2.1 Parasites and animals
The T. spiralis isolate ISS534 used in this study was maintained by serial infection of SD rats from the Animal Parasite Laboratory of Jilin Agricultural University. BALB/c mice (female, 4–6 weeks old) and SD rats were purchased from Beijing Fukang Biotechnology company. All animal husbandry was maintained in Jilin Agricultural University Animal Experiment Center under the care of a professional breeder with a light/dark cycle of 12 hours (h) and with sterile food and water. Furthermore, all experiments in this study were performed in accordance with the Chinese Animal Management Ordinance. The animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Jilin Agricultural University.
2.2 Isolation of T. spiralis adults, new-born larvae and muscle larvae
Muscle larvae, adults and new-born larvae were obtained from rats as previously described [18]. In brief, BALB/c mice were orally inoculated with 300 muscle larvae of T. spiralis and sacrificed 7 days post-infection (dpi). The intestine was exposed, and the entire small intestine was harvested and washed by flushing in sterile saline to remove any intestinal contents before the tissue was cut into 3-cm pieces. The small intestine sections were transferred to a separation screening cloth of a nematode larva separator (200 mesh), which was put in a small beaker containing sterile saline (preheated at 37 °C) and incubated at 37 °C for 4 h. T. spiralis adults were collected from the bottom of the beaker and counted. The adults were incubated and washed in Roswell Park Memorial Institute-1640 medium (RPMI-1640, Gibco) with 20% foetal bovine serum (FBS, Gibco), 100 U/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco) at 37 °C in a humidified incubator with 5% CO2. The culture solution was filtered with a 280-mesh sterile mesh so that the new-born larvae were collected. Muscle larvae of T. spiralis were obtained by standard pepsin digestion (1% pepsin and 1% HCl at 37 °C for 2 h) to release larvae from the muscles of the rats infected at 35 days.
2.3 Animal experimental design
Forty-five healthy female BALB/c mice were randomly divided into three groups: TS+MCC950, TS and control. TS+MCC950 mice were intraperitoneally (IP) injected daily with the specific NLRP3 inhibitor MCC950 (10 mg/kg) after inoculation with T. spiralis (350 larvae per mouse), whereas the TS group mice were injected with an equal volume of PBS (i.p.). The mice in the control group received PBS solution. Five mice in each group were killed on the 7th, 14th and 35th days after infection with T. spiralis by cervical dislocation. The blood samples were placed in a sterile EP tube for 1 h at 37 ℃ and stored overnight in a 4 ℃ refrigerator. Serum was obtained by centrifuging the EP tube at 3000 rpm for 10 min at 4 ℃. Single-cell suspensions of the spleen (SPL) or mesenteric lymph nodes (MLN) were analysed by flow cytometry as previously described [19]. The small intestinal and masseter muscles were extracted as pathological sections to detect inflammation. Western blotting was utilized to detect changes in NLRP3 protein levels.
2.4 Enzyme-linked immunosorbent assay
The production of IgG1, IgG2, IL-1β and IL-18 in the serum of infected mice was determined in the control group, TS group, and TS+MCC950 group at 7, 14, and 35 dpi. Enzyme-linked immunosorbent assay (ELISA) kits (Abcam) were used.
2.5 Western blot
Small intestinal was pulverized using a mortar and pestle in liquid nitrogen and homogenized in ice-cold RIPA buffer (Thermo Scientific™). The protein concentration of the extracts was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific™). Total protein was resolved on 12% SDS–PAGE gels and then transferred to polyvinylidene difluoride membranes (PVDF). The membrane was blocked in TBST (10 mM Tris HCl, 0.15 M NaCl containing 0.05% Tween 20) with 5% nonfat skim milk for 3 h at RT and incubated with the corresponding primary antibody at 4 °C overnight. After washing for 10 min with TBST three times, the membranes were incubated with secondary antibodies at room temperature for 1 hour and washed again. Bands were visualized with ECL chromogenic reagent. The primary antibodies used were anti-NLRP3 (Abcam, 1:2000 dilution) and anti-mouse β-actin ( Proteintech, China,1:2000 dilution).
