The 8-12-week-old C57BL/6 male mice (weight, 20-24 g) were provided by Laboratory Animal Center of Chongqing Medical University (Chongqing, China). Thereafter, all animals were raised in the specific pathogen-free (SPF) environment under 24 °C, 50%-60% relative humidity (RH) and 12 h/12 h light/dark cycle conditions. Each mouse was allowed to drink water and eat standard food. Each animal was healthy and infection-free throughout the experiment.
All mice were treated following the Guidelines for the Care and Use of Laboratory Animals in China. The Institutional Animal Care and Use Committee of Chongqing Medical University approved our study protocol.
"Double-Hit" Mice Model
CLP and intratracheal injection of S. aureus or P. aeruginosa were carried out as the first and second hits, respectively. Briefly, each mouse was given intraperitoneal injection of ketamine (1 mg/ml) and 100 μl xylazine (20 mg/ml) contained within PBS for anesthesia, followed by cecal ligation and puncture using the 26G needle (non-severe CLP, resulting in the mortality rate of 5%–10% in WT mice). Later, we put back the cecum into peritoneal cavity, followed by incision closure using the surgical staples. All mice were given subcutaneous administration of 0.9% sterile normal saline at the dose of 5 ml/100 g body weight (BW) preheated at 37 °C for replacing the 3rd space loss; thereafter, the warm pad was prepared for resuscitation .
At 3 days after CLP, the xylazine/ketamine mixture was administered into the surviving mice for anesthesia. Then, each mouse was placed in the "head-up" position, and the trachea was exposed, followed by intratracheal injection (i.t.) with P. aeruginosa (5 × 107 colony-forming units [CFUs] within 50 μL PBS) or S. aureus (5 × 107 CFUs within 50 μL PBS) .
In vivo administration of basil polysaccharides
For in vivo basil polysaccharides treatment, each mouse was administered i.p. with 75 mg/kg of basil polysaccharides  (Shanxi kingreg Biotech. Ltd., China) or IgG 2 h after the second hit. With regard to CCL4 exposure in vivo, all animals were given 500 ng IgG or recombinant mouse CCL4 (R&D Systems, USA) i.p. at the time of the second hit of S. aureus.
Lung Tissue and Bronchoalveolar Lavage Fluid Collection
At 24 h following S. aureus or P. aeruginosa i.t., the animals were killed under anesthesia. Lungs were extracted, and tissues were harvested, followed by the immediate collection of bronchoalveolar lavage fluid (BALF). After chest clapping, right bronchial bundling and left lung lavage were carried out. In addition, after resecting right lung, we obtained the right upper lobe to count the bacterial numbers, whereas the rest right lung tissues were preserved under −70°C at once for further analysis. Then, BALF was subjected to 10 min of centrifugation at 400 g for separating cells from supernatants. Afterwards, we resuspended cell pellets into the pre-chilled PBS, then the grid hemocytometer was adopted for measuring total cell numbers. Later, we collected 100 µL cells for cytocentrifugation (Cytospin; Cytopro Wescor; Syracuse, NY); after cells were dried in air, they were stained using Giemsa Diff-Quik II stain (Baxter Scientific Products; McGaw Park, IL). Thereafter, a light microscope was utilized to determine differential cell numbers (minimal number, 300 cells). Specifically, the cell numbers were presented in the manner of lymphocytes, neutrophils and monocytes/macrophages.
Determination of Lung and Plasma Bacterial Burdens
Plasma samples were obtained at specific time periods. Meanwhile, we also resected the right upper lung lobe under aseptic condition, followed by homogenization within 1 mL sterile saline using the tissue homogenizer by the use of vented hood. Later, we diluted plasma and lung homogenate at serial concentrations. For every dilution, 10 ml sample was added on the predried tryptic soy-base blood agar plates, followed by overnight incubation under 37°C. Afterwards, CFUs were counted and expressed as total CFU per lung or per milliliter of plasma.
Measurement of inflammatory mediators
Blood samples were collected in heparinized tubes via the ophthalmic vein. Inflammatory mediators, such as CCL4, CXCL-1, TNF-α, IL-1β, IL-6, and IL-17A, were assessed by the Mice Cytokine Magnetic Bead Panel Kit (eBioscience, USA) following the manufacturer's protocol.
