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Animals and ethics

Female C57BL/6 mice (6–8 weeks old) were purchased from the Beijing Experimental Animal Center. Conditional Dnmt1 knockout mice (Dnmt1^fl/fl Itgax-Cre) and their littermate controls (Dnmt1^fl/fl) were generated by crossing Dnmt1^fl/fl mice (Jackson Laboratory, Stock No. 021429) with Itgax-Cre mice (Jackson Laboratory, Stock No. 008068). All experimental protocols were approved by the Animal Care and Use Committee (IACUC) of Shanxi Medical University (Approval Number: A20230086) and complied with the guidelines of the National Institutes of Health (NIH) for the care and use of laboratory animals.

Induction of airway allergy (AA)

Mice were sensitized to induce airway allergy using dust mite extract (DME). On days 0 and 7, mice received subcutaneous injections of 50 µg DME (Dermatophagoides pteronyssinus; Greer Laboratories, Cat. No. XPB82D3A2.5) emulsified in 2 mg Imject^® Alum (Thermo Fisher Scientific). From days 14 to 18, mice were challenged intranasally (i.n.) with 25 µg DME in 40 µL phosphate-buffered saline (PBS); control mice received PBS alone. Following DME challenge, clinical symptoms of AA (sneezing, nasal rubbing, and dyspnea) were scored to assess disease severity.

Drug administration

Mice were treated with 5-azacytidine (5AZA; Sigma-Aldrich, Cat. No. A2385) or PBS (vehicle control) to inhibit DNA methyltransferases. Daily intraperitoneal (i.p.) injections of 5AZA (0.5 mg/kg body weight) or PBS were administered from day 0 to day 18 of the experimental timeline.

Cell isolation and culture

Bone marrow-derived DCs (BMDCs)

Bone marrow cells were flushed from the femurs of mice and cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech), and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) for 7 days to generate BMDCs.

BMDC stimulation

Mature BMDCs were treated with DME (10–50 µg/mL) in the presence or absence of lipopolysaccharide (LPS; 1 µg/mL, E. coli O111:B4; Sigma-Aldrich) for 24 h to assess cytokine production and epigenetic changes.

Molecular biology techniques

Reverse transcription quantitative PCR (RT-qPCR)

Total RNA was extracted from cells using TRIzol reagent (Invitrogen), and complementary DNA (cDNA) was synthesized with SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). Quantitative PCR was performed using SYBR Green Master Mix (Bio-Rad) on a CFX96 Real-Time PCR System (Bio-Rad) to quantify the expression of Il12b, Il12a, Tim4, Il4, and Gapdh (housekeeping gene). Primer sequences are listed in Table 1.

Enzyme-linked immunosorbent assay (ELISA)

Cytokine levels (IL-4, IL-5, IL-13, IFN-γ, TNF-α) in bronchoalveolar lavage fluid (BALF) were measured using DuoSet ELISA kits (R&D Systems) according to the manufacturer’s instructions. Absorbance was read at 450 nm, and cytokine concentrations were calculated using standard curves.

Methylation-specific quantitative PCR (MS-qPCR)

Genomic DNA was extracted from DCs and bisulfite-converted using the EZ DNA Methylation-Lightning Kit (Zymo Research) to distinguish methylated from unmethylated cytosines. The methylation status of the Il12b promoter was analyzed by MS-qPCR using primers specific for methylated (MSP) or unmethylated (USP) sequences: MSP-F: 5’-TTGGGATTTTTCGCGATTC-3’, MSP-R: 5’-GAACCCGACGAACTCCG-3’; USP-F: 5’-TTGGGATTTTTTGTGATTTG-3’, USP-R: 5’-CAACCCCACAAACTCCA-3’. Reactions were performed with SYBR Green Master Mix (Bio-Rad) on a CFX96 Real-Time PCR System.

Flow cytometry

Lung immune profiling

Lung tissues were processed into single-cell suspensions, which were stained with fluorescently conjugated antibodies: CD11c (APC), MHC-II (PE-Cy7), Ly6G (FITC), Siglec-F (BV421), CD3 (PerCP), CD4 (AF700), and CD8 (PE) (all from BD Biosciences). Stained cells were acquired on a BD LSRFortessa™ flow cytometer, and data were analyzed using FlowJo software (v10).

Intracellular cytokine staining

DCs were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin for 4 h. Cells were then fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with antibodies against IL-12b (PE) or IL-12a (AF488) (BD Biosciences) for intracellular cytokine detection.

Histology

Lungs were inflated with 4% paraformaldehyde, fixed overnight, and embedded in paraffin. Paraffin blocks were sectioned into 5-µm slices, which were stained with hematoxylin and eosin (H&E) to assess tissue inflammation. Stained sections were imaged using a Leica DMi8 microscope, and histological changes were evaluated by a blinded pathologist.

Airway hyperresponsiveness (AHR)

AHR was measured using a flexiVent system (SCIREQ) to assess airway resistance. Mice were anesthetized, intubated, and exposed to aerosolized methacholine (0–50 mg/mL) for 3 min. Airway resistance was recorded continuously during and after methacholine exposure, and results were normalized to baseline values.

Co-culture experiments

DME-primed BMDCs were co-cultured with naïve CD4⁺ T cells (isolated from mouse spleens using MACS columns; Miltenyi Biotec) at a 1:5 (DC: T cell) ratio in RPMI-1640 medium supplemented with 10% FBS and antibiotics. After 72 h of co-culture, Il4 mRNA expression in CD4⁺ T cells was quantified by RT-qPCR to assess Th2 polarization.

Ubiquitination assays (Cross-ELISA)

BMDCs were treated with 50 ng/mL recombinant IL-12b (rIL-12b; BioLegend) in the presence or absence of 10 µM MG132 (a proteasome inhibitor; Sigma-Aldrich) for 6 h. Cells were lysed in RIPA buffer, and ubiquitinated TIM4 was detected using a cross-ELISA method. Briefly, plates were coated with an anti-TIM4 antibody (Ab1), and bound ubiquitinated TIM4 was detected with an anti-ubiquitin antibody (Ab2); protocols were adapted from Luo et al. [10] and otherwise consistent with standard ELISA procedures.

Chromatin immunoprecipitation (ChIP)

DCs were crosslinked with 1% formaldehyde for 10 min at room temperature, and crosslinking was quenched with 125 mM glycine. Cells were lysed, and chromatin was sheared by sonication to generate 200–500 bp fragments. Immunoprecipitation was performed overnight at 4 °C using antibodies against DNMT1 (Abcam), H3K4me3 (Cell Signaling Technology), or RNA polymerase II (RNA Pol II; Santa Cruz Biotechnology); normal IgG was used as a negative control. Immunoprecipitated DNA was purified and quantified by qPCR using primers targeting the Il12b promoter to assess enrichment of the target proteins.

Statistical analysis

All data are presented as the mean ± standard deviation (SD). Statistical comparisons between two groups were performed using Student’s t-test. For multiple groups, one-way analysis of variance (ANOVA) with Tukey’s post hoc test was used. Analyses were conducted using R (v4.5.1) software. Significance was defined as p < 0.05 (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****).