Colon stroma mediates an inflammation-driven fibroblastic response controlling matrix remodeling and healing

Abstract

Chronic inflammation is often associated with the development of tissue fibrosis, but how mesenchymal cell responses dictate pathological fibrosis versus resolution and healing remains unclear. Defining stromal heterogeneity and identifying molecular circuits driving extracellular matrix deposition and remodeling stands to illuminate the relationship between inflammation, fibrosis, and healing. We performed single-cell RNA-sequencing of colon-derived stromal cells and identified distinct classes of fibroblasts with gene signatures that are differentially regulated by chronic inflammation, including IL-11-producing inflammatory fibroblasts. We further identify a transcriptional program associated with trans-differentiation of mucosa-associated fibroblasts and define a functional gene signature associated with matrix deposition and remodeling in the inflamed colon. Our analysis supports a critical role for the metalloprotease Adamdec1 at the interface between tissue remodeling and healing during colitis, demonstrating its requirement for colon epithelial integrity. These findings provide mechanistic insight into how inflammation perturbs stromal cell behaviors to drive fibroblastic responses controlling mucosal matrix remodeling and healing.

Fig 1. A cellular census of stromal cells in healthy and inflamed mucosal tissues.

(A) Masson’s trichrome staining of colon from water- and chronic DSS (3 rounds)-fed mice. Collagen accumulation in blue, as demarcated by yellow arrows. Leukocyte infiltrates, as demarcated by yellow arrowheads. n = 2, Scale bar, 300 μm. representative of 2 experiments. (B) IF staining of colon from water- and chronic DSS-fed mice. DAPI (blue), Pdpn (red), ER-TR7 (green). ECM deposition, as demarcated by white arrowhead. Pdpn⁺ cell expansion, as demarcated by white arrow. Scale bar, 150 μm. n = 3. (C) Workflow depicts colon processing, epithelial strip, mechanical and enzymatic digestion dissociation, and sorting to enrich for stromal cells. (D) Gating strategy for FACS to enrich for colon stromal cells (DAPI⁻ CD45⁻ Ter119⁻ EpCAM⁻) prior to performing single-cell transcriptomics. (E) Single-cell atlas of the murine colonic stroma. UMAP of approximately 34,000 single-cell (dots) profiles colored by cell type assignment. (F) Expression of common stromal marker genes across cell type subsets. Color represents average expression of marker gene within clusters; diameter represents percentage expression of marker gene within cluster. (G) A dendrogram of cell subset relationships based on the single-cell transcriptomic data. Numbers at nodes represent score of how closely related the clusters are, with the higher the number indicating transcriptional similarity. DSS, dextran sulfate sodium; ECM, extracellular matrix; IF, immunofluorescence; UMAP, uniform manifold approximation and projection.

Fig 2. Intestinal inflammation elicits a coordinated transcriptional response in the vascular endothelium.

(A) Single-cell atlas of colon endothelial cells. UMAP of endothelial cell (dots) profiles colored by cell type assignment. (B) Expression of canonical markers across endothelial cell clusters. Color represents average expression of marker gene within clusters; diameter represents percentage expression of marker gene within cluster. (C) Volcano plots depicting DEGs between water- and DSS-treated samples for select endothelial cell clusters. Each dot represents an individual gene, and dot colors represent statistical significance. Gray, not differentially expressed; orange, differentially expressed but gene name is not annotated; blue, differentially expressed and gene name is annotated. Significant DEGs had FDR <0.05 using MAST (see Methods). (D-F) Violin plots of expression levels of select novel (D), chemokine (E), and adhesion (F) transcripts across endothelial cell clusters in water- and DSS-treated samples. Normalized gene expression levels are plotted on the y-axis. Significant DEGs had FDR <0.05, and respective adjusted p-values derived using MAST (see Methods); *p < 0.05, **p < 0.001, ***p < 1E-10. DEG, differentially expressed gene; DSS, dextran sulfate sodium; FDR, false discovery rate; LEC, lymphatic endothelial cell; UMAP, uniform manifold approximation and projection.

Fig 3. Single-cell profiling reveals functional and spatial heterogeneity of colonic fibroblasts.

(A) Single-cell atlas of colon fibroblasts. UMAP of fibroblast (dots) profiles colored by cell type assignment. (B) Expression of canonical and newly characterized markers across fibroblast clusters. Color represents average expression of marker gene within clusters; diameter represents percentage expression of marker gene within cluster. (C) Expression of genes involved in maintaining colon crypt architecture. Color represents average expression of marker gene within clusters; diameter represents percentage expression of marker gene within cluster. (D) GO enrichment of DEGs for each fibroblast cluster at baseline (water-fed mice). (E) IF staining of 3 fibroblasts classes from water-fed mouse colon. Boxes zoom onto fibroblast subsets: green box- fibroblast class 1, orange box- fibroblast class 2, and blue box- fibroblast class 3. Pcolce2 (red), CD55 (white), Procr (green), DAPI (blue). Scale bar, 25 μm. n = 3. (F) IF and FISH of various markers for fibroblast subsets. Fibroblast 1 (orange box): Grem1 (FISH). Fibroblast 2 (green box): Adamdec1 (IF), Agt (FISH), and Sox6 (IF). Fibroblast 3 (blue box): Pi16 (FISH), C3 (IF). Denoted stain (white), DAPI (blue). Scale bar, 50 μm. n = 3. (G) Proposed distribution of 3 fibroblast classes within the colon. BMP, bone morphogenetic protein; DEG, differentially expressed gene; FISH, fluorescence in situ hybridization; GO, gene ontology; IF, immunofluorescence; IstF, interstitial fibroblast; MAF, mucosa-associated fibroblast; UMAP, uniform manifold approximation and projection.

