NLRP3-encoding gene knockout (NLRP3-/-) mice were kindly provided by N. Fasel (University of Lausanne, Lausanne, Switzerland) . Mice with knockout of gene encoding myeloid differentiation primary response 88 (MyD88-/-) were originally provided by S. Akira and K. Takeda (Osaka University, Osaka, Japan) . Breeding between heterozygous mutants (+/-) on a C57BL/6 background were used to maintain mouse colonies. Mice were compared only between littermates. Animal experiments were performed in accordance with all relevant national rules and were authorized by the local research ethical committee.
Tissue collection and isolation of blood vessels
Animals were euthanized by inhalation of isofluorane and perfused with ice-cold phosphate-buffered saline. The brain was removed and divided. The left hemisphere was immediately fixed in 4% paraformaldehyde (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in phosphate-buffered saline and embedded in paraffin for immunohistochemistry. The cortex and hippocampus were carefully dissected from the right hemisphere, snap-frozen in liquid nitrogen and stored at -80°C for biochemical analysis. Cortex and hippocampus were also used for isolation of brain vessel fragments according to the published protocol . Briefly, brain tissues were homogenized in HEPES-contained Hanks' balanced salt solution (HBSS) and centrifuged at 4,400 g in HEPES-HBSS buffer supplemented with dextran from Leuconostoc spp. (molecular weight ~ 70,000; Sigma-Aldrich) to delete myelin. The vessel pellet was re-suspended in HEPES-HBSS buffer supplemented with 1% bovine serum albumin (Sigma-Aldrich) and filtered with 20 µm -mesh. The blood vessel fragments were collected on the top of filter and frozen at -80°C for further biochemical analysis.
Histological image acquisition and analysis
Serial 40-μm-thick sagittal sections were cut from the paraffin-embedded hemisphere. Four serial sections per mouse with 400µm of interval between two neighboring sagittal sections were stained with rabbit anti-PDGFRβ monoclonal antibody (clone: 28E1; Cell Signaling Technology Europe, Frankfurt am Main, Germany) and Alexa488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Darmstadt, Germany). The coverage of PDGFRβ staining-positive cells in the whole hippocampus and cortex was estimated with the Cavalieri method on a Zeiss AxioImager.Z2 microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany) equipped with a Stereo Investigator system (MBF Bioscience, Williston, VT, USA). The grid size was set at 10 µm, which provided coefficient of error estimates of < 0.05.
To quantify vasculature in the brain, our established protocol was used . Briefly, 4 serial paraffin-embedded sections per mouse were deparaffinized, heated at 80°C in citrate buffer (10mM, pH = 6) for 1 hour and digested with Digest-All 3 (Pepsin) (Thermo Fisher Scientific) for 20 minutes. Thereafter, brain sections were stained with rabbit anti-collagen IV polyclonal antibody (Catalog: # ab6586; Abcam, Cambridge, UK) and Alexa488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific). After being mounted, the whole brain including hippocampus and cortex was imaged with Microlucida (MBF Bioscience). The length and branching points of collagen type IV staining-positive blood vessels were analyzed with a free software, AngioTool (http://angiotool.nci.nih.gov) . The parameters of analysis for all compared samples were kept constant. The length and branching points were adjusted with area of interest.
Western blot analysis of PDGFRβ and CD13 in cerebral blood vessels
Isolated blood vessels were lysed in RIPA buffer (50mM Tris [pH 8.0], 150mM NaCl, 0.1% SDS, 0.5% sodiumdeoxy-cholate, 1% NP-40, and 5mM EDTA) supplemented with protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) on ice. The tissue lysate was sonicated before being loaded onto 10% SDS-PAGE. For Western blot detection, rabbit monoclonal antibodies against PDGFRβ and CD13/APN (clone: 28E1 and D6V1W, respectively; Cell Signaling Technology Europe) were used. In the same sample, β-actin was detected as a loading control using rabbit monoclonal antibody (clone: 13E5; Cell Signaling Technology Europe). Western blots were visualized via the ECL method (PerkinElmer LAS GmbH, Rodgau, Germany). Densitometric analysis of band densities was performed with ImageJ software (https://imagej.nih.gov/ij/). For each sample, the protein level was calculated as a ratio of target protein/β-actin.
