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InvivoGen anti ifnγ neutralizing antibody

LPS and <t>IFNγ</t> both generate stimulus-specific de novo enhancers in human macrophages; however, only IFNγ-induced enhancers are durable. (A) Schematic of experimental design: Human macrophages were stimulated with either IFNγ (100 ng/ml), LPS (100 ng/ml), or LPS in the presence of 1 µM ruxolitinib for 8 h. Cells were subsequently washed and cultured for an additional 88 h. H3K4me1 CUT&Tag was performed at each time point. (B) Heatmap of Z-scored reads within H3K4me1 peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Top enriched motifs in clusters from B. (D) Box/whisker plot quantifying log2 cpm of reads within IFNγ-induced peaks before and after cytokine washout. (E) Box/whisker quantifying log2 cpm of reads within LPS-induced peaks before and after cytokine washout. (F) Box/whisker quantifying log2 cpm of reads within peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (G) Z-scored heatmap of reads within CUT&Tag peaks of only IFNγ-induced peaks before and after cytokine washout. (H) Boxplot of log2 cpm of reads within peaks for each cluster identified in G. (I) Examples of genome browser tracks for each cluster in E. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. *P < 0.05; ****P < 0.0001.

Anti Ifnγ Neutralizing Antibody, supplied by InvivoGen, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself"

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20250976

LPS and IFNγ both generate stimulus-specific de novo enhancers in human macrophages; however, only IFNγ-induced enhancers are durable. (A) Schematic of experimental design: Human macrophages were stimulated with either IFNγ (100 ng/ml), LPS (100 ng/ml), or LPS in the presence of 1 µM ruxolitinib for 8 h. Cells were subsequently washed and cultured for an additional 88 h. H3K4me1 CUT&Tag was performed at each time point. (B) Heatmap of Z-scored reads within H3K4me1 peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Top enriched motifs in clusters from B. (D) Box/whisker plot quantifying log2 cpm of reads within IFNγ-induced peaks before and after cytokine washout. (E) Box/whisker quantifying log2 cpm of reads within LPS-induced peaks before and after cytokine washout. (F) Box/whisker quantifying log2 cpm of reads within peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (G) Z-scored heatmap of reads within CUT&Tag peaks of only IFNγ-induced peaks before and after cytokine washout. (H) Boxplot of log2 cpm of reads within peaks for each cluster identified in G. (I) Examples of genome browser tracks for each cluster in E. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. *P < 0.05; ****P < 0.0001.

Figure Legend Snippet: LPS and IFNγ both generate stimulus-specific de novo enhancers in human macrophages; however, only IFNγ-induced enhancers are durable. (A) Schematic of experimental design: Human macrophages were stimulated with either IFNγ (100 ng/ml), LPS (100 ng/ml), or LPS in the presence of 1 µM ruxolitinib for 8 h. Cells were subsequently washed and cultured for an additional 88 h. H3K4me1 CUT&Tag was performed at each time point. (B) Heatmap of Z-scored reads within H3K4me1 peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Top enriched motifs in clusters from B. (D) Box/whisker plot quantifying log2 cpm of reads within IFNγ-induced peaks before and after cytokine washout. (E) Box/whisker quantifying log2 cpm of reads within LPS-induced peaks before and after cytokine washout. (F) Box/whisker quantifying log2 cpm of reads within peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (G) Z-scored heatmap of reads within CUT&Tag peaks of only IFNγ-induced peaks before and after cytokine washout. (H) Boxplot of log2 cpm of reads within peaks for each cluster identified in G. (I) Examples of genome browser tracks for each cluster in E. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. *P < 0.05; ****P < 0.0001.

Techniques Used: Cell Culture, Generated, Whisker Assay

Ruxolitinib blocks LPS and IFNγ-induced Janus kinase signaling. (A) Human macrophages were pre-treated with increasing concentrations of ruxolitinib for 15 min and subsequently stimulated with IFNγ (100 ng/ml) or LPS (100 ng/ml) for 3 h. Whole-cell lysate western blots showing effect of ruxolitinib on STAT1 and STAT2 phosphorylation by each stimulus. Blot is representative of two replicates from separate human donors. (B and C) Quantification of pSTAT2 and (C) pSTAT1 band intensities in A. Source data are available for this figure: .

Figure Legend Snippet: Ruxolitinib blocks LPS and IFNγ-induced Janus kinase signaling. (A) Human macrophages were pre-treated with increasing concentrations of ruxolitinib for 15 min and subsequently stimulated with IFNγ (100 ng/ml) or LPS (100 ng/ml) for 3 h. Whole-cell lysate western blots showing effect of ruxolitinib on STAT1 and STAT2 phosphorylation by each stimulus. Blot is representative of two replicates from separate human donors. (B and C) Quantification of pSTAT2 and (C) pSTAT1 band intensities in A. Source data are available for this figure: .

Techniques Used: Western Blot, Phospho-proteomics

IFNγ induces long-lasting transcription factor activity and chromatin accessibility after washout. Macrophages were treated with LPS, IFNγ, and LPS in the presence of ruxolitinib for 8 h, as in . Cells were washed and cultured for an additional 88 h. ATACseq was performed after 8 h of stimulation and 4 days after washout. (A) Heatmap of Z-scored reads within ATAC peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (B) Top enriched motifs in clusters from A. (C) Boxplot quantifying log2 cpm of reads within IFNγ-induced ATAC peaks before and after cytokine washout. (D) Boxplot quantifying log2 cpm of reads within LPS-induced ATAC peaks before and after cytokine washout. (E) Boxplot quantifying log2 cpm of reads within ATAC peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (F) Barplot quantifying percent of transcription factor-bound motifs within STAT1 and IRF1 (IFNγ) and IRF1 and NF-κB (LPS) within induced ATAC peaks in C and D for unstimulated, IFNγ/LPS-stimulated macrophages, and stimulated macrophages 4 days after washout. Motif binding predicted using TOBIAS ATACseq footprinting analysis. Results are average of two technical replicates from a single subject; error bars display standard deviation. (G) Human macrophages were stimulated with IFNγ (100 ng/ml), LPS (100 ng/ml), or IFNβ (10 ng/ml) for 8 h, washed, and then cultured for an additional 66 h. Cells were collected, and whole cell western blotting for phosphorylated STAT1 was performed at the indicated time points. Blot is representative of three replicates from two separate human donors. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001. Source data are available for this figure: .

Figure Legend Snippet: IFNγ induces long-lasting transcription factor activity and chromatin accessibility after washout. Macrophages were treated with LPS, IFNγ, and LPS in the presence of ruxolitinib for 8 h, as in . Cells were washed and cultured for an additional 88 h. ATACseq was performed after 8 h of stimulation and 4 days after washout. (A) Heatmap of Z-scored reads within ATAC peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (B) Top enriched motifs in clusters from A. (C) Boxplot quantifying log2 cpm of reads within IFNγ-induced ATAC peaks before and after cytokine washout. (D) Boxplot quantifying log2 cpm of reads within LPS-induced ATAC peaks before and after cytokine washout. (E) Boxplot quantifying log2 cpm of reads within ATAC peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (F) Barplot quantifying percent of transcription factor-bound motifs within STAT1 and IRF1 (IFNγ) and IRF1 and NF-κB (LPS) within induced ATAC peaks in C and D for unstimulated, IFNγ/LPS-stimulated macrophages, and stimulated macrophages 4 days after washout. Motif binding predicted using TOBIAS ATACseq footprinting analysis. Results are average of two technical replicates from a single subject; error bars display standard deviation. (G) Human macrophages were stimulated with IFNγ (100 ng/ml), LPS (100 ng/ml), or IFNβ (10 ng/ml) for 8 h, washed, and then cultured for an additional 66 h. Cells were collected, and whole cell western blotting for phosphorylated STAT1 was performed at the indicated time points. Blot is representative of three replicates from two separate human donors. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001. Source data are available for this figure: .

