Mesenchymal stromal cells-derived extracellular vesicles reprogramme macrophages in ARDS models through the miR-181a-5p-PTEN-pSTAT5-SOCS1 axis

Abstract

Rationale: A better understanding of the mechanism of action of mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) is needed to support their use as novel therapies for acute respiratory distress syndrome (ARDS). Macrophages are important mediators of ARDS inflammatory response. Suppressor of cytokine signalling (SOCS) proteins are key regulators of the macrophage phenotype switch. We therefore investigated whether SOCS proteins are involved in mediation of the MSC effect on human macrophage reprogramming.

Methods: Human monocyte-derived macrophages (MDMs) were stimulated with lipopolysaccharide (LPS) or plasma samples from patients with ARDS (these samples were previously classified into hypo-inflammatory and hyper-inflammatory phenotype) and treated with MSC conditioned medium (CM) or EVs. Protein expression was measured by Western blot. EV micro RNA (miRNA) content was determined by miRNA sequencing. In vivo: LPS-injured C57BL/6 mice were given EVs isolated from MSCs in which miR-181a had been silenced by miRNA inhibitor or overexpressed using miRNA mimic.

Results: EVs were the key component of MSC CM responsible for anti-inflammatory modulation of human macrophages. EVs significantly reduced secretion of tumour necrosis factor-α and interleukin-8 by LPS-stimulated or ARDS plasma-stimulated MDMs and this was dependent on SOCS1. Transfer of miR-181a in EVs downregulated phosphatase and tensin homolog (PTEN) and subsequently activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) leading to upregulation of SOCS1 in macrophages. In vivo, EVs alleviated lung injury and upregulated pSTAT5 and SOCS1 expression in alveolar macrophages in a miR181-dependent manner. Overexpression of miR-181a in MSCs significantly enhanced therapeutic efficacy of EVs in this model.

Conclusion: miR-181a-PTEN-pSTAT5-SOCS1 axis is a novel pathway responsible for immunomodulatory effect of MSC EVs in ARDS.

Keywords: ARDS; macrophage biology.

Figure 1

Upregulation of SOCS1 protein expression in human macrophages is critical for the paracrine effect of MSCs. SOCS1 upregulation is accompanied by the activation of STAT5 phosphorylation. (A) Levels of TNF-α and IL-8 in MDM conditioned medium after 24 hours of exposure to LPS (measured by ELISA) (n=4–7). (B) Immunoblot for protein expression levels of SOCS1 and β-actin in human MDM lysates after stimulation with LPS for 24 hours. Immunoblots were quantified by densitometry and normalised using β-actin expression (n=5). (C) Immunoblot of human MDM lysates, after MDMs were transfected with SOCS1 or scrambled siRNA and stimulated with LPS for 24 hours. Immunoblots were quantified by densitometry and normalised using β-actin expression (n=6). (D) Levels of TNF-α in the conditioned medium of MDMs transfected with scrambled or SOCS siRNA after LPS stimulation for 24 hours, measured by ELISA (n=4). (E) On the left, schematic image showing the co-culture of human MDMs with MSCs using a Transwell system which involved exposure to patient with ARDS bronchoalveolar lavage fluid (BALF) for 24 hours. On the right, phosphokinase array data from human MDMs cell lysates after exposure to ARDS BALF with and without MSC co-culture. Red arrows showed only STAT5a and STAT5b in MDMs could be upregulated by MSCs (n=1). (F) Immunoblots of different STAT proteins in human MDM cell lysates. (G) Immunoblot of pSTAT5 and STAT5 in human MDM lysates at 24 hours after MSC-CM treatment and LPS stimulation. Immunoblots of pSTAT5 were quantified by densitometry and normalised using total STAT5 protein expression (n=6). Data are represented as mean±SD. Kruskal-Wallis test with post-hoc Dunn’s test (A, B, D, G), Mann-Whitney test (C). ARDS, acute respiratory distress syndrome; CM, conditioned medium; IL, interleukin; LPS, lipopolysaccharide; MDM, monocyte-derived macrophages; MSC, mesenchymal stromal cells; pSTAT, phosphorylated STAT; siRNA, small interfering RNA; SOCS1, suppressor of cytokine signalling 1; STAT, signal transducers and activators of transcription; TNF, tumour necrosis factor.

