Exploring the mechanism of bone marrow mesenchymal stromal cell exosomes in respiratory syncytial virus infection based on miRNA sequencing

Abstract

The role of Bone marrow mesenchymal stem cells (BMSCs) and their exosomes in regulating the host response to viral infections has garnered significant attention, yet research on their specific mechanisms in response to respiratory syncytial virus (RSV) infection remains limited. This study analyzes changes in cytokine levels and exosomal miRNA expression profiles in BMSCs supernatants following RSV infection. The findings reveal that RSV infection leads to a significant decrease in IL-4 levels in BMSCs supernatants, alongside notable increases in IL-6, IL-12, and IFN-γ levels. Additionally, expressions of RSV F protein, G protein, and N gene were detected in the exosomes. Further in vivo experiments demonstrated that exosomes from RSV-treated BMSCs significantly enhanced the inflammatory response in RSV-infected mice, indicated by elevated serum inflammatory cytokines, lung dysfunction, airway inflammation, and increased mucus secretion. In contrast, exosomes from untreated BMSCs showed minimal effects on airway inflammation and damage in infected mice. miRNA sequencing analysis of the exosomes identified differential miRNAs enriched in multiple key signaling pathways, suggesting that RSV infection alters the functional characteristics of BMSCs exosomes, shifting their role from anti-inflammatory and repair mechanisms to a pro-inflammatory function. This transformation may be mediated by changes in the miRNA expression profile.

Keywords: Exosomes; High-throughput sequencing; Mesenchymal stem cells; Respiratory syncytial virus infection; miRNA.

Fig. 1

Identification of BMSCs and their exosomes. (A) Expression of surface markers in BMSCs; more than 95% of the cells were negative for CD34, and 90% of the cells were positive for CD90 and CD44, consistent with the characteristics of BMSCs. (B) Photo of exosomes for TEM, shown as classic tea holder; scale bar is 200 nm. The particle size range of both groups was within 150 nm. (C) The expression of exosomal marker proteins in BMSCs-Exo-RSV and BMSCs-Exo was consistent with that of exosome surface proteins, and both CD81 and CD63 proteins were positive. Negative for Calnexin.

Fig. 2

Effect of RSV on supernatant and exosomes of BMSCs in vitro. (A) BMSCs supernatant concentration of interleukin (IL)-4, IL-6, IL-12, and interferonγ (IFN-γ). Control is the supernatant of blank BMSCs and Model is the supernatant of BMSCs after RSV intervention. (B) Expression of F protein, G protein, and N genes in exosomes. The internal reference gene was M-GAPDH, and the sequence and expression are shown in the supplementary file.

Fig. 3

Detection of differential let-7i-5p and let-7a-5p expression in exosomes and tracheal tissues by RT-PCR, **P < 0.01, *P < 0.05.

Fig. 4

GO enrichment analysis of differential genes between BMSCs-Exo-RSV and BMSCs-Exo. (The Y-axis in the graph is the negative logarithm of p-value, and the higher the height of the bar graph, the smaller the corresponding p-value. Different colour distributions correspond to BP, CC, and MF.)

Fig. 5

Top 30 most significantly up-regulated enriched KEGG signalling pathways between BMSCs-Exo-RSV and BMSCs-Exo. (KEGG bubble chart is depicted, where the X-axis represents the enrichment degree, and the Y-axis denotes the enriched pathways. Larger dots on the graph indicate a higher number of genes in each pathway, with the colour of the bubbles transitioning from purple to blue, green, and finally red. A smaller enrichment p-value indicates greater significance.)

Fig. 6

Impact of exosome nasal drop on lung function, serum inflammatory factor expression, airway inflammation infiltration, and collagen deposition in mice. (A) The values of lung function indicators CDYN, PEF, FVC, and RL in each group of mice. (B) The concentration of serum IL-4, IL-6, IL-12, and IFN-γ in each group of mice. (C) HE staining and (D) Masson’s trichrome staining of each group of mice, all groups were observed at 200 × . N = 6, **P < 0.01, *P < 0.05.