Bacterial strains, cell lines and culture conditions
Pasteurella multocida strains used in this study include strain HuN001 (GenBank accession no. CP073238) and C09. Strain HuN001 was isolated from the sputum of a patient with pneumonia [14], while strain C09 was isolated from the pharyngeal swab of a cat with respiratory symptoms. Both strains are capsular type A and do not produce Pasteurella multocida toxin (PMT), a dermonecrotic toxin. Unless specified otherwise, P. multocida was cultured on tryptic soy agar (TSA; Becton, Dickinson and Company, MD, USA) or in tryptic soy broth (TSB; Becton, Dickinson and Company, MD, USA) supplemented with 5% newborn bovine serum (Tianhang, Hangzhou, China) at 37 °C for a least 12 h. Human brain microvascular endothelial cells (hBMECs) were maintained in RPMI 1640 medium (Gibco, ThermoFisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, USA) in 5% CO2 atmosphere at 37 °C.
Mouse experiment and ethics statements
To investigate the potential of P. multocida to induce BBB disruption in vivo, mouse experiments were conducted at the Laboratory Animal Center at Huazhong Agricultural University (Wuhan, China). The study received approval from the University Ethics Committees (approval no. HZAUMO-2023–0235). The experimental design involved 5–6-week-old female mice divided into three groups: G1, G2, and G3, each consisting of eight mice. In G1, mice were intranasally inoculated with P. multocida HuN001 at a dose of 50 colony-forming units (CFU) per mouse. In G2, mice were intranasally inoculated with P. multocida strain C09 at a dose of 5 × 107 CFU per mouse. As a control, mice in G3 received an intranasal administration of PBS at a volume of 50 µL per mouse (Figure 1A). The doses for challenge were determined based on the lethal doses of these two strains tested on mice in our laboratory previously. At 48 h post-inoculation (hpi), three mice from each group were euthanized, and their brain tissues were collected for histological examination and bacterial recovery. Immunohistochemical examinations were also conducted on brain tissues using a von Willebrand factor (vWF) antibody (1:100) (Abcam, UK), following previously described methods [15]. Another five mice from each group received an injection of Evans Blue dye (Sigma, USA) at a dose of 30 mg/kg body weight through the tail vein routine. After 40 min, all mice were euthanized, and the dye in the brains was extracted using formamide (2 mL) at 55 °C for 24 h (Figure 1A). The changes in BBB permeability were assessed by measuring the absorbance values (optical density at 620 nm [OD620]) of the extracted solutions [16].
In vivo tests in mouse models assessing the influence of Pasteurella multocida infection on the blood–brain barrier. A Study design of the mouse experiments. Mice were inoculated with different P. multocida strains or PBS. Evans blue (EB) was injected at 48 h post inoculation, and after 20 min, the mice were euthanized. Murine brains were collected to quantify EB dyes by measuring the absorbance values of optical density at 620 nm [OD620]. B Pathological damages (marked with black arrows) in the brains of mice inoculated with different P. multocida strains or PBS, as characterized by histological examinations (bar = 200 μm). C Expression of von Willebrand factor (vWF) (indicated by black arrows) in the brains of P. multocida-infected mice (HuN001, C09) and PBS treated mice, as characterized by immunohistochemical examinations (bar = 200 μm). D Recovery of P. multocida HuN001 from the brains of bacterium-infected mice (HuN001) and PBS treated mice. E Recovery of P. multocida C09 from the brains of bacterium-infected mice (C09) and PBS treated mice. F Visualization of the brains obtained from mice inoculated with P. multocida strains (HuN001 and C09), or the control (PBS), showing brain staining with EB dye. G Quantification of EB in the brain obtained from P. multocida-infected mice and control mice. PM refers to P. multocida.
