Succinate exacerbates mastitis in mice via extracellular vesicles derived from the gut microbiota: a potential new mechanism for mastitis

Animals and experimental design

The 6–8 weeks old SPF grade BALB/c mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. After feeding for one week to adapt to the environment, the mice were housed in a 3:1 ratio of male to female, and pregnant female mice were taken for experiments.

For the succinate supplementation experiment, pregnancy mice were separated into four groups randomly, referred to as (1) Control group (C): Mice drinking normal water; (2) Succinate group (S): mice treated with 2.5% succinate (Solarbio, Beijing, China) in water for 28 days (From gestational day 1.5 to postnatal day 10); (3) Low-grade endotoxemia group (L): mice were injected intraperitoneally with LPS (2 µg/mice daily) for 10 days from the first day of delivery to simulate low-grade endotoxemia at the time of SARA; (4) Low-grade endotoxemia + Succinate group (LS): mice were injected intraperitoneally with LPS for 10 days from the first day of delivery accompanied with succinate treatment as mentioned above. At the day 10 after delivery, mice were sacrificed and mammary gland, colon, feces, serum and liver were harvested and stored at -80 ℃ until detection.

For the mEVs oral treatment experiment, pregnancy mice were separated into six groups randomly referred to as (1) Control group (C): Administer 100 µL of PBS orally; (2) In control gut microbiota-derived extracellular vesicles group (CEV): Control group 100 mg of intestinal contents were homogenized in 0.02 μm filtered PBS and mEVs were separated by differential centrifugation. The model was established according to previously described methods [26]. In brief, CEV was administered orally to mice every day for 4 weeks (From gestational day 1.5 to postnatal day 10); (3) In succinate gut bacteria-derived extracellular vesicles group (SEV): Succinate group 100 mg of intestinal contents were homogenized in 0.02 μm filtered PBS and mEVs were separated by differential centrifugation. Treatment as mentioned above CEV; (4) LPS group: Experimental treated as above mice were treated intraperitoneally with LPS (2 µg/mice daily); (5) LPS + CEV group: Mice were injected intraperitoneally with LPS while undergoing oral treatment with CEV as described above; (6) LPS + SEV group: Mice were injected intraperitoneally with LPS while undergoing oral treatment with SEV as described above. On day 10 after delivery, mice were sacrificed and mammary gland colon, feces, serum and liver were harvested and stored at -80 ℃ until detection. For biodistribution of orally administrated mEVs, experimental grouping as above. After oral administration of PKH26 fluorescent dye labeled mEVs, female mice were euthanized at 16 h time points, and the intestine, liver, and mammary gland were collected to monitor the distribution of mEVs. PKH26 fluorescent signals were detected and measured using the laser confocal microscopy.

For the mEVs intravenous injection treatment experiment, pregnancy mice were separated into four groups randomly referred to as (1) C group: Intravenous injection of 100 µL PBS; (2) CEV group: The model was established according to previously described methods [22]. In brief, mice were tail vein injected with CEV twice per week; (3) SEV group: Mice were tail vein injected with SEV twice per week; (4) Low-concentration succinate gut bacteria-derived extracellular vesicles group (LSEV): LSEV group with similar dose as CEV, treatment as mentioned above CEV. Collection of the mammary gland, colon, feces, serum and liver samples after 4 weeks.

Cell culture and treatment

MMECs (HC11cells) and RAW 264.7 cells were obtained from ATCC and cultured in complete Dulbecco’s modified Eagle’s medium (Hyclone, USA) containing 10% FBS (Bovogen Biologicals), 1% Penicillin and Streptomycin at 37 ℃ with 5% CO2. MMECs (1 × 106 cells) and RAW 264.7 cells were preincubated in 6-well plates for 24 h and then treated with mEVs (including the CEV, SEV and LSEV) for the next 24 h to collect the cells. For TLR4 inhibition, cells were pretreated with resatorvid (MedChemExpress, China) at a concentration of 10 nM for 2 h, and then mEVs (including the CEV, SEV and LSEV) were treated for 24 h. The supernatants were collected for cytokines detection by ELISA, and the cells were collected for protein determination by western blotting.

