Animal experiments

Female mice with the C57BL/6 background were used in this study. 5×FAD (Tg6799) mice were kindly provided by Dr. Ming Xiao (Nanjing Medical University, Jiangsu, China). Gsdmd−/− mice and Cx3cr1-Cre mice were gifts from Dr. Shuo Yang (Nanjing Medical University, Jiangsu, China). We crossed 5×FAD mice with Gsdmd−/− mice to generate Gsdmd−/−5×FAD mice. To obtain myeloid cell conditional knockout mice (Gsdmdfl/flCx3cr1-Cre), Gsdmdfl/fl mice were generated via conditional gene targeting methods as described previously [22]. These mice were crossed with Cx3cr1-Cre mice. Then, Gsdmdfl/flCx3cr1-Cre mice were crossed with 5×FAD mice to generate myeloid cell-conditional GSDMD-depleted AD mice (Gsdmdfl/flCx3cr1-Cre;5×FAD). All 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 Tongji University School of Medicine. Mice were bred in a barrier facility with a 12-h light/dark cycle at a temperature of 18–23 °C and a humidity of 40–60%.

Behavior and short-term memory analysisMorris water maze (MWM)

A circular pool with a diameter of 150 cm and a height of 60 cm was filled with water to a depth of 40 cm. Food grade titanium dioxide powder (nontoxic) was added to the water for opacity. The pool was divided into four quadrants. The white escape platform was located in the center of one quadrant and submerged 1 cm below the water surface. Animals were subjected to four training trials each day for a total of 5 days as the learning phase. Each animal was given 60 s to locate the hidden platform; if the animal could not find the platform within 60 s, they were placed on the platform for 10 s. After 5 days of training, the hidden platform in the target quadrant was removed. On the sixth day, the mice were allowed to swim in the pool for 60 s, and the number of crossings over the previous position of the platform and the time spent in the target quadrant were recorded.

Y maze test (Y-maze)

The Y-maze test was used to assess short-term working memory. The Y-maze consists of three arms (marked as A, B, and C). The task was divided into two stages (training and testing). In the training stage, food was placed in one of the three arms. Then, the mice were placed at the end of the starting arm facing the wall and allowed to explore the maze for 10 min. Twenty-four hours later, the food in the target arm was removed, and the test stage was performed in which the mice were permitted to explore the three arms for 5 min. The number of target arm crossings was recorded, and the ratio of time spent in the target arm compared to the other arms was recorded.

Novel object recognition (NOR) and novel location recognition (NLR) tests

The NOR and NLR test apparatus was a white opaque cube. The experiment was divided into three stages. The first stage was the adaptation period in which the mice were allowed to move freely within the apparatus (without objects) for 10 min. The second stage was the familiarization period with two objects (same color, shape, and size) at the bottom of the apparatus. Mice were placed in the apparatus facing one sidewall and allowed to explore freely for 10 min. Mouse exploration time and the number of times on each object were recorded. The third stage was the testing period, during which memory was tested and evaluated as a timed interval. It took place 1 h after the second stage. For the NOR test, one of the two identical objects was replaced by a different object. For the NLR test, the location of one object was changed. Then, the mice were placed in the apparatus with their backs facing the object from an equal distance and allowed them to explore freely for 5 min. The preference for novel and familiar objects or location was recorded to calculate the novel object/location discrimination index.

