Generation of the mouse model of sepsis-induced ALI

All animal experiments followed institutional ethical guidelines and were approved by the Institutional Animal Care and Use Committee of Shanghai Pulmonary Hospital (Approval number: K21-354). Male C57BL/6 mice (6-8 weeks old) were purchased from Charles River Laboratories and housed under specific pathogen-free conditions with a 12-h light-dark cycle and ad libitum access to food and water.

Sepsis was induced via cecal ligation and puncture (CLP). Mice were anesthetized with 3% isoflurane in oxygen, and a midline abdominal incision was made to expose the cecum. The cecum was ligated just below the ileocecal valve and punctured twice using a 21-gauge needle, followed by gentle extrusion of fecal material. The cecum was repositioned, and the peritoneum and skin were sutured. Mice were resuscitated with 1 mL subcutaneous saline. Sham-operated mice underwent the same procedure without cecal ligation or puncture.

Issaeva and colleagues applied Thioguanine (ThG) at 1.5 mg/kg in a mouse model on tumors (Issaeva et al. 2010). Since this study focused on septic lung injury, ThG was administered via intratracheal injection to achieve local delivery to the lungs. After several attempts, we identified ThG (Sigma-Aldrich) administration at 3 mg/kg/day for 5 consecutive days as an optimal regimen to alleviate inflammatory response and improve lung pathology in mice without causing significant advert effects. Therefore, this regimen was used in subsequent experiments.

Unless otherwise specified, ThG (3 mg/kg/day) was dissolved in dimethyl sulphoxide (DMSO) and administered via intratracheal instillation for 5 consecutive days, starting on the day of CLP surgery. Control mice received an equivalent volume of vehicle solution via the same route.

For gene knockdown studies, adeno-associated virus (AAV9)-encapsulated short hairpin RNA (shRNA) targeting DNMT1 was delivered via intratracheal injection (5 × 1011 vg per mouse) 3 d before CLP induction. Mice were monitored every 6 h for signs of morbidity, and humane endpoints were applied in accordance with approved protocols.

At 7 d post-CLP, mice were euthanized using intraperitoneal overdose of nembutal, and lung tissues were collected for histopathological, molecular, and immunological analyses. Bronchoalveolar lavage fluid (BALF) was harvested for assessment of immune cell infiltration and cytokine secretion.

BALF collection and analysis

BALF was obtained by inserting a sterile 22-gauge catheter into the trachea, and 1 mL of sterile PBS was instilled and retrieved gently to avoid cellular damage. This procedure was repeated twice, and the total fluid recovered was centrifuged at 300 g for 10 min at 4°C. Afterward, the supernatant was collected and stored at –80°C for further cytokine analysis, and cell pellets were resuspended in 100 μL PBS for cell counting and differential cell analysis.

Total cells in the BALF were counted by a hemocytometer. For differential cell counting, cytospin was prepared by centrifuging 100 μL of BALF cell suspension at 500 g for 5 min onto microscope slides using a Shandon Cytospin III centrifuge (Thermo Fisher Scientific). Slides were air-dried, fixed in methanol, and stained with Giemsa stain (Sigma-Aldrich) to differentiate macrophages, neutrophils, eosinophils, and lymphocytes. A total of 300 cells per slide were counted by two operators blinded to the grouping details, and the relative percentages of each cell type were calculated.

Cytokine assessment using Enzyme-linked immunosorbent assay

Cytokine concentrations in the BALF supernatant, including tumor necrosis factor-α (TNF-α), IL-1β, monocyte chemotactant protein-1 (MCP-1), IL-10, and TGF-β1 were assessed using the corresponding ELISA kits (Thermo Fisher Scientific). All procedures were conducted strictly adhering to the manufacturer's protocols. Briefly, BALF samples were diluted 1:2 with assay buffer and loaded to 96-well plates precoated with capture antibodies for 2 h. After washing, detection antibodies were applied, followed by incubation with streptavidin-HRP conjugate. Tetramethylbenzidine substrate solution was then added, and the reaction was terminated with sulfuric acid. Optical density at 450 nm was read using a microplate reader (BioTek Instruments). A standard curve for each cytokine was generated using recombinant cytokines provided in the kits, and concentrations were examined by interpolation.

Evans blue dye extravasation assay

To determine pulmonary vascular permeability, mice were intravenously injected with 1% Evans Blue dye (20 mg/kg, Sigma-Aldrich) 1 h before euthanasia. Following euthanasia, lungs were perfused through the right ventricle with 10 mL of PBS to remove intravascular dye. Lungs were excised, weighed, and homogenized in 1 mL of formamide (Sigma-Aldrich). Samples were incubated at 60 °C for 18 h to extract the dye. After centrifugation at 12,000 g for 15 min, OD value was read at 620 nm using a spectrophotometer. Evans Blue concentration was quantified using a standard curve and normalized to lung tissue weight.

