Fig 1: Distinct DNA and H3K4 methylation patterns at Ifng and Tbx21 promoter regions in CD4SP thymocytes from IL27p28-deficient mice.(A) DNA methylation analysis of nine CpG sites in the Ifng promoter using sodium bisulfite-treated genomic DNA from GFP+CD4+CD8-CD44lo CD4SP thymocytes. Each row represents a sequenced allele (n=10 clones from one of the three independent experiments). Filled (●) and open (○) circles denote methylated and unmethylated cytosine, respectively. (B) Left: Percent methylation at individual CpG sites from one representative experiment. Right: Average methylation of three adjacent site groups (group1: −205,–190, –170; group2: −53,–45, –34; group3:+16,+96,+120) and all CpG sites (mean ± SD, n=3). (C) DNA methylation analysis of five CpG sites upstream of the Il4 transcription start site using sodium bisulfite-treated genomic DNA from GFP+CD4+CD8-CD44lo CD4SP thymocytes. Each row represents a sequenced allele (n=10 clones from the two independent experiments). Filled (●) and open (○) circles denote methylated and unmethylated cytosine, respectively. (D) Left: graphs show the percentage of methylation at each individual site (left panel) or all CpG sites (right panel). (E–G) Histone trimethylation analysis in freshly isolated CD4SP thymocytes from IL27p28-deficient and WT mice. ChIP-qPCR was performed using antibodies against H3K4me3 (E), H3K27me3 (F), and H3K9me3 (G). qPCR primers targeted promoter and trans-regulatory regions of Ifng, Tbx21, Il4, and Gata3. Data: mean ± SEM (n=3, duplicates). Significance: * p<0.05; ** p<0.01 (Student’s t-test). Abbreviation: pro., promoter.
Fig 2: Enhanced IFN-γ production and T-bet expression in Il27ra-/- mice initiated at the CD4SP thymocytes stage.(A) CD4SP thymocytes and naive CD4+ T cells were isolated from Il27ra-/- and WT mice, stimulated with plate-coated anti-CD3 (2 µg/mL) and soluble anti-CD28 (1 µg/mL) for 12 hours. mRNA levels of Ifng, Il4, Il2, Tbx21, and Gata3 were determined by qPCR. Data: mean ± SD (n=3, duplicates). (B) CD4SP thymocytes and CD4+ naive T cells were cultured under Th0 conditions for 3 days. The frequency of IFN-γ-producing CD4+ T cells were analyzed by intracellular staining. Representative dot plots (left) and quantification (right, mean ± SD, n=3). Significance: * p<0.05, ** p<0.01 (Student’s t-test).
Fig 3: Elevated IFN-γ production and T-bet expression in Cd11c-p28f/f mice initiated from CD4SP thymocytes stage.(A) CD4SP (GFP+CD4+CD8-CD44lo) thymocytes, CD4+ RTEs (GFP+CD4+CD8-CD25-CD44lo) and CD4+ naive (GFP-CD4+CD8-CD25-CD44lo) T cells were sorted from 6- to 8-week-old Cd11c-p28f/f mice and WT littermates, stimulated with plate coated anti-CD3 (2 µg/mL) and soluble anti-CD28 (1 µg/mL) for 12 hr. mRNA levels of Ifng, Il4, and Il2 were determined by qPCR. Data: mean ± SD (n=4, duplicates). (B) Sorted cells were cultured under Th0 conditions for 3 days. The frequency of IFN-γ-producing CD4+ T cells were measured by intracellular staining. Representative dot plots (left) and statistical data (right, mean ± SD, n=3). (C) Supernatants from 3-day cultures were analyzed for IFN-γ and IL-4 by ELISA. Data: mean ± SD (n=3). (D) mRNA levels of Tbx21 and Gata3 in sorted cells were determined by qPCR. Data: mean ± SD (n=4, duplicates). (E–F) T-bet protein levels were assessed by western blot (E) and flow cytometry (F) after 3-day culture. Data: mean ± SD (n=3). (G) Freshly sorted cells were lysed in Trizol, and Ifng and Tbx21 mRNA levels were determined by qPCR. Data: mean ± SD (n=4, duplicates). Statistical differences: * p<0.05, ** p<0.01, *** p<0.001 (Student’s t-test). Figure 1—source data 1.PDF file containing original western blots for Figure 1E, indicating the relevant bands and treatments. Figure 1—source data 2.Original files for western blot analysis displayed in Figure 1E.
Fig 4: Myeloid-specific Scap deficiency attenuates HDM-induced M2 activation and allergic airway inflammation.Scapf/f and LysMCre-Scapf/f mice were administered intratracheally with HDM to induce allergic airway inflammation. 72 h after the last HDM exposure, A–D M2 macrophages (A, B) and Th2 cells (C, D) were detected by FACs analysis (n = 5–9 per group). E–H The relative mRNA level of Il4 (E) and Il13 (G) in the lungs was measured by Q-PCR, and the secretion of IL4 (F) and IL13 (H) in the BALF supernatants was determined by ELISA (n = 3–7 per group). I The number of eosinophils in the BALF was assessed (n = 6–10 per group). J, K Representative images (J) and semi-quantification (K) of lung sections stained with H&E (n = 5–8 per group, scale bar: 200 μm). Data are presented as mean ± SEM; Differences were identified using one-way ANOVA.
Fig 5: Mature SREBP2 transcriptional modulates IRF4 expression and interactes with it to promote M2 activation.A, B RNA-seq analysis for the expression of Irf4 in IL4-treated BMDMs stimulated with 10 μM Betulin, or 10 μM 25-HC, or 50 μM PF429424, or 50 μM 1,10-Phen for 24 h. C BMDMs were transfected with Srebf2 or Scap siRNA for 24 h, then treated with IL4 (100 ng/ml) for an additional 24 h, and the cell extracts were harvested for measuring the expression of Retnla by Q-PCR. D–M BMDMs were treated with 10 μM Fatostatin (D, E), or 10 μM Betulin (F, G), or 10 μM 25-HC (H, I), or 50 μM PF429424 (J, K), or 50 μM 1,10-Phen (L, M) together with IL4 (100 ng/ml) for 24 h, the relative mRNA levels of Irf4 were measured by Q-PCR (D, F, H, J, L), while the protein levels of IRF4 were detected by western blotting (E, G, I, K, M). N IL4-treated BMDMs were analyzed for co-localization of IRF4 with SREBP2 by confocal microscopic imaging. O HEK293T cells were transfected with GFP-Irf4 and control GFP plasmid for 72 h, and GFP immunoprecipitates were analyzed for IRF4 and mature SREBP2 by western blotting. P BMDMs were transfected with Irf4 siRNA for 24 h, then treated with IL4 (100 ng/ml) for an additional 24 h, and cell extracts were collected for detection of canonical SREBP2-target gene expression by Q-PCR. Q ChIP analysis of the occupancy of Irf4 promoter in mature SREBP2-overexpressing THP-1 cells. Data are representative of three independent experiments and presented as mean ± SEM; Differences between the two groups were identified using the Student’s t-test (P, Q) and multiple groups (C, D, F, H, J, L) using one-way ANOVA.
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