30 asthma patients were enrolled into the study. They were all inpatients of Qilu Hospital of Shandong University, and were diagnosed with asthma according to the Global Initiative for Asthma (2019 edition). We also recruited 30 subjects who visited our hospital for a routine medical checkup but did not have any respiratory condition as no asthmatic controls. All subjects (asthmatics and controls) had no history of heart failure, renal failure, autoimmune diseases, lung disease, tumor and infection within 2 weeks. The study was approved by the Ethics Review Committee for Human Studies at Qilu Hospital of Shandong University(Grant NO. KYLL-2017(ks)-112).
Cell isolation and culture
Primary macrophages (PMs) were prepared referred to previous methods. 12-week-old C57BL/6 mouse was intraperitoneally (i.p.) injected with 2ml of 3.0% sterile thioglycolate medium. 96 hours later, mouse was euthanized, lavished intraperitoneally with 5-10 ml of Dulbecco's Modified Eagle Medium (DMEM) for 3 consecutive times to collect peritoneal exudate cells. Then, the eluent was centrifuged at 1000rpm for 5 min at 4℃, and the cell pellet was resuspended in 5-10 ml of DMEM supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin and 0.1 mg/ml streptomycin. Then cells were seeded in six-well culture plates at a density of 1 ×105 cells per well. After incubation for 2 h at 37°C in a 5% CO2 humidified atmosphere, cells were washed with PBS for 3 times to remove the unattached cells, and the remaining cells were further cultured in complete medium as peritoneal macrophages for subsequent experiments. Purity and viability of PMs (both over 95%) was evaluated microscopically by Wright–Giemsa staining and Trypan blue exclusion, respectively.
C57BL/6 mice, 6-8 weeks of age, were maintained under standard laboratory conditions in the Animal Experimental Center of Shandong University for 1 week before experiments. All protocols were approved by the Institutional Animal Care and Use Committee of Shandong University. Fstl1flox/+ mice were generous gifts from Xiang Gao (Nanjing University, Nanjing, China). Fstl1+/- mice were generated as previously described.
Model and Grouping
WT mice were randomly divided into 5 groups (n = 6/group) as follows: control group, OVA group, FSTL1 intranasal group, PBS intranasal group and FSTL1+MCC950 group. FSTL1+/- mice were randomly divided into 2 groups (n = 6/group): control group and OVA group. OVA-induced allergic airway inflammatory model was established as previously reported, with some modifications (Fig. 2a). Briefly, mice were sensitized by i.p. injection of 50 ug OVA (grade V, Sigma, St. Louis, MO, USA) without adjuvant in 100ul PBS on days 0, 7, 14 and followed by intranasal administration of 10 ug OVA in 40 ul PBS on days 21–28. Control mice were treated in corresponding manner with isometric PBS alone. Mice of FSTL1 group were intranasally administered with 10ug FSTL1 (Sino Biological, Beijing, China) in 40 ul PBS daily for 15 d, and PBS intranasal mice were treated with PBS instead. FSTL1+MCC950 group were i.p. injected with 200ug MCC950 (Target Molecule, MA, USA) 2h before each FSTL1 administration.
Assessment of AHR
24 hours after the final treatment, mice were anesthetized (i.p. by 1% pentobarbital, 50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA), intubated, exposed to aerosolized methacholine (Sigma-Aldrich, St. Louis, MO, USA) at increasing concentration of 0, 4, 8, 12 or 16mg/ml, and were respectively measured using the FlexiVent system (SCIREQ, Canada). The results were expressed as changes from baseline data.
