IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration

The influence of aging on intestinal stem cells and their niche can explain underlying causes for perturbation in their function observed during aging. Molecular mechanisms for such a decrease in the functionality of intestinal stem cells during aging remain largely undetermined. Using transcriptome-wide approaches, our study demonstrates that aging intestinal stem cells strongly upregulate antigen presenting pathway genes and over-express secretory lineage marker genes resulting in lineage skewed differentiation into the secretory lineage and strong upregulation of MHC class II antigens in the aged intestinal epithelium. Mechanistically, we identified an increase in proinflammatory cells in the lamina propria as the main source of elevated interferon gamma (IFNγ) in the aged intestine, that leads to the induction of Stat1 activity in intestinal stem cells thus priming the aberrant differentiation and elevated antigen presentation in epithelial cells. Of note, systemic inhibition of IFNγ-signaling completely reverses these aging phenotypes and reinstalls regenerative capacity of the aged intestinal epithelium.

f) qRT-PCR analysis of MHCII genes, stem and secretory cell marker genes expression in intestinal organoids treated as indicated.IFN used at 0.2 ng/ml and Baricitinib at 2 µM.n = 4 mice per group were analyzed.Error bars represent the SD.P value was calculated by Welch's t test.

Supplementary Tables:
Table S1.Primers for for Real-time qPCR.Related to Methods.S2.

Figure S1 .
Figure S1.Mouse intestinal crypts show transcriptional alterations during aging.a) Point plot of the Euclidean sample-to-sample distance in the RNA-seq datasets as in Figure 1a.Error bars represent the SD.P value was calculated by t test.m = months.n = 4 mice per group were analyzed.b) Bar chart showing the number of DEGs in aging intestinal crypts measured by RNA-seq as in Figure 1a.m = months.c) Venn diagram of the DEGs found in the RNA-seq of the intestinal crypts (young versus old animals).P value was calculated by hypergeometric distribution test.d) Left panel: Hierarchical clustering and heatmap of the gene overlap correlation of the top 120 GO pathways found enriched in intestinal crypts during aging.The clustering revealed four main clusters of pathways that can be used for subdividing the pathways according to the main GO category (signaling, metabolism, inflammation and another mixed category composed of stress response, and epigenetics related pathways).Right panel: Bar chart showing the -log10 enrichment p value (x-axis) and activation Zscore (color coded) of the GO pathways.e) Box plot of the geneset enrichment analysis of the antigen presentation pathway in mouse intestinal crypts from specified ages and in both genders.f) RT-qPCR validation of the indicated gene expression levels in young (2 months) and old (>20 months) female (F) and male (M) mice.At least n = 3 mice per group were analyzed.Error bars represent the SD.P value was calculated by Welch's one-tail t test.* = p < 0.05; ** = p < 0.01.

Figure S2 .
Figure S2.Aged mouse intestinal crypts show gender-specific transcriptional alterations.a) Scatter plot of delta Z-scores (old minus young Z-scores mean) from the geneset enrichment analysis of the GO pathways found significantly enriched both in the Lgr5 hi cells and whole crypt cells.r = Pearson correlation coefficient.b) Bar chart showing the percentage of GO pathways in each of the indicated main GO categories.Immunosystem and inflammation related pathways are those most commonly regulated between different genders.c) Representative FACS plots showing percentages of the MHCII + cells within the Olfm4 + cells isolated from the intestine of young (upper panel) and old (lower panel) mice.d) Box plot of geneset enrichment analysis of the marker datasets of the indicated different intestinal secretory cell populations within the Lgr5 hi intestinal stem cells purified from female (F) and male (M), young (Y) and old (O) mice.P value was calculated by Wilcoxon paired test.

Figure S3 .
Figure S3.Major Histocompatibility Complex class II (MHCII) is strongly overexpressed during aging in the whole intestinal crypts.a) Heatmap of the expression of the top 5 markers of each of the 10 clusters identified in the scRNA-seq analysis.b) Scatter plot of PC1 and PC2 values of the single cell RNA-seq (scRNA-seq) dataset (both the young and the old RNA-seq merged).White-to-red color code represents the expression value of the indicated markers representative of the specified cellular cluster.c) Line-plot of the cell population density of each of the indicated clusters along the PC1 (upper panel) or PC2 (lower panel) measured by the scRNA-seq experiment.d) Line-plot of the average Z-score distribution of the indicated geneset markers along the PC1 (left panel) or PC2 (right panel) measured by the scRNA-seq experiment.Z-score is calculated by gene base scaling the count data.e) Scatter plot of PC1 and PC2 values of the single cell RNA-seq (scRNA-seq) dataset (both the young and the old RNA-seq merged).White-to-red color code represents the expression value of the two indicated markers representative of the antigen presentation pathway.f) Box plot of the geneset enrichment analysis of the antigen presentation pathway in the specified cell populations from young (Y) or old (O) mouse intestinal crypts.g) Bar chart indicating the percentage of cells in young or old animals belonging to the indicated clusters in the scRNA-seq.Aging increases the secretory progenitor cluster (cl.3) and reduces stem and TA cells (cl.1-2).h) Representative FACS plots (upper panels) and relative quantification (bottom panel) showing percentages of the Lrg5 + cells in the intestine of mice at the indicated ages.n ≥ 5 mice per group were analyzed.Error bars represent the SD.P value was calculated by Wilcoxon paired test.i) Line-plot of the cell population density of the Lgr5-expressing cells (Lgr5 + ) along the PC2 axis measured by the scRNA-seq experiment.

