Animal and Ethics Statement
60 Female BALB/c nude mice (6-8 weeks old) were maintained in a light and temperature- controlled room. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Bioduro (China) Co., Ltd under SPF protocol. The ethic number was HRJ-FFS-ON-20200127-01.
Mouse primary cells isolation and culture condition
Mouse primary alveolar epithelial cells (MAE) and AM were isolated from health BALB/C mice，Lung were excised and stored at cold PBS before used. The lung single cell suspensions (SCS) were prepared by Lung Dissociation Kit, mouse (130-095-927, Miltenyi). The AM were selected by CD45 dynabeads(11153D, Thermo) and purified by maintained in RPMI-1640(22400-097, Thermo) with M-CSF (10ng/mL) for two weeks. And MAE were negative selected by CD45 dynabeads. MAE was maintained in DMEM (11965-175, Thermo) medium. 10% fetal bovine serum (16000-044, Gibco) and with 10,000 units penicillin and 10 mg streptomycin/mL (V900929, Sigma-Aldrich) were supplemented into all above medium. The cells were incubated in humidified atmosphere containing 5% CO2 at 37℃.
SWI-ALI models were established via intratracheal instillation 100 mL seawater to each mouse. The seawater was configured by added sea salt (S9883, Sigma-Aldrich) into sterile ddH2O. Control group were received intratracheal instillation PBS with the same volume. Five mice were sacrificed at D1, D3, D7 and D14.
Serum cytokine detection
Serum sample were exacted from jaws vein of mice. The serum sample were stored at -80 ℃ before used. IL-17a detection kit were purchased from Biolegend (432504). ELISA procedure were tested as standard protocol. Each sample were tested three replicates.
SWI-ALI in vitro model
SWI-ALI in vitro model was established by MAE and AM co-culture. 20000 MAE were seeded into each well and allowed to growth over night. The wound was created in the center of each well using a scratcher. AM were added into macrophage group and macrophage+IL-17a group after wound made at 1:10 ratio of effect: target (E:T). IL-17a（10ng/mL）were added into IL-17a group and macrophage+IL-17a group and control group. The wounds were photographed and measured every three hours for two days. After two days the supernatants were collected and detected by fluorescence-activated cell sorting (FACS).
Macrophage polarization detection
The SCS from the lung of SWI-ALI model at D1, D3, D7, D14 and supernatants form in vitro SWI-ALI model were detected by BD Celesta. M1 were defined by F4/80+ (565411, BD) and Ly6C+ (560593, BD).
Statistical significant analysis of the data was using the analysis of variance and the Student’s t-test. And all data were presented as the mean ± standard deviation (SD). ELISA was calculated by 4 parameters logistic regression. The tests were conducted by using the GraphPad Prism 7.0 software (GraphPad software, USA).