Culture of pericytes
Human primary brain vascular pericytes (HBPC) were immortalized by infecting cells with tsSV40T lentiviral particles . The selected immortalized HBPC clone 37 (hereafter referred to as HBPC/ci37) was used for our study. HBPC/ci37 cells were cultured at 33 °C with 5% CO2/ 95% air in pericyte medium (Catalog: # 1201; Sciencell Research Laboratories, Carlsbad, CA, USA) containing 2% (v/v) fetal bovine serum, 1% (w/v) pericyte growth factors, and penicillin-streptomycin. Culture flasks and plates were treated with Collagen Coating Solution (Catalog: # 125-50; Sigma-Aldrich). HBPC/ci37 cells were used at 40 ~ 60 passages in this study.
Analysis of pericyte proliferation and apoptosis
Pericytes were seeded at 1.0 × 104 cells on 96-well plate /100µl (day 0), and cultured in pericyte medium containing NLRP3 inhibitor, MCC950 (Catalog: # PZ0280; Sigma-Aldrich), at 0, 25, 50 and 100 nM. The cell survival was detected with MTT-based Cell Proliferation Kit I (Catalog: # 11465007001; Sigma-Aldrich) on days 1, 2, 3, 4, 5, 6 and 7. In order to further detect cell death and proliferation of pericytes, cells were cultured in 12-well plate at 5.0 × 105 cells/well, and treated with MCC950 as described in MTT assay. After 24 hours, pericytes were collected and lysed in RIPA buffer. Quantitative Western blot was used with rabbit monoclonal antibody against cleaved caspase-3 (clone: 5A1E; Cell Signaling Technology Europe), mouse monoclonal antibody against proliferating cell nuclear antigen (PCNA) (clone: PC10; Cell Signaling Technology Europe) and rabbit monoclonal antibody against Ki-67 (clone: SP6; Abcam). α-tubulin and β-actin were detected as an internal control with mouse monoclonal antibody (clone: DM1A; Abcam) and rabbit monoclonal antibody (clone: 13E5; Cell Signaling Technology Europe), respectively.
Treatments of pericytes for detection of PDGFRβ and CD13 and phosphorylated kinases
Pericytes were cultured in 12-well plate at 5.0 × 105 cells/well. Before experiments, we replaced culture medium with serum-free pericyte medium and cultured cells at 37°C for 3 days to facilitate cell differentiation . Thereafter, pericytes were treated for 24 hours with MCC950, at 0, 25, 50 and 100 nM, recombinant human IL-1β (Catalog: # 201-LB; R&D Systems, Wiesbaden, Germany) at 0, 5, 10 and 50 ng/ml, or AKT Inhibitor VIII (Catalog: # 124018; Sigma-Aldrich) at 0, 0.5, 1 and 5 µM. Cell lysate was prepared in RIPA buffer supplemented with protease inhibitor cocktail (Roche Applied Science) and phosphatase inhibitors (50 nM okadaic acid, 5 mM sodium pyrophosphate, and 50 mM NaF; Sigma-Aldrich). For Quantitative Western blot, the following antibodies were used: rabbit monoclonal antibodies against PDGFRβ, CD13/APN, phosphorylated AKT (Ser473), phosphorylated ERK1/2 (Thr202/Tyr204), phosphorylated NFkB p65 (S536), NFkB p65, β-actin, GAPDH (clone: 28E1, D6V1W, D9E, D13.14.4E, 93H1, D14E12, 13E5, and 14C10, respectively; Cell Signaling Technology Europe), rabbit polyclonal antibodies against AKT and phosphorylated GSK-3β (Ser9) (Catalog: # 9272 and Catalog: # 9336, respectively; Cell Signaling Technology Europe) and mouse monoclonal antibodies against ERK1/2 and GSK-3β (clone: L34F12 and 3D10, respectively; Cell Signaling Technology Europe) and α-tubulin (clone: DM1A; Abcam).
Data was presented as mean ± SEM for mice and mean ± SD for cells. For multiple comparisons, one-way or two-way ANOVA followed by Bonferroni or Tukey post hoc test. Two independent-samples Students t test was used to compare means for two groups of cases. All statistical analyses were performed with GraphPad Prism 5 version 5.01 for Windows (GraphPad Software, San Diego, CA, USA). Statistical significance was set at p < 0.05.