2.6 Flow cytometry
The relative ratios of IL-4 and INF-γ on CD3+ CD4+ T cells in the spleen (SPL) and mesenteric lymph nodes (MLN) were analysed by flow cytometry. Single-cell suspensions of the SPL and mesenteric lymph nodes (MLN) of all groups of mice were prepared as previously described (Shi et al., 2016) at Days 7, 14, and 35 after infection. Briefly, single cells were counted and seeded in 48-well plates at 2.0 × 106 cells/well in 500 µl RPMI-1640 medium (100 U/ml penicillin, 100 μg/ml streptomycin, 10% heat-inactivated FBS) containing ionomycin (1 µg/mL), Golgi plug (10 µg/mL) and PMA (20 ng/mL) and were then incubated at 37 °C in a 5% CO2 incubator for 6 hours. After treatment, the cells were collected into EP tubes by centrifugation (2000 rcf for 5 min at 4 °C) and discarded from the supernatant.
Harvested cells were resuspended in 100 μL of cold PBS and incubated with various combinations of fluorochrome-labelled antibodies (CD3, CD4 along with isotype controls) according to the manufacturer's instructions (BD Stemflow). Anti-mouse CD3 (PerCP-CY5.5, BD Biosciences) and anti-mouse CD4 (FITC, BD Biosciences) were used to label CD3+ T cells and CD4+ T cells at 4 °C for 1 h in the dark. After staining for cell surface markers, the cells were fixed and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer’s instructions and washed two times with 500 μl of cold BD Perm/WashTM buffer (BD Biosciences). Intracellular staining was required to determine the percentage of IL-4+ and INF-γ+ cells. Next, treatment with anti-mouse IL-4 (APC, BD Biosciences) and anti-mouse INF-γ (PE, BD Biosciences) was performed at 4 °C for 1 h in the dark. Following this, all stained cells were washed three times with cold PBS to remove unbound antibodies, suspended in 300 µl of PBS, and then examined using a LSR FortessaTM (BD Biosciences). Data were analysed with Flow Jo software (ver 7.6.1, Tree Star Inc., USA).
2.7 Histopathological evaluation and immunofluorescence
At 7, 14, and 35 dpi, lingual muscle and small intestine samples were obtained from each group and fixed in 4% formalin for 48 h. The tissue was subjected to washing, dehydrated in gradual ethanol (70-100%), made transparent with xylene, processed, embedded with paraffin wax, sliced (3 μm), placed in warm water (42 ℃) for spreading, collected using slides, baked, and prepared into paraffin tissue sections. The tissue slices were baked at 80 ℃ for 1 h and placed in xylene for 8 min twice. For histopathological evaluation, the sections were stained with haematoxylin and eosin and visualized with a microscope (Leica). The number of goblet cells per ten randomly selected villus–crypt units (VCU) was determined by microscopy from at least two sections per animal as previously described[20]. Twenty nonoverlapping representative fields of the tissue were examined microscopically using a 400X objective, and the number of inflammatory cells infiltrating the masseter muscle was observed [21].
2.8 Bone marrow-derived macrophage (BMDM) isolation, culture and stimulation
Mouse bone marrow-derived macrophages (BMDMs) were generated from five-week-old BALB/c mice as previously described [22]. In the in vitro experiments, the BMDMs were randomly divided into the PBS negative control, ATP positive control, adult protein group, neonatal larvae protein group, muscle larvae protein group, and MCC950 group. ELISA was used to detect changes in IL-18 and IL-1β levels, and indirect immunofluorescence was used to detect the expression of NLRP3.
BMDMs were primed with LPS for 3 h and then treated with DMSO (Beyotime) or DMSO+MCC950 (7.5 nm/mL) for 30 min, followed by stimulation with T. spiralis protein (50 mg/mL) of new-born larvae, muscle larvae and adult stages for 6 hours. Protein extraction was performed as previously described (Gómez-Morales, et al., 2018). After treatment, the BMDMs were washed with PBS, fixed with 80% cold acetone for 30 min at RT and then washed three times with PBS. After blocking with 3% BSA in PBS for 30 min at RT, the BMDMs were incubated with an antibody against NLRP3 (Sigma) for 1 h at 4 °C. After washing with PBST, the BMDMs were incubated with secondary antibody (Alexa Fluor® 488 (green). Proteintech) for 40 min at RT. After washing, nuclei were stained with DAPI (blue. Sigma) for 10 min in the dark. Imaging analysis was performed using a fluorescence microscope.
2.9 Statistical analysis
Data were statistically analysed by using GraphPad Prism 9.0 software. The Shapiro–Wilk test was used to analyse normality. For comparisons of only two groups, data were analysed using Student’s t test, while for comparisons of three or more groups, we performed one-way ANOVA with Bonferroni’s multiple comparison test as indicated. The Mann–Whitney U test was used for nonnormally distributed data. The statistically significant differences between the means are indicated by asterisks (*P< 0.05, **P< 0.01, ***P< 0.001). Statistical data are expressed as the mean value ± SD.