Participants
We used the criteria of the National Institute on Aging and the Alzheimer’s Association for AD diagnosis [20], and published criteria [21] for amnestic mild cognitive impairment (aMCI) diagnosis. We recruited 33 patients with AD and 33 patients with aMCI from the Department of Neurology, Affiliated Brain Hospital of Nanjing Medical University, from January 2019 to April 2020. For controls, we recruited 20 age-matched control participants with (i) no history of infectious, inflammatory, and autoimmune diseases; (ii) no history of psychiatric and memory disorders; and (iii) no history of prescription and non-prescription drug use. All participants or their legal guardians provided informed written consent. This study was approved by the Institutional Review Board of the Affiliated Brain Hospital of Nanjing Medical University.
Collection of PBMCs from blood
Blood samples were collected in the morning and processed within 4 h. Approximately 3 mL of anti-coagulated whole blood was centrifuged at 3500 rpm for 3 min at 4°C, and the plasma was collected and stored at -80°C for the measurement of cytokines. To obtain PBMCs, 2 mL of whole blood was diluted with phosphate-buffered saline (PBS, 1:1) and transferred to centrifuge tubes containing 3 mL of Ficoll Paque (GE Healthcare, Uppsala, Sweden). After centrifuging at 400 × g for 20 min at room temperature, PBMCs were collected and washed twice in 10 mL of PBS. Cell pellets were re-suspended in TRIzol or lysis buffer for the isolation of RNA or protein, respectively.
Quantitative polymerase chain reaction (qPCR) analysis
Total RNA extraction, cDNA synthesis, and qPCR reactions were performed according to the manufacturer’s instructions. TRIzol reagent (Invitrogen, CA, USA) was used to extract RNA from PBMCs, and RNA was used for subsequent cDNA synthesis. qPCR was performed using SYBR Premix Ex Taq (Takara Bio, Dalian, China). The following primers were used: Il-1b (5´-TGTAGTGGTGGTCGGAGATT-3´, forward;5´-ATGATGGCTTATTACAGTGGC-3´, reverse), Nlrp3 (5´-AGGGCGTTGTCACTCAGGT-3´, forward; 5´-TCGGAGATTGTGGTTGGG-3´, reverse), Gsdmd (5´-AGTGCCAGGGAGGCGTAGAGT-3´, forward; 5´-TGGGTCTTGCTGGACGAGTG-3´, reverse), Caspase1 (5´-GGAAGAGCAGAAAGCGATAA-3´, forward; 5´-TTGAAGGACAAACCGAAGG-3´, reverse), Caspase4 (5´-TGCCAGGAAAGAGGTAGAAA-3´,forward; 5´-TCGGAAGGTACAGCAATCA-3´, reverse), Nlrc4 (5´-GACTAATGCTGGATCAGGTAG-3´, forward; 5´-TTTGGCGGGAAATCGTGT-3´, reverse), Aim2 (5´-TCAGTACCATAACTGGCAAA-3´, forward; 5´-AGAAATGATGTCGCAAAGC-3´, reverse), Nlrp1 (5´-AACGTAGAACTCCGAGAAC-3´, forward; 5´-CGAATCCACAAGCCACCC-3´, reverse), and Gapdh (5′-GAAGGTGAAGGTCGGAGTC-3′, forward; 5′-GAAGATGGTGATGGGATTTC-3′, reverse). The relative expression level of each target gene was calculated using a standard curve and normalized against Gapdh expression level.
Immunofluorescence staining
PBMCs were collected, fixed with 4% paraformaldehyde (PFA) in 1.5-mL centrifuge tubes at 4 ℃ for 30 min, washed twice in PBS, blocked with 3% goat serum, and incubated with primary antibody: rabbit anti-cleaved N-terminal GSDMD (1:50, Abcam, JHY, UK), that were diluted with PBS containing 3% serum and 2 mM EDTA at 4 ℃ for 12 h, followed by treatment with secondary antibody: Alexa Fluor 555 anti-rabbit (1:500, Invitrogen, CA, USA). For animal experiments, mouse brains were fixed with 4% PFA, dehydrated with 30% sucrose, embedded in optimal cutting temperature compound, and sectioned at 25-µm thickness. Tissue slices were blocked, incubated with primary antibodies: rabbit anti-IBA1 (1:500, Wako, TKY, JPN), mouse anti-GFAP (1:500, Sigma, CA, USA), or mouse anti-NeuN (1:200, MilliporeSigma, CA, USA), and treated with secondary antibodies: Alexa Fluor 488 anti-rabbit (1:500, Invitrogen, CA, USA), and Alexa Fluor 555 anti-mouse (1:500, Invitrogen, CA, USA), followed by staining with 4′,6-diamidino-2-phenylindole (DAPI) or thioflavin S (Sigma-aldrich, MO, USA).
