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

Figure 2.

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.