IL-17RA signaling promotes the dedifferentiation of Paneth progenitors through ADAM17 to regenerate gut epithelium post-irradiation

Domain: biology
Published: 2026-03-17
Editorial outcome: major_revision

Abstract
Paneth cells and their antimicrobial products are critical in mediating small intestinal host defense under homeostatic conditions and after injury or infection. In addition, Paneth cells have also been shown to gain stem-like properties and repropagate intestinal crypts after intestinal injury. The specific role of intestinal IL-17A or its receptor (IL-17RA) signaling in Paneth cells to gain stem-like features has yet to be investigated. Using Paneth cell-specific IL-17RA (Il17rafl/fl;Defa6-cre) knockout mice, anti-IL-17A neutralizing studies and lineage tracer (Defa6-cre;mT/mG) mice, we show that after injury IL-17RA signaling is required for Paneth cell to gain stem-like properties to regenerate the intestinal epithelium. Increased susceptibility of Il17rafl/fl;Defa6-cre mice is associated with reduced expression Adam17 in the terminal ileum. Adam17 overexpression in Il17rafl/fl;Defa6-cre mice rescues the epithelial regeneration defect in these mice. IL-17A induces Nox1 in Paneth cells and H O induces ADAM17 enzymatic activity. Finally, using Paneth cell-specific Adam17 (Adam17fl/fl;Defa6-cre) knockout mice, we show that ADAM17 in Paneth cells is required for tissue regeneration. Collectively, our data reveal an essential role of the IL-17RA-ADAM17 pathways in Paneth cells for tissue regeneration.

Introduction
Paneth cells are highly specialized cells vital for regulating microbiota homeostasis, instituting protective antimicrobial processes, and providing a niche for developing stem cells (1–4). They are localized at the base of intestinal crypts. Patients with Crohn’s Disease (CD), a form of inflammatory bowel disease (IBD), exhibit abnormalities and impaired antimicrobial processes within their Paneth cells (5). A hallmark of Paneth cells is their large, dense secretory granules. These granules store various antimicrobial peptides used to regulate the host microbiota and defend against invading pathogens (6–8). Paneth cells also play a vital role in maintaining the intestinal stem cell (ISC) niche. They produce epidermal growth factor (EGF) and selective WNTs, which help support the self-renewal and survival of Lgr5+ ISCs (2, 7). After intestinal injury, it has been reported that other intestinal epithelial cell types, including Paneth cells, can dedifferentiate and replenish the intestinal crypt (9–12). This process is dependent on the activation of multiple pathways. Notch signaling through notch intracellular domain (NICD) activation has been implicated in Paneth cell dedifferentiation and proliferation (10, 12). Simultaneously, it was reported that stem cell factor (SCF) signaling to c-Kit in response to inflammation causes Paneth cell dedifferentiation through activation of the PI3K/AKT signaling axis (9). While IL-17RA is expressed on the intestinal epithelium, including Paneth cells (13, 14), it is not known if immune cell-derived factors such as IL-17A regulate Paneth cell dedifferentiation.

The IL-17 family of cytokines is widely studied and plays an important role in immunity and inflammation (15). While the family consists of 6 signaling molecules (IL-17A-F), IL-17A and IL-25 (formerly known as IL-17E) are the best characterized (15, 16). IL-17A signals primarily through the IL-17RA/RC co-receptor complex. IL-17RA is expressed widely across mammalian tissue, while IL-17RC expression is restricted to epithelial, mesenchymal cells and few other cell types (17). Since IL-17A can signal to many cell types throughout the body, its expression and production are tightly controlled. Increased expression of IL-17A can be a contributing factor to autoimmune diseases, such as plaque psoriasis (18, 19). Increased amounts of IL-17A are also found in the intestinal mucosa of patients suffering from IBD (20). Anti-IL-17A or anti-IL-17RA therapies commonly used to treat plaque psoriasis patients were unsuccessful in treating IBD and, in some instances, worsened the disease (21, 22). We and others have shown that IL-17A is vital in maintaining intestinal barrier integrity after injury and promoting gut microbiota homeostasis (23–26). Recently, we have shown that IL-17A signaling within the gut, specifically to Lgr5+ stem cells, promotes secretory lineage commitment at a steady state (13). While a beneficial role for IL-17A signaling within the gut has been established, the role of IL-17A signaling to specific intestinal epithelial cell types is still an area of active research. After injury, several epithelial cell types, including Paneth cells, endocrine, and enterocytes, gain stem-like properties to regenerate the gut epithelium (9, 27, 28). Almost nothing is known at present whether IL-17A promotes or inhibits epithelial cell stem-like properties.

Our findings suggest that while, loss of IL-17RA signaling to Paneth cells is insignificant under homeostasis, however, the ability of Paneth cells to dedifferentiate and replenish the intestinal crypt after injury depends on IL-17RA. Utilizing our whole-body IL-17RA knockout (Il17raKO) and Il17rafl/fl;Villin-cre mice, we show a lack of IL-17RA signaling to the intestinal epithelium leads to systemic bacterial dissemination to the spleen and liver after lethal doses of irradiation. This is compounded by significant structural damage and inflammation within the gut of these mice. This phenotype is recapitulated within our Il17rafl/fl;Defa6-cre mice, showing the importance of IL-17RA signaling to Paneth cells in recovering from injury. Neutralizing IL-17A in irradiated Paneth cell lineage tracer (Defa6-cre;mT/mG) mice reduces stemness and epithelial regeneration. We show that continuous stimulation of organoids grown from Paneth cell lineage tracer mice with IL-17A increases the number of Paneth cells in organoid buds after irradiation. Furthermore, a subset of these Paneth cells gains stemlike properties and can develop into new organoids after passage. We found reduced expression of Adam17 in the terminal ileum of irradiated Il17rafl/fl;Defa6-cre+ mice. Furthermore, sorted Paneth cells from irradiated Paneth cell lineage tracer organoids treated with IL-17A showed increased expression of Adam17 and Nox1. NOX1 regulates epithelial redox, and H O enhances ADAM17 enzymatic activity. Using Adam17fl/fl;Defa6-cre+ mice, mechanistically, we show that epithelium, including Paneth cell-based, intestinal regeneration is linked to the IL-17A-ADAM17 axis.

Results
Loss of intestinal IL-17RA signaling promotes intestinal bacterial dissemination to the spleen and liver.
Radiation injury is a well-established model to study Lgr5+ stem cell-independent epithelial regeneration as it ablates actively proliferating stem cells (27). C57BL/6 (WT) and whole-body IL-17RA knockout (Il17raKO) mice were irradiated at 12 Gy and returned to their cages. This dose of irradiation promotes intestinal epithelial cell damage and widespread death of Lgr5+ ISCs and transit-amplifying (TA) cells within crypts (29). Mice were sacrificed 5 days post-irradiation and tissues were collected. Hematoxylin and eosin (H&E) staining of terminal ileal tissue from irradiated Il17raKO mice revealed greater structural damage and inflammation compared to WT mice. Specifically, we measured the length of the crypt/villi structure. We measured from the base of the crypt to the top of the villi and observed shortening of the crypt/villi structure in irradiated Il17raKO mice as compared to WT mice (Fig. 1A). Their liver and spleen were harvested, homogenized, and plated. After 48 hours, colonies were counted. We observed significant growth of bacterial colonies from spleen and liver tissues taken from Il17raKO mice when compared to tissues from WT mice (Fig. 1B). Along with this, there was increased Lipocalin-2 (LCN2) in the cecal contents of Il17raKO mice compared to WT mice (Fig. 1C). LCN2 is a siderophore known to help control microbiota dysbiosis and used as a marker for intestinal inflammation (30). While LCN2 can be induced by IL-17A, it can also be induced by a plethora of inflammatory stimuli (e.g., LPS) and cytokines (e.g., IL-6, IL-10, and IL-22) (31). To determine whether the loss of IL-17RA signaling affected proliferation within the intestinal villi, we performed immunofluorescence (IF) staining and stained tissue for proliferating cell nuclear antigen (PCNA). Results showed reduced proliferation in the ileum of Il17raKO mice compared to WT mice (Fig. 1D). To rule out hematopoietic failure contribution to an observed phenotype, bone marrow from non-irradiated WT and Il17raKO mice were transplanted into their irradiated counterparts 2 hours post-irradiation (Fig. 1E). We observed a significant increase in systemic bacterial dissemination in Il17raKO mice compared to their WT counterparts (Fig. 1F). To further explore IL-17RA signaling within the intestines, we used intestinal epithelial-specific IL-17RA knock-out mice (Il17rafl/fl;Villin-cre+/-). Irradiated Il17rafl/fl;Villin-cre+ mice similarly showed greater structural damage and increased bacterial dissemination to the spleen and liver than their cre- counterparts (Fig. 1G & 1H). We also observed a trend towards increased LCN2 in the cecal content of irradiated Il17rafl/fl;Villin-cre+ mice (Fig. 1I). Lastly, we also observed reduced cellular proliferation in the ileum of cre+ mice as compared to cre- mice (Fig. 1J). Taken together, these observations show that lack of intestinal IL-17RA signaling resulted in gut barrier or epithelial regenerative defects.

Rapid depletion of Lgr5⁺ stem cells does not compromise intestinal regeneration following irradiation-induced injury.
A prior study showed that Lgr5+ cells are indispensable for tissue recovery after radiation-induced injury (32). It is also well established in the literature that radiation readily kills Lgr5+ stem cells (29, 33, 34). Utilizing Lgr5-EGFP-creERT2;RosaLSLTdtomato mice, we confirmed that radiation ablated Lgr5+ cells in the small intestine, which then reappear 7 days post irradiation (Fig. 2A & B). Once the involvement of Lgr5+ stem cells was ruled out, we then investigated which other cell type may mediate IL-17RA-induced protection. To do this, we screened for changes in RT-qPCR data from post-irradiated Il17raKO mice. Results revealed decreased expression of the Paneth cell marker Lyz1. We saw no differences in the expression of markers for other intestinal epithelial cells such as Muc2 (goblet cells), clusterin (Clu), a marker for a type of quiescent intestinal stem cells, and fetal-like markers including Anxa1, Anxa3, Anxa5, Ly6A, and Ly6D. (Fig. 2C). To confirm this expression data, we stained Il17raKO and Il17rafl/fl;Villin-cre+ mice for Lyz1. We saw reduced amounts of Lyz1 in the ileum of both subsets of mice as compared to their WT and cre- counterparts (Fig. 2D & E). Since we observed decreased expression of Lyz1 in irradiated Il17raKO mice, followed by decreased amounts of Lyz1 in both Il17raKO and Il17rafl/fl;Villin-cre+ mice, we decided to study the role of IL-17RA signaling in Paneth cells.

