Reagents
PDX was purchased from Cayman (Ann Arbor, MI, USA). Ly6G antibody was purchased from BD Biosciences (New Jersey, USA). LPS (Escherichia coli 055: B5) and antibodies against phosphor-47phox-ser345 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibody against phospho-38, phospho-ERK1/2, phospho-NF-κB p65 were both purchased from CST (Cell Signaling Technology MA, USA). GAPDH antibody, second antibodies and other reagents were purchased from Antgene Biotechnology (Wuhan, China).
Animals
All animals used in this experiment is male C57BL/6 mice, with a basic state of 6-8 weeks and 20-25g, provided by Beijing Vital River Laboratory Animal Technology and maintained in animal center which located in Huazhong University of Science and Technology. All animals were raised in the specific pathogen free (SPF) environment with food and water provided ad libitum following a strict 12h light-dark cycle, where the temperature should be controlled at 22-24°C and the humidity at 60-65%. All animal experiments met the requirements of the National Institutes of Health (NIH) guidelines and obtained ethical approval from the Laboratory Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China).
Induction of sepsis mouse model and PDX administration
The septic model was made by referring to the previous methods (Rittirsch et al., 2009). Mice were anesthetized by intraperitoneal injection of xylazine (8 mg/kg) and ketamine (120 mg/kg). Then, a longitudinal incision was performed in abdomen to show the cecum. Puncture the cecum with a 20-gauge needle and squeeze out a drop of feces. The cecum in sham group was showed intactly. Finally, the cecum was retracted into the abdominal cavity and the incision was sewn. After surgery, mice were subcutaneously injected with 1ml saline (37° C) to resuscitate. 500ng or 1000ng PDX diluting to 100ul of phosphate buffered saline (PBS) was administered intraperitoneally at 1h after surgery.
Neutrophil isolation and culture
The Clinical Research Committee of Union Hospital approved the human experimental protocol. Neutrophils were isolated from healthy donors who have signed inform using percoll density gradient centrifugation (Kjeldsen et al., 1994). 1×106 cells were cultivated in10% FBS containing RPMI 1640 medium. Isolated neutrophils were cultured in standard condition (37℃, 5% CO2 ).
Experimental design
The mice were divided into the following four groups (7 in each) using random number: (1) Sham group: mice received all surgical operations without ligating and piercing cecum; (2) CLP group: 100ul of 0.038% PBS was injected intraperitoneally at 1h after sepsis model of mice was established; (3) CLP+PDX 500ng group: 500ng PDX dissolved in 100ul PBS was injected intraperitoneally 1h after CLP; (4) CLP+PDX 1000ng group: 1000ng PDX dissolved in 100ul PBS was injected intraperitoneally 1h after CLP.
Neutrophils were divided into the following five groups: (1) Control group: cells were added with 0.038% PBS (PDX solvent); (2) LPS group: RMPI 1640 medium contained 1ug/ml LPS; (3) PDX 10nM group: cells were treated with 1ug/ml LPS for an hour and the final concentration of PDX was 10nM. (4) PDX 100nM group: cells were treated with 1ug/ml LPS for an hour and the final concentration of PDX was 100nM. (5) PDX 1000nM group: cells were treated with 1ug/ml LPS for an hour and the final concentration of PDX was 1000nM. At least 3 wells have be set in different treatment groups.
Survival rate analysis and PaO2 assessment
Survival information was observed and recorded for 8 days after the operation. Mice were anesthetized by intraperitoneal injection of xylazine (8 mg/kg) and ketamine (120 mg/kg). After the mice were fixed in supine position, the neck hair was scraped off and fully disinfected. Dissected and exposed the right common carotid artery, and 0.1-0.2ml of arterial blood was extracted which was immediately detected by blood gas machine.
Histological analysis of lung tissues
The left lung was fixed with 10% paraformaldehyde and embedded in paraffin. Then, the embedded lung tissue was cut into 4µm sections and subjected to hematoxylin-eosin (HE) staining. The sections were
observed under the microscope and lung injury scores were evaluated according to American Thoracic Society (Matute-Bello et al., 2011).
Leukocyte counts and evaluation of pulmonary oedema
The right lung was rinsed three times with 0.5 ml of phosphate-buffered saline (PBS). Broncho alveolar lavage fluid (BALF) was collected and centrifuged at 12000 rpm for 5 min at 4°C using a centrifuge (Sigma, USA). We measured the protein concentration of the supernatant using the BCA assay kit (Thermo Scientific). The precipitate was resuspended and the cell numbers were counted by a hematocytometer. We counted 300 cells under a light microscope for analysis. The different leukocyte subgroups were identified by Wright-Giemsa staining.
Cytokine detection
The expression levels of different inflammatory cytokines (TNF-α, IL-6, IL-1β, and MIP-2) in the supernatant of BALF were detected by ELISA kits (RayBiotech, Inc. Norcross, GA, USA) following the instructions.
SOD, ROS, MPO and MDA activity assays
The superoxide dismutase (SOD), ROS and methylene dioxy amphetamine (MDA) activities were detected using specific assay kits (Jiancheng Bioengineering Institute, Nanjing, China). Besides, the myeloperoxidase (MPO) activity was measured by ELISA kit (Cloud-Clone Corp, USA). All of these indicators were measured by a spectrophotometer.
Immunohistochemistry
First, paraffin sections were dewaxed and incubated in 3% hydrogen peroxide and 5% goat serum albumin for 30 and 20 minutes separately. Then, they were incubated with Ly6G antibody (1:100) overnight at 4°C. The second antibody was added and the sections were incubated at 4℃ for 1 hour the next day. Finally, the diaminobenzidine and haematoxylin staining were performed for microscopic observation.
Immunofluorescence
Sections were first incubated with Ly6G antibody (1:200) and then incubated with the fluorescently labeled secondary antibody (Alexa Fluor 594, 1:400). Finally, Ly6G+ cells were examined under the Olympus fluorescence microscope.
Western Blotting
Lung tissue and neutrophils cultured in vitro were lysed with the lysate including protease inhibitor and phosphatase inhibitor. The lysate was then centrifuged at 12500 rpm for 10 min at 4°C and the supernatant was stored in eppendorf tubes. The BCA kit (Thermo, IL) was used to measure the protein concentration. After making 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, 30ug proteins were used for electrophoresis and transferred to the polyvinylidene fluoride (PVDF) membrane blocked by blocking buffer for an hour. Then, primary antibodies (1:1000) were added to PVDF membrane at 4 °C overnight. TBST was used to washed the membranes for 3 times (10 minutes / time) and the membranes were incubated with second antibodies(1:3000) for 1h at 37℃. Chemiluminescence reagents (Beyotime Institute of Biotechnology, Shanghai, China) was used to measure the optical density by Image J image analysis software after the membranes were washed 3 times (10 minutes / time).
Statistical analysis
Data are presented as mean ± standard error of mean (SEM). We used Graphpad Prism (7.0, USA) to perform the data statistics. One-way analysis of variance (ANOVA) was used for multiple comparisons. Kaplan-Meier survival curves and log-rank statistics were used for survival-rate. P < 0.05 was considered statistically significant.