Figures & data
Figure 1. Analysis of fecal microbiota from healthy and CD patients and selected samples for mouse colonization.
(a) Alpha diversity indices of the fecal microbiome from healthy controls (HCs) and Crohn’s disease patients with ileitis (CD_L1) and ileocolitis (CD_L3). (b) Beta diversity (nonmetric multidimensional scaling; NMDS) analysis of HC, CD_L1 and CD_L3 fecal samples. (c) Compositional depiction of the most abundant genera in HC, CD_L1 and CD_L3 fecal samples. (d) Relative abundance of selected genera used for selection of samples for inoculation of GF mice. (e) Comprehensive analysis of differentially abundant ASVs in samples selected for GF mouse inoculation using DESeq2.
Figure 2. Analysis of the established microbiomes in GF mice humanized with HC and CD_L3 fecal samples.
(a) Alpha diversity indices of fecal and ileal microbiomes from xGF mice humanized with microbiota from HC and CD_L3 patients. (b) Beta diversity (NMDS) analysis of fecal and ileal microbiomes from xGF mice humanized with microbiota from HC and CD_L3 patients. (c) DESeq2 analysis of differentially abundant ASVs in fecal samples from HC and CD_L3 humanized mice. (d) Relative abundance of the six most abundant genera established in the fecal samples of HC and CD_L3 humanized mice.
Figure 3. Histological features of mild to moderate colitis in xGF mice humanized with HC and CD_L3 fecal microbiota.
Representative H&E-stained images of the proximal (a-b) and distal (c-d) colon of humanized mice. (e-g) Expression of the p65 subunit of the NFκB complex in the mucosa of the proximal and distal colon in HC and CD_L3 humanized mice. (h) Relative body weight change in xGF mice. (i) Unbiased histological pathology scoring of the proximal and distal colons of xGF mice humanized with HC and CD_L3 fecal microbiota.
Figure 4. Inflammatory signature of the colonic transcriptome profiling of xGF mice humanized with HC and CD_L3 fecal microbiota.
(a-b) PCA and volcano plots demonstrating the extent of differential gene expression in mice colonized with HC and CD_L3 microbiota. (c) GSEA of the chemokine signaling pathway and leukocyte transendothelial migration in the proximal colon. (d) Heatmap of gene expression data of leukocyte-mediated immunity KEGG pathways in the proximal colon. (e) GSEA of cytokine‒cytokine receptor interactions and chemokine signaling pathways in the distal colon. (f) Heatmap of gene expression data of the innate immune response KEGG pathway in the distal colon. (g-h) Other hallmark and KEGG pathways significantly enriched in the proximal colonic transcriptome of mice humanized with the CD_L3 microbiota. (i) Heatmap of gene expression data of the type II interferon signaling pathway in the proximal colon.
Figure 5. The colonic transcriptomic profile of CD_L3 recipients shows an enrichment in the IBD pathway.
(A) GSEA of the proximal colonic transcriptome of humanized mice. (B) Hierarchical clustering and heatmap of genes associated with the IBD pathway differentially expressed in the proximal and distal colon of HC and CD_L3 recipients (with Padj<0.05 without fold-change filtering).
Figure 6. Increased T-cell infiltration and increased expression of epithelial CD74 and CD14 in the inflamed colonic mucosa of xGF mice humanized with HC and CD_L3 fecal microbiota.
(a) CD3+ T cells (green) in the colonic mucosa were analyzed by IF, and their numbers in the intraepithelial and lamina propria compartments of the proximal and distal colon were summarized and statistically analyzed by T test (b-c; **p < .01 ****p < .0001). (d) CD74 mRNA expression in the colons of humanized mice. (e) IF analysis of CD74 protein (in green; scale bars represent 100 μm). (f) CD14 expression was analyzed by IF in the proximal and distal colon. All scale bars are 50 μm. (g) Summary of the CD14 signal intensity (MFI-mean fluorescent intensity) in the surface and crypt colonic epithelial cells in the proximal and distal colon (T test; **p < .01 ****p < .0001). (H) CD44 mRNA expression in the colons of humanized mice.
Figure 7. Paneth cell-like signature in the proximal colon of xGF mice humanized with HC and CD_L3 fecal microbiota.
(a-b) MMP7 immunostaining in the proximal and distal colon of humanized mice. (c) Mmp7 mRNA expression in the proximal colon of humanized mice. (d) GSEA results indicating enrichment of transcripts associated with the Paneth-cell-like phenotype described in scRNAseq analysis of human immature small intestine.Citation68 (e) Volcano plot of 260 Paneth cell-like gene markers from Gao et al.Citation68 analyzed in the proximal colon of humanized mice. (f) Hierarchical clustering and heatmap of a selection of 13 genes from the Paneth cell-like signature with a fold change >3 in humanized mice. (g) Hierarchical clustering and heatmap of proximal colonic transcripts in humanized mice overlapping with Paneth cell signature genes from scRNA projects from PanglaoDB (consensus of scRNA sequencing data from mouse and human). All genes reported in the heatmap with Padj<0.05 (no fold-change adjustment).
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