Extracellular Vesicles Derived from Allergen Immunotherapy-Treated Mice Suppressed IL-5 Production from Group 2 Innate Lymphoid Cells

Abstract

Allergen immunotherapy (AIT), such as subcutaneous immunotherapy (SCIT), is a treatment targeting the causes of allergic diseases. The roles of extracellular vesicles (EVs), bilayer lipid membrane blebs released from all types of cells, in AIT have not been clarified. To examine the roles of EVs in SCIT, it was analyzed whether (1) EVs are phenotypically changed by treatment with SCIT, and (2) EVs derived from SCIT treatment suppress the function of group 2 innate lymphoid cells (ILC2s), which are major cells contributing to type 2 allergic inflammation. As a result, (1) expression of CD9, a canonical EV marker, was highly up-regulated by SCIT in a murine model of asthma; and (2) IL-5 production from ILC2s in vitro was significantly decreased by the addition of serum EVs derived from SCIT-treated but not non-SCIT-treated mice. In conclusion, it was indicated that EVs were transformed by SCIT, changing to a suppressive phenotype of type 2 allergic inflammation.

Keywords: airway hyperresponsiveness; allergy; asthma; extracellular vesicle; group 2 innate lymphoid cell; interleukin-5; subcutaneous immunotherapy.

Figure 1

Graphical abstract of this study. Extracellular vesicles (EVs) in the bloodstream were transformed by subcutaneous immunotherapy (SCIT), acquiring ability to inhibit interleukin 5 (IL-5) production from group 2 innate lymphoid cells (ILC2s).

Figure 2

Effects of subcutaneous immunotherapy (SCIT) on airway hyperresponsiveness (A) and an amount of interleukin 5 (IL-5) in bronchoalveolar lavage fluid (BALF) (B) of ovalbumin (OVA)-induced asthmatic mice. BALB/c mice were sensitized with intraperitoneal (i.p.) OVA + Al(OH)₃ on days 0 and 14, followed by intratracheal OVA challenges on days 35, 36, 37, and 40 at a dose of 5 μg/animal. SCIT treatment was applied by subcutaneous injection of OVA (1 mg/animal) on days 21, 23, and 25. Twenty-four hours after the 4th OVA challenge (day 41), the development of airway hyperresponsiveness (AHR) was analyzed. Respiratory compliance (Crs) is shown as the maximum value after each methacholine challenge. Following analysis of AHR, bronchoalveolar lavage was conducted. IL-5 concentration in BALF was quantified by ELISA. Each point and column represent the mean ± SEM of 4 or 5 animals. **: p < 0.01 and ***: p < 0.001 versus only sensitization. †: p < 0.05 and ††: p < 0.01 versus sensitization + challenges.

Figure 3

Particle sizes (A), and expression levels of CD9 (B) and CD63 (C) of extracellular vesicles (EVs) derived from the non-subcutaneous immunotherapy (SCIT)-treated and SCIT-treated asthmatic mice. Twenty-four hours after the 4th challenge, sera were collected. EVs were obtained from sera by ExoQuick. The particle sizes of EVs were detected using dynamic light scattering. The expression levels of CD9 and CD63 were analyzed by flow cytometry.

Figure 4

Effects of extracellular vesicles (EVs) derived from sera of the non-subcutaneous immunotherapy (SCIT)-treated or SCIT-treated asthmatic mice on interleukin 5 (IL-5) production from group 2 innate lymphoid cells (ILC2s). All lung lobes were collected 24 h after the 4th ovalbumin (OVA) challenge. ILC2s in the dispersed lung tissue were detected and sorted as lineage⁻ CD45⁺ CD278⁺ CD90.2⁺ ST2⁺ cells by flow cytometry. The sorted ILC2s were cultured for 72 h in the presence of IL-33 and TSLP with or without EVs. After the culture, cell culture supernatants were collected, followed by measurement of IL-5 by ELISA. Each column represents the mean ± SEM of ratio to the control (IL-33/TSLP—stimulated, without EVs) of 5 experiments. *: p < 0.05 and ***: p < 0.001.

Figure 5

Schedule for sensitization, subcutaneous immunotherapy (SCIT), and challenges. Mice were sensitized by intraperitoneal (i.p.) injection with ovalbumin (OVA) + Al(OH)₃ on days 0 and 14. Sensitized mice were treated with subcutaneous (s.c.) injection of OVA at a dose of 1 mg/animal on days 21, 23, and 25. The mice were challenged by intratracheal (i.t.) administration of OVA at a dose of 5 μg/animal on days 35, 36, 37, and 40 under inhalation anesthesia with isoflurane.