BALF collection and analysis
BALF was collected to analyze cellular components and cytokines in supernatants. As previous mentioned[26, 27]，after ligating one side of the bronchus, we flushed the other side of lungs with 0.5mL ice-cold PBS for 3 times via tracheal catheter. Approximately, over 80% of the instilled volume was recovered. Then, the BALF samples were centrifuged, supernatants were collected and stored at -80℃ for the following measurement of cytokines. The cell pellets were resuspended in 1ml PBS, and 0.1 ml suspension was taken out to count the total number of nucleated cells under a cytometer. The differential cell counts were smeared on slides and processed by Wright–Giemsa staining (Sigma-Aldrich, St. Louis, MO, USA). At least 200 cells were classified and counted for each slide by brightfield microscope according to cells morphology, and the counting researchers were blinded to the grouping situations. The other side of lungs without lavage were stored in the Ultra-low temperature freezer or fixed in 10 % formalin for use.
The lung tissues of mice were fixed in 10% formalin for more than 24h, then dehydrated, paraffin‑embedded, and cut into 5μm sections. The sections were dewaxed, rehydrated and stained with Hematoxylin-eosin (H&E) staining and Periodic acid-schiff (PAS) staining. According to previously described methods, the severity of peribronchial and perivascular inﬂammation was graded by lung inﬂammatory scores of 0-4 , and the goblet cell hyperplasia was evaluated by numerical scores of 0-4 determined by PAS-positive cell in each airway.
Sections were deparaffinized, rehydrated, pretreated with 10mM sodium citrate (pH 6.1) for
antigen recovery, prevented from the endogenous peroxidase by 3% H2O2 and blocked with 5% bovine serum. Next, the slides were incubated with primary antibodies of anti-FSTL1 antibody (l:200; Abcam, USA), anti-NLRP3 antibody (l:200; Abcam, USA), anti-pro-caspase 1 antibody (l:200; Proteintech Group Inc., Wuhan, China), anti-Muc5AC antibody (l:250; Boster, China) respectively or PBS overnight at 4˚C. After extensive wash, the sections were incubated with the corresponding HRP-conjugated secondary antigens for 1 hour at room temperature and then developed with DAB solution (Boster, China). At last, the slides were counterstained with hematoxylin for about 2 min. The positive area of the target protein was measured by Image-Pro Plus 6.0 software (Media Cybernetics, USA) at ×400 magniﬁcation.
The levels of IL-4, IL-5, IL-13，IL-1β in BALF supernatant of mice were detected using ELISA kits(CUSABIO, China) according to the manufacturer's instructions. FSTL1 and IL-1β levels in human serum were also measured by ELISA (Abcam, USA). All the calibrations and analyses were performed in duplicate.
Western blot analysis
Prepared lung tissues and PBS washed adherent cells were homogenized by grinding and lysing using ice-cold RIPA buffer in the presence of protease inhibitors. Then, the homogenate was centrifuged at 12000 rpm for 10 min at 4 °C, and soluble supernatants were taken for protein concentration determination by BCA protein assay kit (Boster, China). After that, equal amounts of protein samples (25μg of total protein each) were boiled at 95 °C for 5 min and separated onto SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non‑fat milk for 1 h at room temperature, and then incubated with diluted primary antibodies against NLRP3 (1:1000; CST, MA, USA), pro-caspase1 (1:1,000; Abcam, USA), IL-1β(1:1000; Abcam, USA), GAPDH (1:5000; Abcam, USA) overnight at 4˚C, followed by incubation with HRP‑conjugated secondary antibody for 2h at room temperature. The binding of all the antibodies was detected using an enhanced chemiluminescence (ECL) kit (Pierce Biotechnology, USA). All experiments were repeated in triplicate.
All data are presented as mean ± standard deviation (mean ± SD). The quantitative analysis of ﬁgures of IHC, PAS staining was performed using GraphPad Prism 6 (GraphPad Software Inc, San Diego, CA, USA). The quantitative analysis of IHC was performed using ImageJ. Student’s t-test and one-way analysis of variance (ANOVA) were applied to assess differences among groups. Post hoc analysis of SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) was used to evaluate statistical significance. Results were considered statistically significant at p value less than 0.05 (P < 0.05).