Extracellular Vesicles Induce Nuclear Factor-κB Activation and Interleukin-8 Synthesis through miRNA-191-5p Contributing to Inflammatory Processes: Potential Implications in the Pathogenesis of Chronic Obstructive Pulmonary Disease

Abstract

Extracellular vesicles (EVs) play a pivotal role in a variety of physiologically relevant processes, including lung inflammation. Recent attention has been directed toward EV-derived microRNAs (miRNAs), such as miR-191-5p, particularly in the context of inflammation. Here, we investigated the impact of miR-191-5p-enriched EVs on the activation of NF-κB and the expression of molecules associated with inflammation such as interleukin-8 (IL-8). To this aim, cells of bronchial epithelial origin, 16HBE, were transfected with miR-191-5p mimic and inhibitor and subsequently subjected to stimulations to generate EVs. Then, bronchial epithelial cells were exposed to the obtained EVs to evaluate the activation of NF-κB and IL-8 levels. Additionally, we conducted a preliminary investigation to analyze the expression profiles of miR-191-5p in EVs isolated from the plasma of patients diagnosed with chronic obstructive pulmonary disease (COPD). Our initial findings revealed two significant observations. First, the exposure of bronchial epithelial cells to miR-191-5p-enriched EVs activated the NF-kB signaling and increased the synthesis of IL-8. Second, we discovered the presence of miR-191-5p in peripheral blood-derived EVs from COPD patients and noted a correlation between miR-191-5p levels and inflammatory and functional parameters. Collectively, these data corroborate and further expand the proinflammatory role of EVs, with a specific emphasis on miR-191-5p as a key cargo involved in this process. Consequently, we propose a model in which miR-191-5p, carried by EVs, plays a role in airway inflammation and may contribute to the pathogenesis of COPD.

Keywords: DLCO; chronic obstructive pulmonary disease; extracellular vesicles; inflammation; interleukin-6; interleukin-8; microRNA-191; nuclear factor-κB.

Figure 1

Graphical overview of the experimental design. Bronchial epithelial cells (16HBE) were transfected with miR-191-5p mimic or inhibitor. Subsequently, supernatants were collected for EV isolation and miR-191-5p quantification. Additionally, cells were harvested for miRNA extraction. The bronchial epithelial cells were then exposed to EVs (EV_m: EV derived from 16HBE transfected with miR-191-5p mimic; EV_i: EV derived from 16HBE transfected with miR-191-5p inhibitor; EV_c: EV derived from 16HBE transfected with miRNA scramble; EV_nt: EV derived from 16HBE not transfected) and subsequently collected for miR-191-5p quantification, NF-kκb activation and IL-8 evaluation. The figure was created through “biorender.com” website license agreement number EX2679WEP4, access date 11 December 2024.

Figure 2

Expression of miR-191-5p in 16HBE cells (A) and in EVs (B) after transfection with miRNA mimic of miR-191-5p (mimic-miR-191-5p) and miR-191-5p inhibitor (inhibitor -miR-191-5p). The mimic scramble was used as control (Ctrl) in each experiment. Non-transfected cells represent the not-treated group (Untreated). Expression of miR-191-5p in 16HBE cells (C) after incubation with EVs shed from bronchial epithelial cells transfected with mimic-miR-191-5p and inhibitor-miR-191-5p (EV_m: EV derived from 16HBE transfected with miR-191-5p mimic; EV_i: EV derived from 16HBE transfected with miR-191-5p inhibitor; EV_c: EV derived from 16HBE transfected with miRNA scramble; EV_nt: EV derived from 16HBE not transfected). Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s test selected data (* p < 0.05 vs. Ctrl).

Figure 3

Modulation of NF-κB and IL-8 by miR-191-5p-enirched EVs. (A) Activation of NF-κB in 16HBE cells exposed to EV derived from Ach-stimulated 16HBE cells treated as described in Figure 3A (EV_m: EV derived from 16HBE transfected with miR-191-5p mimic; EV_i: EV derived from 16HBE transfected with miR-191-5p inhibitor; EV_c: EV derived from 16HBE transfected with miRNA scramble; EV_nt: EV derived from 16HBE not transfected). Then, nuclear proteins were extracted and assessed by ELISA to measure the DNA-binding activity of the p65 NF-κB subunit. (B) IL-8 secretion by 16HBE cells incubated with EVs derived from Ach-stimulated 16HBE cells, treated as described in Figure 3A. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test (*** p < 0.001 vs. 16HBE + EV_c).

Figure 4

Correlation of EV-derived miR-191-5p and clinical parameters. Correlation analysis was performed to assess the relationships between EV-derived miR-191-5p levels and specific clinical parameters, including IL-6 (A) and DLCO (B). For technical reasons, IL-6 level was not available in one patient. The analysis was conducted using simple linear regression analysis.

Figure 5

Predictive-network-based analysis involving the activation of NF-κB (A), IL-6 (B), and IL-8 (C). The modulation of the three targets predicted a positive correlation with miR-191-5p, as well as with other nodes related to disease, including IL-1, CRP, and TNF-α, among others. The bioinformatic survey pinpointed a signaling pathway consistent with expression results and reconstructed the molecular neighborhood around targets with several connection nodes that are valuable for a more comprehensive evaluation of the role of miR-191-5p in the pathogenesis of COPD.

Figure 6

Overview of the study main findings. The figure was created through “biorender.com” website license agreement number HP2679WJTA, access date 11 December 2024.