Dextran-based trans-well permeability assay
To evaluate the impact of P. multocida infection on the barrier function of hBMECs, a dextran-based transwell permeability assay was conducted following a previously described protocol [15]. Briefly, approximately 1 × 105 hBMECs in 200 µL of antibiotic-free RPMI 1640 medium were seeded onto 24-well cell culture inserts (Labselect, Hefei, China). The cells were cultured for 36 h in a 5% CO2 atmosphere at 37 °C. Subsequently, the cells were incubated with 200 µL of antibiotic-free RPMI 1640 containing P. multocida (HuN001 or C09) at a multiplicity of infection (MOI) of 200. Additionally, 10 μM of 70-kDa fluorescein isothiocyanate (FITC; Sigma, St. Louis, MO, USA) was added to the medium. The cells were then incubated at 37 °C for 12 h under 5% CO2 atmosphere. After incubation, 100 µL of the medium from the basal chamber was transferred to a black well plate (Greiner Bio-One, Germany). The permeability of dextran was determined based on the results obtained from the plate in a Victor Nivo multimode plate reader (PerkinElmer, Waltham, MA, USA), measuring the fluorescence intensity at excitation/emission wavelengths of 490 nm/520 nm.
Quantitative real-time PCR
HBMEC monolayers were infected with P. multocida HuN001 (200 MOI) or C09 (200 MOI) and incubated at 37 °C under 5% CO2 for 4 h (for NF-κB) or 12 h. As a control, cells treated with PBS under the same conditions were included. After washing three times with PBS, total RNAs was extracted using the TRIzol reagent protocol (Invitrogen, Thermo Fisher, Waltham, MA, USA). Subsequently, cDNAs was synthesized using a PrimeScript RT reagent kit with gDNA Eraser (TAKARA, Japan), and the synthesized cDNAs was used as a template for quantitative real-time PCR (qPCR) assays to detect the transcriptional levels of various genes, including NF-κB, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGFA), tight junctions (ZO1, claudin-5, occludin), adherens junctions (E-cadherin), and chemokines (IL-1β, IL-6, TNF-α). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. Primers used for qPCR are listed in Additional file 1. All experiments were repeated thrice.
Western-blotting
To examine the expression of proteins, hBMEC monolayers were inoculated with P. multocida HuN001 (200 MOI), C09 (200 MOI), or PBS and incubated at 37 °C under 5% CO2 for 4 h (for phosphorylated P65, p-P65) or 12 h. Cells were then lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) containing protease inhibitors. The lysates were centrifuged at 4 °C, 12 000 rpm for 10 min. The protein concentration in the harvested lysates was quantified using a commercial BCA Protein Assay Kit (Beyotime, China). The proteins were separated on 10% or 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). The membranes were washed with Tris-buffered saline with Tween 20 (TBST) for five times then blocked with 5% BSA in TBST for 3 h at room temperature. Subsequently, the membranes were incubated overnight at 4 °C with specific antibodies, including HIF-1α antibody (1:1000) (catalog no. NB100-134; Novus Biologicals, USA), VEGFA polyclonal antibody (1:5000) (catalog no. 19003–1-AP; Proteintech, China), ZO1 polyclonal antibody (1:5000) (catalog no. 21773–1-AP; Proteintech, China), E-cadherin monoclonal antibody (1:1000) (catalog no. P12830; Cell Signaling, USA), Phospho-NF-κB P65 (1:1000) (catalog no. #3033; Cell Signaling, USA), and GAPDH monoclonal antibody (1:20,000) (catalog no. 60004–1-lg; Proteintech, China). Following another round of TBST washing, the membranes were incubated with species-specific horseradish peroxidase-conjugated antibodies for 1 h at room temperature. Finally, the blots were visualized using enhanced chemiluminescence (ECL) reagents (Beyotime, China), and the bands were quantified using ImageJ software (v1.8.0). The results were analyzed as the relative immunoreactivity of each protein, normalized to the respective loading controls. Additional requirements for the examination of HIF-1α have been previously described [15].
siRNA transfection and bacterial infection
To investigate the influence of HIF-1α and NF-κB on barrier function changes in hBMECs induced by P. multocida, specific small interfering RNAs (siRNAs) against HIF-1α or NF-κB (Additional file 1) were synthesized. These siRNAs were transfected into hBMECs using Lipofectamine 2000 reagent (Invitrogen, USA) following the manufacturer’s instructions. A scrambled RNA sequence at the same concentration (100 nM) was transfected as a control. The efficacy of the siRNAs in suppressing the expression of the target genes was examined using qPCR. Monolayers of both siRNA-transfected cells and control cells were then infected with P. multocida HuN001 and C09 at a MOI of 200 for 12 h. The transcriptional levels of different genes, including ZO1, E-cadherin, HIF-1α, NF-κB, TNF-1α, IL-β, and IL-6, were detected using qPCR. Additionally, the expression of ZO1 and E-cadherin was determined using western blotting, as described above.
