CXCR6-dependent ILC3 migration to the intestinal stem cell compartment promotes epithelial regeneration after conditioning-related intestinal injury Free

Interleukin-22 (IL-22) has been shown to promote epithelial regeneration, protect the intestinal stem cell (ISC) compartment from damage after allogeneic bone marrow transplantation, and reduce the severity of gastrointestinal graft vs. host disease (GI GVHD). The endogenous tissue-protective IL-22 response is thought to be driven by host-derived group 3 innate lymphoid cells (ILC3s), which are lost in GVHD. However, it is unknown how IL-22 produced by cells in the lamina propria (LP) and discrete lymphoid follicles (LFs) reaches the crypt base epithelial stem cell compartment. Furthermore, the mechanisms regulating intramucosal localization of IL-22 production are unclear, and the role of ILC migration in intestinal regeneration is poorly understood.To investigate epithelial regeneration prior to ILC3 elimination, we performed 3D microscopy on full-thickness ileum from wild-type (WT) C57BL/6 (B6) mice exposed to 11 Gy split-dose total body irradiation (TBI), modeling experimental pre-transplant conditioning. While some crypts were severely damaged, regenerative crypts showed elongated morphology and increased Ki67⁺ proliferating epithelial cells at day 3 post-TBI, which returned to baseline by day 7, indicating early epithelial regeneration.To evaluate how the localization of IL-22⁺ lymphocytes influences regeneration, we used IL-22-Cre-Rosa26-tdTomato (tdT) fate-mapping reporter mice, in which IL-22⁺ cells are permanently labeled. At baseline, ileal IL-22-tdT⁺ cells were dispersed throughout the LP and clustered in intestinal LFs. Live 2-photon imaging indicated that LP IL-22-tdT⁺ cells were highly mobile, while the LF cells were largely stationary. By day 3 post-TBI, coinciding with regeneration, IL-22-tdT⁺ cells accumulated near the crypt base ISC compartment. We then used IL-22-GFP reporter mice to identify cells actively producing IL-22. At baseline, ileal IL-22-GFP⁺ cells were primarily confined to LFs, with little presence near crypts. By day 3 post-TBI, IL-22⁺ cells appeared outside LFs and localized near the crypt base, consistent with fate-mapping results. CD3 staining indicated that the IL-22⁺ crypt base infiltrate consisted of CD3⁻ ILCs rather than T cells, and their accumulation near the ISC compartment during peak regenerative activity suggested their localization may contribute functionally to epithelial regeneration after damage.Mouse intestinal ILC3s include 2 main subsets: natural cytotoxicity receptor (NCR)⁺ ILC3s, scattered in the LP and expressing NKp46, and CCR6⁺ lymphoid tissue inducer (LTi)-like ILC3s clustered in LFs. Using 3D microscopy, few CD3⁻RORγt⁺ ILC3s were detected near the crypt base at baseline. In contrast, NKp46⁺ ILC3 crypt base density increased markedly by day 3 post-TBI, suggesting that NCR⁺ ILC3s migrated to the ISC compartment after injury. To test if this accumulation resulted from local migration or recruitment via the circulation, we performed parabiosis between WT and constitutive GFP⁺ mice. After 2 months, GFP⁺ cells were found in WT ileum, and GFP⁻ cells were found in GFP⁺ ileum, confirming shared circulation. Consistent with previous data, day 3 post-TBI, ILC3 density near the crypt base was higher in irradiated pairs than unirradiated controls. Moreover, crypt base ILC3s post-TBI were GFP⁻ in WT mice and GFP⁺ in GFP mice, indicating that ILC3 relocalization to the ISC compartment resulted from local intramucosal migration.To identify pathways regulating ILC3 migration, we analyzed single-cell RNA sequencing of intestinal ILCs from WT mice (GSE132273) and found higher Cxcr6 expression in NKp46⁺ ILC3s than CCR6⁺ LTi-like ILC3s. To then test CXCR6's role in migration, we assessed ILC3s in WT mice, CXCR6-deficient (CXCR6^-/-) mice, and heterozygous CXCR6^+/- littermates before and after TBI. While ILC3s were present in the villi at baseline in all genotypes, only WT and CXCR6^+/- controls showed ILC3 relocalization to the crypt base after TBI. ILC3s failed to migrate to the ISC compartment in CXCR6-deficient mice, which also exhibited impaired crypt regeneration, increased crypt damage, and reduced ISC proliferation.These findings demonstrate that IL-22-producing NCR⁺ ILC3s migrate to the ISC compartment after radiation injury in a CXCR6-dependent manner, a process essential for optimal epithelial regeneration. Preserving host ILCs and their ability to migrate to the ISC compartment post-transplant may therefore be integral to reducing transplant-related GI toxicity.