IL-33 Induces Paneth Cell Production of EGF and Soluble ST2, Regulating Epithelial Regeneration after Intestinal Injury

Crypt base intestinal stem cells (ISCs) maintain the epithelial lining of the intestines, but their frequencies are reduced in experimental models of graft vs. host disease (GVHD). Paneth cells provide an epithelial contribution to the stem cell niche, but their frequencies are also reduced in GVHD mouse models and in patients with GVHD. Mechanisms regulating ISCs and their niche after damage remain poorly understood. Interleukin-33 (IL-33) is an immunomodulatory alarmin typically thought to target lymphocytes such as T cells and innate lymphoid cells. In experimental models of allogeneic bone marrow transplantation, IL-33 can promote or attenuate GVHD, depending on the cellular target and the timing of the exposure. A secreted isoform of the IL-33 receptor, termed soluble ST2 (sST2), acts as a negative regulator by blocking IL-33 from binding to the membrane-bound isoform that mediates intracellular signaling. sST2 also has diagnostic and prognostic utility, serving as a biomarker for GVHD severity. While IL-33 has well-described effects on lymphocytes populations, there is limited understanding of the role of IL-33 signaling within the intestinal mucosa after damage. Utilizing experimental radiation injury to model conditioning-related intestinal pathology, we investigated the role of the IL-33/ST2 axis in epithelial damage and regeneration. Wild-type (WT) mice increased IL-33 mRNA and protein expression in the small intestine within days after 10 Gy total body irradiation (TBI). Intestinal sST2 expression also increased quickly in WT mice after TBI, but not in IL-33 -/- mice, suggesting that IL-33 itself was a regulator of sST2. While the ileal mucosa appeared similar at baseline in wild-type and IL-33 -/- mice, the IL-33 -/- mice demonstrated more severe radiation injury 5 days after 10 Gy TBI, with more crypt loss, loss of ISCs and Paneth cells, and an attenuated regenerative response with smaller crypts post-TBI containing fewer Ki67 + cells. 3-D whole-mount confocal microscopy of ileal tissue from IL-33-GFP reporter mice indicated that the crypt epithelium did not express IL-33 at baseline, but Olfm4 + ISCs adjacent to Paneth cells began to express IL-33 following radiation injury (Fig 1). Anti-Thy1-mediated lymphocyte depletion prior to irradiation of WT and IL-33 -/- mice did not abrogate the increased radiation sensitivity of IL-33 -/- mice, indicating a role for IL-33 beyond immunomodulation and suggesting that IL-33 could have direct effects within the epithelium. Intestinal organoid cultures confirmed epithelial expression of IL-33, confirmed increased radiation sensitivity of IL-33 -/- epithelium, and identified impaired epidermal growth factor (EGF) expression in the absence of IL-33. Furthermore, treatment of intestinal organoids with IL-33 induced EGF mRNA and protein expression, both of which were abrogated by the p38 inhibitor SB202190. Treatment of purified Lysozyme-DsRed + Paneth cells indicated that IL-33 directly promoted their production of EGF. In vivo, TBI-induced EGF upregulation was absent in IL-33 -/- mice. To further investigate this newly identified epithelial IL-33/ST2/EGF pathway in the ISC compartment, RiboTag mice, which facilitate analysis of lineage-specific proteome-relevant gene expression with minimal tissue processing and artifact introduction, were crossed to Olfm4-Cre and Lysozyme-Cre transgenics. Lineage-specific ribosome targeting was confirmed by immunostains and qPCR, and this approach also confirmed ISC upregulation of IL-33 following radiation injury and Paneth cell upregulation of EGF and sST2. Finally, to investigate the importance of this radiation-induced EGF upregulation, WT mice were treated with the EGFR inhibitor gefitinib following irradiation, which resulted in more severe radiation-induced ISC loss, similar to IL-33 deficiency. Furthermore, epithelial regeneration and ISC recovery were augmented in IL-33 -/- mice after TBI by treatment with exogenous EGF (Fig 2). These findings reveal an unknown pathway of stem cell niche regulation and crypt regeneration whereby the Paneth cell niche responds dynamically upon injury by upregulating EGF and the stem cells orchestrate regeneration by regulating their niche with IL-33. This regenerative circuit also highlights the breadth of IL-33 activity beyond immunomodulation and the therapeutic potential of EGF administration for treatment of intestinal injury.