During homeostasis loss of IL-17RA signaling to Paneth cells does not alter their function.
To study the importance of IL-17RA signaling in Paneth cells, we generated Il17rafl/fl;Defa6-cre mice, which lack IL-17RA specifically on Paneth cells (Fig. S1A) (13). We extensively characterized naïve Il17rafl/fl;Defa6-cre mice. RNA-seq data from ileum tissues Il17rafl/fl;Defa6-cre+/- mice showed no significant alterations in the expression of antimicrobial peptides or stem cell genes (Fig. S1B). To validate this data, we performed RT-qPCR of Paneth cell-related genes from ileum tissue of Il17rafl/fl;Defa6-cre+/- mice, which did not show significant differences in expression between cre-/+ mice (Fig. S1C). Furthermore, IF staining for Lyz1 in Il17rafl/fl;Defa6-cre+/- mice showed no differences in the number of Lyz1+ Paneth cells (Fig. S1D). We show that Paneth cells of Il17rafl/fl;Defa6-cre+/- mice display similar morphology and secretory vesicle number via transmission electron microscopy of ileal tissues (Fig. S1E). Acid Urea (AU) PAGE electrophoresis was performed to determine changes in cryptidin levels between Il17rafl/fl;Defa6-cre+/- mice. AU-PAGE is a unique form of protein electrophoresis that separates proteins based on their charge. Results indicated no differences in cryptidin protein levels between cre- and cre+ mice (Fig. S1F). Lastly, 16S microbial sequencing revealed no changes in microbiota between naïve cre- and cre+ mice (Fig. S1G). Collectively, our data indicate that loss of IL-17RA signaling to Paneth cells does not alter their function under homeostatic conditions.

IL-17RA signaling, specifically to Paneth cells, promotes intestinal integrity after irradiation-induced injury.
We then characterized Il17rafl/fl;Defa6-cre mice after irradiation-induced injury. Our results were similar to those observed in Il17raKO and Il17rafl/fl;Villin-cre mice. There was shortening of crypt/villi length within the ileum of cre+ mice 3 days post irradiation (Fig. 3A). Five days post irradiation injury, there was increased bacterial dissemination to the spleen and liver in Il17rafl/fl;Defa6-cre+ mice as compared to cre- mice (Fig. 3B). This was coupled with increased LCN2 concentrations within cre+ cecal contents (Fig. 3C). Utilizing an anaerobic chamber, we also observed increased anaerobic bacterial dissemination to the spleen and liver in Il17rafl/fl;Defa6-cre+ mice (Fig. S1H). The presence of cecal albumin was indicative of gut barrier injury (Fig. S1I). Immunofluorescence staining of PCNA revealed decreased cellular proliferation in Il17rafl/fl;Defa6-cre+ mice compared to cre- mice post-irradiation injury (Fig. 3D). We next wanted to determine if the regeneration canonical ISCs was impaired in Il17rafl/fl;Defa6-cre+ mice after irradiation. As expected, we saw reduced amounts of Olfm4+ crypts in mice 5 days post irradiation as compared to non irradiated mice. Interestingly, we also observed decreased amounts of Olfm4+ crypts in irradiated Il17rafl/fl;Defa6-cre+ mice as compared to irradiated cre- mice. This revealed that canonical ISCs regeneration was indeed impaired in Il17rafl/fl;Defa6-cre+ mice as compared to cre- mice 5 days post irradiation (Fig. 3E). We did not observe a difference in the number of Lyz1+ Paneth cells between cre- and cre+ mice post irradiation (Fig. S1J). Immune profiling revealed a lack of differences between populations of adaptive immune cells in the lamina propria of irradiated cre- and cre+ mice (Fig. S2A). However, total cell numbers of ILC1s were significantly greater in the lamina propria of irradiated cre+ mice as compared to cre- mice (Fig. S2B). We also show that there are equivalent amounts of IL-17A producing ILC3s and CD3+ cells within the lamina propria of irradiated cre- and cre+ mice (Fig. S2C & S2D). Furthermore, there was no difference in key IL-17A inducible chemokine (Cxcl5, Ccl2 and Cxcl1) expression within the ilea of irradiated cre- and cre+ mice (Fig. S2E). Commensal microbiota have been shown to promote intestinal regeneration (35). Subsets of commensal microbiota expand at sites of intestinal injury and promote wound healing (36). We first compared the Il17rafl/fl;Defa6-cre+/- mice microbiota in the non-irradiated control group. When observing beta diversity at the phylum, family, genus, and species level, there was no distinct clustering between groups. This indicates that with the Weighted UniFrac metric these groups are more similar than dissimilar at each phylogenetic level. Further, when performing LEfSe, no phylum, family, genus, or species were identified as significantly different in abundance between the cre+/- non-irradiated mice, even with an LDA cut-off of 1, and a p-value of 0.10 (Fig. 3F-I). While the microbiota composition changed after irradiation, the difference was subtle for both cre- and cre+ mice (Fig. 3J-M). These results suggest that IL-17RA signaling to Paneth cells is vital in limiting damage in response to irradiation independent of microbiota regulation.

Subsets of Paneth progenitors express high levels of IL-17RA and IL-17RC.
We have previously shown via single-cell RNA-sequencing (scRNA-seq) of enteroids that a small subset of Paneth cells expresses IL-17RA, while secretory progenitors express the highest levels of IL-17RA (13). To investigate this deeper, we analyzed a previously published single-cell RNA sequencing data set enriched for primary murine Paneth cells (PMID: 29144463), which identified three distinct subsets of Paneth cells (Paneth 1, Paneth 2, Paneth Progenitor) (14). The analysis of these data suggested no significant difference in the average level of IL-17RA expression over the course of Paneth cell differentiation, while IL-17RC expression decreased as Paneth cells matured (Fig. 4A). A more detailed analysis, however, demonstrated unique subsets of Paneth progenitors characterized by intermediate levels of Lyz1 and high expression of IL-17RA (Fig 4B) or IL-17RC (Fig 4C), consistent with our previous results. Utilizing PanethYFP (Defa6-cre;YFP) mice, we were able to show two distinct Paneth cell populations. The high YFP-positive cells represent matured Paneth cells, and cells expressing low levels of YFP mark Paneth progenitors (Fig. 4D). RT-qPCR analysis of these two populations was consistent with previous results (Fig. 4B & 4C), demonstrating that low YFP expressing Paneth cells expressed higher levels of Il17rc, Il17ra, and Olfm4 (a marker for stemness) (Fig. 4E). Interestingly, we analyzed a previously published single-cell RNA sequencing data (PMID: 29887318) which showed that Il17rc and Il17ra are upregulated in Paneth cells after irradiation (Fig. 4F) (10). Taken together, we show that Paneth progenitors, a subset of Paneth cells, express high levels of Il17ra, Il17rc and stem cell markers. Furthermore, post irradiation Paneth cells express higher levels of Il17ra and Il17rc.

IL-17RA signaling promotes Paneth cell stemness after irradiation injury.
To better understand the mechanism by which Paneth cells participate in the regeneration of the intestinal crypt after irradiation-induced injury, we generated and validated Paneth cell lineage tracer mice (Defa6-cre;mT/mG). All Paneth cells and their lineage in these mice express GFP, while all other cells express RFP. Consistent with published literature (9, 10), we were able to show that Paneth cells gain stemness post-irradiation, as revealed by the appearance and counting of ribbons of GFP+ cells in the villus 5 days post irradiation (Fig. 5A). Immunofluorescence staining for GFP and MUC2 (goblet cell-specific marker) or DCLK1 (Tuft cell marker) or ChGA (enteroendocrine cell marker), or I-FABP (enterocyte cell marker) in these mice showed co-staining of GFP and these distinct cell markers, which supports the assertion that Paneth cells can dedifferentiate and repair/regenerate the intestinal crypt (Fig. 5A and S3A, respectively). Small intestinal crypt from non-irradiated Defa6-cre;mT/mG mice forms a viable organoid (Fig. 5B top panel). We next sorted GFP+CD24+ Paneth cells from non-irradiated and irradiated Defa6-cre;mT/mG mice. Non-irradiated sorted GFP+CD24+ Paneth cells do not form organoid. Green organoids (indicating organoids arising from Paneth cell lineage) only grew from Paneth cells sorted from irradiated mice (Fig. 5B bottom panel). To confirm the importance of IL-17A signaling in modulating Paneth cell stemness, we injected Defa6-cre;mT/mG mice with anti-IL-17A or IgG antibody, on day 0 and day 2 post irradiation (Fig. 5C). We then counted the number of GFP+ cells in the base of the crypt and in the villi between the two treatment groups 5 days post irradiation. We also counted the number of GFP+ clones, which we define as at least 4 GFP+ cells in a row, in the villi between the two treatment groups 5 days post irradiation. The results showed decreased GFP+ cells and clones in the villi of mice treated with anti-IL-17A as compared to IgG-treated mice (Fig. 5D). Furthermore, there was decreased crypt/villi length indicating structural damage in the terminal ilea of mice treated with anti-IL-17A compared to IgG treated mice (Fig. 5E). We also observed reduced proliferation in the ileum of mice treated with anti-IL-17A as compared to IgG-treated mice (Fig. 5F). This result demonstrates a strong connection between IL-17A signaling and Paneth cells ability to dedifferentiate and repair the intestinal crypt after radiation injury. We then established an organoid irradiation and regeneration model and identified that 6 Gy is an optimal dose of irradiation (Fig. S3B). Prior literature has also shown that doses lower than 6 Gy show minimal effects on organoid growth, while at 6 Gy, the initial loss of organoid budding was followed by renewed organoid regeneration (37). To confirm the importance of IL-17A in the regeneration of the intestinal epithelium, we supplied external recombinant IL-17A to organoids grown from Defa6-cre;mT/mG mice post-irradiation and observed increased GFP+ cells after IL-17A treatment (Fig. 5G & H). We have previously reported that IL-17A signaling on ISCs promotes secretory lineage commitment (13). Thus, the increased GFP+ cells we observe after IL-17A treatment may be increased amounts of mature Paneth cells or stem-like Paneth cells. To confirm presence of stem-like Paneth cells, we passaged these organoids and observed if any fully green organoids grew. Interestingly, when these organoids were passaged, we saw a higher percentage of fully green organoids from the wells that were treated with IL-17A, indicating that IL-17A may directly increase Paneth cell stemness after irradiation (Fig. 5I). Since IL-25 and IL-17C also signal through IL-17RA, we tested whether recombinant IL-25 or IL-17C can induce green organoids after irradiation. Our results showed no significant increase in green organoid development from passaged Defa6-cre;mT/mG mice post-irradiation after IL-25 or IL-17C treatment (Fig. S3C). We then stained the IL-17A treated green organoids for MUC2 and ChGA. MUC2 marks terminally differentiated goblet cells while ChGA marks terminally differentiated enteroendocrine cells. Since the staining revealed that these green organoids were composed of both GFP+/MUC2+ double positive cells and GFP+/ChGA+ double positive cells, we can ascertain that these green organoids arise from Paneth cells and are mature organoids composed of terminally differentiated epithelial cells (Fig. 5J). Collectively, our data show that IL-17A promotes Paneth cell dedifferention.