Collection of cerebrospinal fluid (CSF)
CSF was collected by lumbar puncture in the morning at the L3/L4 or L4/L5 level. The first 20 drops of CSF were discarded, followed by the collection of approximately 2 mL of CSF in a polypropylene tube. The samples were centrifuged for 10 min at 2000 × g at room temperature to eliminate cells and other insoluble materials, aliquoted, and stored at -80°C until further processing.
Enzyme-linked immunosorbent assay (ELISA)
Plasma and CSF samples were collected from participants to determinate the protein levels of AD biomarkers and IL-1β. The levels of AD biomarkers including Aβ1–40, Aβ1–42, p-tau-181, t-tau were measured with commercially available ELISA kits (INNOTEST, Fujirebio, Ghent, Belgium) according to the test procedure in manufacturer's protocols. Levels of IL-1β in plasma and CSF were measured with commercial ELISA kits (Jingmei, Yancheng, China). A microplate reader (Antobio, Zhengzhou, China) was used to obtain absorbance readings. For animal experiments, serum and brain tissue were collected from mice and used to detect the protein level of IL-1β.
Western blot analysis
Approximately 1.5 × 106 PBMCs were mixed with 70 µL of loading buffer (Yeasen, Shanghai, China) and then boiled in a water bath set to 99 ℃ for 10 min. The protein samples and PageRuler Prestained Protein Ladder were loaded onto SDS-polyacrylamide gels, and then blotted onto PVDF membranes. The blots were incubated overnight at 4 ℃ with the following primary antibodies: mouse anti-NLRP3 (1:1000, Adipogen Corporation, CA, USA), rabbit anti-caspase-1 (1:1000, Abcam, JHY, UK), rabbit anti-GSDMD (1:200, Novus, Littleton, USA), mouse anti-IL-1β (1:1000, R&D Systems, Minneapolis, USA), mouse anti-β-actin (1:4000, Sigma-aldrich, MO, USA). The membranes were then washed three times with Tris-buffered saline and incubated with secondary antibodies: IRDye 800CW goat-anti-mouse/rabbit (1:3000, LI-COR Biosciences, Lincoln, USA). Image J software was used to analyze the intensities of the bands. In the animal study, mouse spleens were harvested and homogenized 1:10 in homogenization buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM sodium vanadate, 1% Triton X-100, 1 mg/mL leupeptin, and protease inhibitor cocktail, pH 7.5). The concentrations of the protein samples were quantified with the bicinchoninic acid method (Bio-Rad, Hercules, CA, USA) after centrifuging at 12000 × g for 10 min. The protein samples were then subjected to western blotting analysis as indicated above.
RNA-seq analysis
For RNA-seq, PBMCs were obtained. RNA isolation, cDNA library construction, and RNA-seq were performed with the BGISEQ-500 system (Beijing Genomic Institution). Clean reads were mapped to the human genome (hg38) by HISAT2. Thereafter, the matched reads were calculated and normalized to FPKM. Fold changes were calculated for all possible comparisons. To select genes with significant changes in expression, a 1.5-fold cutoff was used. Gene Ontology biological process (GO-BP) pathway analyses were performed using the R package. Target genes were filtered using significantly different gene expression (P < 0.05). Raw data files and processed files have been deposited in the Gene Expression Omnibus under accession no. GSE265984.
Treatment of mice with lipopolysaccharides
Animals and treatments
We used 5-month-old 5×FAD mice, kindly provided by Dr. Ming Xiao (Nanjing Medical University, Jiangsu, China), which carried mutations in both the human amyloid precursor protein (APP695) and presenilin-1 (PSEN1) genes. The APP gene contains three familial AD (FAD) mutations: Swedish (K670N, M671L), Florida (I716V), and London (V717I). The PSEN1 gene contains two FAD mutations: M146L and L286V. Low-dose lipopolysaccharides (LPS; 500 µg/kg) or PBS control was injected intraperitoneally 48 hours before the mice were sacrificed. In GSDMD inhibitor treatment experiments, disulfiram (50 mg/kg) or PBS was injected intraperitoneally 4 hours before challenge with LPS. Serum, brain, and spleen tissues were collected for ELISA, western blotting, and immunofluorescence analyses, as described above (n = 3/group). Animal experiments were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and all animal procedures were approved by the Ethical Review Committee for Laboratory Animal Welfare of Nanjing Medical University.
Statistical analysis
Data are shown as the means ± standard error of the mean (SEM). Statistical analysis was performed with one-way ANOVA followed by Sidak's multiple comparisons test or unpaired t test. The correlative analysis was performed using a linear regression model. Chi-square test was used for the analysis of discrete variable such as sex. All statistical analyses in this study were performed in GraphPad Prism 6.0 software (Graph Pad Software Inc., San Diego, CA, USA). P values are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001.