Determination of chemokine CCL4 produced by neutrophils
The neutrophils were sorted from the bronchoalveolar lavage using magnetic separation (Miltenyi Biotec) and were suspended in 10% FBS (Sigma, USA), RPMI1640 (Sigma, USA), and then inoculated on the culture plate. To determine whether basil polysaccharides promote the secretion of CCL4 by neutrophils, we supplemented basil polysaccharide  (100 ug/ml, kingreg Biotech, China) or PBS to the culture. After incubation for 48 h, the chemokine CCL4 in the supernatant was quantified by ELISA using kits (R&D, USA) following specific protocols. The absorbance of each sample was read at 450 nm.
Lung injury index assessment
(1) Morphological evaluation: as for the right upper lung lobe, it was subjected to 10% formalin fixation, paraffin embedding, and sectioning into 4-µm sections. Then, the sections were deparaffinized, dehydrated, and stained by hematoxylin and eosin (H&E) to carry out histological examinations. Mikawa’s method was adopted to estimate lung injury score by adopting the 4 indicators below: 1. alveolar hyperemia; 2. hemorrhage; 3. neutrophil or interstitial aggregation or infiltration; 4. hyaline membrane formation or alveolar septal thickening, where 0-4 marks indicated no/very mild, mild, moderate, severe and very severe damage, respectively. All scores were added up as the final score, and the ARDS pathological score was indicative of increases in lesion number. Lung injury was rated according to the 0-4 scale based on lesion severity of every indicator, where 0-4 points indicated normal results, mild (<25%), moderate (25-50%), severe (50-75%), and very severe (>75%) lung involvements, separately. A greater score was indicative of the more severe lesion. The light microscope (Olympus, Japan) was utilized to evaluate the abnormal histological results. (2) Albumin assessment: Albumin for lung permeability assessment was performed using Albumin Quantification Kit (Bethyl Laboratories, Montgomery, TX) following specific protocols. (3) Myeloperoxidase (MPO) measurement: the MPO activity in tissue was measured to quantify lung neutrophil infiltration. In brief, we homogenized lung tissues with the 20 mmol/L PBS (pH 7.4), followed by 10 min of centrifugation at 4 °C and 10,000g. Later, pellets were resuspended with 50 mmol/L PBS (pH 6.0) contained within 0.5% hexadecyltrimethylammonium bromide (Sigma), and then the homogenate was treated with 4 freeze-thawing cycles, followed by 40 s of sonication for disruption. Afterwards, the samples were subjected to 5 min of centrifugation for 40 s at 10,000g and 40,000ion. The sample was assayed for the myeloperoxidase activity according to previous description, with tetramethylbenzidine (Sigma) being the substrate. Later, we detected the absorbance (OD) values at 460 nm and adjusted them based on tissue weights (fold change FC relative to control). (4) Wet/dry weight: After dissecting left lung, we weighed the wet weight. The lung was incubated, then dried in an oven at 60°C for 3–4 days and re-weighed as dry weight. Then, the wet weight was divided by dry weight to calculate the wet-to-dry (W/D) weight ratio .
In situ Cell Apoptosis Detection Kit I, POD (Roche, Switzerland) was utilized to measure cell apoptosis rate by TUNEL assay following specific instructions. In brief, after xylene deparaffinage, the 4-μm sections were subjected to gradient ethanol rehydration. Thereafter, 3% hydrogen peroxide (H2O2) was used to block endogenous peroxidase activity for a period of 10 min; afterwards, 10–20 μg/mL proteinase K solution was utilized to digest sections under 37 °C for 15 min. After PBS washing, terminal deoxynucleotidyl transferase diluted at 1:20 supplemented within the reaction buﬀer (digoxigenin-labeled nucleotides) was used to react with sections for 2 h under 37°C. Thereafter, the stop/wash buﬀer was used to rinse slides for 2 min thrice. Subsequently, antidigoxin antibody previously diluted at 1:100 was used to incubate sections under 37 °C for 30 min, and later ABC was employed to further incubate sections for 30 min under 37 °C. Apoptosis was measured through incubating sections using 3,3 diaminobenzidine chromogen for about 20 min, followed by hematoxylin counter-staining. Later, 5 ﬁelds of view (FOVs) were selected randomly from every section (×400 magniﬁcation). Then, TUNEL-positive cell proportion per field was recorded at ×400 magnification in 5 random fields.