Figure 2

MSC-EVs but not EV-free MSC conditioned medium are responsible for the inhibition of LPS-induced cytokine secretion and upregulation of pSTAT5 and SOCS1 expression in human MDMs. STAT5 phosphorylation is critical for SOCS1 upregulation by EVs. (A) Levels of TNF-α and IL-8 in MDM conditioned medium (measured by ELISA) after LPS stimulation for 24 hours (n=3–4). (B) Immunoblot for protein expression levels of pSTAT5, STAT5, SOCS1 and β-actin in human MDMs lysates after stimulation with LPS for 24 hours. Immunoblots were quantified by densitometry and normalised using STAT5 protein expression for pSTAT5 or β-actin for SOCS1 (n=5). (C) Immunoblot of pSTAT5, STAT5, SOCS1 and β-actin in MDM lysates after MDMs were pre-treated with pharmacological STAT5 inhibitor AC-4–130. Immunoblots were quantified by densitometry and normalised using total STAT5 expression levels for pSTAT5 or β-actin expression levels for SOCS1 (n=4). (D) Levels of TNF-α in MDM conditioned medium after pre-treatment with STAT5 inhibitor and exposure to LPS for 24 hours (n=3–4). Data are represented as mean±SD. Kruskal-Wallis test with post-hoc Dunn’s test (A, B, C, D). CM, conditioned medium; DMSO, dimethyl sulfoxide; IL, interleukin; EVs, extracellular vesicles; IL, interleukin; LPS, lipopolysaccharide; MDM, monocyte-derived macrophages; MSC, mesenchymal stromal cells; pSTAT, phosphorylated STAT; SOCS1, suppressor of cytokine signalling 1; STAT, signal transducers and activators of transcription; TNF, tumour necrosis factor.

Figure 3

MSC EVs regulate pro-inflammatory cytokine secretion and pSTAT5 and SOCS1 expression in MDMs exposed to plasma samples from patients with ARDS in vitro. (A) Levels of TNF-α and IL-8 in MDMs supernatants (measured by ELISA) after stimulation with pooled healthy or hypo-inflammatory or hyper-inflammatory ARDS plasma for 24 hours (n=3–5). (B) Immunoblot of pSTAT5, STAT5, SOCS1 and β-actin protein expression in human MDM lysates after MDM exposure to healthy or ARDS plasma for 24 hours (n=4). Immunoblots were quantified by densitometry and normalised using total STAT5 expression for pSTAT5 or β-actin expression for SOCS1. Data are represented as mean±SD. Kruskal-Wallis test with post-hoc Dunn’s test (A, B). ARDS, acute respiratory distress syndrome; CM, conditioned medium; IL, interleukin; EV, extracellular vesicles; IL, interleukin; MDM, monocyte-derived macrophages; MSC, mesenchymal stromal cells; pSTAT, phosphorylated STAT; SOCS1, suppressor of cytokine signalling 1; STAT, 5, signal transducers and activators of transcription 5; TNF, tumour necrosis factor.

Figure 4

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.

Figure 5

miRNA-181a transfer through MSC EVs negates the pro-inflammatory response in human MDMs when stimulated with LPS or hyper-inflammatory ARDS plasma. (A) Immunoblot of PTEN, pSTAT5, STAT5 and β-actin in human MDM lysates after stimulation 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=4). (B) Levels of TNF-α secretion by MDMs after stimulation with LPS for 24 hours (n=3–4). (C) Levels of TNF-α secretion by MDMs after stimulation with healthy or pooled hypo-inflammatory or hyper-inflammatory ARDS plasma for 24 hours (n=3–6). (D) Immunoblot of PTEN, pSTAT5, STAT5, SOCS1 and β-actin in human MDMs lysates after stimulation with healthy or hypo-inflammatory or hyper-inflammatory ARDS plasma for 24 hours. Immunoblots were quantified by densitometry and normalised using total STAT5 expression for pSTAT5 or β-actin expression for PTEN/SOCS1 (bottom panel, n=2). Data are represented as mean±SD. Kruskal-Wallis test with post-hoc Dunn’s test (A, B, C). ARDS, acute respiratory distress syndrome; EVs, extracellular vesicles; LNA, locked nucleic acid; LPS, lipopolysaccharide; MDM, monocyte-derived macrophages; miRNA, micro RNA; MSCs, mesenchymal stromal cells; pSTAT, phosphorylated STAT; PTEN, phosphatase and tensin homolog; SOCS1, suppressor of cytokine signalling 1; STAT, signal transducers and activators of transcription ;TNF, tumour necrosis factor.

