Targeting ANGPTL4 improves Treg/Th17 cell imbalance and alleviates inflammation in allergic rhinitis by suppressing the Notch signaling pathway

Figures

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  Fig. 1. Presence of Th17–Treg imbalance in OVA-induced AR in mice. C57BL/6 mice were subjected to OVA challenge for 4 weeks to generate AR models. A, B Number of nasal rubbing and sneezing events within 15 min. C AR scores in mice evaluated using behavioral scoring. D HE staining to assess pathological changes in the nasal mucosa. E PAS staining to analyze mucus production in the nasal mucosa. F Serum levels of IL-10, IL-17, and TGFB1 levels in mice determined using ELISA kits. G, H Populations of Treg (CD4+CD25+Foxp3+) and Th17 (CD4+CD25+IL17+) cells in the lymph nodes of mice determined using FACS. Each group contained 6 mice. Data are shown in dot-and-whisker format. Differences were compared by unpaired t test (A–C, D–H) or two-way ANOVA followed by Tukey’s post hoc examination (F). **p < 0.01, ****p < 0.0001

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  Fig. 2. Notch pathway is activated in OVA-challenged mice and associated with the Th17–Treg imbalance. A, B qPCR and WB assays to detect the mRNA and protein levels of NOTCH1, the ligand JAGGED1, and the downstream product HES1 in the nasal mucosa of mice with or without OVA challenge. C, D Number of nasal rubbing and sneezing events within 15 min. E AR scores in mice evaluated using behavioral scoring. F Serum levels of IL-10, IL-17, and TGFB1 levels in mice determined using ELISA kits. G, H Populations of Treg (CD4+CD25+Foxp3+) and Th17 (CD4+CD25+IL17+) cells in the lymph nodes of mice determined using FACS. Each group contained 6 mice. Data are shown in dot-and-whisker format. Differences were compared by unpaired t test (C-E, G-H) or two-way ANOVA followed by Tukey’s post hoc examination (A, B, F). ***p < 0.001, ****p < 0.0001

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  Fig. 3. ANGPTL4 is markedly upregulated in OVA-challenged mice. A, B Volcano plots (A) and heatmap (B) for differentially expressed genes in the nasal mucosa of AR and sham-operated mice determined using RNA-seq analysis. C mRNA expression of the top 10 differentially expressed RNAs determined using qPCR analysis. D Protein level of ANGPTL4 in the nasal mucosa of mice determined using WB analysis. Two shRNAs (shRNA-#1 and shRNA#2) targeting ANGPTL4 were administered to the mice from the 8th day after AR modeling. E, F mRNA (E) and protein (F) levels of ANGPTL4 in the nasal mucosa of mice after shRNA administration determined using qPCR and WB analyses, respectively. Each group contained 6 mice. Data are shown in dot-and-whisker format. Differences were compared by unpaired t test (D), or by the one-way (E, F) or two-way (C) ANOVA followed by Tukey’s post hoc examination. ***p < 0.001, ****p < 0.0001

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  Fig. 4. ANGPTL4 silencing relieves AR-like symptoms of OVA-induced mice. OVA-challenged mice were administered two shRNAs targeting ANGPTL4 from day 8 after AR modeling. A, B Number of nasal rubbing and sneezing events within 15 min. C AR scores in mice evaluated using behavioral scoring. D HE staining to assess pathological changes in the nasal mucosa. E PAS staining to analyze mucus production in the nasal mucosa. F Serum levels of IL-10, IL-17, and TGFB1 levels in mice determined using ELISA kits. Data are shown in dot-and-whisker format. Differences were compared by one-way ANOVA followed by Tukey’s post hoc examination (A–E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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  Fig. 5. ANGPTL4 silencing reduces OVA-induced inflammatory responses. A, B ELISA analysis of IL-10, IL-17, and TGF-β1 levels in serum and NALF of AR mice after ANGPTL4 silencing. C FACS analysis of the numbers of neutrophils, eosinophils, lymphocytes, and macrophages in the NALF of AR mice after ANGPTL4 silencing. D, E ELISA analysis of IgE, IgG1, and IgG2a levels in serum and NALF of mice. F, G IgG2a/IgG1 ratio in the serum or NALF of mice. Each group contained 6 mice, and data are presented as dot and whiskers. Differences were compared by the one-way (F, G) or two-way (A–E) ANOVA followed by Tukey’s post hoc examination. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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  Fig. 6. ANGPTL4 silencing improves Th17–Treg balance in AR mice. A, B Populations of Treg (CD4+CD25+Foxp3+) and Th17 (CD4+CD25+IL17+) cells in the lymph nodes of mice determined using FACS. C, D Populations of Treg (CD4+CD25+Foxp3+) and Th17 (CD4+CD25+IL17+) cells in the NALF of mice determined using FACS. E, F mRNA and protein levels of Foxp3, IL10, ROR-γt, and IL17 in the nasal mucosa of mice determined using qPCR and WB assays. Each group contained 6 mice, and data are presented as dot and whiskers. Differences were compared by the one-way (A–D) or two-way (E, F) ANOVA followed by Tukey’s post-hoc examination. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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  Fig. 7. ANGPTL4 silencing improves Treg/Th17 balance by inactivating the Notch signaling pathway. A, B qPCR and WB assays to detect the mRNA and protein levels of NOTCH1, JAGGED1, and HES1 in the nasal mucosa of mice after ANGPTL4 silencing. AR mice treated with the ANGPTL4 shRNA-2# were additionally treated with the Notch signaling activator VA. C, D Number of nasal rubbing and sneezing events within 15 min. E AR scores in mice evaluated using behavioral scoring. F, G Concentrations of IL-10, IL-17, and TGFB1 levels in the serum and NALF of mice determined using ELISA kits. H, I Populations of Treg (CD4+CD25+Foxp3+) and Th17 (CD4+CD25+IL17+) cells in the lymph nodes or NALF of mice determined using FACS. Each group contained 6 mice. Data are shown in dot-and-whisker format. Differences were compared by unpaired t test (C–E, H, I) or two-way ANOVA followed by Tukey’s post hoc examination (A, B, F, G). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001