Histological analysis

Mouse lung tissues were fixed overnight in 4% paraformaldehyde at 4 °C, dehydrated, and embedded in paraffin. Sections were cut at 5 μm thickness using a rotary microtome (Leica RM2235) and mounted on glass slides. The prepared sections were subjected to hematoxylin and eosin (H&E) staining to evaluate to assess histological changes including alveolar wall thickening, edema, and inflammatory cell infiltration. Additionally, mucus production was evaluated using and periodic acid-Schiff (PAS) staining. The results were evaluated by two pathologists blinded to the groups using a light microscope (Leica DM5000B). A semiquantitative scoring system was used to evaluate lung injury based on the extent of alveolar collapse, infiltration of inflammatory cells, and hemorrhage.

Immunofluorescence staining for FOXP3+ Tregs

Lung sections were deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer (pH 6.0) at 95 °C for 20 min. Afterward, sections were sealed with 5% bovine serum albumin for 1 h and incubated overnight with FOXP3 antibody (1:100, Abcam) at 4 °C and probed with fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h. Nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich), and slides were mounted with Fluoromount-G (SouthernBiotech). Immunofluorescence images were captured using a Zeiss LSM 880 confocal microscope. of the FOXP3+ Tregs were quantified by counting positive cells in 10 randomly selected fields (200 x) using ImageJ software.

Generation of DNMT1-CD4-knockout (ko) mice

DNMT1fl/fl mice (Jackson Laboratory) were allowed to mate with CD4-Cre mice to generate DNMT1-CD4-ko offspring. Genotyping was conducted by polymerase chain reaction (PCR) using genomic DNA isolated from tail biopsies. DNMT1fl/fl littermates were utilized as controls. Mice at 6-8 weeks of age were used for sepsis-induced ALI studies as described above.

Treg differentiation from CD4+ T cells

CD4+ T cells were isolated from the spleens of DNMT1fl/fl or DNMT1-CD4-ko mice using magnetic-activated cell sorting with a CD4+ T Cell Isolation Kit (Miltenyi Biotec). The cells were purified using flow cytometry, making sure a purity exceeding 95% prior to subsequent experiments. Naive CD4+ T cells were stimulated with plate-bound anti-CD3 (2 μg/mL) and anti-CD28 (2 μg/mL) antibodies in the presence of TGF-β1 (5 ng/mL) and IL-2 (10 ng/mL) for 72 h to induce differentiation into Tregs. Cells were harvested for RNA sequencing (RNA-seq) and protein analysis.

Isolation of Tregs from mice and co-culture experiments

To evaluate the role of DNMT1 in Treg-mediated immunosuppressive activity, CD4+CD25+ Tregs were isolated from the spleens and lymph nodes of either DNMT1fl/fl or DNMT1-CD4-KO mice using magnetic-activated cell sorting (MACS). Single-cell suspensions were prepared by mechanical dissociation and filtration through a 70 μm cell strainer. CD4+CD25+ Tregs were enriched using a CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat# 130-091-041) following the manufacturer's protocol. Cell purity (>90%) was confirmed by flow cytometry. Responder CD4+CD25- conventional T cells (Tcon) were isolated from wild-type C57BL/6 mice and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Thermo Fisher, Cat# C34554) at a final concentration of 5 μM in PBS at 37 °C for 10 min. The labeling reaction was quenched with RPMI-1640 medium containing 10% FBS and washed twice. For suppression assays, CFSE-labeled Tcon cells (1 × 105 per well) were co-cultured with Tregs from DNMT1fl/fl or DNMT1-CD4-ko mice in round-bottom 96-well plates. Cells were stimulated with anti-CD3 (2 μg/mL, BioLegend, Cat# 100314) and anti-CD28 (1 μg/mL, BioLegend, Cat# 102112) in the presence of irradiated splenocytes as antigen-presenting cells. After 72 h, cells were harvested and analyzed by flow cytometry. Tcon proliferation was quantified based on CFSE dilution.

RNA-seq analysis

Total RNA was extracted from differentiated Tregs using the RNeasy Mini Kit (Qiagen). RNA quality was assessed using an Agilent Bioanalyzer 2100, and samples with RNA integrity numbers (RIN) greater than 7 were used for library preparation. RNA-seq libraries were generated with the NEBNext Ultra RNA Library Prep Kit for Illumina, and sequencing was carried out on an Illumina NovaSeq 6000 platform. Differential expression analysis was performed using DESeq2, with genes showing adjusted p-values < 0.05 considered significantly differentially expressed.

Quantitative PCR (qPCR) analysis

qPCR was conducted to validate RNA-seq results. Total RNA was reverse-transcribed using the PrimeScript RT Reagent Kit (Takara), and qPCR was performed using SYBR Green PCR Master Mix on a CFX96 Real-Time PCR System (Bio-Rad). Primers for FOXO1 and GAPDH (used as an internal control) were designed and synthesized by Sangon Biotech. Relative gene expression levels were calculated using the 2−ΔΔCt method.

