Background: Intestinal epithelial restitution is a rapid, highly coordinated response required to restore barrier integrity after injury. Although the early phases of wound closure rely on migration, cytoskeletal remodelling, and matrix interaction, the upstream signalling pathways that initiate and sustain restitution remain incompletely defined. Adenosine signalling through the A2B adenosine receptor (A2BAR) is elevated during inflammation and hypoxia, suggesting a potential role in epithelial repair, but its mechanistic contribution to wound closure has not been fully characterised. We investigated A2BAR‑dependent restitution using human intestinal epithelial monolayers and a physiologically relevant organoid injury model.
Methods: T84 monolayers were scratch‑wounded and treated with the selective A2BAR agonist BAY 60‑6583 (1 μM). Wound closure was quantified over 24 h (n = 6 independent experiments). Proliferation was assessed by EdU incorporation (n = 4). Migration was analysed using 12 h time‑lapse imaging (n = 4). RNA‑seq was performed on wounded monolayers ± BAY 60‑6583 at 6 h (n = 3 biological replicates per condition; FDR < 0.05). Matrix‑remodelling genes (MMP1, MMP7, MMP10) were validated by qPCR (n = 5) and Western blotting (n = 3 independent lysates). Murine ileal organoids were generated from C57BL/6 mice (n = 4 mice; HPRA licence compliant). A single‑cell laser ablation assay was developed using targeted 405 nm injury, with restitution quantified by live imaging (n = 12 organoids per condition).
Results: BAY 60‑6583 significantly accelerated wound closure compared with vehicle (62.3 ± 4.1% vs 41.7 ± 3.8% closure at 24 h; p < 0.01, two‑tailed t‑test). EdU incorporation increased by 1.8‑fold (p < 0.05), indicating enhanced S‑phase entry. Migration velocity increased from 0.42 ± 0.05 μm/min to 0.67 ± 0.06 μm/min (p < 0.01). RNA‑seq identified 266 differentially expressed genes enriched for Neuregulin signalling, epithelial migration, and wound‑healing pathways. Upstream regulator analysis predicted activation of ELK1, MYC, and STAT5. A focused seven‑gene signature linked A2BAR signalling to migration‑associated repair. MMP1 was the most strongly induced transcript (11.4‑fold; FDR < 0.001), consistent with its elevated expression in ulcerative colitis and Crohn’s disease datasets. BAY 60‑6583 increased MMP1 (6.2‑fold), MMP10 (3.9‑fold), and context‑dependent MMP7 (2.1‑fold) expression by qPCR (p < 0.05 for all). Western blots (n = 3) confirmed increased MMP1 protein. Mechanistic interrogation suggested modulation of AP‑1/FOS family transcription factors rather than a purely cAMP/PKA‑dependent pathway, supported by increased FOSL1 and JUNB expression (RNA‑seq and qPCR; p < 0.05). Murine ileal organoids expressed canonical epithelial lineage markers (Lgr5, Muc2, ChgA) and matrix‑remodelling genes. Single‑cell laser ablation produced immediate membrane blebbing and rapid neighbouring cell movement into the damaged site. Restitution occurred within 8.3 ± 1.1 min (n = 12), establishing a tractable ex vivo platform for quantifying epithelial repair.
Conclusion: A2BAR activation promotes epithelial restitution by enhancing proliferation, migration, and AP‑1‑dependent matrix remodelling, with MMP1 emerging as a key downstream effector. The organoid laser‑wounding assay provides a physiologically relevant system for future mechanistic and therapeutic studies.