Histology and immunohistochemistry

After completing the behavioral tests, mice were anesthetized and sacrificed, brain and spleen tissues were fixed with 4% paraformaldehyde and embedded in paraffin. For immunohistochemistry, and after heat-induced antigen retrieval, the sections were blocked with 3% H2O2 for 15 min and 5% goat serum for 30 min at room temperature. The sections were then incubated with primary antibody overnight at 4 °C and treated with horseradish peroxidase (HRP)-labeled secondary antibody followed by diaminobenzidine (DAB) staining. For immunofluorescence, the sections were permeabilized in PBS supplemented with 0.25% Triton X-100 (PBST) for 15 min, blocked, and then incubated with primary antibodies diluted in 0.1% PBST overnight at 4 °C. The next day, the sections were incubated with the corresponding fluorescence-labeled secondary antibodies and treated with 4′,6-diamidino-2-phenylindole (DAPI) or thioflavin S (Sigma-Aldrich, St. Louis, MO, USA). Primary antibodies were specific for: β-Amyloid (4G8; 1:500; SIG-39200; Biolegend, San Diego, CA, USA), GSDMD (1:500; ab219800; Abcam, Cambridge, MA, USA), caspase-1 (1:200; AG-20B-0042; AdipoGen, San Diego, CA, USA), caspase-11 (1:100; NB120-10454; Novus, Littleton, CO, USA), caspase-8 (1:100; sc-5263; Santa Cruz, CA, USA), IBA1 (1:500; 019-19741; Wako, Osaka, Japan), IBA1 (1:500; GB12105; Servicebio, Wuhan, China), GFAP (1:500; G3893; Sigma-Aldrich), NeuN (1:300; MAB377; Merck Millipore, Billerica, MA, USA), CD11b (1:300; MA1-80091; Invitrogen, Carlsbad, CA, USA), CD3 (1:200; GB12014; Servicebio), CD8 (1:200; GB114196; Servicebio), TMEM119 (1:300; ab209064; Abcam), P2RY12 (1:500; 55043; AnaSpec, Fremont, CA, USA), PD-1 (1:1000; 66220-1-Ig; Proteintech, Rosemont, IL, USA), and MHC-II (1:200; 107650; Biolegend). The secondary antibodies used were: Alexa Fluor 488-conjugated anti-rabbit (1:500; 111-545-144; Jackson ImmunoResearch, West Grove, PA, USA), Alexa Fluor 555-conjugated anti-mouse (1:500; GB21301; Servicebio), and Alexa Fluor 555-conjugated anti-rat (1:500; A-21434; Invitrogen) antibodies. Images were acquired with a Leica DM3000 microscope and processed using Image-Pro Plus software.

Western blot analysis

Mouse brains and spleens were quickly collected on ice and homogenized at 1:10 (w/v) in NP-40 lysis buffer (50 mM Tris–HCl, pH 7.4, containing 150 mM NaCl, 1% [vol/vol] Igepal, 10% [wt/vol] glycerol, 50 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol, and 1 mM phenylmethylsulphonyl fluoride, supplemented with protease inhibitor cocktail), and then incubated at 4 °C for 1 h. Equal amounts of protein were mixed, dissolved in 5× SDS/PAGE sample buffer, heated for 10 min at 100 °C, separated on SDS–polyacrylamide gels and blotted onto polyvinylidene difluoride (PVDF) membranes. For clinical samples, approximately 1.5 × 106 PBMCs were combined with 70 µl of sample buffer, processed as above, and PVDF membranes incubated overnight at 4 °C with the following primary antibodies specific for: caspase-1 (1:1000; ab179515; Abcam), GSDMD (1:1000; ab209845; Abcam); Cleaved-caspase-8 (1:1000; 8592; Cell Signaling Technology, Danvers, MA, USA), caspase-11 (1:1000; NB120-10454; Novus), IκB-α (1:1000; 9242; Cell signaling Technology), p-IκB-α (Ser32/Ser36) (1:1000; 9246; Cell signaling Technology), NF-κB (p65) (1:1000; 8242; Cell Signaling Technology), p-NF-κB (p-p65) (1:1000; 3033; Cell Signaling Technology), PD-1 (1:1000; 66220-1-Ig; Proteintech), and β-Actin (1:1000; 4967; Cell Signaling Technology). The next day, after being washed three times in TBST, the membranes were incubated with the following secondary antibodies: HRP-conjugated anti-rabbit (1:1000; 7074; Cell Signaling Technology), HRP-conjugated anti-rat (1:1000; 7077; Cell Signaling Technology), and HRP-conjugated anti-mouse (1:1000; 7076; Cell Signaling Technology). The intensity of target protein bands was analyzed using ImageJ software.

Enzyme‑linked immunosorbent assay

Brain tissue samples and serum were collected from the mice to measure inflammatory factors. The levels of IL-1β, IL-6, and TNF-α were measured with ELISA kits (Abcam). Plasma was collected from AD patients and age-matched subjects to measure the levels of PD-1 (USCN, Wuhan, China) and IL-1β (Jingmei, Yancheng, China). CSF specimens were used to measure the protein levels of AD biomarkers, including Aβ1–40, Aβ1–42, p-tau-181, and t-tau, using commercially available ELISA kits (INNOTEST, Fujirebio, Ghent, Belgium). A microplate reader (Antobio, Zhengzhou, China) was used to obtain the absorbance.