Techniques Used: Activity Assay, Cell Culture, Generated, Binding Assay, Footprinting, Standard Deviation, Western Blot, Whisker Assay

Cell surface–bound IFNγ mediates persistent signaling regardless of cytokine manufacturer and can be degraded by trypsinization. (A) Quantification of pSTAT1 intensity normalized to 3H IFNγ in . (B) Human macrophages were treated with Escherichia coli sourced IFNγ purchased from PeproTech and mammalian-sourced IFNγ purchased from Sigma-Aldrich and ACRO. Cells were treated at 100 ng/ml for 8 h, washed, and cultured for an additional 48 h in regular media prior to collection. Cells were collected for immunoblot at the indicated times. Representative blot of duplicates from one human subject. (C) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in B. (D) Human A549 airway epithelial cells were stimulated with 100 ng/ml IFNγ, washed, and cultured for an additional 72 h. Cells were collected for immunoblot at the indicated timepoints. Representative blot of two replicates. (E) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in D. (F) Human macrophages were treated with 100 ng/ml IFNγ for 8 h, washed, and lifted by scraping after incubation with either PBS, 0.5 mM EDTA in PBS, or trypsin. Cells were replated and cultured for an additional 24 h in regular media. Cells were collected for immunoblot at the indicated times. (G) Quantification of pSTAT1 band intensity normalized to GAPDH for each condition in F. Statistical tests were determined by single-tailed t test. *P < 0.05; **P < 0.01. Source data are available for this figure: .

Figure Legend Snippet: Cell surface–bound IFNγ mediates persistent signaling regardless of cytokine manufacturer and can be degraded by trypsinization. (A) Quantification of pSTAT1 intensity normalized to 3H IFNγ in . (B) Human macrophages were treated with Escherichia coli sourced IFNγ purchased from PeproTech and mammalian-sourced IFNγ purchased from Sigma-Aldrich and ACRO. Cells were treated at 100 ng/ml for 8 h, washed, and cultured for an additional 48 h in regular media prior to collection. Cells were collected for immunoblot at the indicated times. Representative blot of duplicates from one human subject. (C) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in B. (D) Human A549 airway epithelial cells were stimulated with 100 ng/ml IFNγ, washed, and cultured for an additional 72 h. Cells were collected for immunoblot at the indicated timepoints. Representative blot of two replicates. (E) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in D. (F) Human macrophages were treated with 100 ng/ml IFNγ for 8 h, washed, and lifted by scraping after incubation with either PBS, 0.5 mM EDTA in PBS, or trypsin. Cells were replated and cultured for an additional 24 h in regular media. Cells were collected for immunoblot at the indicated times. (G) Quantification of pSTAT1 band intensity normalized to GAPDH for each condition in F. Statistical tests were determined by single-tailed t test. *P < 0.05; **P < 0.01. Source data are available for this figure: .

Techniques Used: Cell Culture, Western Blot, Incubation

Cell surface–bound IFNγ mediates persistent JAK/STAT signaling even after cytokine washout. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h, washed, and then cultured in regular media or media containing ruxolitinib (1 µM) or increasing concentrations of anti-IFNγ neutralizing antibody for an additional 28 h. Cells were collected, and whole cell western blotting for phosphorylated STAT1 and IRF1 was performed at indicated time points. Representative blot of duplicates from two separate subjects. (B) Human macrophages were stimulated with 100 ng/ml IFNγ for 3 h, washed, and then cultured in either ruxolitinib (1 µM), anti-IFNγ neutralizing antibody (10 µg/ml), or isotype control antibody (10 µg/ml) for 2 h. Samples were collected at the indicated times for immunoblot. Representative blot of duplicates from two separate subjects. (C) Quantification of pSTAT1 band intensities from B. (D) Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and cultured for an additional 88 h in regular media. Supernatants from stimulated macrophages were collected after the 8-h stimulation and 88 h after washout. This supernatant was used to stimulate fresh macrophages for 1 h in the presence/absence of ruxolitinib (1 µM) or anti-IFNγ neutralizing antibody (10 µg/ml). As a control, fresh macrophages were stimulated with media supplemented with 1 ng/ml IFNγ for 1 h. Representative blot of duplicates from two separate subjects. (E) Macrophages were left in regular media or pre-treated with 10 µg/ml CHX for 15 min and stimulated with 100 ng/ml IFNγ for 3 h. Treated macrophages were washed and subsequently cultured for 2 h in regular media, media supplemented with 10 µg/ml CHX, or anti-IFNγ neutralizing antibody (10 µg/ml) and collected for immunoblot. Duplicates from one subject are shown. (F) Quantification of pSTAT1 band intensities in E normalized to band intensity of macrophages treated with IFNγ for 3 h. (G) Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and cultured in regular media or media supplemented with 1 µM ruxolitinib for 16 h. After 16 h, cells were washed again and cultured in regular media for an additional 24 h. Cells were collected for immunoblot at indicated times. Representative blot of four replicates from two subjects. (H) Quantification of pSTAT1 band intensities in G normalized to band intensity of macrophages treated with IFNγ for 3 h. Statistical tests were determined by a single-tailed t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Source data are available for this figure: .

Figure Legend Snippet: Cell surface–bound IFNγ mediates persistent JAK/STAT signaling even after cytokine washout. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h, washed, and then cultured in regular media or media containing ruxolitinib (1 µM) or increasing concentrations of anti-IFNγ neutralizing antibody for an additional 28 h. Cells were collected, and whole cell western blotting for phosphorylated STAT1 and IRF1 was performed at indicated time points. Representative blot of duplicates from two separate subjects. (B) Human macrophages were stimulated with 100 ng/ml IFNγ for 3 h, washed, and then cultured in either ruxolitinib (1 µM), anti-IFNγ neutralizing antibody (10 µg/ml), or isotype control antibody (10 µg/ml) for 2 h. Samples were collected at the indicated times for immunoblot. Representative blot of duplicates from two separate subjects. (C) Quantification of pSTAT1 band intensities from B. (D) Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and cultured for an additional 88 h in regular media. Supernatants from stimulated macrophages were collected after the 8-h stimulation and 88 h after washout. This supernatant was used to stimulate fresh macrophages for 1 h in the presence/absence of ruxolitinib (1 µM) or anti-IFNγ neutralizing antibody (10 µg/ml). As a control, fresh macrophages were stimulated with media supplemented with 1 ng/ml IFNγ for 1 h. Representative blot of duplicates from two separate subjects. (E) Macrophages were left in regular media or pre-treated with 10 µg/ml CHX for 15 min and stimulated with 100 ng/ml IFNγ for 3 h. Treated macrophages were washed and subsequently cultured for 2 h in regular media, media supplemented with 10 µg/ml CHX, or anti-IFNγ neutralizing antibody (10 µg/ml) and collected for immunoblot. Duplicates from one subject are shown. (F) Quantification of pSTAT1 band intensities in E normalized to band intensity of macrophages treated with IFNγ for 3 h. (G) Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and cultured in regular media or media supplemented with 1 µM ruxolitinib for 16 h. After 16 h, cells were washed again and cultured in regular media for an additional 24 h. Cells were collected for immunoblot at indicated times. Representative blot of four replicates from two subjects. (H) Quantification of pSTAT1 band intensities in G normalized to band intensity of macrophages treated with IFNγ for 3 h. Statistical tests were determined by a single-tailed t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Source data are available for this figure: .

Techniques Used: Cell Culture, Western Blot, Control

Extracellular IFNγ signaling sustains chromatin accessibility and ISG expression even after cytokine washout. (A) Schematic of experiments: Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and then cultured for an additional 88 h in regular media or media with 1 µM ruxolitinib for an additional 88 h. Cells were collected for ATACseq and RNAseq at the indicated time points. (B) Heatmap of Z-scored reads within ATAC peaks induced by IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation for 4 days after washout when cultured in regular media or media with 1 µM ruxolitinib. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Boxplot of log2CPM of reads within each peak for each cluster in B. (D) Heatmap of Log2 fold change in RNAseq reads of genes induced at least fivefold after 8 h of IFNγ stimulation. Log2 fold changes are shown after washout for cells cultured in regular media and media containing 1 µM ruxolitinib. Genes are clustered by persistent level of expression after washout (CPM after wash as percent of CPM at 8-h simulation). (E) Boxplot showing Log2 fold changes of individual genes by cluster in D. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. (F) Macrophages were stimulated and washed as above in A; after washout, cells were cultured in media alone, media with 1 µM ruxolitinib, or 10 µg/ml anti-IFNγ neutralizing antibody for 88 h. Cells were collected 88 h after washout, and qPCR was performed for IDO1. Boxplots indicate 2 ΔΔCt normalized to HPRT. Error bars indicate standard deviation. Statistical tests determined by ordinary one way ANOVA. (G) qPCR for IRF1 as in F. ** P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure Legend Snippet: Extracellular IFNγ signaling sustains chromatin accessibility and ISG expression even after cytokine washout. (A) Schematic of experiments: Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and then cultured for an additional 88 h in regular media or media with 1 µM ruxolitinib for an additional 88 h. Cells were collected for ATACseq and RNAseq at the indicated time points. (B) Heatmap of Z-scored reads within ATAC peaks induced by IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation for 4 days after washout when cultured in regular media or media with 1 µM ruxolitinib. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Boxplot of log2CPM of reads within each peak for each cluster in B. (D) Heatmap of Log2 fold change in RNAseq reads of genes induced at least fivefold after 8 h of IFNγ stimulation. Log2 fold changes are shown after washout for cells cultured in regular media and media containing 1 µM ruxolitinib. Genes are clustered by persistent level of expression after washout (CPM after wash as percent of CPM at 8-h simulation). (E) Boxplot showing Log2 fold changes of individual genes by cluster in D. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. (F) Macrophages were stimulated and washed as above in A; after washout, cells were cultured in media alone, media with 1 µM ruxolitinib, or 10 µg/ml anti-IFNγ neutralizing antibody for 88 h. Cells were collected 88 h after washout, and qPCR was performed for IDO1. Boxplots indicate 2 ΔΔCt normalized to HPRT. Error bars indicate standard deviation. Statistical tests determined by ordinary one way ANOVA. (G) qPCR for IRF1 as in F. ** P < 0.01; ***P < 0.001; ****P < 0.0001.

