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Mesenchymal-Stem-Cell-Derived Extracellular Vesicles Attenuate Brain Injury in Escherichia coli Meningitis in Newborn Rats.

ABSTRACT: We recently reported that transplantation of mesenchymal stem cells (MSCs) significantly reduced bacterial growth and brain injury in neonatal meningitis induced by Escherichia coli (E. coli) infection in newborn rats. As a next step, to verify whether the MSCs protect against brain injury in a paracrine manner, this study was designed to estimate the efficacy of MSC-derived extracellular vesicles (MSC-EVs) in E. coli meningitis in newborn rats. E. coli meningitis was induced without concomitant bacteremia by the intra-cerebroventricular injection of 5 × 10² colony-forming units of K1 (-) E. coli in rats, at postnatal day 11. MSC-EVs were intra-cerebroventricularly transplanted 6 h after the induction of meningitis, and antibiotics were administered for three consecutive days starting at 24 h after the induction of meningitis. The increase in bacterial growth in the cerebrospinal fluid measured at 24 h after the meningitis induction was not significantly reduced following MSC-EV transplantation. However, an increase in brain cell death, reactive gliosis, and inflammation following meningitis were significantly attenuated after MSC-EV transplantation. Taken together, our results indicate that MSCs show anti-apoptotic, anti-gliosis, and anti-inflammatory, but not antibacterial effects, in an EV-mediated paracrine manner in E. coli-induced neonatal meningitis.

SUBMITTER: Kim YE

PROVIDER: S-EPMC9319453 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Mesenchymal-Stem-Cell-Derived Extracellular Vesicles Attenuate Brain Injury in Escherichia coli Meningitis in Newborn Rats.

Kim Young-Eun YE Ahn So-Yoon SY Park Won-Soon WS Sung Dong-Kyung DK Sung Se-In SI Yang Mi-Sun MS Chang Yun-Sil YS

Life (Basel, Switzerland) 20220711 7

We recently reported that transplantation of mesenchymal stem cells (MSCs) significantly reduced bacterial growth and brain injury in neonatal meningitis induced by Escherichia coli (E. coli) infection in newborn rats. As a next step, to verify whether the MSCs protect against brain injury in a paracrine manner, this study was designed to estimate the efficacy of MSC-derived extracellular vesicles (MSC-EVs) in E. coli meningitis in newborn rats. E. coli meningitis was ...[more]

PMID: 35888118

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Project description:BackgroundAcute lung injury (ALI) following pneumonia involves uncontrolled inflammation and tissue injury, leading to high mortality. We previously confirmed the significantly increased cargo content and extracellular vesicle (EV) production in thrombin-preconditioned human mesenchymal stromal cells (thMSCs) compared to those in naïve and other preconditioning methods. This study aimed to investigate the therapeutic efficacy of EVs derived from thMSCs in protecting against inflammation and tissue injury in an Escherichia coli (E. coli)-induced ALI mouse model.MethodsIn vitro, RAW 264.7 cells were stimulated with 0.1 µg/mL liposaccharides (LPS) for 1 h, then were treated with either PBS (LPS Ctrl) or 5 × 107 particles of thMSC-EVs (LPS + thMSC-EVs) for 24 h. Cells and media were harvested for flow cytometry and ELISA. In vivo, ICR mice were anesthetized, intubated, administered 2 × 107 CFU/100 µl of E. coli. 50 min after, mice were then either administered 50 µL saline (ECS) or 1 × 109 particles/50 µL of thMSC-EVs (EME). Three days later, the therapeutic efficacy of thMSC-EVs was assessed using extracted lung tissue, bronchoalveolar lavage fluid (BALF), and in vivo computed tomography scans. One-way analysis of variance with post-hoc TUKEY test was used to compare the experimental groups statistically.ResultsIn vitro, IL-1β, CCL-2, and MMP-9 levels were significantly lower in the LPS + thMSC-EVs group than in the LPS Ctrl group. The percentages of M1 macrophages in the normal control, LPS Ctrl, and LPS + thMSC-EV groups were 12.5, 98.4, and 65.9%, respectively. In vivo, the EME group exhibited significantly lower histological scores for alveolar congestion, hemorrhage, wall thickening, and leukocyte infiltration than the ECS group. The wet-dry ratio for the lungs was significantly lower in the EME group than in the ECS group. The BALF levels of CCL2, TNF-a, and IL-6 were significantly lower in the EME group than in the ECS group. In vivo CT analysis revealed a significantly lower percentage of damaged lungs in the EME group than in the ECS group.ConclusionIntratracheal thMSC-EVs administration significantly reduced E. coli-induced inflammation and lung tissue damage. Overall, these results suggest therapeutically enhanced thMSC-EVs as a novel promising therapeutic option for ARDS/ALI.

