a0965645205af473e120b05b8c154d8a 303544.pdf f1e0ac87561c50500f9379a319ab0aba52f8f642 303544.pdf 01c5eb2cc2e7c259fd9782c311099ef285d97db250812fa758b134a7c49145c8 303544.pdf Title: Priming Mesenchymal Stem Cells with Lipopolysaccharide Boosts the Immunomodulatory and Regenerative Activity of Secreted Extracellular Vesicles Subject: Background: Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) have been proposed as an alternative to live-cell administration for Acute Respiratory Distress Syndrome (ARDS). MSC-EVs can be chiefly influenced by the environment to which the MSCs are exposed. Here, lipopolysaccharide (LPS) priming of MSCs was used as a strategy to boost the natural therapeutic potential of the EVs in acute lung injury (ALI). Methods: The regenerative and immunemodulatory effect of LPS-primed MSC-EVs (LPS-EVs) and non-primed MSC-EVs (C-EVs) were evaluated in vitro on alveolar epithelial cells and macrophage-like THP-1 cells. In vivo, ALI was induced in adult male rats by the intrapulmonary instillation of HCl and LPS. Rats (n = 8 to 22/group) were randomized to receive a single bolus (1 108 particles) of LPS-EVs, C-EVs, or saline. Lung injury severity was assessed at 72 h in lung tissue and bronchoalveolar lavage. Results: In vitro, LPS-EVs improved wound regeneration and attenuated the inflammatory response triggered by the P. aeruginosa infection, enhancing the M2 macrophage phenotype. In in vivo studies, LPS-EVs, but not C-EVs, significantly decreased the neutrophilic infiltration and myeloperoxidase (MPO) activity in lung tissue. Alveolar macrophages from LPS-EVs-treated animals exhibited a reduced expression of CXCL-1, a key neutrophil chemoattractant. However, both C-EVs and LPS-EVs reduced alveolar epithelial and endothelial permeability, mitigating lung damage. Conclusions: EVs from LPS-primed MSCs resulted in a better resolution of ALI, achieving a greater balance in neutrophil infiltration and activation, while avoiding the complete disruption of the alveolar barrier. This opens new avenues, paving the way for the clinical implementation of cell-based therapies. Keywords: acute lung injury; mesenchymal stem cells; extracellular vesicles; LPS priming;immunomodulation; regeneration Author: Aina Areny-Balagueró, Marta Camprubí-Rimblas, Elena Campaña-Duel, Anna Solé-Porta, Adrián Ceccato, Anna Roig, John G. Laffey, Daniel Closa and Antonio Artigas Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Fri Oct 11 09:32:36 2024 CEST ModDate: Fri Oct 11 09:44:35 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 22 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 6586446 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JYPLJJ+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 OWQKLJ+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 MEKZEW+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 BXOWMO+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 UBTAZT+CMSY10 Type 1 Builtin yes yes yes 32 0 SWRFCF+VnURWPalladioL Type 1 Custom yes yes yes 37 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 57 0 KESBIZ+EURM10 Type 1 Builtin yes yes yes 81 0 MPUDFL+PazoMath Type 1 Builtin yes yes yes 95 0 STKLCB+TeX-tipa10 Type 1 Builtin yes yes yes 164 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-11-26 02:45:09 CET RepresentationInformation: 303544.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-11-25 13:06:15 CET Size: 6586446 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 376 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Background Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Isolation, Culture and Preconditioning of MSCs Destination: subsection.2.1 Item: Title: Characterization of C-MSCs and LPS-MSCs Destination: subsection.2.2 Item: Title: C-EVs and LPS-EVs Isolation and Characterization Destination: subsection.2.3 Item: Title: Assessment of MSCs-Derived EVs’ Therapeutic Effect In Vitro Destination: subsection.2.4 Item: Title: In Vivo MSCs-Derived EVs’ Therapeutic Evaluation Destination: subsection.2.5 Item: Title: Statistics for In Vitro and In Vivo Studies Destination: subsection.2.6 Item: Title: Results Destination: section.3 Children: Item: Title: Characterization of Isolated MSCs Destination: subsection.3.1 Item: Title: Characterization of MSCs-Derived EVs Destination: subsection.3.2 Item: Title: LPS-EVs Accelerate Wound Healing and Cell Proliferation versus C-EVs In VItro Destination: subsection.3.3 Item: Title: LPS-EVs Produce a Major Anti-Inflammatory Effect Compared to C-EVs in P. aeruginosa-Infected Macrophage-like THP-1 Cells In Vitro Destination: subsection.3.4 Item: Title: C-EV and LPS-EV Therapy Ameliorates Lung/Body Weight Ratio In Vivo Destination: subsection.3.5 Item: Title: LPS-EVs and C-EVs Reduce Lung Permeability, but Only LPS-EVs Significantly Decrease Neutrophil Infiltration In Vivo Destination: subsection.3.6 Item: Title: MSCs-EVs Modulate Lung Inflammation but LPS-EVs Further Decrease AM’s Chemotaxis Activity In Vivo Destination: subsection.3.7 Item: Title: C-EVs and LPS-EVs Restored Lung Architecture In Vivo Destination: subsection.3.8 Item: Title: Discussion Destination: section.4 Item: Title: References Destination: appendix.A. Info: Title: Priming Mesenchymal Stem Cells with Lipopolysaccharide Boosts the Immunomodulatory and Regenerative Activity of Secreted Extracellular Vesicles Author: Aina Areny-Balagueró, Marta Camprubí-Rimblas, Elena Campaña-Duel, Anna Solé-Porta, Adrián Ceccato, Anna Roig, John G. Laffey, Daniel Closa and Antonio Artigas Subject: Background: Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) have been proposed as an alternative to live-cell administration for Acute Respiratory Distress Syndrome (ARDS). MSC-EVs can be chiefly influenced by the environment to which the MSCs are exposed. Here, lipopolysaccharide (LPS) priming of MSCs was used as a strategy to boost the natural therapeutic potential of the EVs in acute lung injury (ALI). Methods: The regenerative and immunemodulatory effect of LPS-primed MSC-EVs (LPS-EVs) and non-primed MSC-EVs (C-EVs) were evaluated in vitro on alveolar epithelial cells and macrophage-like THP-1 cells. In vivo, ALI was induced in adult male rats by the intrapulmonary instillation of HCl and LPS. Rats (n = 8 to 22/group) were randomized to receive a single bolus (1 108 particles) of LPS-EVs, C-EVs, or saline. Lung injury severity was assessed at 72 h in lung tissue and bronchoalveolar lavage. Results: In vitro, LPS-EVs improved wound regeneration and attenuated the inflammatory response triggered by the P. aeruginosa infection, enhancing the M2 macrophage phenotype. In in vivo studies, LPS-EVs, but not C-EVs, significantly decreased the neutrophilic infiltration and myeloperoxidase (MPO) activity in lung tissue. Alveolar macrophages from LPS-EVs-treated animals exhibited a reduced expression of CXCL-1, a key neutrophil chemoattractant. However, both C-EVs and LPS-EVs reduced alveolar epithelial and endothelial permeability, mitigating lung damage. Conclusions: EVs from LPS-primed MSCs resulted in a better resolution of ALI, achieving a greater balance in neutrophil infiltration and activation, while avoiding the complete disruption of the alveolar barrier. This opens new avenues, paving the way for the clinical implementation of cell-based therapies. 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