Techniques Used: Expressing, Cell Culture, RNA sequencing, Generated, Whisker Assay, Standard Deviation

$Macrophages from a second human donor show reversibility of IFNγ-induced chromatin accessibility and de novo enhancers. Human macrophages from a second human subject were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media or media supplemented with 1 µM ruxolitinib. ATAC and H3K4me1 CUT&Tag was performed after 8 h of stimulation and 88 h after cytokine washout. (A) Boxplot quantifying log2 fold changes of reads within IFNγ-induced ATAC peaks after 8 h of IFNγ stimulation and after washout for each condition (L2FC > 2, FDR < 0.01). (B) Heatmap of Z-scored reads within ATAC peaks induced IFNγ after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Boxplot quantifying log2 fold changes of reads within IFNγ-induced H3K4me1 CUT&Tag peaks after 8 h of IFNγ stimulation and after washout for each condition (L2FC > 2, FDR < 0.01). (D) Barplot showing fraction of IFNγ-induced H3K4me1 peaks at 8 h that persist 4 days after washout in each condition. Persistence was defined as L2FC ≥ 0, FDR < 0.01. (E) Heatmap of Z-scored reads within H3K4me1 peaks induced IFNγ after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (F) Boxplot of log2CPM of reads within each peak for each cluster in E. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001.$
Figure Legend Snippet: Macrophages from a second human donor show reversibility of IFNγ-induced chromatin accessibility and de novo enhancers. Human macrophages from a second human subject were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media or media supplemented with 1 µM ruxolitinib. ATAC and H3K4me1 CUT&Tag was performed after 8 h of stimulation and 88 h after cytokine washout. (A) Boxplot quantifying log2 fold changes of reads within IFNγ-induced ATAC peaks after 8 h of IFNγ stimulation and after washout for each condition (L2FC > 2, FDR < 0.01). (B) Heatmap of Z-scored reads within ATAC peaks induced IFNγ after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Boxplot quantifying log2 fold changes of reads within IFNγ-induced H3K4me1 CUT&Tag peaks after 8 h of IFNγ stimulation and after washout for each condition (L2FC > 2, FDR < 0.01). (D) Barplot showing fraction of IFNγ-induced H3K4me1 peaks at 8 h that persist 4 days after washout in each condition. Persistence was defined as L2FC ≥ 0, FDR < 0.01. (E) Heatmap of Z-scored reads within H3K4me1 peaks induced IFNγ after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (F) Boxplot of log2CPM of reads within each peak for each cluster in E. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001.

Techniques Used: Cell Culture, Generated, Whisker Assay

$Durability of IFNγ-induced de novo enhancers is dependent on continued JAK/STAT signaling by IFNγ. (A) Schematic of experimental design: Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media or media supplemented with 1 µM ruxolitinib or 8 µg/ml anti-IFNγ neutralizing antibody. H3K4me1 CUT&Tag was performed at each time point. (B) Boxplot quantifying log2 fold changes of reads within IFNγ-induced H3K4me1 CUT&Tag peaks after 8 h of IFNγ stimulation and after washout for each condition. (C) Barplot showing fraction of IFNγ-induced H3K4me1 peaks at 8 h that persist 4 days after washout in each condition. Persistence was defined as L2FC ≥ 0, FDR < 0.01. (D) . Heatmap of Z-scored reads within H3K4me1 peaks induced IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (E) Representative genome browser tracks of peaks from each cluster in D. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ***P < 0.001; ****P < 0.0001.$
Figure Legend Snippet: Durability of IFNγ-induced de novo enhancers is dependent on continued JAK/STAT signaling by IFNγ. (A) Schematic of experimental design: Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media or media supplemented with 1 µM ruxolitinib or 8 µg/ml anti-IFNγ neutralizing antibody. H3K4me1 CUT&Tag was performed at each time point. (B) Boxplot quantifying log2 fold changes of reads within IFNγ-induced H3K4me1 CUT&Tag peaks after 8 h of IFNγ stimulation and after washout for each condition. (C) Barplot showing fraction of IFNγ-induced H3K4me1 peaks at 8 h that persist 4 days after washout in each condition. Persistence was defined as L2FC ≥ 0, FDR < 0.01. (D) . Heatmap of Z-scored reads within H3K4me1 peaks induced IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (E) Representative genome browser tracks of peaks from each cluster in D. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ***P < 0.001; ****P < 0.0001.

Techniques Used: Cell Culture, Generated, Whisker Assay

IFNγ exposed macrophages exhibit potentiated inflammatory gene expression upon LPS restimulation. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 12 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS genes potentiated by IFNγ before treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes defined as those reaching fivefold increase in reads after LPS stimulation and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS. Genes are clustered by expression level 88 h after IFNγ washout. The top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given genes between PBS and IFNγ treated is quantified for each gene in F. (D) Example of CPM for a potentiated gene that showed basal expression equivalent to that of PBS-treated cells: CSF3 . (E) Example of CPM for a potentiated gene that showed basal expression higher than that of PBS-treated cells: IDO1 . (F) IFNγ-induced H3K4me1 CUT&Tag peaks (as defined in ) were linked to protein-coding genes within ±20 kb of a gene’s TSS. Promoter-proximal (±1 kb of TSS) peaks were excluded from analysis. Analysis was limited to LPS-induced genes. The mean “IFNγ potentiation” of each gene was calculated (defined as the average of the delta log2 fold change for each gene in C between IFNγ-trained and untrained conditions across all time points). The mean IFNγ potentiation value was plotted against the fold change of the enhancer induced 4 days after IFNγ washout. (G) Mean IFNγ potentiation and enhancer fold change in the presence of ruxolitinib as calculated in F.

Figure Legend Snippet: IFNγ exposed macrophages exhibit potentiated inflammatory gene expression upon LPS restimulation. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 12 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS genes potentiated by IFNγ before treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes defined as those reaching fivefold increase in reads after LPS stimulation and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS. Genes are clustered by expression level 88 h after IFNγ washout. The top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given genes between PBS and IFNγ treated is quantified for each gene in F. (D) Example of CPM for a potentiated gene that showed basal expression equivalent to that of PBS-treated cells: CSF3 . (E) Example of CPM for a potentiated gene that showed basal expression higher than that of PBS-treated cells: IDO1 . (F) IFNγ-induced H3K4me1 CUT&Tag peaks (as defined in ) were linked to protein-coding genes within ±20 kb of a gene’s TSS. Promoter-proximal (±1 kb of TSS) peaks were excluded from analysis. Analysis was limited to LPS-induced genes. The mean “IFNγ potentiation” of each gene was calculated (defined as the average of the delta log2 fold change for each gene in C between IFNγ-trained and untrained conditions across all time points). The mean IFNγ potentiation value was plotted against the fold change of the enhancer induced 4 days after IFNγ washout. (G) Mean IFNγ potentiation and enhancer fold change in the presence of ruxolitinib as calculated in F.

