The role of extracellular vesicles in murine asthma model: insights into IgE-independent mast cell activation within animal science

Abstract

Asthma, a prevalent respiratory condition in animal science, involves bronchial inflammation, leading to symptoms like coughing and difficulty breathing. Mast cells and macrophages, pivotal immune cells, contribute to the inflammatory response triggered by various factors. Extracellular vesicles (EVs), including exosomes, play crucial roles in intercellular communication and have been implicated in murine asthma. We hypothesize that murine asthma-derived EVs modulate immune cell responses in animals. Our study investigates the role of these EVs in immune cell activation, shedding light on asthma pathogenesis. Using a murine asthma model induced by ovalbumin (OVA), we assessed serum EVs via Nanoparticle Tracking Analysis (NTA). Serum EVs from normal and asthmatic mice were subjected to Enzyme-linked immunosorbent assay (ELISA) and proteomic analysis for component identification. Elevated EV concentration and inflammatory cytokines, such as Interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α, were found in asthma-derived EVs. Additionally, variations in immunoglobulin light chain types were identified. Surprisingly, murine asthma EVs failed to activate T lymphocytes, B lymphocytes, eosinophils, and macrophages but stimulated mouse bone marrow-derived mast cells (mBMMCs) via enhanced degranulation and MAP kinase phosphorylation. These results suggest murine asthma-derived EVs as potential therapeutic targets for managing asthmatic symptoms in animal science. Further research into their mechanisms and clinical applications is warranted.

Keywords: Asthma; Extracellular vesicles; MAP kinase phosphorylation; Mast cells.

Fig. 1.. Characterization of asthma serum-derived EVs.

(A) The timeline for murine asthma model. (B) Histologic analysis of lung tissues in normal and asthma model-induced group. (C) Population of macrophage and eosinophil in lung tissue. (D) Serum EV concentrations (n = 10) and (E) sizes (n = 10) were determined by using NTA. Magnifications are 100× or 200×. Student t-tests were used to compare differences between groups. Data are mean ± SD. ^***p < 0.001. EVs, extracellular vesicles.

Fig. 2.. The increase of exosome marker proteins and inflammatory cytokines in asthma-derived EVs.

(A) Representative images of normal/asthma-derived EVs were shown by using Bio-TEM analysis. (B) Exosome marker proteins, CD63, TSG101, CD9, CD81, and ALIX were expressed by using the immunoblot analysis. (n=3) (C) The levels of pro-inflammatory cytokines IL-6, IL-8, and TNF-α were measured by ELISA. Student t-tests were used to compare differences between groups. Data are mean ± SD. ^**p < 0.01, ^***p < 0.001. EVs, extracellular vesicles.

Fig. 3.. Proteomics results of normal EVs and asthma EVs.

(A) The diagram illustrating the number of proteins analyzed in normal EVs and asthma EVs. (B) Proteins that are only present in the asthma EVs group are listed in the table, with immunoglobulins represented in red. (C) The levels of various kappa free light chain and lambda free light chain were determined by ELISA (n = 6). Student t-tests were used to compare differences between groups. Data are mean ± SD. ^**p < 0.01, ^***p < 0.001. EVs, extracellular vesicles.

Fig. 4.. Immune cell activation by asthma-derived EVs.

(A) The population of T cells, B cells, and eosinophils were quantified using flow cytometric analysis (n = 3). (B) The relative expression level of M1 macrophage marker genes, IL-1β and iNOS, and M2 macrophage marker genes, Arginase 1 and Retnla, was measured by real-time qPCR (n = 3). (C and D) Representative immunoblotting images and quantitative graph of phospho-JNK and -p38 (n = 3) in bone marrow-derived macrophages treated by normal EVs and asthma EVs. One-way ANOVA tests were used to compare differences between groups. Data are mean ± SD. ^***p < 0.001. PBS, phosphate-buffered saline; EVs, extracellular vesicles; LPS, lipopolysaccharide; qPCR, real-time quantitative polymerase chain reaction.

Fig. 5.. Mast cell activation by asthma-derived EVs.

Bone marrow-derived mast cells were stimulated by normal EVs and asthma EVs. (A) The percentage of β-hexosaminidase was determined as the ratio of released β-hexosaminidase to total β-hexosaminidase (n = 4). Representative immunoblotting (B) images and (C–E) densitometric quantification graph of phospho-Erk, -JNK and -p38 (n = 3). One-way ANOVA tests were used to compare differences between groups. Data are mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. PBS, phosphate-buffered saline; EVs, extracellular vesicles; LPS, lipopolysaccharide.

Fig. 6.. Illustrations showing the mechanisms by which asthma-derived EVs influence the asthmatic environment in airways.

EVs, extracellular vesicles.