mEVs isolation and nanoparticle tracking analysis (NTA)

100 mg of feces was homogenized in 0.02 μm filtered PBS and mEVs isolated by differential centrifugation, as described previously [27, 28]. Briefly, the homogenate was centrifuged at 340 × g for 10 min at 4 °C. Supernatant was then centrifuged at 10,000 × g for 20 min at 4 °C followed by 18,000 × g for 45 min at 4 °C, filtered through a 0.22 μm filter and centrifuged at 100,000 × g for 2 h at 4 °C. mEVs pellet was resuspended in sterile PBS. mEVs particle size and concentration were assessed by NTA on a Nanosight NS300 (Malvern Instruments Limited). NTA 3.2 software was used to calculate mEVs concentration and size distribution.

1 mL of plasma was pooled from mice. To collect mEVs from plasma, plasma (0.5 mL) was diluted with 2 mL sterile PBS and passed through a 0.22 μm filter. The supernatant was then subjected to ultracentrifugation at 100,000 × g for 4 h at 4 °C with a SW60 Ti swinging-bucket rotor (Beckman Coulter). After ultracentrifugation, both plasma mEVs pellets were assessed by NTA, as shown above.

In vivo mEVs trafficking assays

To monitor mEVs transport, we used the PKH26 Fluorescent Cell Linker Kit (Cat. No. PKH26GL-1KT, Sigma) to label CEV and SEV with PKH26 fluorescent dye. After PKH26 staining, CEV and SEV were washed with PBS and collected by ultracentrifugation (100,000 × g, 2 h) at 4 °C. Finally, the PKH26-labeled CEV and SEV were resuspended in sterile PBS to be used. For the mEVs trafficking assays experiment in mice, mice were tail vein injected PKH26-labeled CEV and SEV. 16 h after CEV and SEV injection, portions of the liver, colon, and mammary gland were collected for detection of the appearance of PKH26 red fluorescence.

Electron microscopy

Samples were deposited on Formvar carbon coated, glow-discharged grids. Then the grids were placed on 2% gelatin at 37 oC for 20 min, rinsed with 0.15 M glycine/PBS and the sections blocked using 1% cold water fish-skin gelatin. Grids were viewed using a JEOL 1200EX II (JEOL) transmission electron microscope and photographed using a Gatan digital camera (Gatan).

Histological analysis

The mammary gland tissue was collected and fixed with 4% formaldehyde 24 h. The mammary gland tissue was dehydrated, embedded in paraffin, and cut into 5-µm sections. After staining with hematoxylin and eosin (H&E), the sections were observed under a light microscope.

Mammary cytokine detection

The mammary gland tissue was collected and the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the mammary gland tissue were measured using ELISA kits according to the manufacturer’s instructions (BD Biosciences).

Myeloperoxidase (MPO) activity assay

The mammary gland tissue was taken and homogenized on ice with reaction buffer (weight to volume ratio 1:19). The MPO activity assay was performed according to the manufacturer’s instructions (Nanjing Jiancheng Institute of Biological Engineering, Nanjing, 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 [29]. The sections were further stained using an Alcian Blue Stain Kit according to the manufacturer’s instructions (Solarbio, Beijing, China).

Serum cytokines and LPS determination

Serum LPS levels from mice were detected as previously described [30]. In brief, the serum was 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 cytokines in mice were detected using mice TNF-α and IL-1β ELISA kits (Biolegend, CA, USA).

The limuius amebocyte lysate (LAL)

We quantified the mEVs related LPS by the LAL test [31]. The LPS activity was performed according to the manufacturer’s instructions (Associates of Cape Cod, Massachusetts, USA).

