FLASH radiotherapy drives intestinal regeneration via macrophage-derived WNT4 signaling and attenuated T-cell inflammation

Abstract

Ultra-high dose rate radiotherapy (FLASH-RT) reduces normal-tissue toxicity, yet how elevated dose rate improves tissue repair remains unclear. Here we combined whole-abdominal FLASH-RT and conventional radiotherapy (CONV-RT) in mice with single-cell transcriptomics, trajectory analysis, cell-cell communication inference, and in vivo perturbation experiments to define the intestinal repair ecosystem. Compared with CONV-RT, FLASH preserved Lgr5+ intestinal stem cells and Fabp1+ absorptive epithelial cells. Trajectory analysis showed that FLASH maintained stem-cell differentiation toward mature epithelial states enriched for fatty-acid metabolism. During a day-3 repair window, FLASH was accompanied by enrichment of Mrc1 macrophages, increased WNT4-associated signaling involving Fzd5Lrp5/6-expressing stem cells, and early transient {beta}-catenin activation. In vivo augmentation of Wnt4 signaling promoted crypt regeneration after CONV-RT. FLASH also reduced Tcf7 and Tbx21 CD4 T-cell populations and TNF- levels, consistent with attenuation of the pro-inflammatory CD4 T-cell response during intestinal recovery. Together, these findings indicate that FLASH-associated tissue sparing involves coordinated remodeling of regenerative and inflammatory immune states, suggesting that radiation dose rate influences not only initial tissue injury but also the immune microenvironment of subsequent recovery. Targeting this immune-stem cell crosstalk may provide a strategy to mimic FLASH-like radioprotection.