrast, exosome inhibitor treatment induced accumulation of miR-1a, miR-9 and miR-155 but not miR-146a in LPS-stimulated primary CPE cells (Fig 3E–H). These data show that CPE cells secrete miRNA-containing exosomes into the supernatant in response to LPS. Figure 3. Effect of exosome inhibition on EV and miRNA secretion of primary CPE cells stimulated with LPS A. In vitro quantification of EVs isolated from conditioned medium of LPS-stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 (n = 3). B–D. TaqMan assay quantification of the miRNAs miR-9 (B), miR-146a (C), and miR-155 (D) in supernatant of LPS-stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion (n = 3). miR-1a levels were below detection limit. E–H. TaqMan assay quantification of the miRNAs miR-1a (E), miR-9 (F), miR-146a (G), and miR-155 (H) in cell lysates of LPS-stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion (n = 3). Data information: Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *0.01 ≤ P < 0.05; **0.001 ≤ P < 0.01. Download figure Download PowerPoint CPE cells are the main source of miRNA-containing EVs that are released into the CSF upon systemic inflammation in vivo Transthyretin (TTR) is a protein that consists of four identical subunits of 14 kDa in a tetrahedral symmetry (Ingenbleek & Young, 1994). Plasma TTR originates primarily from the liver, whereas brain TTR is exclusively produced, secreted, and regulated by the choroid plexus (Herbert et al, 1986; Aldred et al, 1995). Interestingly, Western blot analysis of EV samples isolated from CSF revealed the presence of TTR (Fig EV3). Clearly, EVs isolated from CSF contain a choroid plexus specific marker, suggesting that the choroid plexus is an important source of the EVs that are present in the CSF. Click here to expand this figure. Figure EV3. Western blot analysis of choroid plexus cell lysates and EVs isolated from CSFChoroid plexus (CP) tissue was isolated, pooled from three mice, lysed, and analyzed by SDS–PAGE. Similarly, EVs were isolated from ~25 μl CSF and analyzed by SDS–PAGE. Detection was done with an anti-TTR antibody (green) and an anti-β-actin antibody (red) using the Odyssey Imaging system. Download figure Download PowerPoint To study whether CPE cells show changes in EV production upon i.p. LPS injection in vivo, similar to what we observed in vitro, we analyzed choroid plexus gene expression of different EV markers. Several of the tested markers were strongly altered in the CPE cells in vivo after 6 h LPS treatment: Hspa1a, Cd63, and Anxa5 were up-regulated while Cd9 and Cd81 were down-regulated (Fig EV4A–E, indicating an effect on the exosome machinery). Furthermore, we performed immunofluorescence analysis of different EV markers, namely CD63, RAB5, and ANXA2, on brain sections of naive mice and 4 and 8 h after LPS injection. This revealed a strong induction of all tested EV proteins early upon stimulation with LPS (Fig 4A). CD63 was mainly observed in the perinuclear area in basal conditions and early upon LPS stimulation there is an increased signal at the apical side, close to the CSF. At a later time point, high CD63 levels are observed both at the perinuclear area and at the apical side of the choroid plexus epithelial cells. Similarly, RAB5 can be detected in the choroid plexus of naive mice and LPS stimulation results in higher levels of RAB5 both in the cytoplasm and at the apical side of the choroid plexus epithelial cells. Although ANXA2 expression was less homogeneous throughout the choroid plexus, this marker is expressed at basal conditions and is strongly induced upon LPS stimulation. Click here to expand this figure. Figure EV4. Analysis of the exosomal machinery in CPE cells upon systemic inflammation A–E. QPCR gene expression analysis of exosomal markers Cd9 (A), Cd81 (B), Hspa1a (C), Cd63 (D), and Anxa5 (E) in the choroid plexus before and after LPS treatment (n = 4). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *0.01 ≤ P < 0.05; **0.001 ≤ P < 0.01. F–K. Representative TEM images from choroid plexus tissue isolated 0 (F), 1 (G), 2 (H), 3 (I), 4 (J), or 6 (K) h after LPS injection. Black arrow heads point to exosomes present in MVBs. Scale bars, 9 μm. Mv, microvilli; Nu, nucleus. L–N. In situ hybridization (ISH) analysis of miR expression in CPE cells in vivo. LNA™-ISH of miR-146a (L), miR-9 (M), and miR-155 (N) on brain sections. Scale bars, 30 μm. Download figure Download PowerPoint Figure 4. Systemic inflammation activates the exosomal machinery in the choroid plexus A. Representative confocal images of CD63, RAB5, and ANXA2 (red) in the choroid plexus (CP) at 0, 4, and 8 h after LPS treatment. Hoechst (blue) was used to stain the nucleus. The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image. Scale bars, 100 μm. B, C. Representative TEM images showing the presence of MVBs in the CPE cells before (B) and 6 h after (C) LPS administration in vivo. Black arrow heads point to exosomes present in MVBs. Scale bars, 9 μm. D–F. Quantification of number of MVBs per cell section (D), number of exosomes per MVB (E), and number of exosomes per cell section (F), based on TEM analysis of several adjacent cells (0 h, n = 20; 3 h, n = 21; 4 h, n = 13; 6 h, n = 23). G–J. Quantitative real-time polymerase chain reaction (qPCR) analysis of miR-1a (G), miR-9 (H), miR-146a (I), and miR-155 (J). Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay (0 h, n = 4; 1 h, n = 5; 6 h, n = 5; 24 h, n = 3). K. NanoSight analysis of CSF isolated from LPS-injected mice followed by icv injection of vehicle or GW4869, a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 8). L. qPCR analysis of the expression of miR-1a, miR-9, miR-146a, and miR-155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle (black) or GW4869 (gray) (n = 4). M. NanoSight analysis of the supernatant of choroid plexus explants from PBS- or LPS-injected mice (n = 6). Data information: Data in (D–M) are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *0.01 ≤ P < 0.05; **0.001 ≤ P < 0.01; ***0.0001 ≤ P < 0.001; ****P < 0.0001. Download figure Download PowerPoint Moreover, TEM of the choroid plexus revealed a huge increase in amount of exosomes in the MVBs of LPS-treated mice (Fig 4C) compared to MVBs in the choroid plexus of unchallenged mice (Fig 4B). We quantified both the amount of MVBs per c
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