Immunohistochemistry

The paraffin-embedded glass slides were dewaxed in xylene and different concentrations of alcohol. The antigen is restored with 0.01 M citrate buffer. Incubate in 3% H2O2 and then in diluted goat serum. The sections were incubated with primary antibodies: Claudin-3 (#AF0129, Affinity Biosciences, USA) and ZO-1 (#AF5145, Affinity Biosciences, USA) antibodies at 4 °C. After washing 3 times, the sections were incubated with HRP-labeled goats with anti-rabbit secondary antibody (ZS-Bio, Beijing, China) at 37 °C for 15 min. This section was stained with DAB and observed under a microscope.

Immunofluorescence staining

Parts of the liver, colon, and mammary gland of CEV or SEV mice were snap-frozen at optimum cutting temperature (O.C.T., Fisher Healthcare) with dry ice. 6-µm cryo-sections of tissue sections were cut and fixed with pre-cold acetone for 20 min. After washing, nuclei were stained with DAPI for 10 min at room temperature. Mounting media and coverslips were then added to slides for imaging to track mEVs ectopic conditions. RAW. 264.7 monolayers were fixed in 4% paraformaldehyde for 20 min at room temperature. They were then blocked with 2% BSA and were performed with a primary anti-TLR4 rabbit monoclonal antibody (Invitrogen, Rockford, USA) 2.5 µg/mL and a secondary Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, Rockford, USA) 1 µg/mL for 1 h at room temperature. Nuclei were stained with DAPI for 10 min at room temperature. Final imaging was performed. Imaging was performed using a Leica DMI 60,000 microscope coupled to an Andor DSD2 confocal scanner and a Zyla 5.5 CMOS camera.

Western blotting

The mammary gland tissue was collected. The total proteins from the mammary gland tissue were extracted by tissue protein extraction solution (T-PER, Pierce). Equal amounts of proteins were separated by 12% SDS-PAGE and transferred onto 0.22 mm PVDF membranes. After blocking with 5% nonfat milk for 2 h, the membranes were probed with primary antibodies, TLR4 (#AF7017, Affinity Biosciences, USA), p-p65 (#AF2006, Affinity Biosciences, USA), p65 (#BF8005, Affinity Biosciences, USA), p-IκB (#AF2002, Affinity Biosciences, USA), IκB (#AF5002, Affinity Biosciences, USA), Claudin-3, Occludin (#DF7504Affinity Biosciences, USA), ZO-1, and β-actin (YT0099, Immnoway Biotechnology, USA) at 4 °C overnight. The membranes were washed three times and probed with horseradish peroxidase-labeled secondary antibodies. Finally, PVDF membranes were visualized using the ECL plus western blotting detection system from Tanon (Shanghai, China).

16 S rRNA gene sequencing and bioinformatics

Fecal samples from succinate treated mice (n = 12, 6 mice from each of the control and succinate groups) were used for bacterial DNA extraction and microbial analysis. Total genomic DNA from the fecal samples was extracted using the CTAB method. DNA concentration and purity were monitored on 1% agarose gel. DNA was diluted to 1 ng/µL using sterile water. The 16 S rRNA genes of the V3-V4 region were amplified using primer pairs 338 F (5′-ACT CCT ACG GGA GGC AGC AG-3′) and 806R (5′-GGA CTA CHVGGG TWT CTAAT-3′) 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 1 × 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 bppaired-end reads were generated. Shannon, Sobs, and Simpson indices were performed for alpha diversity analysis. Principal coordinate analysis (PCoA) was performed to identify microbial structures and Linear discriminant analysis effect size (LEfSe) was performed to identify bacterial taxa that were differentially enriched in different treatment groups. The Circos plot was performed to reveal microbial composition by R (v 3.2.0). Spearman correlation analysis was performed to examine the relationships between different gut microbial taxa and host parameters, utilizing Wekemo Bioincloud (https://www.bioincloud.tech).

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. Other specific statistical analyses are mentioned in the figure legends.

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