Overexpression of Adam17 rescued intestinal regeneration defect in Il17rafl/fl;Defa6-cre+ mice after radiation-induced injury independent of EGFR ligands.
We next investigated mechanisms of IL-17A-induced Paneth cell stem-like properties. We performed RT-qPCR analysis in the terminal ileum of Il17rafl/fl;Defa6-cre mice for a range of genes involved in tissue regeneration pathways. These genes were selected because epithelial regeneration has been shown to be mediated via WNT, ADAM17, and Notch pathways (9, 38, 39) . Furthermore, the annexin family genes (Anxa 1, Anxa3, Anxa5), Ly6 family genes (Ly6a and Ly6d) and YAP family genes (Yap and Hbegf) are markers for the fetal-like reversion regenerative pathway (40–42). We showed decreased expression of Adam17, Dll1, and Wnt9b in Il17rafl/fl;Defa6-cre+ mice after radiation injury compared to cre- mice. Interestingly, Ly6a but not Ly6d, Anxa1, Anxa3, Anxa5, Yap, and Hbegf expression was increased in the small intestine of cre+ mice as compared to cre- mice (Fig. 6A & S4A). The increased expression of Ly6a may be occurring as an alternate regenerative mechanism to compensate for the decrease in expression of Adam17, Dll1, and Wnt9b after injury. The increased ligand expression of Ly6a does not appear to result in noticeable down stream effects. We also grew organoids from our Paneth cell lineage tracer mice and irradiated them for 24 hours as described prior. Next, we treated these organoids with IL-17A for 4 hours and sorted Paneth cells (Fig. 6B & S3D). RT-qPCR analysis of the sorted Paneth cells showed that 4 hour treatment of IL-17A directly increased the expression of Adam17, specifically in Paneth cells (Fig. 6C). ADAM17 is a metalloprotease involved in the proteolysis of ~80 proteins. Loss of ADAM17 has been shown to cause intestinal regenerative defects and is associated with chronic intestinal injury (39, 43, 44). Increased degradation of ADAM17 has also been shown to cause hair follicle stem cell exhaustion due to reduced Notch signaling (45). Hif1 has been shown to promote Adam17 expression (46, 47). Thus, we explored if the expression of Hif1 and its target genes (Hk2, Slc2a1, Pgk1) were also reduced in in the terminal ileum of irradiated Il17rafl/fl;Defa6-cre+ mice. No difference in expression was observed (Fig. 6D). Furthermore, RNA sequencing data (PMID: 29887318) also showed no difference in the expression of Hif1 related genes in Paneth cells post irradiation (Fig. 6E) (10). ADAM17 has also been shown to be modulated via NADPH oxidase 1 (Nox1). Specifically, the stability of ADAM17 has been shown to be mediated through Nox1. Nox1 has been shown to protect ADAM17 from ubiquitin-mediated degradation through direct interactions (48). Nox1 is also an important producer of intestinal epithelial ROS. Depletion of Nox1 significantly reduces intestinal epithelial ROS production (49). Oxidative stress and redox agents have been shown to activate ADAM17 and induce shedding of ADAM17 targets (50, 51). Thus, we explored if Nox1 may play a role in our observations. Expression of Nox1 was reduced in both naïve and irradiated Il17rafl/fl;Defa6-cre+ as compared to cre- mice, while expression patterns of other proliferative pathway genes remained unchanged between naïve Il17rafl/fl;Defa6-cre+ and cre- mice (Fig. S1K & Fig. 6F). Furthermore, immunohistochemistry (IHC) staining for Nox1 showed decreased Nox1 protein amounts in Il17rafl/fl;Defa6-cre+ as compared to cre- post irradiation (Fig. S4B). RT-qPCR analysis of the sorted Paneth cells showed that 4 hour treatment of IL-17A also directly increased expression of Nox1 specifically in Paneth cells (Fig. 6G). Analysis of a previously published single cell RNA sequencing data (PMID: 29887318) also showed increased expression of Adam17, Nox1, and Olfm4 in Paneth cells post irradiation (Fig. 6H) (10). Since Nox1 is a major producer of ROS, we next show that exogenous redox agents such as H O is able to directly induce ADAM17 activity (Fig. 6I). To confirm the importance of ROS in modulating Paneth cell stemness, we gave Paneth cell lineage tracer mice 4% n-acetylcysteine (NAC) in their drinking water after irradiation (52). NAC is an antioxidant, which has been shown to be readily metabolized within the small intestine (53). Mice were kept on 4% NAC water until euthanasia 5 days post irradiation (Fig. 6J). We then quantified the number of GFP+ cells in the crypt and villi between the two treatment groups. The results showed decreased GFP+ cells and clones in the villi of mice given on 4% NAC water as compared to mice given untreated water (Fig. 6K). We also observed a trend towards decreased ADAM17 activity in the ileum of mice given on 4% NAC water as compared to mice given untreated water (Fig. S4C). Next, to study the role of ADAM17 in our observations, we utilized an ADAM17 adenovirus (Ad-Adam17) to overexpress Adam17 (Fig 6L & Fig. S4D). Overexpression of ADAM17 rescued Il17rafl/fl;Defa6-cre+ mice from structural damage as well, while not affecting Paneth cell number (Fig. 6M & Fig. S4E). Increased cellular proliferation was observed in the ileum of Il17rafl/fl;Defa6-cre+ after over expression of ADAM17 (Fig 6N). Overexpression of ADAM17 in Il17rafl/fl;Defa6-cre+ showed reduced bacterial burden in their liver post irradiation injury (Fig. 6O). This was coupled with a trend towards decreased cecal LCN2 in these mice as well (Fig. S4F). Collectively, our data show that ADAM17 is required for intestinal epithelial regeneration.

ADAM17 expression in Paneth cells contributes to intestinal regeneration after radiation-induced injury.
We confirmed that both naïve and irradiated Paneth cells express ADAM17 by immunostaining the terminal ileum of control and irradiated Defa6-cre;mT/mG mice (Fig. S5A & S5B). We next generated Paneth (Defa6) cell-specific Adam17 knockout mice. Reduced staining of ADAM17 in Paneth cells in the ilea of Adam17fl/fl;Defa6-cre+ mice confirmed the specificity of the deletion (Fig. S6). We then irradiated Adam17fl/fl;Defa6-cre+/- mice. Five days after irradiation Adam17fl/fl;Defa6-cre+ mice had greater structural damage than their cre- counterparts (Fig. 7A). We also observed increased cecal LCN2 and increased hepatic and splenic bacterial dissemination in irradiated cre+ mice (Fig. 7B & 7C). Irradiated cre+ mice also had reduced proliferation as compared to their cre- counterparts (Fig. 7D). To determine whether ADAM17 expression on Paneth cells was important for Paneth cell specific proliferation, we stained irradiated Adam17fl/fl;Defa6-cre+/- mice for PCNA and Lyz1. We saw decreased amounts of proliferating Paneth cells in irradiated Adam17fl/fl;Defa6-cre+ mice as compared to cre- mice (Fig. 7E). While Paneth cell specific proliferation was decreased in Adam17fl/fl;Defa6-cre+, overall proliferation remains was not effected. The intestinal epithelium has been shown to have high plasticity where a wide variety of epithelial cells can dedifferentiate to regenerate the epithelium in the absence of ISCs (28, 54). Thus, we believe if Paneth cell dedifferentiation is inhibited, other cell types would then help regenerate the intestine. While RT-qPCR analysis of regenerative markers within the terminal ileum yielded no significant differences between irradiated Adam17fl/fl;Defa6-cre+/- mice, we did see a strong trend towards decreased Nox1 expression in Adam17fl/fl;Defa6-cre+ mice (Fig. 7F). To validate these finding, we stained for Nox1 in the ileum of irradiated Adam17fl/fl;Defa6-cre+/- mice. Indeed, we saw decreased Nox1+ crypts/villi in Adam17fl/fl;Defa6-cre+ mice as compared to cre- mice (Fig. 7G). Nox1 has been show to stabilize and promote ADAM17 activation, which in turn can then activate the EGFR-ERK1/2 pathway. (48, 55, 56). The EGFR-ERK1/2 pathway modulates ATF-1 which has been shown to promote NOX1 signaling (57). Thus, Adam17 expression and Nox1 expression may result from a positive feedback loop and rely upon each other. We next wanted to determine if Paneth cell specific ADAM17 played a role in intestinal epithelial barrier integrity. First, we stained for ZO-1, a common epithelial cell tight junction protein, in the terminal ilea of irradiated Adam17fl/fl;Defa6-cre+ and cre- mice. We then looked at expression levels of two other tight junction proteins, Ocln and Cldn4, in the ilea of these mice. There was no difference in gap junction protein amounts or expression between Adam17fl/fl;Defa6-cre+ and cre- mice post irradiation (Fig. 7H & Fig. 7I). Taken together, these results show that Paneth cell specific ADAM17 signaling is vital in limiting damage in response to irradiation via promoting cellular proliferation rather than modulating gut barrier proteins.

Methods
Study Design
Animal studies were conducted with the approval and under all relevant ethical regulations of Stony Brook University's Institutional Animal Care and Use Committee (IACUC protocol number 968871). The objective of this study was to determine the significance of IL-17RA signaling to Paneth cells in response to irradiation induced injury. Various genetically modified mice were utilized for this study. An irradiation-based injury model was utilized to study morphological, immunological, and regenerative changes within the small intestine. Sample sizes and replicates used were based on prior literature and experience with various tissue Il17ra knockout mice (12,13). Euthanasia time points were determined based on prior literature (34).

Mice
C57BL/6 (WT), Villin-Cre (C57BL/6 background), Gt(ROSA)26Sortm1(EYFP)Cos/J (C57BL/6 background) and ROSAmT/mG (C57BL/6 background) mice were purchased from The Jackson Laboratory. Il17RAKO mice were obtained from Dr. Jay Kolls, Tulane University. Defa6-Cre mice were obtained from Dr. Richard Blumberg, Brigham and Women’s Hospital, Harvard. Lgr5-EGFP-creERT2 , Lgr5-EGFP-creERT2;RosaLSLTdtomato and Villin-creERT2 mice were obtained from Dr. Vincent W. Yang, Stony Brook University. Adam17fl/fl mice were obtained from Dr. Rebecca K. Martin, Virginia Commonwealth University. Generation and characterization of IL-17-Floxed (Il17rafl/fl) mice were performed as previously described (26). Il17rafl/fl mice were bred with Villin-Cre or Defa6-Cre to develop entire gut epithelium and Paneth cell specific IL-17RA knockout mice. Defa6-Cre mice were bred with Gt(ROSA)26Sortm1(EYFP)Cos/J or ROSAmT/mG mice to develop PanethYFP and Paneth cell lineage tracing mice, respectively. Adam17fl/fl mice were bred with Defa6-Cre mice to develop Paneth cell specific ADAM17 knockout mice. Mice of both genders were used for all experiments unless indicated in the figures. Mice ages for each experiment are provided in the figure legends. Littermate controls were used for experiments, thus all cre- and cre+ mice were age matched. Sex was not considered in this study design and analysis as it was not relevant to our findings. All mice were housed in specific pathogen-free conditions at Stony Brook University, Stony Brook, NY. All animal studies were conducted with the approval of Stony Brook University Institutional Animal Care and Use Committee.

Animal Treatment
Anti-17A or IgG control (250g/mouse) was administered intraperitoneally to mice on same day of irradiation and 2 days post irradiation for select experiments. Mice were given 4% n-acetylcysteine in their drinking water directly after irradiation continuously until euthanasia on day 5 post irradiation for select experiments.

Cell Lines and Organoid Cultures
L-WRN cell line was obtained from Dr. Thaddeus Stappenbeck, Washington University. HEK-293 cell line was obtained from Dr. Vincent W. Yang, Stony Brook University.

Irradiation Treatment
Mice were irradiated at 12 Gy in a gamma cell irradiator (GammaCell 40; AEC Ltd). WT (C57BL/6) and Il17raKO mice bedding was mixed 1 week prior to gamma-irradiation to help ensure mixing of gut microbiota. Mice were euthanized at various time points (3 and 5 days) post irradiation and tissue was collected for experiments. Organoids were initially irradiated at increasing doses of gamma cell irradiation in a gamma cell irradiator (GammaCell 40; AEC Ltd) to develop our model. Once we confirmed 6 Gy as our optimal dose, all successive organoids were irradiated at 6 Gy. Organoids were then imaged and collected for RT-qPCR varying days post irradiation (3, 5, 6 days).

