recombinant mouse tnf alpha  (R&D Systems)

Journal: Nature Microbiology

Article Title: Weaning drives microbiome-mediated epigenetic regulation to shape immune memory in mice

doi: 10.1038/s41564-026-02295-6

Figure Lengend Snippet: a , Time-course analysis of Cd74 methylation and expression in GF colonic organoids treated with a low dose of IFNγ (0.1 ng ml −1 ) demonstrates rapid demethylation and concomitant gene activation. Data are presented as mean ± s.e.m. of 2 independent experiments. b , DNA demethylase TET3 mediates IFNγ-induced epigenetic reprogramming of Cd74 . Shown is the Cd74 locus with its genomic location, ATAC-seq peaks from sorted Lgr5-GFP + ISCs ( GSE83394 ), the microbial-induced DMR (dashed box) and the positions of primer sets used to assess STATs and TETs binding by ChIP–qPCR. Compared with a non-DMR control region (ChIP-1), STAT3 and TET3 showed an IFNγ-dependent increase in binding at the DMR/enhancer (ChIP-2 and ChIP-3). c , Combined treatment with IFNγ and the hypomethylation agent DAC synergistically enhanced IFNγ-induced demethylation and transcriptional activation. In contrast, treatment with TNF, sodium butyrate (NaB) or LPS alone did not alter Cd74 methylation. d , Experimental design for testing transcriptional memory in organoids with (primed) or without (naïve) previous IFNγ exposure. Pretreated organoids were rested for 7 days without IFNγ and then restimulated with IFNγ or TNF. Memory was indicated by faster and stronger induction of Cd74 expression. e , Before restimulation, organoids exposed to IFNγ for 3 passages (primed) showed nearly complete loss of Cd74 methylation. f , IFNγ (left) or TNF (right) restimulation of primed organoids accelerated and enhanced Cd74 expression compared to naïve organoids. g , Experimental design to determine whether microbiota drive methylation-dependent transcriptional memory of epithelial MHC-II. h , Methylation of MHC-II genes in organoids derived from adult GF, SPF and GF→ SPF (converted at weaning) mice at 15 weeks of age, with methylation assessed after passage 2. i , IFNγ stimulation induced methylation-dependent, memory-like transcriptional activation of the MHC-II genes Cd74 , H2-Eb1 , H2-Aa and Ciita in SPF and GF→ SPF organoids, measured by RT–qPCR. In b , c , e , f , h and i , all data are presented as mean ± s.e.m. of at least 3 independent experiments. In b , c , e and h , P values were calculated using unpaired (two-tailed) t -test.

Article Snippet: For single-agent treatment, organoids were treated for 48 h with 0.1 ng ml −1 IFNγ, 5 ng ml −1 TNF (R&D Systems, 410-MT-010), 2.5 μg ml −1 LPS (Thermo Fisher, 00-4976-03), 2 mM sodium butyrate (Sigma-Aldrich, 303410) or 0.08 μM DAC (Sigma-Aldrich, A3656).

Techniques: Methylation, Expressing, Activation Assay, Binding Assay, ChIP-qPCR, Control, Derivative Assay, Quantitative RT-PCR, Two Tailed Test