Single-cell profiling reveals stromal niche and α5β1 regulation of neutrophil phenotypes in ulcerative colitis.

Authors

Yuxi Zhou, Jorge Canas, Jiayu Ye, Jacqueline N Hoang, Sonia Ferkel-Soltau, Derek R Holman, Raoul Sojwal, Touran Fardeen, Gustavo Vazques, Yuting Huang, Kathryn Peterson, Garry Nolan, Sidhartha Sinha, Stephan Rogalla, John Gubatan

Published in

bioRxiv : the preprint server for biology. Sep 23, 2026. Epub Sep 23, 2026.

Abstract

Fibroblast-neutrophil interactions are implicated in ulcerative colitis (UC) treatment resistance, but whether fibroblasts modify neutrophil inflammatory activity remains unclear. We investigated responses to fibroblast exposure, stromal depletion, and α5β1-directed treatment.
We integrated human and mouse colonic single-cell RNA sequencing (scRNA-seq), spatial protein profiling (CODEX), fibroblast-neutrophil coculture, and dextran sulfate sodium (DSS) mouse colitis with fibroblast activation protein (FAP) ablation or α5β1-directed treatment. Readouts included cellular representation and protein fluorescence via flow cytometry, neutrophil extracellular trap (NET)-associated elastase activity, RNA programs and epithelial responses using scRNA-seq.
Inflamed UC tissue contained higher fractions of inflammatory fibroblasts and oncostatin M (OSM)- and CXCR4-associated neutrophils. Inflammatory fibroblasts had higher FAP-α5β1 module scores, and proximity to FAP-high fibroblasts was associated with higher neutrophil OSM and CXCR4 fluorescence. UC fibroblasts increased neutrophil OSM, CXCR4 and myeloperoxidase (MPO) fluorescence and NET-associated elastase activity relative to control fibroblasts, while α5β1-directed treatment attenuated these responses. Both mouse FAP ablation and α5β1 blockade in DSS colitis reduced histologic inflammation and overall neutrophil frequency. Ablation broadly reduced recovered neutrophil representation, whereas blockade increased OSM-positive and PADI4-positive percentages within neutrophils despite lower MPO fluorescence within these subsets. Both interventions were associated with lower epithelial chemokine scores but distinct absorptive and mucus/secretory responses.
Fibroblast exposure modifies neutrophil inflammatory phenotype and effector-associated activity, identifying candidate therapeutic pathways in UC. Evaluating these responses alongside neutrophil representation and tissue inflammation could inform pharmacodynamic assessment of stromal-directed therapies.
Background and Context: Fibroblast-neutrophil interactions are associated with treatment resistance in ulcerative colitis. Whether fibroblast exposure modifies neutrophil inflammatory phenotype and effector activity, beyond its established recruitment role, remains unclear.New Findings: UC fibroblasts increased neutrophil inflammatory proteins and NET-associated elastase activity; α5β1-directed treatment attenuated these responses. FAP ablation and α5β1-directed treatment reduced colitis and overall neutrophil frequency. α5β1-directed treatment lowered MPO within OSM-positive and PADI4-positive neutrophils despite higher marker-positive percentages. Epithelial single-cell RNA sequencing revealed reduced chemokine programs with both interventions but distinct barrier-associated and regenerative expression patterns.Limitations: Cellular drug targets remain unresolved. Spatial associations and elastase activity do not establish direct contact, NET formation, or clinical efficacy.Clinical Research Relevance: Fibroblast-associated pathways offer candidate therapeutic opportunities to modify neutrophil inflammatory phenotype and effector activity in UC. Coculture responses were pharmacologically modifiable, and stromal interventions reduced experimental colitis. Measuring neutrophil frequency, subset proteins, and effector activity alongside tissue inflammation could guide pharmacodynamic assessment. Clinical efficacy and treatment-selection utility require validation.Basic Research Relevance: Human coculture provides experimental evidence that fibroblast exposure modifies neutrophil inflammatory proteins and NET-associated elastase activity. FAP+ stromal ablation and α5β1-directed treatment produced distinct patterns of neutrophil representation and subset proteins in colitis. Epithelial single-cell RNA sequencing revealed reduced chemokine programs with both interventions but differing barrier-associated and regenerative expression patterns. Together, these findings motivate mechanistic studies of how stromal recruitment cues, soluble signals, and matrix-associated interactions regulate neutrophil responses, and whether these changes contribute to reduced epithelial inflammation and tissue repair.Lay Summary: Fibroblasts, support cells in the intestine, increased inflammatory activity in immune cells called neutrophils. Blocking a pathway involved in cell attachment reduced these responses in cultures. Targeting fibroblast-associated pathways also reduced experimental colitis, with changes in intestinal lining gene activity related to inflammation, barrier function, and repair. These findings suggest potential approaches to treating ulcerative colitis.

PMID:
42818174
Bibliographic data and abstract were imported from PubMed on 01 Oct 2026.

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