Bacterial Dissemination
Mice were euthanized 5 days post irradiation and their livers and spleens were harvested, weighed, homogenized, and plated on Luria broth (LB) or brain heart infusion (BHI) plates. After 48 hours, colonies were counted. Homogenates were also incubated in an anaerobic chamber (GasPak anaerobic container) for 48 hours and then counted to determine the dissemination of anaerobic bacteria.

Bone Marrow Transplantation
Wild type (WT) and Il17raKO mice were irradiated at 12 Gy. Non-irradiated WT and Il17raKO mice were euthanized and bone marrow was extracted from their femurs. Extracted bone marrow was then transplanted into their irradiated counterparts 2 hours post irradiation (1 million cells). Mice were then euthanized 5 days post irradiation and livers and spleens were harvested to measure bacterial dissemination as previously described and mesenteric lymph node was isolated for flow cytometry to ensure transplantation.

Induction of gene knockouts
To induce the knockout of genes, tamoxifen (1 mg/mouse) was administered intraperitoneally from day 0 to day 5 to Lgr5-EGFP-creERT2;RosaLSLTdtomato mice. On day 5 mice were irradiated and euthanized at multiple time points and tissues were harvested for bacterial dissemination.

Crypt Isolation
Ileums were harvested and flushed with ice-cold 1x PBS. They were then cut open longitudinally and Peyer’s patches were removed and cut into ~2 cm sections. Tissues were then washed in 30 mL of ice cold 1x PBS 3 times. Tissues were then incubated in 1x ice-cold PBS supplemented with 3.75 mM EDTA on an orbital rocket (60 rpm) for 30 minutes at 4C. Tissues were then washed twice with 50 mL of 1x ice-cold PBS. Tissues were then moderately shaken in 25 mL of ice cold 1x PBS and filtered through 70 micron filters to extract crypts. This was repeated three times to maximize crypt accumulation.

Organoid Culture
Isolate crypts were pelleted (300 x g) and re-suspended in Matrigel Matrix (Corning) at a concentration of ~100 crypts per 30 L Matrigel Matrix. 24 well plates were then seeded with 30 L/well of crypt suspension. The seeded plates were then incubated at 37C, 5% CO for 15 minutes to allow for Matrigel polymerization. The polymerized domes were then immersed in 500 L of enriched organoid growth medium (1:1 mixture of L-WRN and DMEM/F12) containing 1x penicillin-streptomycin-glutamine (Invitrogen), 1x B27 (Invitrogen), 1x N2 (Invitrogen), 50 ng/mL of murine EGF (R&D system), 1 mM of N-acetylcysteine (Sigma-Aldrich), 10 nM of Gastrin-Leu15 (Sigma-Aldrich), 500 nM of A83-01 (Torics), 10 mM of Y-27632 (Sigma-Aldrich) and 10 mM of CHIR99021 (Tocris). After 2 days, enriched organoid growth medium was replaced with supplemented organoid growth medium (no Y-27632 and CHIR99021). Supplemented organoid growth medium was continuously replaced every 2 days. Organoids were treated with IL-17A (50 ng/ml) every 2 days and harvested on day 6 for RT-PCR. Images were taken on days represented in figures with an Olympus CKX41 microscope.

For passaging, organoids were lifted in 500 L of cell recovery solution and incubated on an orbital shaker for 5 minutes at room temperature. Organoids were then pelleted (300g) and incubated in TrypLE Express (Thermo Fisher) for 5 minutes to make a single cell solution. Cells were then resuspended in Matrigel Matrix and plated in a 24 well plate (30 L/well). Domes were then resuspended in enriched organoid growth medium and cultured as described above.

RT-qPCR
RNA was extracted from terminal ileum tissues via Trizol-based extraction or from organoids via RNeasy Mini Kit (QIAGEN). RNA was reverse transcribed into cDNA using Bio-Rad iScript kits according to the manufacturer’s protocol. RT-PCR was performed by mixing 5 L of SsoAdvanced Universal Probes Supermix (Bio-Rad) or SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), 4.5 L of cDNA and 0.5 L of primer per reaction. All analysis were done relative to Gapdh or Hprt. Primer sequences can be found in Supplemental Table S1.

ELISA
Cecal samples were collected from mice and stored at -80C until needed. Samples were prepared for ELISA as previously reported (68). Briefly, frozen cecal samples were thawed and homogenized in PBS containing 0.1% Tween 20 at a concentration of 100 mg/mL. Homogenates were then centrifuged for 10 minutes and 12,000 rpm to remove solid debris. Clear supernatant was collected and utilized for experimentation with Mouse Lipocalin-2/NGAL DuoSet ELISA kit (R&D Systems) or Mouse Albumin ELISA Kit (RayBiotech) as per manufacture’s protocol.
Tissue samples were collected, immediately flash frozen in liquid nitrogen, and stored at -80C until needed. Samples were homogenized for ELISA in tissue protein extraction buffer containing 100mM Tris (pH 7.4), 150mM NaCl, 1mM EDTA, 1% triton, 0.5% sodium deoxycholate, 1x protease inhibitor, phosphate inhibitor, and 1mM Phenylmethylsulfonyl fluoride (PMSF). Homogenates were then centrifuged at 17,000 x g for 20 minutes at 4C and clear supernatant was collected. Protein concentration was determined via Pierce™ BCA Protein Assay Kits (23225). Equivalent amounts of protein was the utilized for experimentation with Mouse Epiregulin ELISA Kit (RayBiotech) as per manufacture’s protocol.

Histopathology
Terminal ileum tissue samples were prepared into Swiss roles, fixed in 10% formalin overnight and embedded in paraffin blocks. Paraffin embedded blocks were cut into 5 micron sections and adhered onto charged slides. Samples were then deparaffinized via Xylene and rehydrated via a descending ethanol gradient (100%, 95%, 70%, pure dH2O). For hematoxylin and eosin (H&E) staining, tissues were submerged in hematoxylin (VWR) for 5 minutes. Slides were then submerged in 3% acetic acid for 1 minute. Slides were then washed under running tap water for approximated 15 minutes. Tissues were then submerged in eosin (VWR) for approximately 1 minute. Slides were then submerged in 95% and 100% ethanol successively for 2 minutes. Slides were then mounted using toluene and imaged using an Olympus CKX41 microscope. Crypt lengths were measured via the program ImageJ.

Immunofluorescence
Terminal ileum tissue samples were prepared into Swiss roles, fixed in 10% formalin overnight and embedded in paraffin blocks. Paraffin embedded blocks were cut into 5 micron sections and adhered onto charged slides. Samples were then deparaffinized via Xylene and rehydrated via a descending ethanol gradient (100%, 95%, 70%, pure dH2O). Antigen retrieval was performed by heating slides in a steamer for 40 minutes. Tissues were then submerged under 1x PBS containing 0.1% Triton X-100 (Sigma-Aldrich) for 10 minutes and then washed three times with 1X PBS containing 0.01% Tween 20 (VWR) to be permeabilized. Tissued were then blocked by 1x PBS containing 5% fetal bovine serum at 34C for 1 hour. Following blocking, tissues were submerged in 1x PBS containing 5% fetal bovine serum with primary anti-bodies against LYZ1-FITC (1:200) (Dako: F037201), PCNA (1:200) (Santa Cruz: sc-56), GFP (1:100) (Cell Signaling: 295SS), MUC2 (1:200) (Abcam: ab272692), DCLK1 (1:200) (Cell Signaling: 62257S), ADAM17 (1:200) (Invitrogen: MA5-32572), RFP (1:200) (Rockland: 200-101-379), UEA1-Dylight 649 (1:200) (Vector: DL-1068), IL-17RA (1:200) (Abcam: ab180904), ChGA (1:200) (Santa Cruz: sc-393941), I-FABP (1:200) (Santa Cruz: sc-374482) and ZO-1 (1:200) (Invitrogen: 61-7300) at 4C overnight. The next day, slides were washed three times with 1x PBS, the sections were incubated for 30 minutes at room temperature with Alexa Fluor® 488 AffiniPure™ Goat Anti-Rabbit IgG (H+L) (1:200) (Jackson ImmunoResearch: 111-545-144), Alexa Fluor® 647 AffiniPure™ F(ab')₂ Fragment Goat Anti-Rabbit IgG (H+L)(1:200) (Jackson ImmunoResearch: 111-606-144), Alexa Fluor® 488 AffiniPure™ F(ab')₂ Fragment Goat Anti-Mouse IgG (H+L) (1:200) (Jackson ImmunoResearch: 115-546-003), or Alexa Fluor® 647 AffiniPure™ F(ab')₂ Fragment Donkey Anti-Goat IgG (H+L) (1:200) (Jackson ImmunoResearch: 705-606-147). Slides were then mounted with DAPI hard mount (Southern Biotech) and images were acquired using an Olympus CKX41 microscope.

Organoids were lifted using ice cold 1X PBS. Organoids were fixed in 4% paraformaldehyde for 15 minutes. Organoids were then washed with 1X PBS and permeabilized with .01% triton in 1X PBS for 10 minutes. After permeabilization, organoids were washed with 1X PBS and then blocked in 1X PBS containing 5% BSA for 1 hour at 34C. After blocking, organoids were incubated overnight at 4C in 1X PBS containing 5% BSA with primary antibodies against GFP (1:100) (Rockland: 600-901-215), Muc2 (1:200) (Santa Cruz: sc-515032), or ChGA (1:200) (Santa Cruz: sc-393941). The next day, organoids were washed with 1X PBS and then incubated for 30 minutes at 37C in 1X PBS containing 5% BSA with secondary antibodies Alexa Fluor® 488 AffiniPure™ F(ab')₂ Fragment Donkey Anti-Chicken IgG (H+L) (1:100) (Jackson ImmunoResearch: 703-544-155) and Alexa Fluor® 647 AffiniPure™ F(ab')₂ Fragment Donkey Anti-Mouse IgG (H+L) (1:200) (Jackson ImmunoResearch: 715-605-150). Organoids were then washed with 1X PBS and mounted on slides with DAPI hard mount (Southern Biotech) and images were acquired using an Olympus CKX41 microscope.

Live fluorescent murine organoids endogenously expressed fluorescence and thus were directly imaged in culture using an EVOS M5000 imaging system.

Immunohistochemistry
Paraffin embedded blocks were cut into 5 micron sections and adhered onto charged slides. Samples were then deparaffinized via Xylene and rehydrated via a descending ethanol gradient (100%, 95%, 70%, pure dH2O). Antigen retrieval was performed by heating slides in a steamer for 40 minutes. Tissues were then incubated in hydrogen peroxide block (abcam: 58661) for 10 minutes at room temperature. Sections were then washed in MilliQ water 2 times for 5 minutes followed by 1 wash in 1X TBS-T for 5 minutes. Sections were then blocked for 1 hour at room temperature in 5% BSA in 1X TBS-T. Sections were then washed and avidin biotin block was performed as per manufacturers protocol (Vector: SP-2001). Sections were then incubated overnight at 4C in 1X TBS-T containing primary antibody against Nox1 (1:250) (Proteintech: 17772-1-AP). The next day, section were washed 3 times in 1X TBS-T. Sections were then incubated at room temperature for 30 minutes in horse anti-rabbit IgG (H+L), biotinylated, R.T.U. (Vector: BP-1100). Sections were then washed in 1X TBS-T 3 times. Sections were then incubated in VECTASTAIN Elite ABC reagent, Peroxidase, R.T.U. (PK-7100) for 30 minutes at room temperature. Sections were then washed and then incubated in DAB (Dako: K3467) as per manufacturers protocol. Sections were then rinsed with MilliQ water and washed 3 times in 1X TBS-T for 5 minutes. Sections were then washed in MilliQ water and then counterstained with hematoxylin for 5 minutes. Slides were then washed under running tap water for approximated 15 minutes. Slides were then submerged in 95% and 100% ethanol successively for 2 minutes. Slides were then mounted using toluene and imaged using an Olympus CKX41 microscope. Crypt lengths were measured via the program ImageJ.