Techniques Used: Gene Expression, Cell Culture, RNA sequencing, Expressing

Sustained JAK/STAT signaling is required for long-term IFNγ-induced potentiated and tolerized gene expression responses. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 6 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS-induced genes potentiated by IFNγ treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes are defined as LPS-induced genes reaching at least fourfold increase for macrophages cultured in ruxolitinib and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS in two contiguous time points. Genes are clustered by expression level 88 h after IFNγ washout: the top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given gene between PBS and IFNγ treated is quantified for each gene in F. (D) Boxplot quantifying difference in L2FC for IFNγ pre-treated and PBS-pre-treated cells at each time point. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001. (E) Example of fold change for potentiated gene: CCR7 . (F) Heatmap of log2 fold change in reads of LPS-induced genes tolerized by IFNγ. Tolerance is defined as twofold reduction in transcription in two contiguous time points with IFNγ before treatment and at least fourfold induction by LPS in the presence of ruxolitinib. (G) Heatmap quantifying extent of IFNγ-induced tolerance as in C. (H) Example of fold change for potentiated gene: PTX3 .

Figure Legend Snippet: Sustained JAK/STAT signaling is required for long-term IFNγ-induced potentiated and tolerized gene expression responses. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 6 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS-induced genes potentiated by IFNγ treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes are defined as LPS-induced genes reaching at least fourfold increase for macrophages cultured in ruxolitinib and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS in two contiguous time points. Genes are clustered by expression level 88 h after IFNγ washout: the top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given gene between PBS and IFNγ treated is quantified for each gene in F. (D) Boxplot quantifying difference in L2FC for IFNγ pre-treated and PBS-pre-treated cells at each time point. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001. (E) Example of fold change for potentiated gene: CCR7 . (F) Heatmap of log2 fold change in reads of LPS-induced genes tolerized by IFNγ. Tolerance is defined as twofold reduction in transcription in two contiguous time points with IFNγ before treatment and at least fourfold induction by LPS in the presence of ruxolitinib. (G) Heatmap quantifying extent of IFNγ-induced tolerance as in C. (H) Example of fold change for potentiated gene: PTX3 .

Techniques Used: Gene Expression, Cell Culture, RNA sequencing, Expressing, Whisker Assay

Image Search Results

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: LPS and IFNγ both generate stimulus-specific de novo enhancers in human macrophages; however, only IFNγ-induced enhancers are durable. (A) Schematic of experimental design: Human macrophages were stimulated with either IFNγ (100 ng/ml), LPS (100 ng/ml), or LPS in the presence of 1 µM ruxolitinib for 8 h. Cells were subsequently washed and cultured for an additional 88 h. H3K4me1 CUT&Tag was performed at each time point. (B) Heatmap of Z-scored reads within H3K4me1 peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Top enriched motifs in clusters from B. (D) Box/whisker plot quantifying log2 cpm of reads within IFNγ-induced peaks before and after cytokine washout. (E) Box/whisker quantifying log2 cpm of reads within LPS-induced peaks before and after cytokine washout. (F) Box/whisker quantifying log2 cpm of reads within peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (G) Z-scored heatmap of reads within CUT&Tag peaks of only IFNγ-induced peaks before and after cytokine washout. (H) Boxplot of log2 cpm of reads within peaks for each cluster identified in G. (I) Examples of genome browser tracks for each cluster in E. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. *P < 0.05; ****P < 0.0001.

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Cell Culture, Generated, Whisker Assay

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Ruxolitinib blocks LPS and IFNγ-induced Janus kinase signaling. (A) Human macrophages were pre-treated with increasing concentrations of ruxolitinib for 15 min and subsequently stimulated with IFNγ (100 ng/ml) or LPS (100 ng/ml) for 3 h. Whole-cell lysate western blots showing effect of ruxolitinib on STAT1 and STAT2 phosphorylation by each stimulus. Blot is representative of two replicates from separate human donors. (B and C) Quantification of pSTAT2 and (C) pSTAT1 band intensities in A. Source data are available for this figure: .

Techniques: Western Blot, Phospho-proteomics

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: IFNγ induces long-lasting transcription factor activity and chromatin accessibility after washout. Macrophages were treated with LPS, IFNγ, and LPS in the presence of ruxolitinib for 8 h, as in . Cells were washed and cultured for an additional 88 h. ATACseq was performed after 8 h of stimulation and 4 days after washout. (A) Heatmap of Z-scored reads within ATAC peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (B) Top enriched motifs in clusters from A. (C) Boxplot quantifying log2 cpm of reads within IFNγ-induced ATAC peaks before and after cytokine washout. (D) Boxplot quantifying log2 cpm of reads within LPS-induced ATAC peaks before and after cytokine washout. (E) Boxplot quantifying log2 cpm of reads within ATAC peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (F) Barplot quantifying percent of transcription factor-bound motifs within STAT1 and IRF1 (IFNγ) and IRF1 and NF-κB (LPS) within induced ATAC peaks in C and D for unstimulated, IFNγ/LPS-stimulated macrophages, and stimulated macrophages 4 days after washout. Motif binding predicted using TOBIAS ATACseq footprinting analysis. Results are average of two technical replicates from a single subject; error bars display standard deviation. (G) Human macrophages were stimulated with IFNγ (100 ng/ml), LPS (100 ng/ml), or IFNβ (10 ng/ml) for 8 h, washed, and then cultured for an additional 66 h. Cells were collected, and whole cell western blotting for phosphorylated STAT1 was performed at the indicated time points. Blot is representative of three replicates from two separate human donors. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001. Source data are available for this figure: .

Techniques: Activity Assay, Cell Culture, Generated, Binding Assay, Footprinting, Standard Deviation, Western Blot, Whisker Assay

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Cell surface–bound IFNγ mediates persistent signaling regardless of cytokine manufacturer and can be degraded by trypsinization. (A) Quantification of pSTAT1 intensity normalized to 3H IFNγ in . (B) Human macrophages were treated with Escherichia coli sourced IFNγ purchased from PeproTech and mammalian-sourced IFNγ purchased from Sigma-Aldrich and ACRO. Cells were treated at 100 ng/ml for 8 h, washed, and cultured for an additional 48 h in regular media prior to collection. Cells were collected for immunoblot at the indicated times. Representative blot of duplicates from one human subject. (C) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in B. (D) Human A549 airway epithelial cells were stimulated with 100 ng/ml IFNγ, washed, and cultured for an additional 72 h. Cells were collected for immunoblot at the indicated timepoints. Representative blot of two replicates. (E) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in D. (F) Human macrophages were treated with 100 ng/ml IFNγ for 8 h, washed, and lifted by scraping after incubation with either PBS, 0.5 mM EDTA in PBS, or trypsin. Cells were replated and cultured for an additional 24 h in regular media. Cells were collected for immunoblot at the indicated times. (G) Quantification of pSTAT1 band intensity normalized to GAPDH for each condition in F. Statistical tests were determined by single-tailed t test. *P < 0.05; **P < 0.01. Source data are available for this figure: .

Techniques: Cell Culture, Western Blot, Incubation

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Cell surface–bound IFNγ mediates persistent JAK/STAT signaling even after cytokine washout. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h, washed, and then cultured in regular media or media containing ruxolitinib (1 µM) or increasing concentrations of anti-IFNγ neutralizing antibody for an additional 28 h. Cells were collected, and whole cell western blotting for phosphorylated STAT1 and IRF1 was performed at indicated time points. Representative blot of duplicates from two separate subjects. (B) Human macrophages were stimulated with 100 ng/ml IFNγ for 3 h, washed, and then cultured in either ruxolitinib (1 µM), anti-IFNγ neutralizing antibody (10 µg/ml), or isotype control antibody (10 µg/ml) for 2 h. Samples were collected at the indicated times for immunoblot. Representative blot of duplicates from two separate subjects. (C) Quantification of pSTAT1 band intensities from B. (D) Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and cultured for an additional 88 h in regular media. Supernatants from stimulated macrophages were collected after the 8-h stimulation and 88 h after washout. This supernatant was used to stimulate fresh macrophages for 1 h in the presence/absence of ruxolitinib (1 µM) or anti-IFNγ neutralizing antibody (10 µg/ml). As a control, fresh macrophages were stimulated with media supplemented with 1 ng/ml IFNγ for 1 h. Representative blot of duplicates from two separate subjects. (E) Macrophages were left in regular media or pre-treated with 10 µg/ml CHX for 15 min and stimulated with 100 ng/ml IFNγ for 3 h. Treated macrophages were washed and subsequently cultured for 2 h in regular media, media supplemented with 10 µg/ml CHX, or anti-IFNγ neutralizing antibody (10 µg/ml) and collected for immunoblot. Duplicates from one subject are shown. (F) Quantification of pSTAT1 band intensities in E normalized to band intensity of macrophages treated with IFNγ for 3 h. (G) Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and cultured in regular media or media supplemented with 1 µM ruxolitinib for 16 h. After 16 h, cells were washed again and cultured in regular media for an additional 24 h. Cells were collected for immunoblot at indicated times. Representative blot of four replicates from two subjects. (H) Quantification of pSTAT1 band intensities in G normalized to band intensity of macrophages treated with IFNγ for 3 h. Statistical tests were determined by a single-tailed t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Source data are available for this figure: .