Flow cytometry

Brain and spleen tissues were prepared as single-cell suspensions as previously described [22]. In brief, chopped brain tissues were digested with DNase I (10 U/ml; Roche) and collagenase IV (0.5 mg/ml; Sigma-Aldrich) in RPMI 1640 with agitation (200 rpm) at 37 °C for 60 min. Cell suspensions were filtered through a 70 μm cell strainer and centrifuged through a Percoll density gradient (GE Healthcare, Waukesha, WI, USA). Immune cells were collected from the interface fractions between 37% and 70% Percoll. For splenic tissue, splenocytes were released by mechanical force and red blood cells were lysed with ammonium–chloride–potassium (ACK) lysing buffer. After intensive washing, the cells were labeled with the following antibodies specific for: CD45 (553079), CD8 (553032), CD4 (552051), CD44 (561859), CD19 (551001), PD1 (562671), MHC-II (562363), IFN-γ (560660), and Foxp3 (563101), which were purchased from BD Biosciences. Specific antibodies against CD11b (101212), CD62L (104412), CD11c (117320), F4/80 (123107), MHC-II (107650), and Ki67 (151209) were purchased from Biolegend. An FVD specific antibody (65-0863-14) was purchased from Invitrogen. For intracellular Foxp3 and Ki67 staining, cell samples were fixed and permeabilized using a Transcription Factor Buffer Set (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s instructions and then stained with specific antibodies. For intracellular cytokine staining, cell samples were stimulated for 4 h with PMA, ionomycin, and brefeldin A, and then subjected to IFN-γ staining. Flow cytometry was performed using a FACS Calibur (BD Biosciences, San Jose, CA, USA), and the data were analyzed with FlowJo 7.6.1 software.

RNA-sequencing analysis

For RNA-sequencing (RNA-seq) analysis, splenocytes were collected. RNA isolation and cDNA library construction were performed with the MGISEQ-2000RS system (Beijing Genomic Institution). Clean reads were mapped to the mouse genome (GRCm38.p5) by HISAT2, and matched reads were normalized by FPKM. Fold changes were calculated for all possible comparisons, and a 1.5-fold cutoff was used to select genes with expression changes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene ontology biological process (GO-BP) analysis were performed using the R package, and target genes were filtered for significant differentially expressed genes (P < 0.05). The gene network was analyzed by the Beijing Genomics Institute in-house customized data mining system. Gene set enrichment analysis (GSEA) was performed as previously described [24] using the Gene Ontology Biological Process Database. The sequencing data files have been deposited in the NCBI Sequence Read Archive (SRA) database under the accession code PRJNA1096339.

Quantitative RT-PCR

Total RNA was extracted with TRIzol reagent (Takara, Tokyo, Japan) and then subjected to cDNA synthesis. Real-time quantitative PCR (RT-qPCR) was performed using a TB Green Premix Ex Taq Kit (Takara). The expression of target genes was calculated by the standard curve method and standardized to the expression of Hprt. The primers used were as follows:

Il1b 5′-TCATTGTGGCTGTGGAGAAG-3′, forward.

5′-AGGCCACAGGTATTT TGTCG-3′, reverse.

Il-6 5′-CTTGGGACTGATGCTGGTGAC-3′, forward.

5′ -GCCATTGCACAACTCTTTTCTC-3′, reverse.

Tnfα 5′-TACTGAACTTCGGGGTGATCG-3′, forward.

5′-TCCTCCACTTGGTGGTTTGC-3′, reverse.

Inos 5′-GGGAATCTTGGAGCGAGTTG-3′, forward.

5′-TGTCCAGGAAGTAGGTGAGGG-3′, reverse.

H2-ob 5′-GACAACAGTAATGCTGGAAATGA-3′, forward.

5′-TGA GCCTTGAGATGGATAACAAC-3′, reverse.

H2-Ab1 5′-AGCCCCATCACTGTGGAGT-3′, forward.

5′ -GATGCCGCTCAACATCTTGC-3′, reverse.

Ccl8 5′-TATCCAGAGGCTGGAGAGCTAC-3′, forward.

5′-TGGAATCCCTGACCCATCTCTC-3′, reverse.

Cxcr4 5′-CTCCTCTTTGTCATCACGCTTCC-3′, forward.

5′-GGATGAGGACACTGCTGTAGAG-3′, reverse.

Ccr1 5′-GTTGGGACCTTGAACCTTGA-3′, forward.

5′-CCCAAAGGCTCTTACAGCAG-3′, reverse.

Pdcd1 5′-ACCCTGGTCATTCACTTGGG-3′, forward.

5′-CATTTGCTCCCTCTGACACTG-3′, reverse.