Figure S4 .
Figure S4.Intestinal cell composition changes during aging.a) Representative pictures of anti-Chga staining in young and old intestinal crypts.Scale bar, 20 µm.The dotted lines indicate the crypt structure.b) The bar chart shows the average number of cells positive for Chga in young and old intestinal crypts.n = 5 mice per group were analyzed.Error bars represent the SD.P value was calculated by Welch's t test.c) Box plot of geneset enrichment analysis of the indicated gene datasets in freshly isolated intestinal crypts and intestinal organoids.P value was calculated by Wilcoxon non-paired test.

Figure S5 .
Figure S5.Aging lamina propria immune cells show compositional changes towards more proinflammatory condition.a) t-SNE projection of all immune cells from young and old mice presenting nine different clusters, identified via shared nearest neighbor modularity optimization-based clustering algorithm, followed by merging of similar clusters.b) Heatmap of the expression of the top 5 markers of each of the nine clusters identified in the scRNA-seq analysis.c) Bar chart indicating the percentage of cells in young or old animals belonging to the indicated clusters in the scRNA-seq.Error bars represent the SE.P value was calculated by non-paired t test.d) Gating strategy of FACS analysis for immune cell populations in freshly isolated cells from intestinal lamina propria.

Figure S6 .
Figure S6.In vitro treatment of intestinal organoids with IFN could mimic aging induced changes in intestinal crypt cells.a) Normalized RNA counts for Kit gene in different clusters of scRNA-seq from intestinal crypt cells.b) Percentage (y axis) of cKit + cells within each identified cluster (color legend).c) Similar number of organoids were treated with different concentrations of IFN and after 5 days absolute cell number was quantified for each well.Each dot represents one mouse.d) The bar chart shows the percentage of Olfm4 + cells from intestinal organoids treated with IFN after 24h.n = 7 mice per group were analyzed.Error bars represent the SD.P value was calculated by Welch's t test.e) Experimental scheme of long-term IFN treatment of intestinal organoids and FACS analysis of organoids stimulated with two concentrations of IFN for 21 days to assess viable cells, Lgr5 + cells, MHCII + cells and cKit + cells.n = 4 mice per group were analyzed.Error bars represent the SD.P value was calculated by Welch's t test.f) Bar chart indicating the percentage of cells belonging to the indicated clusters in the scRNA-seq from organoids after IFN treatment for 24 hours with 2ng/ml dose.Error bars represent the SD.P value was calculated by non-paired t test.g) Heatmap of the expression level (scaled by gene in the row) of the MHCII genes in the different clusters found in the scRNA-seq of in vitro treated intestinal organoids with 2 ng/ml IFN for 24h.h) Hierarchical clustering and heatmap of the expression level of all the genes in the whole Antigen Presentation pathway in the different clusters found in the scRNA-seq of in vitro treated intestinal organoids with 2 ng/ml IFN for 24h.

Figure S7 .
Figure S7.IFN regulates intestinal homeostasis.a) qRT-PCR analysis of stem cell markers (Olfm4, Ascl2 and Lgr5) and of secretory cell markers (Muc2, Lyz1, and Dclk1) in intestinal crypts of mice of the indicated conditions.n = 4 mice per group were analyzed.Error bars represent the SD.P value was calculated by Welch's t test.b,c) FACS apoptosis assay using Annexin V staining of cells from organoids treated as indicated.b) representative FACS profile.c) quantification of cells in the 4 gates.n = 4 mice per group were analyzed.Organoids treated with IFN (2 ng/ml) with or without Ruxolitinib (Rux) for 3 days.Error bars represent the SD.P value was calculated by Welch's t test.d) FACS analysis of BrdU + cells in the indicated conditions.n = 4 mice per group were analyzed.Organoids treated with IFN (2 ng/ml) with or without Ruxolitinib (Rux) for 3 days.Error bars represent the SD.P value was calculated by Welch's t test.e) qRT-PCR analysis of stem cell markers (Olfm4, Ascl2 and Lgr5) and of secretory cell markers (Muc2, Lyz1, and Dclk1) in intestinal organoids treated as indicated.IFN used at 0.2ng/ml and Ruxolitinib at 10 µM.n = 4 mice per group were analyzed.Error bars represent the SD.P value was calculated by Welch's t test.