Techniques: Cell Culture, Western Blot, Control

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Extracellular IFNγ signaling sustains chromatin accessibility and ISG expression even after cytokine washout. (A) Schematic of experiments: Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and then cultured for an additional 88 h in regular media or media with 1 µM ruxolitinib for an additional 88 h. Cells were collected for ATACseq and RNAseq at the indicated time points. (B) Heatmap of Z-scored reads within ATAC peaks induced by IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation for 4 days after washout when cultured in regular media or media with 1 µM ruxolitinib. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Boxplot of log2CPM of reads within each peak for each cluster in B. (D) Heatmap of Log2 fold change in RNAseq reads of genes induced at least fivefold after 8 h of IFNγ stimulation. Log2 fold changes are shown after washout for cells cultured in regular media and media containing 1 µM ruxolitinib. Genes are clustered by persistent level of expression after washout (CPM after wash as percent of CPM at 8-h simulation). (E) Boxplot showing Log2 fold changes of individual genes by cluster in D. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. (F) Macrophages were stimulated and washed as above in A; after washout, cells were cultured in media alone, media with 1 µM ruxolitinib, or 10 µg/ml anti-IFNγ neutralizing antibody for 88 h. Cells were collected 88 h after washout, and qPCR was performed for IDO1. Boxplots indicate 2 ΔΔCt normalized to HPRT. Error bars indicate standard deviation. Statistical tests determined by ordinary one way ANOVA. (G) qPCR for IRF1 as in F. ** P < 0.01; ***P < 0.001; ****P < 0.0001.

Techniques: Expressing, Cell Culture, RNA sequencing, Generated, Whisker Assay, Standard Deviation

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Macrophages from a second human donor show reversibility of IFNγ-induced chromatin accessibility and de novo enhancers. Human macrophages from a second human subject were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media or media supplemented with 1 µM ruxolitinib. ATAC and H3K4me1 CUT&Tag was performed after 8 h of stimulation and 88 h after cytokine washout. (A) Boxplot quantifying log2 fold changes of reads within IFNγ-induced ATAC peaks after 8 h of IFNγ stimulation and after washout for each condition (L2FC > 2, FDR < 0.01). (B) Heatmap of Z-scored reads within ATAC peaks induced IFNγ after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Boxplot quantifying log2 fold changes of reads within IFNγ-induced H3K4me1 CUT&Tag peaks after 8 h of IFNγ stimulation and after washout for each condition (L2FC > 2, FDR < 0.01). (D) Barplot showing fraction of IFNγ-induced H3K4me1 peaks at 8 h that persist 4 days after washout in each condition. Persistence was defined as L2FC ≥ 0, FDR < 0.01. (E) Heatmap of Z-scored reads within H3K4me1 peaks induced IFNγ after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (F) Boxplot of log2CPM of reads within each peak for each cluster in E. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001.

Techniques: Cell Culture, Generated, Whisker Assay

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Durability of IFNγ-induced de novo enhancers is dependent on continued JAK/STAT signaling by IFNγ. (A) Schematic of experimental design: Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media or media supplemented with 1 µM ruxolitinib or 8 µg/ml anti-IFNγ neutralizing antibody. H3K4me1 CUT&Tag was performed at each time point. (B) Boxplot quantifying log2 fold changes of reads within IFNγ-induced H3K4me1 CUT&Tag peaks after 8 h of IFNγ stimulation and after washout for each condition. (C) Barplot showing fraction of IFNγ-induced H3K4me1 peaks at 8 h that persist 4 days after washout in each condition. Persistence was defined as L2FC ≥ 0, FDR < 0.01. (D) . Heatmap of Z-scored reads within H3K4me1 peaks induced IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (E) Representative genome browser tracks of peaks from each cluster in D. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ***P < 0.001; ****P < 0.0001.

Techniques: Cell Culture, Generated, Whisker Assay

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: IFNγ exposed macrophages exhibit potentiated inflammatory gene expression upon LPS restimulation. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 12 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS genes potentiated by IFNγ before treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes defined as those reaching fivefold increase in reads after LPS stimulation and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS. Genes are clustered by expression level 88 h after IFNγ washout. The top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given genes between PBS and IFNγ treated is quantified for each gene in F. (D) Example of CPM for a potentiated gene that showed basal expression equivalent to that of PBS-treated cells: CSF3 . (E) Example of CPM for a potentiated gene that showed basal expression higher than that of PBS-treated cells: IDO1 . (F) IFNγ-induced H3K4me1 CUT&Tag peaks (as defined in ) were linked to protein-coding genes within ±20 kb of a gene’s TSS. Promoter-proximal (±1 kb of TSS) peaks were excluded from analysis. Analysis was limited to LPS-induced genes. The mean “IFNγ potentiation” of each gene was calculated (defined as the average of the delta log2 fold change for each gene in C between IFNγ-trained and untrained conditions across all time points). The mean IFNγ potentiation value was plotted against the fold change of the enhancer induced 4 days after IFNγ washout. (G) Mean IFNγ potentiation and enhancer fold change in the presence of ruxolitinib as calculated in F.

Techniques: Gene Expression, Cell Culture, RNA sequencing, Expressing

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Sustained JAK/STAT signaling is required for long-term IFNγ-induced potentiated and tolerized gene expression responses. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 6 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS-induced genes potentiated by IFNγ treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes are defined as LPS-induced genes reaching at least fourfold increase for macrophages cultured in ruxolitinib and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS in two contiguous time points. Genes are clustered by expression level 88 h after IFNγ washout: the top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given gene between PBS and IFNγ treated is quantified for each gene in F. (D) Boxplot quantifying difference in L2FC for IFNγ pre-treated and PBS-pre-treated cells at each time point. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001. (E) Example of fold change for potentiated gene: CCR7 . (F) Heatmap of log2 fold change in reads of LPS-induced genes tolerized by IFNγ. Tolerance is defined as twofold reduction in transcription in two contiguous time points with IFNγ before treatment and at least fourfold induction by LPS in the presence of ruxolitinib. (G) Heatmap quantifying extent of IFNγ-induced tolerance as in C. (H) Example of fold change for potentiated gene: PTX3 .

Techniques: Gene Expression, Cell Culture, RNA sequencing, Expressing, Whisker Assay

a–h Allogeneic B10.BR-into-B6 (Allo) or syngeneic B6-into-B6 (Syn) BMT using Stat1 fl/fl x Villin - Cre ( Stat1 ΔIEC ) or Cre-negative Stat1 fl/fl ( Stat1 WT ) littermate controls. a Experimental design. b Survival curves; n = 3 Syn Stat1 WT , 3 Syn Stat1 ΔIEC , 15 Allo Stat1 WT , 13 Allo Stat1 ΔIEC mice; Log-rank-test (Allo Stat1 WT vs Allo Stat1 ΔIEC , p = 0.0034). c Body weight loss; n = 15 Stat1 WT , 12 Stat1 ΔIEC mice ( p = 0.0179). d-e Representative staining ( d ) and quantification ( e ) of Ki67 + crypt cells, day 7 post-BMT (n = 168 Syn Stat1 WT , 178 Syn Stat1 ΔIEC , 426 Allo Stat1 WT , 528 Allo Stat1 ΔIEC crypts). f Ki67 + crypt cells 14 days post-BMT (n = 253 Allo Stat1 WT , 408 Allo Stat1 ΔIEC crypts). Representative staining ( g ) and quantification ( h ) of crypt Olfm4 + cells 14 days post-BMT (n = 388 Allo Stat1 WT , 703 Allo Stat1 ΔIEC crypts; scale bars = 50μm). i Analysis of crypt Ki67 immunohistochemistry after B10.BR-into-B6 or B6-into-B6 BMT using IFNαR-intact (WT) or IFNαR –/– knockout (KO) recipients (n = 98 Syn IFNαR WT , 86 Syn IFNαR KO , 83 Allo IFNαR WT , 61 Allo IFNαR KO crypts; Allo Ifnar1 WT vs Allo Ifnar1 ΔIEC , p = 0.9835). j–l Analyses after BMT into Ifngr fl/fl x Villin - Cre ( IFNγR ΔEC ) recipients or Cre-negative IFNγ R fl/fl ( IFNγR WT ) littermate controls. j Analysis of crypt Ki67 immunohistochemistry after B10.BR-into-B6 or B6-into-B6 BMT (n = 175 Syn IFNγR WT , 106 Syn IFNγR ΔIEC , 195 Allo IFNγR WT , 318 Allo IFNγR ΔIEC crypts). Frequencies of crypt Ki67 + cells ( k ; n = 248 BM IFNγR WT , 351 BM + T IFNγR WT , 361 BM + T IFNγR ΔIEC crypts) and frequencies of crypt Olfm4 + cells ( l ; n = 585 BM IFNγR WT , 978 BM + T IFNγR WT , 1053 BM + T IFNγR ΔIEC crypts) 10 days after LP-into-B6 BMT with either donor marrow alone (BM) or marrow and T cells (BM + T). Panels ( b , c , e , f , h , k , l ) are combined from two independent experiments; ( d , g , j ) are representative of two independent experiments. Graphs indicate mean and s.e.m.; comparisons performed with two-tailed t tests (two groups) or one-way ANOVA multiple comparison testing (multiple groups); * p < 0.05, ** p < 0.01, *** p < 0.001. The exact p values are p < 0.001 unless specified otherwise.