TEM
Transmission electron microscopy was performed as previously described (69). Briefly, terminal ileum samples were fixed in Karnovsky’s Fixative (Electron Microscopy Sciences) overnight. The next day, tissues were incubated in 1% osmium tetroxide in 0.1 M PBS pH 7.4 and then dehydrated via a descending ethanol gradient. Dehydrated tissues were then embedded in EMbed 812 resin. 80 nm sections were then cut and placed on formvar coated slot copper grids and counter stained using uranyl acetate and lead citrate. A FEI Tecnai12 BioTwinG2 transmission electron microscope was used to visualized tissues and an AMT XR-60 CCD Digital Camera system was used to acquire images.

Acid-urea polyacrylamide gel electrophoresis (AU-PAGE)
Intestinal crypts were isolated as described. Crypts were then resuspended in 30% acetic acid and sonicated. After sonication, lysates were incubated overnight at 4C with agitation. Samples were then diluted threefold and centrifuged with a speed of 100,000 x g at 4C for 2 hours to remove insoluble material. Supernatant was collected and protein concentration was determined via Pierce™ BCA Protein Assay Kits (23225). Equivalent amounts of protein were then lyophilized. The lyophilized protein was then dissolved in 30 L of AU-PAGE loading buffer (3M urea in 5% (v/v) glacial acetic acid in H O and methyl green). Equivalent amounts of protein was then loaded into AU-PAGE gels and run in 5% acetic acid running buffer with reverse polarity. Gels were run at 150V until proteins reached the bottom. Proteins were then visualized via Coomassie blue staining and densitometry was performed via ImageJ.

Lymphocyte isolation
Lymphocytes were isolated as previously described (70). Briefly, the small intestines were opened, Peyer’s patched and luminal contents were removed, and the tissues were cut into ~2cm pieces. To isolate lamina propria (LP) lymphocytes, the tissues were treated with EDTA (Invitrogen) solution at 37°C with shaking at 220 rpm for 30 min to remove intestinal epithelial cells. The tissues were then digested with Type I collagenase (Gibco) at 37°C with shaking at 300 rpm for 40 min and mashed through 70-μm cell strainers. LP lymphocytes were subsequently isolated using a 44%/67% Percoll gradient.

Flow Cytometry
Intestinal crypts were isolated as described. Crypts were incubated in DMEM/F12 supplemented with 10% fetal bovine serum for 20 minutes at 4C. Crypts were then disassociated in TrypLE (Invitrogen) with 10μM Rock inhibitor (Y-27632) and 2.5 μg/ml DNAse 1 (Sigma-Aldrich) for 10 minutes at 37C. Single cells were then washed twice in PBS. The following fluorophore-conjugated antibodies were purchased from Invitrogen: anti-CD24 (M1/69), anti-CD45 (30-F11), and anti-Epcam (G8.8). Live/Dead aqua fluorescent reactive dye was purchased from Invitrogen. Cells were then stained with antibodies for 30 minutes at 4C and filtered to remove clumps. Results were acquired with a BD-LSR-Fortessa or cells were sorted using a FACS ARIA IIIU or a CytoFLEX SRT cell sorter.

Lymphocytes were isolated as described. The following fluorophore-conjugated antibodies were purchased from Biolegend, BD Biosciences or eBioscience: anti-CD45 (30-F11), anti-CD3ε (145-2C11), anti-CD19 (6D5), anti-CD11b (Mac-1), anti-Ly6C (HK1.4), anti-Ly6G (1A8), anti-CD64 (S18017D), anti-CD11c (N418), anti-MHC II (M5/114.15.2), anti-NK1.1 (PK136), anti-CD49b (DX5), anti-CD8α (53-6.7), anti-CD103 (2E7), anti-CD127 (A7R34), anti-RORγt (AFKJS-9), anti-Thy1.2 (30-H12), anti-CD4 (RM4-5) and anti-TCRβ (H57-597). Live/Dead dye was purchased from Invitrogen. Cells were incubated with antibodies for 30 min at 4°C in the dark. For transcriptional factor staining, Foxp3/Transcription Factor Staining Buffer set (Invitrogen) was used according to the manufacturer’s instructions. Samples were acquired on Aurora (Cytek), and data were analyzed using the FlowJo v.10 software (FlowJo, LLC).

Total RNA Sequencing (RNA-seq)
RNA sequencing and data analysis was performed as previously described (69). Before constructing the Illumina TruSeq Total RNA library, total RNA was quantified using the Qubit RNA BR assay kit (Thermo Fisher Scientific: Guide MAN0001987 MP10210, Kit #Q10210). RNA quality was assessed on an Agilent 4150 Tapestation (G2992AA) with the Agilent RNA ScreenTape Analysis (Agilent: Publication Part Number: G2991-S90021, Kit #5067-5576). A total of 2.5 μg of RNA was treated with 1 M Baseline-Zero DNase (Epicentre Cat. No DB0711K) according to the Baseline–Zero DNase protocol (www.epicentre.com, Lit # 263). The RNA was then purified and concentrated using 1.8 × volume Agencourt RNAClean XP Beads (Beckman Coulter A63987) following the standard protocol (Protocol 001298v001). RNA concentration was re-measured with the Qubit BR RNA assay kit to prepare for input into the TruSeq Stranded Total RNA library protocol.

Illumina-compatible cDNA libraries with RiboZero Gold depletion were constructed according to the TruSeq Stranded Total RNA Reference Guide (Illumina Document #1000000040499v00, TruSeq Stranded Total RNA Gold Illumina Kit #20020598). Final cDNA library concentrations were determined using the Qubit dsDNA BR assay kit (Thermo Fisher Scientific: Guide MAN0002325 MP 32850, Kit #Q32850). The quality of the libraries was evaluated by running each on an Agilent 4150 Tapestation using the DNA 1000 Screentape kit (Agilent: Publication Part Number: G2991-90031, Kit #5067-5583). The electropherogram of each library was analyzed using Agilent 4150 Tapestation Analysis Software (Version 3.1) with a range of 200–600 bp to determine the average library size. Average size and concentration were used to calculate the molarity of each library. Libraries were pooled and denatured according to the Illumina Denature and Dilute Libraries Guide for the NextSeq System (Illumina Part #15048776). The denatured libraries were loaded onto an Illumina NextSeq 550 v2.5 Mid 150 output reagent cartridge (Illumina #20024904) at a final concentration of 1.5 pM in HT1 buffer, with 1% PhiX control library v3 (Illumina #FC-110-3001) added. Paired-end 75 bp single-index sequencing was performed, generating approximately 7.5 million paired-end reads per sample.

Sequencing analysis was carried out using RNA-seq for Eukaryotes Analysis v3 by the Banana Slug Genomics Center at the University of California, Santa Cruz. Raw paired-end sequencing reads from the Illumina sequencer were assessed for potential issues and contaminants using FastQC. Adapter sequences and primers were removed from the reads using Trimmomatic, followed by trimming of polyA tails, polyN sequences, and any reads with quality scores below 28 using PRINSEQ. Any reads shorter than 20 bp after trimming were discarded. A second round of quality control with FastQC was performed to compare read quality before and after trimming. The trimmed reads were mapped to the reference genome (GRCm38/mm10) using TopHat2, with NCBI RefSeq annotated genes as the transcriptome index. Read alignment coverage and summary statistics were computed using SAMtools, BEDtools, and UCSC Genome Browser utilities. For abundance estimation and differential expression analysis, the Cufflinks 2.2.0 workflow and read-counting methods using DESeq and edgeR were employed. Specifically, the sequencing reads aligned to RefSeq annotated genes were quantified using Cuffquant, and gene expression levels across the samples were normalized using Cuffnorm with FPKM values computed for sample correlation assessment. Differential expression analysis was performed using Cuffdiff on the results from Cuffquant. Additionally, HTSeq was used to compute raw read counts for the annotated RefSeq genes. Raw read counts were normalized across all samples and used for differential expression analysis with edgeR.

16S Microbial Sequencing
Mouse cecal samples were collected and stored at -80C. Cecal samples were shipped on ice. Fecal DNA was extracted via Qiagen DNeasy PowerLyser PowerSoil Kit (Qiagen). Bead beating step was performed following the manufacturer’s instructions (PowerLyzer 24 Homogenizer, Omni International). The 16S microbial sequencing was performed as previously described (71).

AD-Adam17 and Ad-Y5 Infection
HEK293 cells were infected with 100 MOI of with either Ad-Adam17 or Ad-Y5 overnight and were harvested for RT-PCR to confirm upregulation. Ad-Adam17 and Ad-Y5 were generated as previously described (72). Mice were injected intraperitoneally with either Ad-Adam17 or Ad-Y5 at a concentration of 1x10^9 pfu/mouse. Mice were then irradiated 6 days post injection and processed as described above.

ADAM17 Activity Assay
ADAM17 Fluorogenic Kit (BPS Bioscience) was utilized for ADAM17 activity assays as per manufacture’s protocol. Briefly, ADAM17 protein was incubated at room temperature for 30 minutes with or without 4mM H O . ADAM17 fluorogenic substrate was then added and results were collected every 10 minutes for 250 minutes utilizing a HTS 7000 Bio Assay Reader.

