Experimental design
The experimental design has been shown in Fig. 1. We first investigated the ruminal microbiota profiles in healthy and mastitis cows (Fig. 1A). To indicate the role of the ruminal microbiota in mastitis pathogenesis, RMT from healthy (H-RMT) and mastitis cows (M-RMT) to mice was performed (Fig. 1B). According to the differences identified between the healthy and mastitis groups in cows and RMT mice, we next investigated the role of ruminal dysbiosis-derived low-grade endotoxemia in the pathogenesis of mastitis in mice (Fig. 1C). Finally, we studied whether impaired host anti-inflammatory enzyme by ruminal dysbiosis aggravates pathogen-induced mastitis in mice (Fig. 1D).
Collection of dairy cow samples
Mastitis was diagnosed according to macroscopical inflammatory changes (e.g., redness, edema, fever, and pain) and somatic cell counts (SCC, SCC > 5 × 105 cells/mL) [33]. Healthy cows were defined as no mammary injury and SCC less than 2 × 105 cells/mL [34]. A total of 16 cows (4–6 years) were employed for the dairy sample collection, including 8 healthy and 8 mastitis cows from Qingzhou city, Shandong province, China. All cows were fed a standard diet consisting of hay and grains to meet their energy requirements [8, 33]. All healthy and mastitis cows were not exposed to antibiotics for 6 months before sample collection. The serum, milk, and ruminal samples were harvested aseptically and stored at − 80 °C until determination of systemic inflammation, mastitis, and the ruminal microbiota, respectively.
Animals and RMT treatment
A total of 160 specific pathogen-free (SPF) grade BABL/c mice (22–24 g, 120 females and 40 males) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China). All mice were fed with sufficient standard diet and water under SPF-grade feeding conditions with 12 h of light daily. The mice were further mated at a ratio of three females to one male until pregnancy was confirmed, and then the male mice were separated. The RMT model was established according to previously described methods [22, 23]. In brief, ruminal samples from each group, including the healthy and mastitis groups, were mixed and homogenized with sterile PBS at a final concentration of 50 mg/mL. The prepared homogenate was centrifuged at 100×g and 4 °C for 2 min, and then the supernatant was collected and dispensed based on experimental demand. Mice were transplanted 1 week after pregnancy. These mice were treated with antibiotic (200 mg/kg of ampicillin (Sigma Aldrich, USA), metronidazole (Sigma Aldrich, USA), and neomycin (Sigma Aldrich, USA) and 100 mg/kg vancomycin (Sigma Aldrich, USA)) daily through oral gavage for 5 days to eliminate commensal microbiota. Next, antibiotics were removed for 1 day, and mice were incubated daily with 300 μL/mouse ruminal microbiota for three consecutive days and then twice a week for 3 weeks.
Low-grade LPS, cIAP, and zanamivir treatment
To investigate the role of low-grade LPS on the development of mastitis, mice were treated with LPS (2 μg/mouse or 20 μg/mouse daily, n = 5/group, E. coli 0111:B4, Sigma Aldrich, USA) intraperitoneally for 10 consecutive days from the first day of delivery according to previous studies [24, 32]. For treatment with cIAP and zanamivir, cIAP (20 U/mL, n = 6/group, Biolegend, CA, USA) or zanamivir (0.5 mg/mL, n = 6/group, Yuanyebio, Shanghai, China) was supplemented in the drinking water immediately following the initial LPS treatment and continued for the duration of the study as indicated [30].
Pathogen-induced mouse mastitis model
A mouse mastitis model was established by treatment with E. coli according to our previous description [20]. Briefly, E. coli were cultured in LB medium for 12 h to reach the mid-logarithmic phase. RMT-treated mice or conventional lactating mice 7 days after parturition were then separated from the offspring for three hours and injected with E. coli (107 CFU/mL) through the milk ducts using a 100-μL syringe with a 30-gauge blunt needle. To investigate the protective effects of host ALP on E. coli-induced mastitis in mice, lactating mice 7 days after parturition were pretreated with cIAP (75 U/kg, Biolegend, CA, USA) and zanamivir (500 μg/kg, Yuanyebio, Shanghai, China) for 2 h followed by E. coli infection. Twenty-four hours after the E. coli injection, the mammary gland tissues were harvested and stored at − 80 °C until the detection of inflammatory parameters.
Histological analysis
All tissue samples used for histological analysis, including the mammary glands, ileum, colon, and liver tissues from RMT mice and the mammary glands from LPS- and E. coli-treated mice, were fixed with 4% paraformaldehyde for more than 48 h. The fixed samples were then embedded in paraffin and sliced into 5-μm slices (five slices per mouse). The prepared sections were further stained with hematoxylin and eosin (H&E) and assessed with an optical microscope (Olympus, Tokyo, Japan). The histological score was determined as previously described [20].