Journal: Nature Communications

Article Title: STAT1 regulates immune-mediated intestinal stem cell proliferation and epithelial regeneration

doi: 10.1038/s41467-024-55227-5

Figure Lengend Snippet: a–h Allogeneic B10.BR-into-B6 (Allo) or syngeneic B6-into-B6 (Syn) BMT using Stat1 fl/fl x Villin - Cre ( Stat1 ΔIEC ) or Cre-negative Stat1 fl/fl ( Stat1 WT ) littermate controls. a Experimental design. b Survival curves; n = 3 Syn Stat1 WT , 3 Syn Stat1 ΔIEC , 15 Allo Stat1 WT , 13 Allo Stat1 ΔIEC mice; Log-rank-test (Allo Stat1 WT vs Allo Stat1 ΔIEC , p = 0.0034). c Body weight loss; n = 15 Stat1 WT , 12 Stat1 ΔIEC mice ( p = 0.0179). d-e Representative staining ( d ) and quantification ( e ) of Ki67 + crypt cells, day 7 post-BMT (n = 168 Syn Stat1 WT , 178 Syn Stat1 ΔIEC , 426 Allo Stat1 WT , 528 Allo Stat1 ΔIEC crypts). f Ki67 + crypt cells 14 days post-BMT (n = 253 Allo Stat1 WT , 408 Allo Stat1 ΔIEC crypts). Representative staining ( g ) and quantification ( h ) of crypt Olfm4 + cells 14 days post-BMT (n = 388 Allo Stat1 WT , 703 Allo Stat1 ΔIEC crypts; scale bars = 50μm). i Analysis of crypt Ki67 immunohistochemistry after B10.BR-into-B6 or B6-into-B6 BMT using IFNαR-intact (WT) or IFNαR –/– knockout (KO) recipients (n = 98 Syn IFNαR WT , 86 Syn IFNαR KO , 83 Allo IFNαR WT , 61 Allo IFNαR KO crypts; Allo Ifnar1 WT vs Allo Ifnar1 ΔIEC , p = 0.9835). j–l Analyses after BMT into Ifngr fl/fl x Villin - Cre ( IFNγR ΔEC ) recipients or Cre-negative IFNγ R fl/fl ( IFNγR WT ) littermate controls. j Analysis of crypt Ki67 immunohistochemistry after B10.BR-into-B6 or B6-into-B6 BMT (n = 175 Syn IFNγR WT , 106 Syn IFNγR ΔIEC , 195 Allo IFNγR WT , 318 Allo IFNγR ΔIEC crypts). Frequencies of crypt Ki67 + cells ( k ; n = 248 BM IFNγR WT , 351 BM + T IFNγR WT , 361 BM + T IFNγR ΔIEC crypts) and frequencies of crypt Olfm4 + cells ( l ; n = 585 BM IFNγR WT , 978 BM + T IFNγR WT , 1053 BM + T IFNγR ΔIEC crypts) 10 days after LP-into-B6 BMT with either donor marrow alone (BM) or marrow and T cells (BM + T). Panels ( b , c , e , f , h , k , l ) are combined from two independent experiments; ( d , g , j ) are representative of two independent experiments. Graphs indicate mean and s.e.m.; comparisons performed with two-tailed t tests (two groups) or one-way ANOVA multiple comparison testing (multiple groups); * p < 0.05, ** p < 0.01, *** p < 0.001. The exact p values are p < 0.001 unless specified otherwise.

Article Snippet: Media containing human Interleukin-2 (Proleukin; 12 IE/ml, Prometheus) and, where applicable, anti-IFNγ neutralizing antibodies (50 μg/ml, eBioscience cat. #16-7318-85), were added to the co-cultures.

Techniques: Staining, Immunohistochemistry, Knock-Out, Two Tailed Test, Comparison

a Representative images and quantification of B6 SI organoids co-cultured with allogeneic BALB/c T cells ± anti-IFNγ neutralizing antibodies; culture day 7; frequency: n = 4 wells/group; size: n = 152 (control), 64 ( + T cells), 114 ( + anti-IFNγ) organoids/group; scale bars = 500 μm. b Size of human SI organoids cultured 1:1 with human allogeneic T cells (500 single cells with 500 T cells) ± anti-IFNγ neutralizing antibodies; culture day 7; n = 75 (control), 107 ( + T cells), 99 ( + anti-IFNγ) organoids/group (T cells vs anti-IFNγ, p = 0.0011). c Representative images and quantification of SI organoids ± rmIFNγ for 7 days; n = 139 (0 ng/ml), 76 (0.1 ng/ml), 44 (1 ng/ml) organoids/group; scale bars = 500 μm (0 ng/ml vs 0.1 ng/ml, p = 0.0292). d Ccnd1 qPCR of mouse SI organoids ± rmIFNγ for 24 h; n = 6 wells/group; two-tailed Mann–Whitney analysis ( p = 0.0022). e Size of SI ISC colonies ± rmIFNγ for 3 days; n = 69 (0 ng/ml), 31 (0.1 ng/ml), 73 (1 ng/ml) colonies/group. f Cell-cycle analysis of Lgr5-GFP high cells in SI organoids ± rmIFNγ (0.1 ng/ml) for 24 h; n = 4 wells/group (Control vs rmIFNγ, p = 0.0030 for G0; p = 0.0014 for S/G2/M). g Number of SI organoids +/- decreasing concentrations of IFNγ (culture day 5, n = 4 wells/group; 0 ng/ml vs 0.05 ng/ml, p = 0.0053). h Size of Stat1 WT or Stat1 ΔIEC B6 SI organoids ± “low dose” IFNγ [culture day 5; n = 132 ( Stat1 WT ), 110 (WT + IFNγ), 112 ( Stat1 ΔIEC ), 95 (ΔIEC + IFNγ) organoids/group]. i Quantification of human duodenal organoids +/- “low dose” IFNγ; culture day 7; frequency: n = 9 wells/group; size: n = 100 (0 ng/ml), 144 (0.01 ng/ml) organoids/group, p = 0.0011. Graphs indicate mean and s.e.m.; comparisons performed with two-tailed t tests (two groups) or one-way ANOVA multiple comparison testing (multiple groups), unless stated otherwise; ** p < 0.01, *** p < 0.001. The exact p values are p < 0.001 unless specified otherwise. Data are representative of five ( a ), two ( b – e , g – i ), or three ( f ) independent e xper i ments.