Immunofluorescence
To observe and compare the expression of ZO1 in bacteria-infected cells and PBS-treated cells, monolayers of hBMECs were inoculated with P. multocida HuN001 (MOI = 200), C09 (MOI = 200), or PBS (50 µL) for 12 h at 37 °C under 5% CO2. After washing with precooled PBS to remove free bacteria, the cells were fixed with precooled formaldehyde for 2 h and blocked in 5% BSA at room temperature for 2 h. Subsequently, the cells were incubated overnight at 4 °C with ZO1 polyclonal antibody (1:2000). After washing with precooled PBS, cells were incubated with CoraLite488-conjugated Goat Anti-Rabbit IgG(H + L) (catalog no. SA00013-2; Proteintech, China) at 37 °C for 30 min in dark place. Finally, the cells were incubated with antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, China) for 30 min at room temperature in the dark place. The expression of ZO1 was observed under an inverted fluorescence microscope (Olympus BX53, Japan).
Laser scanning confocal microscope
Laser scanning confocal microscopy was used to examine the influence of P. multocida infection on the phosphorylation of NF-κB P65 in hBMECs. To achieve this, Monolayers of hBMECs were incubated with P. multocida HuN001 (MOI of 200), C09 (MOI of 200), or PBS (50 µL) at 37 °C for 4 h, followed by washing with precooled PBS to remove free bacteria. The cells were then fixed in pre-cooled formaldehyde for 2 h and blocked with 5% BSA for 1 h at room temperature. After washing, the cells were incubated overnight at 4 °C with a Phospho-NF-κB P65 antibody (1:2000) (catalog no. #3033; Cell Signaling Technology). Following washing with precooled PBS, the cells were incubated with CoraLite488-conjugated Goat Anti-Rabbit IgG(H + L) at 37 °C for 1 h in a dark place. Finally, the cells were incubated with antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, China) for 30 min at room temperature in the dark place. The phosphorylation of NF-κB P65 was observed under a Zeiss LSM 800 Confocal Laser Scanning Microscope and was analyzed using NIS-Elements Viewer 4.20 (Nikon, Tokyo, Japan).
Transmission electron microscope
To examine the strategy used by P. multocida to migrate the barrier formed by hBMECs, samples were prepared for transmission electron microscopy following the method described in a previously published article [17]. Briefly, monolayers of hBMECs were incubated with P. multocida HuN001 (MOI = 200) or PBS (50 µL) at 37 °C for 3 h. After washing three times with PBS, the bacterium-infected cells or PBS-treated cells were divided into two groups. One group of cells was fixed using a commercial electron microscope fixative (code: G1102, Servicebio, Wuhan, China) for 5 min in dark place, while another group of cells was treated with kanamycin (100 μg/mL) and ampicillin (100 μg/mL) for 30 min at 4 °C to remove extracellular bacteria. After washing thrice with PBS, antibiotic-treated cells were fixed using a commercial electron microscope fixative for 5 min in the dark. Thereafter, the cells were scraped and collected by centrifugation at 2000 rpm for 5 min. The cells were then resuspended in fresh electron microscope fixative and fixed for 30 min in the dark. The fixed cells were then sent to the National Key Laboratory of Agricultural Microbiology Core Facility at Huazhong Agricultural University for slide preparation. The prepared slides were observed under a 100-kV transmission electron microscope (H-7650, HITACHI, Japan).
Statistical analyses
Statistical analysis was performed using the multiple-t-test strategy in GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Data represent mean ± standard deviation (SD). The significance level was set at a P value of < 0.05 (*), a P value of < 0.01 (**), or a P value of < 0.001 (***).
Add Comment