Statistical Analysis
Bacterial dissemination, RT-qPCR, ELISA and all counting experiments were analyzed via non-parametric Mann-Whitney test, two tailed or Welch’s t-test using GraphPad/Prizim. Paired samples were analyzed via Wilcox test using GraphPad/Prizim. Grouped experiments were performed via two way ANOVA. Outliers were determined by using GraphPad Prism (ROUT analysis, Q=1%). More specific statistical analysis details can be found in the figure legend of each figure. Cell counts were performed from at least 30 crypts per mouse.
Microbiome analysis was performed in R (version 4.3.1). Data was formatted utilizing phyloseq (version 1.44.0). Specifically, to normalize data we filtered taxa with a relative abundance of  1e-4, Total Sum Scaled (TSS) abundances to 1e6, and performed a general log transformation. We calculated the beta diversity metric, Weighted UniFrac, and identified significance, using ADONIS, with the vegan package (version 2.6.6.1). To calculate LEfSe we utilized the microbiomeMarker (version 1.6.0) package. All plots were created with ggplot2 (version 3.5.1).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Kempen et al. reported that IL-17A-IL-17RA signalling in Paneth cells is critical for host defence responses in the small intestine. The authors also revealed that IL-17A-induced Nox1 is necessary for the subsequent tissue regeneration responses mediated by ADAM17 enzymatic activity. To provide in vivo evidence to support their conclusions, the authors used a series of Paneth cell-specific knockout mice, including Il17rafl/fl Defa6-cre and Adam17fl/fl Defa6-cre. This study is likely to attract wide attention from scientists involved in mucosal immunology.\nOverall, the observations and underlying implications described here are both reasonable and significant. The experiments are rigorous, with sufficient replicates and controls. However, it is well known that intestinal epithelial cells are a major IL-17A-targeting cell type in the gut, and that IL-17A is considered a protective cytokine at the mucosal surface because it can induce antibacterial peptides and work synergistically with IL-22. Therefore, these previous reports and existing knowledge compromise the novelty of the current study.\nMy only major concern about this study is that the authors only used Il17rafl/fl Defa6-cre mice to represent a genetic deficiency in IL-17A signalling in Paneth cells. However, IL-17RA is the receptor for all IL-17 family cytokines. IL-17RC deficiency is more relevant to claiming the specific effects of IL-17A/F. IL-17RA deficiency cannot rule out the effects mediated by IL-17C, since IL-17RA forms a heterodimer with IL-17RE in response to IL-17C, and IL-17C-IL-17RA/RE signalling has also been reported to play an important role in maintaining mucosal homeostasis. Therefore, the authors should rule out or include the effects of IL-17C in their study.", "reviewer_2": "The manuscript “Immune cell-derived IL-17A promotes Paneth cell dedifferentiation through the Nox1-ADAM17 axis to regenerate the gut epithelium” by Kempen et al. describes a novel function of IL-17RA signaling in regulating the reprogramming of Paneth cells into multipotent stem-like cells upon irradiation. By selectively knocking out IL-17RA in Defa6-expressing Paneth cells, the authors provide evidence that Paneth cell–specific IL-17RA signaling protects against irradiation-induced intestinal damage.\nUsing Paneth cell lineage-tracing mice, they further show that Paneth cells can acquire stem cell–like features following intestinal injury, a process that can be blocked by neutralizing IL-17A with antibodies. Mechanistically, the study demonstrates that IL-17A increases ADAM17 expression in Paneth cells. Notably, Paneth cell–specific ADAM17 knockout phenocopied the IL-17RA knockout in the context of irradiation-induced injury, while ectopic ADAM17 expression rescued the effects of IL-17RA deficiency.\nThe manuscript also establishes a link between IL-17RA–dependent Nox1 expression, ROS production, and ROS-mediated ADAM17 activation. Pharmacological inhibition of ROS with NAC suppressed the dedifferentiation of Paneth cells into stem-like cells after irradiation, suggesting a central role for ROS in injury-induced Paneth cell plasticity. Finally, the authors indicate that ADAM17 promotes tissue regeneration primarily by enhancing cellular proliferation rather than by modulating gut barrier proteins.\nThe findings are highly exciting and of significant scientific interest, as they identify a novel function of IL-17RA signaling in regulating Paneth cell reprogramming during intestinal injury. It has become increasingly clear that Paneth cells play a central role in maintaining intestinal integrity—not only through their functions as innate immune cells and as stem cell niche supporters but also by serving as an alternative stem cell population that contributes to intestinal regeneration following tissue damage. While defects in Paneth cell function have long been associated with the development of intestinal inflammatory diseases, recent evidence shows that the reacquisition of stem cell features during inflammation can also enable Paneth cells to give rise to intestinal tumors (Verhagen et al., 2024 DOI: 10.1038/s41588-024-01801-y). In light of these diverse roles of Paneth cells in both health and disease, it is critical to better understand how their fate is extrinsically regulated.\nKempen et al. have invested considerable effort in studying the role of IL-17RA signaling in Paneth cells by employing several transgenic mouse lines and ex vivo organoid systems, providing strong support for a role of IL-17A signaling in regulating Paneth cell plasticity. Their findings may also have translational relevance, as they could open new avenues for the treatment of IBD or for supportive care during irradiation therapy. In this regard, the authors should consider performing additional experiments to broaden the translational impact of their study.\nAs IL-17RA signaling appears to be dispensable for Paneth cell dedifferentiation during DSS-induced gastroenteritis (Lin et al., 2022 DOI: 10.1016/j.immuni.2021.12.016), the mechanism described by Kempen et al. does not seem to be universal, but rather context-dependent and influenced by the type of injury. It would therefore be valuable to examine whether Paneth cell–specific IL-17RA signaling is also relevant for intestinal regeneration following chemotherapy, thereby expanding the potential scope of IL-17A–based supportive care.\nMoreover, it would strengthen the study to assess whether a similar mechanism might apply to colonic regeneration. Recent findings indicate that Paneth-like Deep Crypt Secretory (DCS) cells in the colon can revert to stem-like cells to support tissue repair during colitis (Xu et al., 2025 DOI: 10.1038/s41418-025-01471-1). The authors could, for example, evaluate whether DCS proliferation is impaired in irradiated IEC-specific IL-17RA knockout mice (e.g. by co-staining PCNA or Ki67 with DCS markers such as Reg4, c-Kit, or WGA), or at least determine whether colonic damage is exacerbated upon epithelial loss of IL-17RA or blockade of IL-17A signaling with neutralizing antibodies. Demonstrating that IL-17RA signaling also contributes to colonic regeneration would substantially enhance the relevance of the study with relatively limited additional effort.\nBeyond these points, I have additional critical remarks and questions that should be addressed prior to considering this manuscript for publication in Nature Communications. These points are summarized in bullet-point format for clarity:\n1. First, and perhaps the most crucial experiment missing to convincingly demonstrate that IL-17RA directly regulates Paneth cell dedifferentiation, is the analysis of lineage tracing in irradiated IL17RAfl/fl;Defa6-cre;mTmG mice. I am somewhat surprised that the authors did not do this experiment. Although the anti-IL17A antibody treatment causes a reduction of Defa6-lineage tracing it might be due to other indirect mechanisms.\n2. Likewise, irradiation-induced Paneth cell lineage tracing should be assessed in ADAM17fl/fl;Defa6-Cre;mTmG mice. Alternatively, Paneth cell proliferation should be quantified by co-staining proliferation markers (e.g., Ki67 or PCNA) with Paneth cell–specific markers such as lysozyme to support the importance of ADAM17 for Paneth cell dedifferentiation.\n3. As the authors introduce the depletion and re-appearance of canonical ISCs upon irradiation (Fig. 2B), they should address whether the regeneration of canonical ISCs is impaired in IL17RAfl/fl;Defa6-cre mice (e.g. by a simple OLFM4 staining or Lgr5 in situ hybridization).\n4. Kempen and colleagues claim increased intestinal damage in IL17RA whole-body knockout mice or in mice with IL17RA knockout in IECs. Although they demonstrate increased bacterial dissemination in the spleen and liver, the only reported evidence of enhanced tissue damage is a minor reduction in crypt length. Why did the authors not include additional parameters such as villus length, weight loss, quantification of viable crypts (as in Metcalfe et al., 2014 PMID: 24332836), or quantification of PCNA+ cells, as they performed in Fig. 3D for the Paneth cell–specific knockout? The same concern applies to the mice treated with IL17A antibodies (Figure 5) and the mice overexpressing ADAM17 (Figure 6). The inclusion of some additional parameters would provide stronger support for their claim.\n5. In Figure 2C, the authors report reduced Lyz mRNA levels in the intestinal epithelium of IL17RA knockout mice after irradiation, which led them to further investigate IL17RA signaling in Paneth cells. However, it remains unclear whether this reduction is due to decreased lysozyme expression within Paneth cells or to a reduction in Paneth cell numbers upon loss of IL17RA. To address this, Paneth cells should be stained and quantified. Similarly, Paneth cell numbers and lysozyme expression should be evaluated in irradiated IL17RAfl/fl;Villin-cre mice and IL17RAfl/fl;Defa6-cre mice, in order to determine whether a decrease in lysozyme expression can be also observed upon tissue or Paneth cell specific Il17RA knockout.\n6. Regarding the effects on lysozyme expression upon IL17RA knockout, I have another critical remark: in their prior study (Lin et al., 2022 PMID: 35081371), the authors reported no difference of lysozyme expression in the intestinal epithelium of 6-week-old IL17RAfl/fl;Villin-cre mice, while Paneth cell number was reduced in 10-week-old IL17RAfl/fl;Villin-cre mice. In the current manuscript, the only information provided about mouse age is found in the Methods section, where it is stated that animals were between 6 and 15 weeks old. Given the previously described age-dependent effects of IL17RA deletion on Paneth cells, it is essential to indicate the exact age of the mice used in each experiment.\n7. In Figure 4, the authors dedicate an entire section to different Paneth cell populations, mentioning a Defa6-low population, expressing stem cell genes. These cells likely represent Paneth cell progenitors, potentially more prone to dedifferentiate than Defa6-high/OLFM4-low cells. However, no experiments were performed to address this. For example, this could have been tested by analysing the organoid-forming capacity sorted Defa6-YFP high and low or Defa6-GFP high/CD24+ and Defa6-GFP low/CD24+ cells following irradiation. At minimum, the authors should discuss the possibility that the differentiation status of Paneth cells may influence their ability to dedifferentiate upon injury.\n8. The authors show lineage tracing in Defa6-cre;mT/mG mice upon irradiation (Figure 5A) as well as organoid growth from sorted GFP+CD24+ cells from these mice (Figure 5B). However, no quantification is provided for the lineage tracing frequency (i.e. the number of tracings per GFP labelled crypt) between non-irradiated and irradiated mice, nor is there quantification of the number of GFP+ organoids per plated cells to indicate the efficiency of Paneth cells to dedifferentiate. In Figure 5D and Figure 6K, the authors do quantify single GFP+ cells and GFP+ clones in crypts and villi, but it is not explained how these quantifications were performed or what the y-axis represents. This information should be clarified.\n9. In Figure 5G, the authors claim to provide evidence for increased Paneth cell lineage tracing in IL17A-treated organoids. I find this interpretation unconvincing: the images do not show ribbons of GFP+ cells that would indicate lineage tracing but rather single GFP+ cells distributed in organoid buddings, consistent with the normal localization of Paneth cells. A more likely explanation is that IL17A treatment enhances Paneth cell differentiation, similar to what they reported previously (Lin et al., 2022 PMID: 35081371). The authors might argue that organoid passaging results in fully GFP-labelled organoids; however, the images in Figures 5H and 5I are also not convincing in this regard. Instead, they appear more consistent with immature spheres, though this is difficult to assess because no bright-field images were provided to illustrate organoid morphology. Stronger evidence is needed to convincingly demonstrate Paneth cell dedifferentiation ex vivo.