Paneth Cells Provide a Unique Source of EGF for Intestinal Epithelium in Radiation Injury That Is Dispensable in Gvhd.

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Paneth cells (PCs) are a secretory epithelial population located in small intestine (SI) crypts. PCs produce growth factors within the intestinal stem cell (ISC) niche, including WNT3 , DLL1 and EGF, that maintain the intestinal epithelium . In murine models of acute GVHD , reductions in both ISCs and PCs has been reported. In patients with GVHD , PC loss has also been observed and its severity is correlated with poor clinical prognosis. We therefore asked if the loss of PCs and their niche function led to ISC loss, intestinal pathology, and overall GVHD outcomes. To investigate, we treated Lyz1 DTR B6 mice with diphtheria toxin to ablate PCs prior to a MHC-mismatch BMT from B10.BR mice. The elimination of PCs in the SI (Fig 1A) had no impact on survival, clinical scores, and weight loss versus WT mice (Fig 1B). PC ablation also did not affect ISC and crypt loss (not shown). To determine if MHC-mismatched GVHD severity overwhelmed PC support of the SI and overall outcomes, we performed a minor-antigen-mismatched 129-into-B6 BMT. Again, PC ablation had no effect on survival, scoring and weight loss (Fig 1C), indicating experimental GVHD was not driven by PC maintenance of intestinal crypts. Given the lack of impact from PC ablation on crypt injury in GVHD, we examined the expression of niche factors in crypts from WT and Lyz1 DTR BMT recipients. While GVHD reduced the expression of Dll1 and WNT3 , PC ablation had no further effect (Fig 2A) . However, PC ablation reduced crypt EGF expression (Fig 2A). The persistence of niche factors despite PC ablation suggested other potential sources. Indeed, scRNA-sequencing analysis of crypts (Fig. 2CB) identified other epithelial populations expressing these factors. Expression of Wnt3 by non-PC cells was validated by smFISH staining (Fig 2C), and their contribution appeared to increase during GVHD (Fig 2D). These finding indicated that other cells besides PCs may support the ISC niche. While PCs were a non-redundant source of EGF, this had little impact on GVHD pathophysiology . We next sought to study PC niche function in a setting with elevated Egf expression. Recent work has found increased PC EGF expression after 10 Gy, unfractionated, total body irradiation (TBI). In this setting, we found that TBI induced intestinal injury with loss of crypts and OLFM4 + ISCs, and a regenerative response shown by increased crypt height in WT mice. PC-ablated Lyz1 DTR mice, had exacerbated loss of crypts and ISCs, and the absence of crypt regenerome. (Fig 3). Extrinsic EGF treatment of irradiated Lyz1 DTR mice restored crypt and ISC numbers, and crypt regeneration to WT TBI levels (Fig 3). These findings show that PC EGF production supports the intestinal epithelium after radiation injury. Given this function appears dispensable in GVHD, perhaps due to non-PC niche function or direct targeting of ISCs by donor T cells , our findings suggest PC niche function is beneficial in select injury settings.

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