Western blot (WB) analysis

Protein extracts were prepared from Tregs using RIPA buffer. Protein concentration was determined using the BCA Kit (Thermo Fisher Scientific). Equal amounts of protein (30 μg) were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore). Membranes were blocked with 5% non-fat milk in TBST for 1 hour and then probed with primary antibodies against FOXO1 (1:1000, Cell Signaling Technology) and β-actin (1:2000, Abcam) overnight at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibodies, and signals were visualized using ECL detection reagents (Thermo Fisher Scientific)

Chromatin immunoprecipitation (ChIP) assays

ChIP assays were performed using the SimpleChIP Kit (Cell Signaling Technology). Tregs, differentiated from CD4+ T cells, were cross-linked with 1% formaldehyde for 10 min to preserve protein-DNA interactions. Cross-linking was quenched by adding glycine to a final concentration of 0.125 M for 5 min. Cells were lysed in the provided lysis buffer, and chromatin was sheared into 200-600 bp fragments using micrococcal nuclease digestion and sonication (Bioruptor Plus, Diagenode). For immunoprecipitation, 5 μg of sheared chromatin was incubated overnight at 4 °C with 5 μg of either anti-DNMT1 (Cell Signaling Technology) or anti-RUNX1 (Abcam) antibodies, or normal rabbit IgG (negative control). Protein A/G magnetic beads (Cell Signaling Technology) were added, and the complexes were washed with low- and high-salt wash buffers. Cross-links were reversed by incubating with ChIP elution buffer and proteinase K at 65 °C for 4 h. The immunoprecipitated DNA was purified using the kit’s spin columns. ChIP-qPCR was performed using SYBR Green Master Mix on a CFX96 Real-Time PCR Detection System (Bio-Rad). Primers targeting the FOXO1 promoter region, including the CpG island, were designed using Primer3 software. Fold enrichment was calculated relative to input DNA and normalized to the IgG control.

Luciferase reporter assays

The FOXO1 promoter region (including the CpG island) was amplified from mouse genomic DNA and cloned into the pGL3-Basic vector (Promega). The promoter-reporter construct was verified by Sanger sequencing. For reporter assays, 293 T cells were seeded in 24-well plates at 2 × 105 cells per well and transfected using Lipofectamine 3000 with 500 ng of the FOXO1 promoter-pGL3 construct and 50 ng of the Renilla luciferase plasmid (pRL-TK) as a control. Co-transfection experiments were performed with DNMT1 shRNA or RUNX1 overexpression plasmids, along with the FOXO1 promoter reporter construct. DNMT1 shRNA and RUNX1 overexpression plasmids were obtained from Addgene. After 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity, and relative luciferase units were calculated as a percentage of the control group.

Bisulfite sequencing PCR (BSP-qPCR)

Genomic DNA was extracted from Tregs using the DNeasy Blood & Tissue Kit (Qiagen). Bisulfite conversion of unmethylated cytosines to uracil was performed using the EZ DNA Methylation-Gold Kit (Zymo Research). Converted DNA was amplified by PCR with primers targeting the CpG-rich region in the FOXO1 promoter. Primers were designed using MethPrimer software to ensure optimal specificity for bisulfite-treated DNA. The amplified PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and cloned into the pCR4-TOPO vector (Thermo Fisher Scientific) for sequencing. A minimum of 10 individual clones from each sample were sequenced to assess the methylation status of each CpG site. Methylation levels were determined by comparing cytosine to thymine conversions at CpG sites using ClustalW alignment. For quantitative methylation analysis, BSP-qPCR was performed on bisulfite-treated DNA. Primers specific to the FOXO1 CpG island were used in qPCR reactions with SYBR Green PCR Master Mix. Methylation levels were calculated using the 2−ΔΔCt method and normalized to input DNA.

Biological replicates and statistical analysis

Data are presented as the mean ± standard error of the mean (SEM). Unless otherwise specified, for cellular experiments, each experiment was performed using cells isolated from six independent mice, with each measurement conducted in technical triplicates. For animal experiments, each group consisted of six mice, and three independent replications were conducted for each sample. The data from each independent experiment are represented as individual dots in the graphs. Comparisons between two groups were analyzed using unpaired two-tailed Student's t-tests. For multiple group comparisons, one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was applied. Normality of data distribution was assessed using the Shapiro-Wilk test. For non-normally distributed data, non-parametric tests (e.g., Kruskal-Wallis with Dunn’s post hoc test) were used. Statistical analyses were performed using GraphPad Prism 9.0 and R version 4.3.1. A p-value of < 0.05 was considered statistically significant. No data points were excluded, unless due to technical errors.