\n10. The rationale behind the selection of genes analyzed in the context of Paneth cell dedifferentiation (Figure 6A) is not entirely clear. For example, why did the authors examine Wnt9b and Wnt6 to assess Wnt signaling rather than canonical Wnt target genes such as Axin2, CyclinD1, c-Myc, or CD44? Similarly, why were Dll1 and Dll4 measured instead of established Notch target genes such as HES or HEY to evaluate pathway activity? In addition, the authors did not include YAP target genes, despite their own re-analysis of scRNA-seq data showing that YAP targets are upregulated in the same Paneth cell subcluster with the highest expression of ADAM17 and Nox1 (Figure 6H). Likewise, in irradiated IL17RAfl/fl;Defa6-cre mice, the authors measured expression of Hif1α, Hk2, Slc2a1, and Pgk1, but only Hif1α is discussed in the text. What was the rationale for including the other genes?\n11. The title of the manuscript suggests that Paneth cell dedifferentiation is regulated through the NOX1–ADAM17 axis, which is a strong claim given that the only evidence indicating an involvement of NOX1 is the upregulation of NOX1 mRNA upon irradiation and IL-17A treatment, and the downregulation of NOX1 mRNA in irradiated ADAM17fl/fl; Defa6-Cre mice. At a minimum, effects on NOX1 expression should be confirmed at the protein level, ideally by immunostaining in mouse tissues. Additionally, demonstrating reduced ROS production upon Paneth cell–specific IL-17RA knockout (e.g., by ROS analysis in irradiated organoids using indicators such as CellROX or H2DCFDA) would further strengthen the proposed link between IL-17RA signaling and ROS-mediated ADAM17 activation. Even then, the title might falsely indicate regulation by NOX1, as ROS might be increased by multiple other mechanisms upon irradiation.\n12. Regarding the title, I have an additional remark: the authors do not provide direct evidence that the observed effect is specifically mediated by immune cell–derived IL-17A. To be accurate, either immune cells should be removed from the title, or experiments should be included to specifically address their involvement. Notably, IL-17A can be expressed by other cell types, such as fibroblasts, as reported by Lotti et al., 2013 (https://doi.org/10.1084/jem.20131195).\n13. As stated in the main text, ADAM17 is expressed in Paneth cells of both naïve and irradiated mice (Fig. S5A). Based on the images, ADAM17 expression appears to be considerably lower in irradiated mice compared to non-irradiated controls. The authors neither comment on this nor provide further analysis of differential ADAM17 expression between the two conditions. If confirmed, this would contradict the results shown in Figure 6H, where re-analysis of published scRNA-seq data from irradiated mice indicated increased ADAM17 expression. This discrepancy should be addressed and discussed.\n14. Figure S6 is intended to show a Paneth cell–specific ADAM17 knockout; however, ADAM17 expression also appears reduced in other epithelial cells. Here either higher-quality images should be provided to clarify this or it should be commented on why a Paneth cell–specific knockout might lead to a broader reduction of ADAM17 expression.\n15. Figure 6K shows a reduction of lineage tracing in NAC-treated mice. Is it possible to assess ADAM17 activity in these mice? This would help to strengthen the link between ROS production and ADAM17 activation.\n16. Regarding Figure 7F, Kempen et al. state that there is no difference in the tight junction protein ZO-1 in ADAM17fl/fl;Defa6-Cre mice and conclude that ADAM17 limits damage in response to irradiation by promoting cellular proliferation rather than modulating gut barrier proteins. However, based on the images provided in Figure 7F, it appears that ZO-1 is absent in the crypts of Adam17fl/fl;Defa6-cre mice. The authors should either reconsider their statement or provide additional data to support their interpretation.\nIn addition to the above I have some minor remarks:\n1. In Figure 1A and F, Figure 3A, Figure 5E, Figure 6M, Figure 7A, Figure S3A it is not described what the arrows should indicate.\n2. Y-axes in Figure 1A,, 3A, 5E, 6M, 7A, 7D should start from 0.\n3. It is not described how PCNA+ cells in Figure 3D and Figure 7D were quantified (cells per crypt? How many crypts per mouse?)\n4. Authors should describe in more detail how outliers were identified and in which experiments outliers were excluded.\n5. It is not always clear how many times the experiments were repeated or how many samples were analyzed and quantified. For example, the authors state: “Figures 5A, 5B, 5G, and 5I are representative of 2–3 mice. Figures 5B, 5D, 5E, 5G, and 5H are generated from 2–3 independent experiments, and representative images are shown and quantified.” This level of reporting is insufficient for a high-quality journal such as Nature Communications. The exact number of biological and technical replicates, as well as the precise number of mice analyzed in each experiment, must be clearly specified.\n6. The manuscript refers to Figure S6A, however, Figure S6 has no A or B.\nAltogether, in my opinion considerable additional work is required for the manuscript to reach the high standard expected for Nature Communications. Nonetheless, I am confident that the authors can address the points raised, and that, with these revisions the manuscript could make a significant impact in the field."}, "author_response": "Dear Reviewers,\nWe would like to thank the reviewers for their consideration of our manuscript and for\nproviding insightful comments. In addition, we appreciate the opportunity to revise and\nresubmit this manuscript. We found reviewer comments to be helpful and note that the\nreviewers provided many enthusiastic comments. Based on their suggestions, we have\nadded new data that supports our findings. Specifically, we demonstrate that: 1) IL-17C\ndoes not promote fully green organoid development after irradiation or Paneth cell lineage\ntracer organoids, 2) Paneth cell specific proliferation is reduced in ADAM17fl/fl;Defa6-Cre+\nmice, 3) there is reduced cellular proliferation in IL-17RA KO, Il17RAfl/fl;villin-cre+,\nand anti-IL-17A administered lineage tracer mice. Furthermore, cellular proliferation is\nincreased when Adam17 expression is increased, 4) fully green organoids derived from\nirradiated and IL-17A treated Paneth cell lineage tracer organoid contain terminally\ndifferentiated epithelial cells and 5) NOX1 protein is reduced in the ileum of both\nIl17RAfl/fl;Defa6-Cre+, and ADAM17fl/fl;Defa6-Cre+ mice. Also, we made modifications to\nthe text, which are highlighted in yellow. Overall, the reviewers’ comments and\nsuggestions helped us significantly strengthen our manuscript. Below, we provide each\nof the reviewers’ comments followed by a detailed point-by-point response.\nReviewer 1:\nSummary and Significance: In this study, Kempen et al. reported that IL-17A-IL-17RA\nsignalling in Paneth cells is critical for host defence responses in the small intestine.\nThe authors also revealed that IL-17A-induced Nox1 is necessary for the subsequent\ntissue regeneration responses mediated by ADAM17 enzymatic activity. To provide in\nvivo evidence to support their conclusions, the authors used a series of Paneth cell-\nspecific knockout mice, including Il17rafl/fl Defa6-cre and Adam17fl/fl Defa6-cre. This\nstudy is likely to attract wide attention from scientists involved in mucosal immunology.\nOverall, the observations and underlying implications described here are both\nreasonable and significant. The experiments are rigorous, with sufficient replicates and\ncontrols. However, it is well known that intestinal epithelial cells are a major IL-17A-\ntargeting cell type in the gut, and that IL-17A is considered a protective cytokine at the\nmucosal surface because it can induce antibacterial peptides and work synergistically\nwith IL-22. Therefore, these previous reports and existing knowledge compromise the\nnovelty of the current study.\nComment: My only major concern about this study is that the authors only used\nIl17rafl/fl Defa6-cre mice to represent a genetic deficiency in IL-17A signalling in Paneth\ncells. However, IL-17RA is the receptor for all IL-17 family cytokines. IL-17RC deficiency\nis more relevant to claiming the specific effects of IL-17A/F. IL-17RA deficiency cannot\nrule out the effects mediated by IL-17C, since IL-17RA forms a heterodimer with IL-\n17RE in response to IL-17C, and IL-17C-IL-17RA/RE signalling has also been reported\nto play an important role in maintaining mucosal homeostasis. Therefore, the authors\nshould rule out or include the effects of IL-17C in their study.\nResponse I thank the reviewer for the recommendation to consider the effects of IL-\n17C in this study. Following the approach used for IL-25 (Fig. S3C), we tested whether\nrecombinant IL-17C stimulation in irradiated Paneth cell-specific lineage-tracer mice\ncould induce green organoid development after irradiation. We did not observe a\nsignificant increase in green organoid formation following IL-17C treatment (Fig. S3C).\nIn addition, we have revised the manuscript title to emphasize that IL-17RA signaling in\nPaneth cells promotes acquisition of stem-like properties, rather than attributing this\neffect specifically to IL-17A.\nReviewer 2:\nSummary and Significance: The manuscript “Immune cell-derived IL-17A promotes\nPaneth cell dedifferentiation through the Nox1-ADAM17 axis to regenerate the gut\nepithelium” by Kempen et al. describes a novel function of IL-17RA signaling in\nregulating the reprogramming of Paneth cells into multipotent stem-like cells upon\nirradiation. By selectively knocking out IL-17RA in Defa6-expressing Paneth cells, the\nauthors provide evidence that Paneth cell–specific IL-17RA signaling protects against\nirradiation-induced intestinal damage.\nUsing Paneth cell lineage-tracing mice, they further show that Paneth cells can acquire\nstem cell–like features following intestinal injury, a process that can be blocked by\nneutralizing IL-17A with antibodies. Mechanistically, the study demonstrates that IL-17A\nincreases ADAM17 expression in Paneth cells. Notably, Paneth cell–specific ADAM17\nknockout phenocopied the IL-17RA knockout in the context of irradiation-induced injury,\nwhile ectopic ADAM17 expression rescued the effects of IL-17RA deficiency.\nThe manuscript also establishes a link between IL-17RA–dependent Nox1 expression,\nROS production, and ROS-mediated ADAM17 activation. Pharmacological inhibition of\nROS with NAC suppressed the dedifferentiation of Paneth cells into stem-like cells after\nirradiation, suggesting a central role for ROS in injury-induced Paneth cell plasticity.\nFinally, the authors indicate that ADAM17 promotes tissue regeneration primarily by\nenhancing cellular proliferation rather than by modulating gut barrier proteins.\nThe findings are highly exciting and of significant scientific interest, as they identify a\nnovel function of IL-17RA signaling in regulating Paneth cell reprogramming during\nintestinal injury. It has become increasingly clear that Paneth cells play a central role in\nmaintaining intestinal integrity—not only through their functions as innate immune cells\nand as stem cell niche supporters but also by serving as an alternative stem cell\npopulation that contributes to intestinal regeneration following tissue damage. While\ndefects in Paneth cell function have long been associated with the development of\nintestinal inflammatory diseases, recent evidence shows that the reacquisition of stem\ncell features during inflammation can also enable Paneth cells to give rise to intestinal\ntumors (Verhagen et al., 2024 DOI: 10.1038/s41588-024-01801-y). In light of these\ndiverse roles of Paneth cells in both health and disease, it is critical to better understand\nhow their fate is extrinsically regulated.\nKempen et al. have invested considerable effort in studying the role of IL-17RA\nsignaling in Paneth cells by employing several transgenic mouse lines and ex vivo\norganoid systems, providing strong support for a role of IL-17A signaling in regulating\nPaneth cell plasticity. Their findings may also have translational relevance, as they\ncould open new avenues for the treatment of IBD or for supportive care during\nirradiation therapy. In this regard, the authors should consider performing additional\nexperiments to broaden the translational impact of their study.