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. 2022 Aug 10;78(6):617–630. doi: 10.1136/thoraxjnl-2021-218194

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© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/licenses/by/4.0/.

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Transfer of miRNA-181a-5p in MSC EVs modulates LPS-induced secretion of pro-inflammatory cytokines through PTEN-pSTAT5-SOCS1 axis. (A) Heat map of next generation sequencing data comparing BALF, cell and MSC-EV expression of selected micro RNAs. ‘1’, ‘2’, ‘3’ labelling refers to different MSC donors. (B) Representative live microscopy images of human MDMs transfection with Dy574-labelled miRNA181a-5p mimic (left) and miRNA Scramble/Negative control mimic (right) as an indicator of efficiency of transfection, monitored by immunofluorescence. Images were taken using EVOS FL Auto epifluorescent microscope (Objective lens 10×, scale bar=50 µm). (C) Immunoblot of PTEN, pSTAT5, STAT5, SOCS1 and β-actin in human MDMs lysates transfected with miRNA181a-5p mimic and miRNA Scramble (negative control) mimic after LPS stimulation for 24 hours. Immunoblots were quantified by densitometry and normalised using total STAT5 expression for pSTAT5 or β-actin expression for PTEN/SOCS1 (n=4). (D) Levels of TNF-α secretion by MDMs after stimulation with LPS for 24 hours (n=3). (E) Immunoblot of PTEN, pSTAT5, STAT5, SOCS1 and β-actin in human MDMs lysates after MDMs were stimulated with LPS for 24 hours. Immunoblots were quantified by densitometry and normalised using total STAT5 expression for pSTAT5 or β-actin expression for PTEN/SOCS1 (n=3–4). (F) Representative live microscopy images of MSCs (left) and human MDMs (right) transfection with Dy574-labelled miRNA181a-5p mimic. The images were taken using EVOS FL Auto epifluorescent microscope (Objective lens 40×, scale bar=50 µm). Data are represented as mean±SD. Kruskal-Wallis test with post-hoc Dunn’s test (C, D), Mann-Whitney test (E). EV, extracellular vesicles; LPS, lipopolysaccharide; MDM, monocyte-derived macrophages; miRNA, micro RNA; MSC, mesenchymal stromal cells; pSTAT, phosphorylated STAT; PTEN, phosphatase and tensin homolog; siRNA, small interfering RNA; SOCS1, suppressor of cytokine signalling 1; STAT, 5, signal transducers and activators of transcription 5; TNF, tumour necrosis factor.