Milk sample collection
A total of 20 milk samples, including eight normal milk samples and 12 mastitis milk samples, which were collected from the Weigang Animal Diary Company, Jiangsu, China.
S. agalactiae was isolated from fresh milk samples collected from cows with mastitis and injected into the mammary gland tissue of mice to construct a mastitis model. A single colony was isolated from the fresh milk samples using the plate-drawing method and expanded culture to extract the bacterial DNA. After sequencing, multiple pathogenic bacteria (e.g., S. agalactiae) were obtained (primer sequence: F5'-AGTTTGATCCTGGCTCAG-3' and R5'-AGGCCCGGGGAACGTATTCAC-3', Sangon Biotech, Shanghai, China). S. agalactiae was incubated on a shaker for 12 h (37°C at 220 rpm), diluted with PBS, and coated on the plate to achieve the desired concentration (1 × 109 CFU/mL), in accordance with previously published methods . The bacteria was stored for a short time at 4°C. Before use, 1 mL of the diluted bacterial solution was resuscitated for 4 h (37°C at 220 rpm), and centrifuged for 5 min (3000 rpm). The supernatant was removed and the bacterial pellet was resuspended in 1 mL PBS.
A total of 36 female and 15 male KM mice (6–8-weeks-old, weighing 20 g–25 g) were maintained under SPF housing conditions. Both the female and male mice cohabited at a two female to one male ratio for conception, standard rearing, and free drinking water (25°C ± 1°C, humidity 40%–80%). They were housed separately after pregnancy until 12 days after calving. The feeding and management of the experimental animals were carried out in accordance with the Animal Management Regulations issued by the National Science and Technology Commission of the People's Republic of China (November 14, 1988), and with the permission of the Animal Ethics Committee of Anhui Normal University (Approval No. 2015002).
Mouse model of mastitis
Female mice were randomly separated into two groups: 1) S. agalactiae group, one breast was injected with PBS and the other was injected with S. agalactiae after 12 days post-partum; and 2) PBS group, one breast received a blank control and the other was injected with PBS after 12 days post-partum. The pups were removed 2 h prior to the experiment. The female mice were anesthetized with 1% carbrital, and the third and fourth pairs of milk areas were wiped with a cotton ball soaked in 75% alcohol. Slowly, 100 µL of S. agalactiae was injected into the left breast area with a microinjector needle with a diameter of 0.06 mm, and 100 µL of sterile PBS was injected into the right side. At the corresponding time points (24 h, 48 h, and 72 h) after treatment, the mice were sacrificed by cervical dislocation, and the mouse mammary glands were collected under aseptic conditions for subsequent processing and analysis.
The mammary gland samples of the S. agalactiae group and PBS group were collected and fixed in 4% buffered paraformaldehyde for more than 48 h. the samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E), and the pathological changes were observed by microscopy.
Western blot analysis
After mixing 400 µL cell lysate and 4 µL proteinase inhibitor, 40 mg of udder tissue were added, ground for 50 s into a powder on an ultrasonic shaker, and lysed for 2.5 h on a shaker. The lysate was centrifuged at 13,000 × g for 20 min and the entire process was carried out at 4°C. Next, the supernatant was collected into a new centrifuge tube and the supernatant was quantified using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). An appropriate amount of SDS-PAGE loading buffer was added to the protein sample and placed into a metal incubator for 95°C 10 min. The samples were allowed to cool to room temperature and stored at -20°C. After the protein was transferred to the PVDF membrane, it was blocked in 5% BSA for 1 h and incubated with the primary antibody at 4°C overnight. The membranes were then washed three times with TBST for 10 min/wash. The membranes were incubated with a horseradish peroxidase-labeled secondary antibody at 37°C for 1 h. After washing the membrane three times with TBST, an enhanced chemiluminescence solution detection system was used to take pictures and perform an analysis (Tano, Shanghai, China). Densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD). The following antibodies were used in this study: TNFRSF21 (Catalog No: #bs-7678R, BOSTER Biotechnology, Wuhan) 68 kDa, NLRP3 (Catalog No: #sc-134306, Santa Cruz Biotechnology, USA) 120 kDa, IL-1β (Catalog No: #sc-12742, Santa Cruz Biotechnology, USA) 17 kDa, caspase1 (Catalog No: #sc-392736, Santa Cruz Biotechnology, USA) 45 kDa, GAPDH (Catalog No: #AT0002, CMCTAG, USA) 36 kDa, goat anti-mouse IgG secondary antibody (Catalog No: #BA1038, BOSTER Biotechnology, Wuhan), and goat anti-rabbit IgG secondary antibody (Catalog No: #BA1039, BOSTER Biotechnology, Wuhan).
Mammary tissue sections (thickness: 4 µm) were deparaffinized and dehydrated by density and fluorescently stained. Densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD). The methods were performed in accordance with the manufacturer instructions in the TUNEL cell pyroptosis detection kit-CY3 (50T) (BOSTER, Wuhan, China).
Statistical analyses were performed using a Student’s t-test with GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). Data were presented as the mean ± the standard deviation (SD). The fluorescence intensity was determined by defining a circular region-of-interest (ROI) for the entire cell. ROIs were corrected based on the average value of a background ROI defined outside the cell. A colocalization analysis was performed using JACoP Plugin in ImageJ. A threshold of P < 0.05 was considered statistically significant.