Il18 5′-CCGCCTCAAACCTTCCAAAT-3′, forward.

5′-TGTGTTTCTTTTCTGGGTGCC-3′, reverse.

Cd74 5′ -ATGGCGACGAGAACGGTAAC-3′, forward.

5′-CGTTGGGGAACACACACCA-3′, reverse.

Nfkb1 5′-ATGGCAGACGATGATCCCTAC-3′, forward.

5′-TGTTGACAGTGGTATTTCTGGTG′, reverse.

Csk 5′-CGACGAGACTCCTGACGTG-3′, forward.

5′-GCACCTCGGGACCCAAATC-3′, reverse.

Ccr6 5′-TCACGACTCGGATTGCTC-3′, forward.

5′-CTGCTGGGTATGGGACTG-3′, reverse.

Hprt 5′-GTCCCAGCGTCGTGATTAGC-3′, forward.

5′-TGGCCTCCCATCTCCTTCA-3′, reverse.

In vitro dendritic cell and microglia antigen presentation assay

As previously described [19], Aβ1–42 peptide (2 mg per mouse) was emulsified with complete Freund’s adjuvant (including 2 mg/ml M. tuberculosis H37Ra, 100 µl per mouse) and used to subcutaneously immunize 5×FAD mice. Fourteen days later, the mice were boosted with Aβ peptide. Seven days later, CD8+ T cells were sorted from the splenocytes of immunized mice by flow cytometry. A total of 2 × 105 CD8+ T cells were labeled with 5 µM carboxyfluorescein succinimidyl ester (CFSE) and cocultured with WT or Gsdmd−/− dendritic cells (DCs) (CD11c+, 4 × 104) or microglia (CD45lowCD11b+, 1 × 105) in the presence of 10 µM Aβ peptide and IL-2 (10 ng/ml). Anti-PD-1 antibody (10 µg/ml, BE0273, BioXCell, West Lebanon, NH, USA) was added as indicated. CFSE dilution and CD44+ percentages were determined by flow cytometry 5 days later. For IL-1β stimulation, 2 × 105 CD8+ T cells were cultured with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) antibodies, and recombinant IL-1β (rIL-1β, 5 µg/ml) was added as indicated.

GSDMD inhibitor treatment and PD-1 blockade in 5×FAD mice

To chronically suppress the cleavage and activation of GSDMD, 5×FAD mice were intraperitoneally injected with dimethyl fumarate (DMF, 5 mg/kg) every 3 days from 2 to 3.5 months of age. To chronically inhibit PD-1 signaling, PD-1 specific antibodies (500 µg per mouse) were intraperitoneally injected into 5×FAD mice every 5 days from 2 to 3.5 months of age [25]. Behavioral feature, histologic, and flow cytometry analyses were performed as described above at 6 months of age.

Cerebrospinal fluid and plasma were collected from AD patients

The criteria of the National Institute on Aging and the Alzheimer’s Association were used for AD diagnosis [26]. We recruited 84 patients with AD from the Department of Neurology, Affiliated Brain Hospital of Nanjing Medical University, from January 2021 to April 2022. For controls, we recruited 33 age-matched subjects. Blood was collected and centrifuged at 3500 rpm for 10 min at 4 °C, and plasma was collected and stored at − 80 °C for subsequent testing. To obtain PBMCs, PBS diluted whole blood (1:1) was transferred to 15 ml centrifuge tubes containing 3 ml of Ficoll Paque (GE Healthcare, Uppsala, Sweden) and centrifuged at 400 × g for 20 min at room temperature. PBMCs were collected and washed twice in 10 ml of PBS. The cells were resuspended in 1× protein loading buffer for subsequent analysis. CSF was collected by lumbar puncture at the L3/L4 or L4/L5 level in the morning. Approximately 2 ml of CSF was collected in a polypropylene tube, centrifuged at 2000 × g for 10 min at room temperature to eliminate cells and other insoluble materials, aliquoted, and stored at − 80 °C for further processing. All participants or their legal guardians provided informed written consent. The Institutional Review Board of the Affiliated Brain Hospital of Nanjing Medical University approved this study.

Statistical analysis

The data were analyzed by GraphPad Prism 7.0 software (Graph Pad Software Inc., San Diego, CA) and are presented as the mean ± standard error of the mean (SEM). The statistics were analyzed by using an unpaired t test for two groups and one-way ANOVA with Dunnett’s multiple comparisons test or two-way ANOVA with Sidak’s multiple comparisons test for multiple groups. P values are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.