Aquaporin-9 Aggravates Lipopolysaccharides Induced Acute Lung Injury Via Facilitating M1-Like Macrophage Polarization
Aquaporin-9 Aggravates Lipopolysaccharides Induced Acute Lung Injury Via Facilitating M1-Like Macrophage Polarization
Jiajia Wan1,2, Chi Zhang3, Xiaodi Li3, Longkang Liu3, Linxiu Peng3,4, Yuejian Liu2, Yang Qiu2, Guokai Wu2, Bo Zhang3*, Yan Wang3* and Tonghui Ma3*
AQP9 expression is up-regulated in AMs after LPS stimulation. (A) Representative PCR amplification analysis of AQP 0-12 in AMs and neutrophils isolated from C57BL/6J mice. (B) mRNA levels of AQP9 in LPS-treated (500 or 1000 ng/mL) AMs and neutrophils were assessed by RT-qPCR (n = 4). (C) Western blot analysis of AQP9 in LPS-treated (500 or 1000 ng/mL) AMs (n = 3). (D) Representative fluorescence images of AQP9 (red) and macrophage marker F4/80 (green) and quantification of AQP9 in LPS-treated (500 ng/mL) AMs isolated from male AQP9-RFP mice (n = 3). Scale bars, 50 μm. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test for two-group comparisons, two-way ANOVA for comparisons with multiple variables.
Effect of AQP9 deletion on LPS-induced ALI and pulmonary inflammation. (A, B, H) AQP9 KO and WT mice were intratracheally instilled with LPS (5 mg/kg), then sacrificed at days 1, 3, and 6 after surgery for functional analysis. (A) Representative images and (B) lung injury score analysis of H&E staining of lung tissues (n = 5). Scale bars, 50 μm. (C-G) Male WT and AQP9 KO mice were sacrificed at day 3 after LPS (5 mg/kg) administration for functional analysis. (C) Pulmonary Wet/Dry weight ratio (n = 3). (D) Quantity of inflammatory cells in BALF (n = 5). (E) Total protein content in BALF (n = 3). (F) Concentration of IL-6 and IL-10 in BALF (n = 3). (G) mRNA levels of IL-1β and TNF-α in lung tissues (n = 3). (H) Representative CD68+ staining and quantitative analysis in cross-sections of lung tissues (n = 3). Scale bars, 20 μm. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA for comparisons with multiple variables.
Effect of AQP9 deletion on M1-like macrophage polarization in vivo and ex vivo. (A) Representative immunofluorescence images of iNOS (green) and CD206 (red) and quantification of iNOS+ areas in the lung tissues of WT and AQP9 KO mice at days 1, 3, and 6 after LPS intratracheal instillation (5 mg/kg) (n = 6). Scale bars, 50 μm. (B-E) AMs isolated from WT and AQP9 KO mice were stimulated with LPS (500 ng/mL) for 24 h. (B) Representative immunofluorescence images and quantification of CD86 (red) and CD206 (green) in AMs (n = 3). Scale bars, 20 μm. (C) mRNA levels of iNOS and CD206 in AMs were assessed by RT-qPCR (n = 3). (D) The levels of IL-6 and IL-10 in the culture media of AMs were determined by ELISA (n = 3). (E) mRNA levels of IL-1β, IL-6 and IL-10 in AMs (n = 3). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA for comparisons with multiple variables.
Effect of AQP9 deletion on the H2O2/NF-κB axis in AMs. AMs isolated from WT and AQP9 KO mice were treated with LPS (500 ng/mL) for 4 h. (A) Western blot analysis of NOX2 in WT AMs (n=3). (B) Intracellular H2O2 content in WT and AQP9 KO AMs (n=3). (C) Extracellular H2O2 levels in the culture media of AMs (n=3). (D) Representative fluorescent images and quantitative analysis of HyPerRed-transduced WT and AQP9 KO AMs cultured with or without LPS or NAC (2.5 mM) for 4 h (n=3). Scale bars, 10 μm. (E) Representative fluorescent images and quantification of HyPerRed-transduced WT and AQP9 KO AMs incubated with 100 μM H2O2 (n=3). Scale bars, 10 μm. (F, G) Western blot analysis of p-IKK, IKK, IκBα, p-p65, and p65 in LPS-treated AMs (n=3). (H, I) Western blot analysis of nuclear extracts prepared from AMs and analyzed for p65 and Histone H3 (n=3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA for comparisons with multiple variables.