Mammary cytokine detection
The mammary gland tissues from RMT-, LPS-, and E. coli-treated mice were weighed and homogenized (0.1 mg/mL) using PBS. The prepared homogenates were centrifuged at 12,000×g and 4 °C for 10 min, and the supernatants were collected for tumor necrosis factor (TNF)-α and interleukin (IL)-1β detection by using TNF-α and IL-1β ELISA kits (Biolegend, CA, USA).
Myeloperoxidase (MPO) activity determination
The mammary samples from RMT-, LPS-, and E. coli-treated mice were prepared to generate a 10% homogenate using the provided MPO buffer, and MPO activity was detected according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China).
Quantification of bacteria in the mammary glands of mice
To investigate the effects of different treatments on E. coli-induced mastitis, the mammary E. coli load was detected as previously described [35]. In brief, mammary samples were harvested aseptically and homogenized at a final concentration of 0.1 mg/mL using sterile PBS. The prepared homogenates were subjected to tenfold serial dilutions until the desired concentrations were reached, and then the diluted homogenates were plated on MacConkey agar (Hopebio, Qindao, China). The number of E. coli was counted, and the absolute quantities were calculated based on the corresponding dilutions after 24 h of growth.
Serum LPS, ALP, and cytokine determination
Serum LPS levels from cows and RMT- and LPS-treated mice were detected as previously described [23]. In brief, the sera were collected under pyrogen-free conditions, and serum LPS levels were detected using a LPS ELISA kit according to the manufacturer’s instructions (Lanpaibio, Shanghai, China). Serum ALP levels from cows and RMT-treated mice were measured with an ALP assay kit according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China). Serum cytokines in cows and RMT-treated mice were detected using bovine TNF-α and IL-1β ELISA kits (Lanpaibio, Shanghai, China) and mouse TNF-α and IL-1β ELISA kits (Biolegend, CA, USA).
Test to determine pathogens in milk
To test for major mastitis pathogens, including Staphylococcus aureus (S. aureus), Streptococcus, and E. coli, in the milk of healthy and mastitis cows in this study, 50-μL milk samples were plated on tryptone soya broth agar (TSB, Hopebio, Qindao, China), blood agar (Hopebio, Qindao, China), and MacConkey agar (Hopebio, Qindao, China), respectively. After 24 h of incubation, the numbers of bacterial colony-forming units (CFUs) were counted.
Routine blood tests and serum biochemical tests
A routine blood test was performed to detect the changes in blood immune cells in RMT-treated mice. Blood samples from RMT mice were collected into vacuum tubes with an anticoagulant (ethylenediamine tetraacetic acid), and then blood immune cells were detected with an automated hemocyte analyzer according to the manufacturer’s instructions (HB-7510, Sinnowa, China). Serum biochemical tests for cows and RMT-treated mice were performed as previously described [36]. Briefly, blood samples from cows and RMT-treated mice were collected into vacuum tubes with a coagulation activator (thrombin). Biochemical analysis was performed with an automatic analyzer (CS-T240, Dirui Industrial Co., Ltd., China) using the commercial veterinary kit (DiAvTest, Russia).
Lipocalin-2 assay
Fecal samples from the RMT treatment groups were weighed and prepared as 10% homogenates using PBS and then centrifuged at 3000×g and 4 °C for 10 min. The supernatants were collected, and fecal lipocalin-2 levels were detected by ELISA according to the manufacturer’s instructions (Lanpaibio, Shanghai, China).
Alcian blue (AB) staining
Paraffin sections of the colon were prepared as mentioned above. The prepared paraffin sections were dewaxed with xylene and serial alcohol solutions as previously described [28]. The sections were further stained using an Alcian Blue Stain Kit according to the manufacturer’s instructions (Solarbio, Beijing, China).
Immunochemistry
All samples used for immunochemistry were prepared into 5-μm sections and dewaxed as mentioned above, and immunochemistry was performed according to our previous study [20, 37]. Briefly, the dewaxed colon sections were subjected to antigen retrieval using sodium citrate and washed with PBS. The prepared sections were further incubated with endogenous peroxidase blockers (SAP (Mouse/Rabbit) IHC Kit, Maixin Biotechnology, Fuzhou, China) for 30 min and normal nonimmune goat serum (SAP (Mouse/Rabbit) IHC Kit, Maixin Biotechnology, Fuzhou, China) for 30 min at room temperature. The sections were further incubated with mucin-2 antibody (1:200, diluted with 5% goat serum, Affinity Biosciences, USA) overnight at 4 °C, and then the secondary antibody (goat-anti rabbit IgG) was added for 30 min of incubation at room temperature. Next, the sections were incubated with horseradish peroxidase (HRP) (SAP (Mouse/Rabbit) IHC Kit, Maixin Biotechnology, Fuzhou, China) for 20 min at room temperature and washed with PBS. After treatment with DAB color-developing fluid (Maixin Biotechnology, Fuzhou, China), the sections were stained with hematoxylin for 3 min and then differentiated with hydrochloric acid alcohol and ammonium hydroxide. After dehydration, the sections were sealed with neutral resin.