Fig 4. Intestinal inflammation elicits a dynamic fibroblast response.

(A) Volcano plots depicting DEGs between water- and DSS-treated samples for select fibroblast clusters. Each dot represents an individual gene, and dot colors represent the contributing fibroblast subset when that gene is differentially expressed. Significant DEGs had FDR <0.05 using MAST (see Methods). (B) GO enrichment of DEGs for each fibroblast cluster comparing water- and DSS-treated mice. Color represents adjusted p-value of GO enrichment annotation for each fibroblast cluster; diameter represents gene ratio for each fibroblast cluster. (C) Violin plots of expression levels of select IGFBP family members across all stromal clusters in water- and DSS-treated samples. Normalized gene expression levels are plotted on the y-axis. Significant DEGs had FDR <0.05, and respective adjusted p-values derived using MAST (see Methods); *p < 0.05, **p < 0.001. (D) Fibroblast frequency changes between water- and DSS-treated samples. Significant frequency changes derived using Dirichlet multinomial regression; *p < 0.05. (E) Feature plot for IL11 expression in fibroblasts from water- and DSS- treated samples; *p < 0.05 (F) Expression of select IL6 family receptors across fibroblast clusters in water- and DSS-treated samples. Color represents average expression of marker gene within clusters; diameter represents percentage expression of marker gene within clusters. DEG, differentially expressed gene; DSS, dextran sulfate sodium; FDR, false discovery rate; GO, gene ontology; IGFBP, insulin growth factor binding protein; IstF, interstitial fibroblast; MAF, mucosa-associated fibroblast; MyoF, myofibroblast.

Fig 5. The myofibroblast differentiation program confers matrix remodeling function.

(A) Genes that are most often differentially expressed in all stromal clusters between water- and DSS-treated samples and whose expression is enriched in gastrointestinal tissues. The frequency of times they are a DEG is plotted on the y-axis. (B) Violin plot of Adamdec1 across fibroblast clusters in water- and DSS-treated samples. Normalized gene expression levels are plotted on the y-axis. Significant DEGs had FDR <0.05, and respective adjusted p-values derived using MAST (see Methods); *p < 0.05, **p < 0.001, ***p < 1E-10. (C) IF staining of colon from water- and chronic DSS-treated mice. Adamdec1 (green), DAPI (blue). Scale bar, 100 μm. n = 3. (D) Inferred differentiation trajectory for MAFs into myofibroblast subset populations. Each dot represents a cell, and color represents the estimated pseudotime for each cell. (E) Adamdec1 expression overlaid on top of inferred differentiation trajectory for MAF into myofibroblast subset populations. Each dot represents a cell, and color represents Adamdec1 expression. (F) Dynamics of Adamdec1 expression levels as a function of pseudotime. Each dot represents a cell, and color represents the annotated fibroblast subset. (G) Genes identified in association with MAF-to-myofibroblast inferred differentiation trajectory. Color indicates when gene expression peaks along differentiation trajectory along the x-axis (from left to right). DEG, differentially expressed gene; DSS, dextran sulfate sodium; FDR, false discovery rate; IF, immunofluorescence; MAF, mucosa-associated fibroblast; UMAP, uniform manifold approximation and projection.

Fig 6. Adamdec1 is required for matrix remodeling and healing in response to epithelial injury.

(A) ISH of Adamdec1 in WT or Adamdec1 KO colon at basal state (water-fed mice). Scale bar, 200 μm. n = 2 per cohort. (B) Weight loss in Adamdec1 KO versus WT mice administered 2% DSS for 7 days and followed by H₂O for 7 days. n = 4 per cohort. Mann–Whitney test, *p < 0.05. (C) Colon length in Adamdec1 KO versus WT mice administered DSS as described above. n = 8 per cohort. Mann–Whitney test, *p < 0.05. For source data for panels B and C, see S1 Data. (D) HE staining of representative images of colon following DSS in WT and Adamdec1 KO mice. Mice were administered 2% DSS for 7 days, H₂O for 1 day, and killed on day 8. Scale bar, 600 μm. n = 4 per cohort. (E) IF staining was performed on colons from Adamdec1 KO and WT mice with indicated markers. Mice were administered 2% DSS for 7 days and killed on day 7. αSMA (red), ER-TR7 (blue), DAPI (gray). Indicated ECM component (green). (Top row) Collagen type I (green). Yellow arrow denotes submucosal ECM accumulation. (Middle row) Collagen type VI (green). Yellow dotted line denotes submucosal thickening and edema. (Bottom row) Fibronectin (green). Yellow dotted line denotes hyperplastic response. Yellow arrow denotes muscle thickening. Scale bar, 200 μm. n = 3 per cohort, representative of 2 experiments. DSS, dextran sulfate sodium; ECM, extracellular matrix; HE, hematoxylin–eosin; IF, immunofluorescence; ISH, in situ hybridization; KO, knockout; WT, wild-type.