Project description:BackgroundNecrotizing enterocolitis (NEC) is a critical gastrointestinal disease in preterm infants, for which no specific treatment is established. We previously demonstrated that thrombin-preconditioned mesenchymal stromal cell-derived extracellular vesicles (thMSC-EVs) enhance protection against other neonatal tissue injuries. Therefore, this study aimed to evaluate the therapeutic potential of thMSC-EVs in modified in vitro, in vivo, and organoid models of NEC.MethodsIn vitro, the effects of thMSC-EVs and naïveMSC-EVs were compared in hyperosmotic, ischemic, and hypothermic (HIT)-stressed IEC-6 cells and LPS-treated peritoneal macrophages. In vivo, NEC was induced in P4 mouse pups by three cycles of formula feeding, oral LPS administration, hypoxia, and hypothermia, followed by overnight dam care. 2 × 109 thMSC-EVs were intraperitoneally administered daily for three days, and the therapeutic effects were assessed macroscopically, histologically, and biochemically. NEC mouse-derived organoids were established to evaluate the thMSC-EVs' effect in mature enterocytes. LC-MS/MS was performed to analyze the EV proteomics.ResultsIn vitro, compared with naïveMSC-EVs, thMSC-EVs significantly improved cellular viability in HIT-induced IEC-6 cells and reduced pro-inflammatory (IL-1α, IL-1β, TNF-α) but increased anti-inflammatory (TGF-b) cytokine levels in LPS-treated peritoneal macrophages. In vivo, thMSC-EVs significantly attenuated clinical symptoms, reduced intestinal damage, and retained intestinal stem cell markers, showing more significant localization in NEC-induced intestines than in healthy intestines. In NEC mouse-derived organoids, thMSC-EVs significantly increased OLFM4 and claudin-4 expression and reduced stress-related markers such as sucrase-isomaltase, defensin, and chromogranin A. Proteomic analysis revealed that thMSC-EVs were greater enriched in anti-apoptotic, anti-inflammatory, cell adhesion, and Wnt signaling pathways than naïveMSC-EVs.ConclusionthMSC-EVs improved cellular viability, reduced apoptosis, attenuated inflammation, and upregulated key intestinal stem cell markers, collectively suggesting their tissue-protective effects and highlighting their potential as a treatment for NEC.