Journal: Nature Communications

Article Title: STAT1 regulates immune-mediated intestinal stem cell proliferation and epithelial regeneration

doi: 10.1038/s41467-024-55227-5

Figure Lengend Snippet: a Representative images and quantification of B6 SI organoids co-cultured with allogeneic BALB/c T cells ± anti-IFNγ neutralizing antibodies; culture day 7; frequency: n = 4 wells/group; size: n = 152 (control), 64 ( + T cells), 114 ( + anti-IFNγ) organoids/group; scale bars = 500 μm. b Size of human SI organoids cultured 1:1 with human allogeneic T cells (500 single cells with 500 T cells) ± anti-IFNγ neutralizing antibodies; culture day 7; n = 75 (control), 107 ( + T cells), 99 ( + anti-IFNγ) organoids/group (T cells vs anti-IFNγ, p = 0.0011). c Representative images and quantification of SI organoids ± rmIFNγ for 7 days; n = 139 (0 ng/ml), 76 (0.1 ng/ml), 44 (1 ng/ml) organoids/group; scale bars = 500 μm (0 ng/ml vs 0.1 ng/ml, p = 0.0292). d Ccnd1 qPCR of mouse SI organoids ± rmIFNγ for 24 h; n = 6 wells/group; two-tailed Mann–Whitney analysis ( p = 0.0022). e Size of SI ISC colonies ± rmIFNγ for 3 days; n = 69 (0 ng/ml), 31 (0.1 ng/ml), 73 (1 ng/ml) colonies/group. f Cell-cycle analysis of Lgr5-GFP high cells in SI organoids ± rmIFNγ (0.1 ng/ml) for 24 h; n = 4 wells/group (Control vs rmIFNγ, p = 0.0030 for G0; p = 0.0014 for S/G2/M). g Number of SI organoids +/- decreasing concentrations of IFNγ (culture day 5, n = 4 wells/group; 0 ng/ml vs 0.05 ng/ml, p = 0.0053). h Size of Stat1 WT or Stat1 ΔIEC B6 SI organoids ± “low dose” IFNγ [culture day 5; n = 132 ( Stat1 WT ), 110 (WT + IFNγ), 112 ( Stat1 ΔIEC ), 95 (ΔIEC + IFNγ) organoids/group]. i Quantification of human duodenal organoids +/- “low dose” IFNγ; culture day 7; frequency: n = 9 wells/group; size: n = 100 (0 ng/ml), 144 (0.01 ng/ml) organoids/group, p = 0.0011. Graphs indicate mean and s.e.m.; comparisons performed with two-tailed t tests (two groups) or one-way ANOVA multiple comparison testing (multiple groups), unless stated otherwise; ** p < 0.01, *** p < 0.001. The exact p values are p < 0.001 unless specified otherwise. Data are representative of five ( a ), two ( b – e , g – i ), or three ( f ) independent e xper i ments.

Techniques: Cell Culture, Control, Two Tailed Test, MANN-WHITNEY, Cell Cycle Assay, Comparison

a–d Single cell RNA sequencing of small intestine crypt cells from healthy B6 mice (Naive) or five days after B6-into-B6 syngeneic or B10.BR-into-B6 allogeneic BMT. a GSEA of ISC cluster genes filtered for differential correlation with Stat1 expression in ISCs from syngeneic and allogeneic BMT recipients; NES: normalized enrichment score. b Myc expression in crypt epithelial populations, highlighting Myc upregulation in ISCs after allo-BMT; gene expression imputed using MAGIC; (n = 3685 Naive, 3371 Syngeneic, 4670 Allogeneic cells); E: enterocytes, EEC: enteroendocrine cells, G: goblet cells, I: ISCs, P: Paneth cells, T: tuft cells. The boxplots represent three quartiles, and the whiskers indicate 1.5 times the interquartile range. c Correlation of Myc expression with expression of IFNγ-responsive genes or with expression of Stat1 in ISCs during homeostasis and after syngeneic or allogeneic BMT. d Correlation between average MAGIC-imputed expression of Myc target genes (Hallmark MYC_v1 and v2 pathways) and average imputed cell-cycle-related genes (KEGG cell cycle pathway) in ISCs after allogeneic BMT. The plot shows average gene expression (2nd–98th percentiles). e-f Imaging of full-thickness ileum by 3-D whole mount microscopy four days after B10.BR-into-B6 allogeneic BMT using either TCD BM alone, which does not result in GVHD, or BM and T cells, resulting in GVHD. Shown are 2-D optical slices of the ISC compartment in the lower crypt region ( e ) or the very base of the crypt ( f ) from 3-D fluorescent imaging performed after staining with anti-c-Myc (green), anti-Olfm4 (orange glow), and DAPI (blue). Arrows indicate c-Myc + Olfm4 + ISCs; scale bar: 25 μm.

Journal: Nature Communications

Article Title: STAT1 regulates immune-mediated intestinal stem cell proliferation and epithelial regeneration

doi: 10.1038/s41467-024-55227-5

Figure Lengend Snippet: a–d Single cell RNA sequencing of small intestine crypt cells from healthy B6 mice (Naive) or five days after B6-into-B6 syngeneic or B10.BR-into-B6 allogeneic BMT. a GSEA of ISC cluster genes filtered for differential correlation with Stat1 expression in ISCs from syngeneic and allogeneic BMT recipients; NES: normalized enrichment score. b Myc expression in crypt epithelial populations, highlighting Myc upregulation in ISCs after allo-BMT; gene expression imputed using MAGIC; (n = 3685 Naive, 3371 Syngeneic, 4670 Allogeneic cells); E: enterocytes, EEC: enteroendocrine cells, G: goblet cells, I: ISCs, P: Paneth cells, T: tuft cells. The boxplots represent three quartiles, and the whiskers indicate 1.5 times the interquartile range. c Correlation of Myc expression with expression of IFNγ-responsive genes or with expression of Stat1 in ISCs during homeostasis and after syngeneic or allogeneic BMT. d Correlation between average MAGIC-imputed expression of Myc target genes (Hallmark MYC_v1 and v2 pathways) and average imputed cell-cycle-related genes (KEGG cell cycle pathway) in ISCs after allogeneic BMT. The plot shows average gene expression (2nd–98th percentiles). e-f Imaging of full-thickness ileum by 3-D whole mount microscopy four days after B10.BR-into-B6 allogeneic BMT using either TCD BM alone, which does not result in GVHD, or BM and T cells, resulting in GVHD. Shown are 2-D optical slices of the ISC compartment in the lower crypt region ( e ) or the very base of the crypt ( f ) from 3-D fluorescent imaging performed after staining with anti-c-Myc (green), anti-Olfm4 (orange glow), and DAPI (blue). Arrows indicate c-Myc + Olfm4 + ISCs; scale bar: 25 μm.

Techniques: RNA Sequencing Assay, Expressing, Imaging, Microscopy, Staining

a , b Single cell RNA sequencing of B6 small intestine organoids after exposure to IFNγ; E: enterocytes, EEC: enteroendocrine cells, P/G: Paneth/goblet cells, I: ISCs, T: tuft cells. a UMAP plots indicating the experimental culture conditions and showing the cell type annotations for each cluster. Star symbol indicates ISC clusters. b Myc expression: n = 4599 (Ctrl), 3166 (6 h), 4533 (24 h), 6353 (48 h) cells. The boxplots represent three quartiles, and the whiskers indicate 1.5 times the interquartile range. qPCR analysis of mouse organoids ( c , n = 6 wells/group) or ISC colonies ( d , n = 4 wells/group) ± IFNγ (1 ng/ml) for gene expression of Irf1 (d, 0 vs 3 h, p = 0.0360), Myc (c, 3 vs 9 h, p = 0.0045; d, 0 vs 3 h, p = 0.0227), Axin2 , and Ccnd1 (d, 0 vs 6 h, p = 0.0286; 0 vs 9 h, p = 0.0451); Kruskal-Wallis multiple comparison testing. e qPCR analysis of human organoids ± 6 h exposure to IFNγ (n = 3 donors/group); Friedman-tests; 0 vs 1 ng/ml, p = 0.0429 for IRF1 ; p = 0.0429 for MYC ; p = 0.0429 for AXIN2 ; p = 0.0429 for CCND1 . Representative images ( f ) and quantification ( g ) of mouse organoids ± 10058-F4 (μM); culture day 4; frequency: n = 4 wells/group; size: n = 353 (0), 395 (60), 352 (100), 0 (400) organoids/group; one-way ANOVA multiple comparison testing; 60 vs 100 μM, p = 0.0089. Representative images ( h ) and quantification ( i ) of mouse organoids ± IFNγ (ng/ml) ± 10058-F4 (60μM); culture day 4; n = 251 (No IFNγ), 236 (10058-F4), 231 (IFNγ 0.01), 238 (IFNγ 0.01 + 10058-F4), 228 (IFNγ 0.1), 223 (IFNγ 0.1 + 10058-F4) organoids/group; one-way ANOVA multiple comparison testing; No IFNγ vs 10058-F4, p = 0.1087. j qPCR for Ccnd1 gene expression in mouse organoids ± IFNγ + /− 10058-F4 for 6 h; n = 6 wells/group; Kruskal-Wallis multiple comparison testing; No IFNγ vs 0.1 ng/ml, p = 0.0197; IFNγ 0.1 ng/ml + 10058-F4 60 vs 100 μM, p = 0.0197. Graphs indicate mean and s.e.m.; * p < 0.05, ** p < 0.01, *** p < 0.001. The exact p values are p < 0.001 unless specified otherwise. Scale bars = 500 μm. Panels ( c , d , f–g ) are representative of two independent experiments.

