Increased miR-124-3p alleviates type 2 inflammatory response in allergic rhinitis via IL-4Rα

Abstract

Background and objectives: miRNAs play a crucial role in regulating immune responses. However, the effect of miR-124-3p on type 2 inflammation in allergic rhinitis (AR) is unclear. We aimed to study the immune regulation of miR-124-3p in AR and the mechanisms involved.

Methods: The direct interaction between miR-124-3p and IL-4Rα was confirmed through a dual-luciferase reporter assay. In vitro splenic lymphocytes from mice and peripheral blood mononuclear cells (PBMCs) from healthy individuals were cultured and treated with miR-124-3p mimic/inhibitor. Twenty-four female C57BL/C mice were divided into four groups: control, AR model, miR-124-3p agomir, and miR-124-3p antagomir groups (n = 6 per group). The allergic responses were evaluated based on the number of sneezing and nasal scratching, the serum HDM-specific IgE (sIgE) levels, and the degree of nasal mucosa eosinophil infiltration. The expression of IL-4Rα, p-STAT6, and type 2 inflammatory cytokines (IL-4, IL-5 and IL-13) in lymphocytes or nasal mucosa was determined by qPCR, western blotting, flow cytometry, immunohistochemistry and immunofluorescence.

Results: miR-124-3p directly targets the 3'UTR of IL-4Rα. The miR-124-3p mimic lowered the IL-4Rα, p-STAT6, IL-4, IL-5, and IL-13 expression levels in both mouse splenic lymphocytes and human PBMCs in vitro, and the miR-124-3p inhibitor rescued these changes. Furthermore, the miR-124-3p agomir decreased the levels of IL-4Rα and IL-4 in nasal mucosa, Th2 differentiation in spleen, and allergic response in AR mice. Moreover, the miR-124-3p antagonist increased the IL-4Rα and IL-4 levels and further aggravated the allergic responses.

Conclusions: miR-124-3p might attenuate type 2 inflammation in AR by regulating IL-4Rα signaling, and miR-124-3p may be a promising new target in AR treatment.

Keywords: Allergic rhinitis; IL-4Rα; Inflammation; STAT6; miR-124-3p.

Fig. 1

Schematic for the establishment of AR mice and treatment schedule

Fig. 2

miR-124-3p directly interacts with IL-4Rα by binding to its 3′-UTR. a Predicted binding sequences and mutated sequences between miR-124-3p and IL-4Rα. b Relative luciferase activity after cotransfection of mmu-miR-124-3p mimics/controls and the IL-4Rα wild/mutant type (WT/Mut) reporter vector (n = 3). ***p < 0.001

Fig. 3

miR-124-3p decreases the mRNA and protein expression levels of IL-4Rα and type 2 cytokines in vitro (n = 3). The effect of miR-124-3p mimic and inhibitor on mRNA expression of a IL-4Rα, b IL-4, c IL-5 and d IL-13 splenic lymphocytes determined by quantitative RT-PCR. e–g Effect of miR-124-3p mimic on protein expression of IL-4Rα and p-STAT6. e Representative images and histogram of f IL-4Rα and g p-STAT6 after treatment with miR-124-3p mimic. h–j Effect of miR-124-3p inhibitor on protein expression of IL-4Rα and p-STAT6. h Representative images and histogram of i IL-4Rα and j p-STAT6 after treatment with miR-124-3p mimic. k–n Effect of miR-124-3p mimic on protein expression of IL-4, IL-5 and IL-13. k Representative images and histogram of l IL-4, m IL-5 and n IL-13 after treatment with miR-124-3p mimic. o–r Effect of miR-124-3p inhibitor on protein expression of IL-4, IL-5 and IL-13. o Representative images and histogram of p IL-4, q IL-5 and r IL-13 after treatment with miR-124-3p mimic. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 4

miR-124-3p reduces the mRNA and protein expression of IL-4Rα and type 2 cytokines in human PBMCs (n = 3). The effect of miR-124-3p mimic and inhibitor on mRNA expression of a IL-4Rα, b IL-4, c IL-5 and d IL-13 in lymphocytes determined by quantitative RT-PCR. e–g Effect of miR-124-3p mimic on protein expression of IL-4Rα and p-STAT6. e Representative images and histogram of f IL-4Rα and g p-STAT6 after treatment with miR-124-3p mimic. h–j Effect of miR-124-3p inhibitor on protein expression of IL-4Rα and p-STAT6. h Representative images and histogram of i IL-4Rα and j p-STAT6 after treatment with miR-124-3p mimic. k–n Effect of miR-124-3p mimic on protein expression of IL-4, IL-5 and IL-13. k Representative images and histogram of l IL-4, m IL-5 and n IL-13after treatment with miR-124-3p mimic. o–r Effect of miR-124-3p inhibitor on protein expression of IL-4, IL-5 and IL-13. o Representative images and histogram of p IL-4, q IL-5 and r IL-13 after treatment with miR-124-3p mimic. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 5

miR-124-3p decreases IL-4Rα and IL-4 cytokine expression in the nasal mucosa. a Representative images of IL-4Rα (green) expression in the nasal mucosa obtained by immunofluorescence labeling (×400). DAPI (blue), represents nuclear staining. The scale bar represents 10 μm. b Histogram of the percentage of IL-4Rα positive cells in nasal mucosa tissue sections detected by immunofluorescence staining (n = 3). c IL-4Rα mRNA expression in the nasal mucosa detected by quantitative RT-PCR (n = 5). d Representative images of IL-4 expression in the nasal mucosa obtained by immunohistochemical staining (×400). The scale bar represents 20 μm. e Histogram of the number of IL-4 positive cells in nasal mucosa tissue sections detected by immunohistochemistry (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 6

miR-124-3p alleviates Th2 cell differentiation of splenic lymphocytes in vivo (n = 3). a–c Gating strategy for Th1 and Th2 cells. a Gating for lymphocytes. b Gate for CD4⁺ T cells. c Representative dot plots of the percentages of Th1 and Th2 cells. d Percentage of Th2 cells. e Percentage of Th1 cells. f Ratio of Th2 to Th1 subsets. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 7

miR-124-3p protects against HDM-induced allergic response in allergic rhinitis mice. A, B Number of (a) sneezing and (b) nasal scratching after treatment with miR-124-3p agomir/antagomir. (c) HDM-specific IgE levels in serum. (d) Eosinophil counts detected in H&E-stained nasal mucosa tissue sections (200× magnification). (e) Representative H&E staining of the nasal mucosa (400×). The yellow arrows indicate eosinophils, the green arrows indicate mucus glands, and the black arrows indicate goblet cells. The scale bar represents 20 μm. HDM, house dust mite. NS, control group challenged with normal saline (n = 6). AR, allergic rhinitis group challenged with HDM (n = 6). Agomir, AR group treated with miR-124-3p agomir (n = 6). Antagomir, AR group treated with miR-124-3p antagomir (n = 6). Each dot represents one mouse. *p < 0.05, **p < 0.01 and ***p < 0.001