Western blot analysis
The protein extraction kit (Beyotime, China) was utilized to extract total macrophage proteins in accordance with specific protocols. Bicinchoninic acid (BCA) protein assay kit (Pierce, USA) was employed for detecting protein contents. Thereafter, proteins were separated through 10%SDS-PAGE, followed by transfer to the nitrocellulose membranes. After transfer, the membranes were incubated in blocking buffer containing 5% (w/v) skimmed milk supplemented within the Tris-buffered saline that contained 0.05% tween-20, followed by overnight incubation with primary antibody under 4 ° C and then secondary antibody incubation. At last, the ECL detection system was used to visualize protein blots.
After PBS washing, cells were prepared into pellets and analyzed by the flow cytometer. The following monoclonal antibodies including, CD4, CD25, Foxp3, CD11b, Ly6G, F4/80. To stain CD4, CD25, Foxp3, CD11b, Ly6G, F4/80, the Fixation/Permeabilization kit (eBioscience, USA), anti-CD4-FITC, anti-CD11b-APC, anti-Ly6G-FITC, anti-F4/80-FITC anti-CD25-PE and anti-Foxp3-APC (eBioscience, USA) were utilized following specific protocols. The FACScan flow cytometer (Becton Dickinson) was used to collect cells (105), whereas Flow Jo software 7.6 was adopted for analysis.
Cell purification and culture
We adopted the Lymphocyte Separation Medium (GE healthcare, USA) to isolate splenic peripheral blood mononuclear cells (PBMCs) from mice. Thereafter, magnetic activated cell sorting (Miltenyi Biotec) was carried out to isolate naïve CD4+ T cells from PBMCs using the Naïve CD4+ T Cell Isolation Kit II (StemCell, Canada) following specific protocols. Then, flow cytometric analysis was performed to measure the naïve CD4+ T cell purity (> 90%). Then, we cultivated cells within the RPMI 1640 complete medium (Gibco, Grand Island, NY, USA) that contained 10% foetal bovine serum (FBS) and incubated them under 37°C and 5% CO2 conditions.
Treg cell subset generation
In this study, we produced Tregs cell subsets through exposing to 50 mM β-mercaptoethanol, 2 mM L-glutamine, 2 µg/ml anti-CD28, 5 µg/ml anti-CD3, 2.5 ng/ml TGF-β and 50 U/ml IL-2 for a period of 3 days. To determine whether basil polysaccharide was involved in the induction process, we supplemented basil polysaccharide (100 ug/ml, kingreg Biotech, China) to the culture. Flow cytometric analysis was performed to assess intracellular staining and surface marker expression.
Macrophage phagocytosis assays
BALFs were incubated using 0.5 mg/ml FITC (Sigma) under 37 °C for 20 min, so that macrophages adhered to the plastic, FITC-labeled S. aureus for separation. Thereafter, FITC-labeled bacteria (MOI, 100) were used to incubate the separated macrophages under 37°C for 30 min. Then, cells were washed, nuclei were subjected to DAPI (Invitrogen) staining and visualized under the confocal laser scanning microscope (LSM 510, Zeiss). One independent reviewer was responsible for quantifying engulfed bacterial proportion of the 300 cells counted/well. For certain experiments, 100 ug/ml BPS (kingreg Biotech, China) was used to pre-treat bronchoalveolar macrophages before infection with FITC-labeled S. aureus.
Macrophage killing assays
Alive S. aureus (with the multiplicity of infection (MOI) of 10) was used to infect 1 x 105 bronchoalveolar macrophages for 1 h under 37 °C. Later, buffer that contained 100 µg/ml tobramycin was adopted to wash cells for removing extracellular bacteria, whereas lysis buffer (Promega) was used for lysis. Lysate culture was utilized to quantify alive intracellular bacteria so as to assess bacterial uptake as well as intracellular killing (t = 0 and 2, respectively). Killing was determined by colony proportion occurring at t = 2 h in comparison with that at t = 0 h below, 100−CFU number at t = 2 h/CFU number t = 0 h]. In certain experiment, 100 ug/ml BPS (Kingreg Biotech, China) was used to pre-treat bronchoalveolar macrophages prior to alive S. aureus infection.
SPSS19.0 (IBM, Armonk, New York, USA) was employed for statistical analysis. Values were presented in the manner of median (interquartile ranges) or mean ± SD. Differences of two groups were evaluated by Mann-Whitney U tests, whereas those among several groups were evaluated by one-way ANOVA. Log-rank (Mantel-Cox) test was used to analyze survival curves. P<0.05 indicated statistical significance.