recombinant ifn treatment  (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 ) GF colonic organoids treated with increasing concentration of IFNγ for 48 h. Low-dose IFNγ (0.1 ng/ml) had minimal effects on organoid proliferation and viability. Representative bright-field images show dose-dependent morphological changes. PBS, phosphate-buffered saline. Scale bars, 500 μm. ( b ) ChIP-qPCR analysis of GF colonic organoids treated with 0.1 ng/ml IFNγ to induce demethylation of the Cd74 enhancer. Binding of STAT and TET family members was assessed at the enhancer regions showing demethylation (ChIP-2 and ChIP-3) and at a non-DMR control region (ChIP-1). STAT1, TET1, and TET2 showed no detectable enrichment at the demethylated enhancer regions relative to the control. Data are presented as mean ± s.e.m. of 4 independent experiments. P values were calculated using unpaired (two-tailed) t -test.

Article Snippet: For IFNγ treatment, GF-derived organoids were incubated with recombinant mouse IFNγ (R&D Systems, 485-MI-100) at 0.1 ng ml −1 for 0, 24, 48, 72 or 96 h before collection.

Techniques: Concentration Assay, Saline, ChIP-qPCR, Binding Assay, Control, Two Tailed Test

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 IFNγ treatment, GF-derived organoids were incubated with recombinant mouse IFNγ (R&D Systems, 485-MI-100) at 0.1 ng ml −1 for 0, 24, 48, 72 or 96 h before collection.

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

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 ) Representative images show comparable proliferation and viability following IFNγ treatment in organoids derived from GF, SPF, or GF→ SPF (converted at weaning) mice. Scale bars, 500 μm. ( b ) 6 h post-treatment, MHC-II gene activation was enhanced only in organoids from mice with prior microbial exposure, indicating that induction is microbiota- and methylation-dependent, and not due to changes in proliferation. Data are presented as mean ± s.e.m. of 3 independent experiments. P values were calculated using unpaired (two-tailed) t -test.

Article Snippet: For IFNγ treatment, GF-derived organoids were incubated with recombinant mouse IFNγ (R&D Systems, 485-MI-100) at 0.1 ng ml −1 for 0, 24, 48, 72 or 96 h before collection.

Techniques: Derivative Assay, Activation Assay, Methylation, Two Tailed Test