Project description:Mesenchymal stromal cells (MSCs) are potent regulators of immune responses largely through paracrine signaling. MSC secreted extracellular vesicles (MSC-EVs) are increasingly recognized as the key paracrine factors responsible for the biological and therapeutic function of MSCs. We report the first comprehensive study demonstrating the immunomodulatory effect of MSC-EVs on dendritic cell (DC) maturation and function. MSC-EVs were isolated from MSC conditioned media using differential ultracentrifugation. Human monocyte-derived DCs were generated in the absence or presence of MSC-EVs (20 ug/ml) then subjected to phenotypic and functional analysis in vitro. MSC-EV treatment impaired antigen uptake by immature DCs and halted DC maturation resulting in reduced expression of the maturation and activation markers CD83, CD38, and CD80, decreased secretion of pro-inflammatory cytokines IL-6 and IL-12p70 and increased production of anti-inflammatory cytokine TGF-β. MSC-EV treated DCs also demonstrated a diminished CCR 7 expression after LPS stimulation, coupled with a significantly reduced ability to migrate toward the CCR7-ligand CCL21, although they were still able to stimulate allogeneic T cell proliferation in vitro. Through microRNA profiling we have identified 49 microRNAs, which were significantly enriched in MSC-EVs compared to their parent MSCs. MicroRNAs with known effect on DC maturation and functions, including miR-21-5p, miR-142-3p, miR-223-3p, and miR-126-3p, were detected within the top 10 most enriched miRNAs in MSC-EVs, with MiR-21-5p as the third highest expressed miRNA in MSC-EVs. In silico analysis revealed that miR-21-5p targets the CCR7 gene for degradation. To verify these observations, DCs were transfected with miR-21-5p mimics and analyzed for their ability to migrate toward the CCR7-ligand CCL21 in vitro. MiR-21-5p mimic transfected DCs showed a clear trend of reduced CCR7 expression and a significantly decreased migratory ability toward the CCL21. Our findings suggest that MSC-EVs are able to recapitulate MSC mediated DC modulation and MSC-EV enclosed microRNAs may represent a novel mechanism through which MSCs modulate DC functions. As MSCs are currently used in clinical trials to treat numerous diseases associated with immune dysregulation, such as graft-versus-host disease and inflammatory bowel disease, our data provide novel evidence to inform potential future application of MSC-EVs as a cell-free therapeutic agent.

Project description:BACKGROUND:Mesenchymal stem cells (MSCs) have been shown to mitigate vascular permeability in hemorrhagic shock (HS) and trauma-induced brain and lung injury. Mechanistically, paracrine factors secreted from MSCs have been identified that can recapitulate many of the potent biologic effects of MSCs in animal models of disease. Interestingly, MSC-derived extracellular vesicles (EVs), contain many of these key soluble factors, and have therapeutic potential independent of the parent cells. In this study we sought to determine whether MSC-derived EVs (MSC EVs) could recapitulate the beneficial therapeutic effects of MSCs on lung vascular permeability induced by HS in mice. METHODS:Mesenchymal stem cell EVs were isolated from human bone marrow-derived MSCs by ultracentrifugation. A mouse model of fixed pressure HS was used to study the effects of shock, shock + MSCs and shock + MSC EVs on lung vascular endothelial permeability. Mice were administered MSCs, MSC EVs, or saline IV. Lung tissue was harvested and assayed for permeability, RhoA/Rac1 activation, and for differential phosphoprotein expression. In vitro, human lung microvascular cells junctional integrity was evaluated by immunocytochemistry and endothelial cell impedance assays. RESULTS:Hemorrhagic shock-induced lung vascular permeability was significantly decreased by both MSC and MSC EV infusion. Phosphoprotein profiling of lung tissue revealed differential activation of proteins and pathways related to cytoskeletal rearrangement and regulation of vascular permeability by MSCs and MSC EVs. Lung tissue from treatment groups demonstrated decreased activation of the cytoskeletal GTPase RhoA. In vitro, human lung microvascular cells, MSC CM but not MSC-EVs prevented thrombin-induced endothelial cell permeability as measured by electrical cell-substrate impedance sensing system and immunocytochemistry of VE-cadherin and actin. CONCLUSION:Mesenchymal stem cells and MSC EVs modulate cytoskeletal signaling and attenuate lung vascular permeability after HS. Mesenchymal stem cell EVs may potentially be used as a novel "stem cell free" therapeutic to treat HS-induced lung injury.

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Mesenchymal-Stem-Cell-Derived Extracellular Vesicles Attenuate Brain Injury in Escherichia coli Meningitis in Newborn Rats.

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Mesenchymal-Stem-Cell-Derived Extracellular Vesicles Attenuate Brain Injury in <i>Escherichia coli</i> Meningitis in Newborn Rats.

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