Journal: Nature Communications

Article Title: STAT1 regulates immune-mediated intestinal stem cell proliferation and epithelial regeneration

doi: 10.1038/s41467-024-55227-5

Figure Lengend Snippet: a , b Single cell RNA sequencing of B6 small intestine organoids after exposure to IFNγ; E: enterocytes, EEC: enteroendocrine cells, P/G: Paneth/goblet cells, I: ISCs, T: tuft cells. a UMAP plots indicating the experimental culture conditions and showing the cell type annotations for each cluster. Star symbol indicates ISC clusters. b Myc expression: n = 4599 (Ctrl), 3166 (6 h), 4533 (24 h), 6353 (48 h) cells. The boxplots represent three quartiles, and the whiskers indicate 1.5 times the interquartile range. qPCR analysis of mouse organoids ( c , n = 6 wells/group) or ISC colonies ( d , n = 4 wells/group) ± IFNγ (1 ng/ml) for gene expression of Irf1 (d, 0 vs 3 h, p = 0.0360), Myc (c, 3 vs 9 h, p = 0.0045; d, 0 vs 3 h, p = 0.0227), Axin2 , and Ccnd1 (d, 0 vs 6 h, p = 0.0286; 0 vs 9 h, p = 0.0451); Kruskal-Wallis multiple comparison testing. e qPCR analysis of human organoids ± 6 h exposure to IFNγ (n = 3 donors/group); Friedman-tests; 0 vs 1 ng/ml, p = 0.0429 for IRF1 ; p = 0.0429 for MYC ; p = 0.0429 for AXIN2 ; p = 0.0429 for CCND1 . Representative images ( f ) and quantification ( g ) of mouse organoids ± 10058-F4 (μM); culture day 4; frequency: n = 4 wells/group; size: n = 353 (0), 395 (60), 352 (100), 0 (400) organoids/group; one-way ANOVA multiple comparison testing; 60 vs 100 μM, p = 0.0089. Representative images ( h ) and quantification ( i ) of mouse organoids ± IFNγ (ng/ml) ± 10058-F4 (60μM); culture day 4; n = 251 (No IFNγ), 236 (10058-F4), 231 (IFNγ 0.01), 238 (IFNγ 0.01 + 10058-F4), 228 (IFNγ 0.1), 223 (IFNγ 0.1 + 10058-F4) organoids/group; one-way ANOVA multiple comparison testing; No IFNγ vs 10058-F4, p = 0.1087. j qPCR for Ccnd1 gene expression in mouse organoids ± IFNγ + /− 10058-F4 for 6 h; n = 6 wells/group; Kruskal-Wallis multiple comparison testing; No IFNγ vs 0.1 ng/ml, p = 0.0197; IFNγ 0.1 ng/ml + 10058-F4 60 vs 100 μM, p = 0.0197. Graphs indicate mean and s.e.m.; * p < 0.05, ** p < 0.01, *** p < 0.001. The exact p values are p < 0.001 unless specified otherwise. Scale bars = 500 μm. Panels ( c , d , f–g ) are representative of two independent experiments.

Techniques: RNA Sequencing Assay, Expressing, Comparison

a Diagram of the Myc gene locus (NCBI Refseq) with mapping of the genome location, ATAC-seq peaks (blue) from sorted Lgr5 GFP ISCs (GSE83394), ENCODE (Accession EM10E0600598) candidate cis-regulatory elements (cCREs) indicating a proximal enhancer (black) and promoter regions (gray), and location of primers (red) used to detect STAT1 binding by qPCR following anti-STAT1 antibody and isotype control ChIP. b Δ% of input of anti-STAT1/isotype ChIP-qPCR of Irf1, Myc and negative control (NC) Foxp3 loci performed on ISC colonies treated with IFNγ (3 h, 1 ng/ml). c Myc and Ccnd1 qPCR analysis of mouse Stat1 WT or Stat1 ΔIEC SI organoids cultured ± IFNγ (n = 6 wells/group); Stat1 WT vs IFNγ, p = 0.0022 for both Myc and Ccnd1 ; Stat1 ΔIEC vs IFNγ, p = 0.0649 for Myc, p = 0.1255 for Ccnd1 ; combined from two independent experiments. d , e Allogeneic B10.BR-into-B6 BMT using Stat1 ΔIEC recipients or Stat1 WT littermate control recipients. d qPCR analysis of SI crypts harvested day 7 post-BMT (n = 9 Stat1 WT , 8 Stat1 ΔIEC recipients; combined from two independent experiments). e Immunofluorescent staining and quantification of c-Myc (green) in ileum (day 5 post-BMT; n = 9 independent sections each from 3 Stat1 WT and 3 Stat1 ΔIEC recipients; scale bars = 20 μm; two-tailed t test). f Olfm4-Cre x RiboTag (Olfm4-Ribo) mice were treated intraperitoneally with 20μg IFNγ or PBS twice, 48 and 24 h prior to harvest, as well as tamoxifen 20 h prior to harvest. Hemagglutinin-labeled Olfm4 + ISC ribosomes were then isolated from small intestine, and qPCR was performed on the associated RNA transcripts; n = 7 mice/group; combined from three independent experiments. Graphs indicate mean and s.e.m.; comparisons performed with two-tailed Mann–Whitney unless otherwise specified; ** p < 0.01, *** p < 0.001. The exact p values are p < 0.001 unless specified otherwise.

Journal: Nature Communications

Article Title: STAT1 regulates immune-mediated intestinal stem cell proliferation and epithelial regeneration

doi: 10.1038/s41467-024-55227-5

Figure Lengend Snippet: a Diagram of the Myc gene locus (NCBI Refseq) with mapping of the genome location, ATAC-seq peaks (blue) from sorted Lgr5 GFP ISCs (GSE83394), ENCODE (Accession EM10E0600598) candidate cis-regulatory elements (cCREs) indicating a proximal enhancer (black) and promoter regions (gray), and location of primers (red) used to detect STAT1 binding by qPCR following anti-STAT1 antibody and isotype control ChIP. b Δ% of input of anti-STAT1/isotype ChIP-qPCR of Irf1, Myc and negative control (NC) Foxp3 loci performed on ISC colonies treated with IFNγ (3 h, 1 ng/ml). c Myc and Ccnd1 qPCR analysis of mouse Stat1 WT or Stat1 ΔIEC SI organoids cultured ± IFNγ (n = 6 wells/group); Stat1 WT vs IFNγ, p = 0.0022 for both Myc and Ccnd1 ; Stat1 ΔIEC vs IFNγ, p = 0.0649 for Myc, p = 0.1255 for Ccnd1 ; combined from two independent experiments. d , e Allogeneic B10.BR-into-B6 BMT using Stat1 ΔIEC recipients or Stat1 WT littermate control recipients. d qPCR analysis of SI crypts harvested day 7 post-BMT (n = 9 Stat1 WT , 8 Stat1 ΔIEC recipients; combined from two independent experiments). e Immunofluorescent staining and quantification of c-Myc (green) in ileum (day 5 post-BMT; n = 9 independent sections each from 3 Stat1 WT and 3 Stat1 ΔIEC recipients; scale bars = 20 μm; two-tailed t test). f Olfm4-Cre x RiboTag (Olfm4-Ribo) mice were treated intraperitoneally with 20μg IFNγ or PBS twice, 48 and 24 h prior to harvest, as well as tamoxifen 20 h prior to harvest. Hemagglutinin-labeled Olfm4 + ISC ribosomes were then isolated from small intestine, and qPCR was performed on the associated RNA transcripts; n = 7 mice/group; combined from three independent experiments. Graphs indicate mean and s.e.m.; comparisons performed with two-tailed Mann–Whitney unless otherwise specified; ** p < 0.01, *** p < 0.001. The exact p values are p < 0.001 unless specified otherwise.

Techniques: Binding Assay, Control, Negative Control, Cell Culture, Staining, Two Tailed Test, Labeling, Isolation, MANN-WHITNEY

While regulation of ISC Myc expression may be primarily Wnt-driven at baseline, we propose that there is additional STAT1-dependent regulation of Myc expression driven by IFNγ in the setting of GVHD.

Journal: Nature Communications

Article Title: STAT1 regulates immune-mediated intestinal stem cell proliferation and epithelial regeneration

doi: 10.1038/s41467-024-55227-5

Figure Lengend Snippet: While regulation of ISC Myc expression may be primarily Wnt-driven at baseline, we propose that there is additional STAT1-dependent regulation of Myc expression driven by IFNγ in the setting of GVHD.

Techniques: Expressing

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