Figure 6

Transfer of miR181a is critical for the immunomodulatory effects of MSC-EVs in vivo. (A) Total protein concentrations in the BALF samples 24 hours after LPS administration (n=5–7 mice per group). (B) Total leucocyte counts (left graph) and neutrophil cell counts (right graph) in the BALF samples (total cells (n=5–7 mice per group); neutrophils (n=2–4 mice per group)). (C) Representative images of BALF cytospin preparations demonstrating cell recruitment to the airspaces 24 hours after LPS administration. Images were taken using Leica Epifluorescence DM5500 microscope (Objective lens ×20). (D) BALF levels of TNF-α and keratinocyte-derived chemokine (KC, murine analogue of interleukin-8) (n=5–7 mice per group). (E) Representative confocal microscopy of alveolar macrophages, isolated from BALF 24 hours after LPS administration and stained with anti-CD68 (alveolar macrophage marker) and anti-pSTAT5 Ab. Arrows indicate co-localisation of CD68 and pSTAT5. The images were taken using Leica SP8 confocal microscope with a 100× oil-immersion objective (n=3, scale bar=50 µm). Quantitative fluorescence intensity was analysed by Image J software (MFI-pSTAT5). (F) Representative confocal microscopy of alveolar macrophages, isolated from BALF samples 24 hours after LPS administration and stained with anti-CD68 and anti-SOCS1 Ab. Arrows indicate co-localisation of CD68 and SOCS1. Images were taken using Leica SP8 confocal microscope with a 100× oil-immersion objective (scale bar=50 µm). Quantitative fluorescence intensity was analyses by Image J software (MFI-SOCS1). Data are represented as mean±SD. Kruskal-Wallis test with post hoc Dunn’s test (A, B, D, E, F). Ab, antibody; BALF, bronchoalveolar lavage fluid; DAPI, 4',6-diamidino-2-phenylindole; EVs, extracellular vesicles; LNA, locked nucleic acid; LPS, lipopolysaccharide; MFI, mean fluorescent intensity; MSCs, mesenchymal stromal cells; PBS, phosphate-buffered saline; pSTAT, phosphorylated STAT; SOCS1, suppressor of cytokine signalling 1; STAT, signal transducers and activators of transcription ;TNF, tumour necrosis factor.

Figure 7

Overexpression of miR181a in MSC-EVs improves the therapeutic efficacy of MSC-EVs in the in vivo model of LPS-induced lung injury. (A) Total protein concentrations in the BALF samples 24 hours after LPS administration (n=6 mice per experimental group, n=1 sham control). (B) Total leucocyte counts (left graph, n=6 mice per experimental group, n=1 sham control) and neutrophil cell counts (n=1–3, right graph) in the BALF samples). (C) Representative images of the BALF cytospin preparations, 24 hours after LPS administration demonstrating cell recruitment to the airspaces. Images were taken using Leica Epifluorescence DM5500 microscope (objective lens original magnification ×20). (D) BALF levels of TNF-α and keratinocyte-derived chemokine (KC, murine analogue of interleukin-8) (n=6 mice per experimental group, n=1 sham control). (E) Representative confocal microscopy of alveolar macrophages, isolated from BALF 24 hours after LPS administration and stained with anti-CD68 and anti- pSTAT5 Ab. Arrows indicate co-localisation of CD68 and pSTAT5. The images were taken using Leica SP8 confocal microscope with a 100× oil-immersion objective (n=3, scale bar=50 µm). Quantitative fluorescence intensity was analysed by Image J software (MFI-pSTAT5). (F) Representative confocal microscopy of alveolar macrophages stained with anti-CD68 and anti-SOCS1 Ab. Arrows indicate co-localisation of CD68 and SOCS1. The images were taken using Leica SP8 confocal microscope with a 100× oil-immersion objective (scale bar=50 µm). Quantitative fluorescence intensity was analysed by Image J software (MFI-SOCS1). Data are represented as mean±SD. Kruskal-Wallis test with post-hoc Dunn’s test (A, B, D, E, F). Ab, antibody; BALF, bronchoalveolar lavage fluid; DAPI, 4',6-diamidino-2-phenylindol; EVs, extracellular vesicles; LPS, lipopolysaccharide; MFI, mean fluorescent intensity; MSCs, mesenchymal stromal cells; PBS, phosphate-buffered saline; pSTAT, phosphorylated STAT; SOCS1, suppressor of cytokine signalling 1; STAT, signal transducers and activators of transcription ;TNF, tumour necrosis factor.

Figure 8

MSC EVs transfer miR-181a which modulates macrophage intracellular signalling through PTEN-pSTAT5-SOCS1 axis. Left: Stimulation of macrophage with inflammatory stimuli (LPS or ARDS plasma) results in the upregulation of secretion levels of pro-inflammatory cytokines TNF-α and IL-8. Right: miR181a packaged in MSC-EVs reprogrammes macrophage towards anti-inflammatory state through downregulation of PTEN and enhancement of pSTAT5/SOCS1 expression resulting in reduced levels of TNF-α and IL-8 production. ARDS, acute respiratory distress syndrome; EVs, extracellular vesicles; IL, interleukin; MSCs, mesenchymal stromal cells; PTEN, phosphatase and tensin homolog; pSTAT, phosphorylated STAT; SOCS1, suppressor of cytokine signalling 1; STAT, signal transducers and activators of transcription; TNF, tumour necrosis factor.