Total bacterial DNA extraction and Illumina NovaSeq sequencing
Ruminal samples from cows (n = 16, 8 cows from each group of the healthy and mastitis groups) and fecal samples from RMT-treated mice (n = 21, 7 mice from each of the control, H-RMT, and M-RMT groups) were used for bacterial DNA extraction and microbial analysis. Total genomic DNA from the ruminal and fecal samples was extracted using the CTAB method. DNA concentration and purity were monitored on 1% agarose gels. DNA was diluted to 1 ng/μL using sterile water. The 16S rRNA genes of the V4 region were amplified using a 515F-806R primer with the barcode. All PCRs were carried out with 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 2 μM forward and reverse primers, and approximately 10 ng template of DNA. Thermal cycling consisted of initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and elongation at 72 °C for 30 s. Finally, the samples were incubated at 72 °C for 5 min. The same volume of 1X loading buffer (containing SYB green) was mixed with the PCR products, and electrophoresis was performed on a 2% agarose gel for detection. Then, the PCR products were purified with a Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer’s recommendations, and index codes were added. Library quality was assessed on a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina NovaSeq platform, and 250-bp paired-end reads were generated.
Alpha diversity was performed to analyze the complexity of taxa diversity, including observed species, Chao1, ace, and PD_whole tree index. Principal coordinate analysis (PCoA) was performed to identify the microbial structures in the ruminal samples and fecal samples of the RMT mice. Linear discriminant analysis effect size (LEfSe) was performed to identify bacterial taxa that were differentially enriched in the different cows and RMT mice treatment groups [38]. Spearman correlation analysis was performed to analyze the relationship between different ruminal or gut microbial taxa and host parameters using Wekemo Bioincloud (https://www.bioincloud.tech). Tax4Fun analysis was performed to identify bacterial functions that were enriched in cows and RMT mice [39].
Total RNA extraction and quantitative RT-PCR
Total RNA was extracted from colonic tissues from RMT-treated mice (n = 7 per group) using TRIzol (Invitrogen, Carlsbad, CA, USA), and quantitative RT-PCR was performed as described previously [20]. In brief, total RNA was extracted and prepared for cDNA. Furthermore, quantitative RT-PCR was performed using TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) and then FastStart Universal SYBR Green Master Mix (ROX) (Roche, Switzerland, Basel) in a Step One Plus apparatus (Applied Biosystems, Foster City, CA, USA). The 2−ΔΔCt method was utilized to determine the gene expression. GAPDH was used as an endogenous control, and all data were normalized to the control group. The primers used in this study are detailed in Additional file 1: Table S1.
Western blotting
Total protein extraction from mammary and colonic tissues and western blotting were performed according to previous studies [19, 20]. In brief, mammary and colonic samples were weighed and homogenized using tissue protein extract (Thermo Fisher Scientific, USA). After centrifugation at 12,000×g and 4 °C for 10 min, the supernatants were harvested, and protein concentration determination was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). SDS-PAGE (12% or 15%) was performed to separate the targeted proteins according to molecular size. The targeted proteins were transferred to 0. 45-μm PVDF membranes and treated with 5% skim milk for 3 h at room temperature. Furthermore, specific primary antibodies, including p-p65, p-65, p-IκB, IκB, STING, p-TBK1, TBK1, p-IRF3, IRF3, occludin, ZO-1, and claudin-3 from Affinity Biosciences (OH, USA); TLR4, cGAS, NLRP3, ASC, and IL-1β from Cell Signaling Technology (Boston, USA); and β-actin from Immnoway Biotechnology (USA), were added for incubation at 4 °C overnight. After incubating with secondary antibodies (goat anti-rabbit IgG or rabbit anti-mouse IgG, Immnoway Biotechnology, USA) for 2 h at room temperature, the proteins were detected using an ECL plus western blotting detection system.
Statistical analysis
GraphPad Prism 8 (San Diego, CA, USA) was used for statistical analysis, and the data are expressed as the mean ± SD or represented as a boxplot. Significant differences between the two groups were evaluated by the Mann-Whitney U test (nonparametric) or two-tailed unpaired Student’s t test (parametric). For comparisons among more than two groups, a one-way analysis of variance (ANOVA) and a post hoc Tukey test were performed. For all statistical tests, p < 0.05 indicates significance. Other specific statistical analyses are mentioned in the figure legends.