\nMajor Comments\nComment: As IL-17RA signaling appears to be dispensable for Paneth cell\ndedifferentiation during DSS-induced gastroenteritis (Lin et al., 2022 DOI:\n10.1016/j.immuni.2021.12.016), the mechanism described by Kempen et al. does not\nseem to be universal, but rather context-dependent and influenced by the type of injury.\nIt would therefore be valuable to examine whether Paneth cell–specific IL-17RA\nsignaling is also relevant for intestinal regeneration following chemotherapy, thereby\nexpanding the potential scope of IL-17A–based supportive care. Moreover, it would\nstrengthen the study to assess whether a similar mechanism might apply to colonic\nregeneration. Recent findings indicate that Paneth-like Deep Crypt Secretory (DCS)\ncells in the colon can revert to stem-like cells to support tissue repair during colitis (Xu\net al., 2025 DOI: 10.1038/s41418-025-01471-1). The authors could, for example,\nevaluate whether DCS proliferation is impaired in irradiated IEC-specific IL-17RA\nknockout mice (e.g. by co-staining PCNA or Ki67 with DCS markers such as Reg4, c-\nKit, or WGA), or at least determine whether colonic damage is exacerbated upon\nepithelial loss of IL-17RA or blockade of IL-17A signaling with neutralizing antibodies.\nDemonstrating that IL-17RA signaling also contributes to colonic regeneration would\nsubstantially enhance the relevance of the study with relatively limited additional effort.\nResponse: We sincerely thank Reviewer 2 for their thoughtful comment and valuable\ninsight. We agree that investigating whether IL-17RA signaling in Paneth-like Deep\nCrypt Secretory (DCS) cells of the colon may promote reversion to a stem-like state and\ncontribute to tissue repair during colitis is a compelling and important question.\nHowever, we believe this direction falls outside the scope of the current study. Our work\nis specifically focused on the small intestine and bona fide Paneth cells, whose plasticity\nand contribution to epithelial regeneration have been well characterized in prior\nliterature. Our experimental approach, conditional knockout mice, Paneth cells lineage\ntracer mice, resources, and analyses were therefore designed to address this defined\nbiological context. We fully agree that examining IL-17RA signaling in colonic DCS cells\nrepresents an intriguing and valuable avenue for future research, and it may serve as\nthe basis for a separate, dedicated study in the future. We are grateful to the reviewer\nfor highlighting this possibility.\nComment: First, and perhaps the most crucial experiment missing to convincingly\ndemonstrate that IL-17RA directly regulates Paneth cell dedifferentiation, is the analysis\nof lineage tracing in irradiated IL17RAfl/fl;Defa6-cre;mTmG mice. I am somewhat\nsurprised that the authors did not do this experiment. Although the anti-IL17A antibody\ntreatment causes a reduction of Defa6-lineage tracing it might be due to other indirect\nmechanisms.\nResponse: We agree with reviewer 2. Utilizing Il17RAfl/fl;Defa6-cre;mTmG mice to\ndemonstrate that IL-17RA directly regulates Paneth cell dedifferentiation would have\nbeen a very strong piece of data to support our story. We discussed trying to do this,\nunfortunately developing this mice line would be very challenging. Please refer to the\nfollowing reply from The Jackson Laboratory in response to our inquiry of developing\nthese mice:\n“The Il17ra and ROSA26 (insertion site of the mT/mG transgene) are tightly linked on\nmouse chromosome 6. They are only ~7 Mbp apart. If you are going to need to\ngenerate mice that are homozygous for the Il17ra<tm2.1Koll> loxp-flanked allele AND\ncarrying the Gt(ROSA)26Sor,tm4(ACTB-tdTomato,-EGFP)Luo. Transgene (as a\nheterozygote or a homozygote), you will need a meiotic recombination event to put both\nthe Il17ra<tm2.1Koll> and the Gt(ROSA)26Sor,tm4(ACTB-tdTomato,-EGFP)Luo alleles\non the same copy of mouse chromosome 6. As the two loci are so close together, the\nchances of that are low. It may be very difficult (as in needing to generate a lot of mice),\nor not possible.”\nThus, we decided to utilize anti-IL17A treatment and recombinant IL-17A with our\nPaneth cell lineage tracing mice as an alternative approach.\nComment: Likewise, irradiation-induced Paneth cell lineage tracing should be assessed\nin ADAM17fl/fl;Defa6-Cre;mTmG mice. Alternatively, Paneth cell proliferation should be\nquantified by co-staining proliferation markers (e.g., Ki67 or PCNA) with Paneth cell–\nspecific markers such as lysozyme to support the importance of ADAM17 for Paneth\ncell dedifferentiation.\nResponse: We stained and quantified Paneth cell specific proliferation in\nAdam17fl/fl;Defa6-Cre mice via co-staining PCNA and Lyz1 as recommended by the\nreviewer. We observed significant reduction of Lyz1+/PCNA+ double positive cells in\ncre+ mice as compared to cre- mice (Fig. 7E).\nComment: As the authors introduce the depletion and re-appearance of canonical ISCs\nupon irradiation (Fig. 2B), they should address whether the regeneration of canonical\nISCs is impaired in IL17RAfl/fl;Defa6-cre mice (e.g. by a simple OLFM4 staining or Lgr5\nin situ hybridization).\nResponse: We stained for OLFM4 in both naïve and irradiated Il17rafl/fl;Defa6-cre+/-\nmice. As expected, we saw reduced amounts of Olfm4+ crypts in mice 5 days post\nirradiation as compared to non-irradiated mice. Interestingly, we also observed\ndecreased amounts of Olfm4+ crypts in irradiated Il17rafl/fl;Defa6-cre+ mice as\ncompared to irradiated cre- mice. This revealed that canonical ISCs regeneration was\nindeed impaired in Il17rafl/fl;Defa6-cre+ mice as compared to cre- mice 5 days post\nirradiation (Fig. 3E).\nComment: Kempen and colleagues claim increased intestinal damage in IL17RA\nwhole-body knockout mice or in mice with IL17RA knockout in IECs. Although they\ndemonstrate increased bacterial dissemination in the spleen and liver, the only reported\nevidence of enhanced tissue damage is a minor reduction in crypt length. Why did the\nauthors not include additional parameters such as villus length, weight loss,\nquantification of viable crypts (as in Metcalfe et al., 2014 PMID: 24332836), or\nquantification of PCNA+ cells, as they performed in Fig. 3D for the Paneth cell–specific\nknockout? The same concern applies to the mice treated with IL17A antibodies (Figure\n5) and the mice overexpressing ADAM17 (Figure 6). The inclusion of some additional\nparameters would provide stronger support for their claim.\nResponse: Our initial measurements were of the crypt/villus structure. Specifically, we\nmeasure from the base of the crypt to the top of the villi. We have clarified this in our\nmain text. Furthermore, we stained and quantified PCNA+ cells all of our other\nexperiments, as suggested. We observed reduced cellular proliferation in Il17ra-/-, villin-\ncre+, and anti-IL-17A treated lineage tracer mice. Furthermore, cellular proliferation is\nincreased when Adam17 expression is increased (Figs. 1D, 1J, 5F, & 6N). Lastly, we\ndid not record weight loss in any experiments as the mice are irradiated and thus bone\nmarrow ablated and immunodeficient. Thus, handling them may lead to contamination,\nwhich we wanted to minimize.\nComment: In Figure 2C, the authors report reduced Lyz mRNA levels in the intestinal\nepithelium of IL17RA knockout mice after irradiation, which led them to further\ninvestigate IL17RA signaling in Paneth cells. However, it remains unclear whether this\nreduction is due to decreased lysozyme expression within Paneth cells or to a reduction\nin Paneth cell numbers upon loss of IL17RA. To address this, Paneth cells should be\nstained and quantified. Similarly, Paneth cell numbers and lysozyme expression should\nbe evaluated in irradiated IL17RAfl/fl;Villin-cre mice and IL17RAfl/fl;Defa6-cre mice, in\norder to determine whether a decrease in lysozyme expression can be also observed\nupon tissue or Paneth cell specific Il17RA knockout.\nResponse: As recommended, we stained for Lyz1 in tissue from irradiated Il17ra KO,\nIl17rafl/fl;villin-cre+/-, and Il17rafl/fl;Defa6-cre+/- mice. We observed decreased Lyz1+\ncells in the ileum of Il17ra KO and Il17rafl/fl;villin-cre+ mice as compared to their WT and\ncre- counterparts (Figs. 2D & E). There was no difference in Lyz1+ cells in the ileum\nbetween and Il17rafl/fl;Defa6-cre+/- mice (Fig. S1J).\nComment: Regarding the effects on lysozyme expression upon IL17RA knockout, I\nhave another critical remark: in their prior study (Lin et al., 2022 PMID: 35081371), the\nauthors reported no difference of lysozyme expression in the intestinal epithelium of 6-\nweek-old IL17RAfl/fl;Villin-cre mice, while Paneth cell number was reduced in 10-week-\nold IL17RAfl/fl;Villin-cre mice. In the current manuscript, the only information provided\nabout mouse age is found in the Methods section, where it is stated that animals were\nbetween 6 and 15 weeks old. Given the previously described age-dependent effects of\nIL17RA deletion on Paneth cells, it is essential to indicate the exact age of the mice\nused in each experiment.\nResponse: We have added the mice ages of all mice used in the figure legends.\nComment: In Figure 4, the authors dedicate an entire section to different Paneth cell\npopulations, mentioning a Defa6-low population, expressing stem cell genes. These\ncells likely represent Paneth cell progenitors, potentially more prone to dedifferentiate\nthan Defa6-high/OLFM4-low cells. However, no experiments were performed to address\nthis. For example, this could have been tested by analysing the organoid-forming\ncapacity sorted Defa6-YFP high and low or Defa6-GFP high/CD24+ and Defa6-GFP\nlow/CD24+ cells following irradiation. At minimum, the authors should discuss the\npossibility that the differentiation status of Paneth cells may influence their ability to\ndedifferentiate upon injury.\nResponse: We have discussed the possibility that the differentiation status of Paneth\ncells may influence their ability to dedifferentiate upon injury. We have added this in the\ndiscussion section (lines 439-442). Specifically, we state: “We believe these Paneth\nprogenitor cells drive Paneth cell specific regeneration post irradiation injury rather than mature\nPaneth cells. Paneth progenitor cells have a unique expression profile (high Il17ra/rc and\nOlfm4) which allows them to be primed for rapid dedifferentiation in response to injury and\nincreased IL-17A signaling.” Furthermore, we have changed our title to explicitly state that we\nbelieve Paneth progenitors drive regeneration in our study.\nComment: The authors show lineage tracing in Defa6-cre;mT/mG mice upon\nirradiation (Figure 5A) as well as organoid growth from sorted GFP+CD24+ cells from\nthese mice (Figure 5B). However, no quantification is provided for the lineage tracing\nfrequency (i.e. the number of tracings per GFP labelled crypt) between non-irradiated\nand irradiated mice, nor is there quantification of the number of GFP+ organoids per\nplated cells to indicate the efficiency of Paneth cells to dedifferentiate. In Figure 5D and\nFigure 6K, the authors do quantify single GFP+ cells and GFP+ clones in crypts and\nvilli, but it is not explained how these quantifications were performed or what the y-axis\nrepresents. This information should be clarified.\nResponse: We have quantified lineage tracing in Defa6-cre;mT/mG mice upon\nirradiation (Fig. 5A). Furthermore, we have explained how we quantified figures 5D and\n6K in the text. Specifically, we state: “We then counted the number of GFP+ cells in the\nbase of the crypt and in the villi between the two treatment groups 5 days post\nirradiation. We also counted the number of GFP+ clones, which we define as at least 4\nGFP+ cells in a row, in the villi between the two treatment groups 5 days post\nirradiation.” We also updated the y-axis titles so they are more easily understood.\nComment: In Figure 5G, the authors claim to provide evidence for increased Paneth\ncell lineage tracing in IL17A-treated organoids. I find this interpretation unconvincing:\nthe images do not show ribbons of GFP+ cells that would indicate lineage tracing but\nrather single GFP+ cells distributed in organoid buddings, consistent with the normal\nlocalization of Paneth cells. A more likely explanation is that IL17A treatment enhances\nPaneth cell differentiation, similar to what they reported previously (Lin et al., 2022\nPMID: 35081371). The authors might argue that organoid passaging results in fully\nGFP-labelled organoids; however, the images in Figures 5H and 5I are also not\nconvincing in this regard. Instead, they appear more consistent with immature spheres,\nthough this is difficult to assess because no bright-field images were provided to\nillustrate organoid morphology. Stronger evidence is needed to convincingly\ndemonstrate Paneth cell dedifferentiation ex vivo.\nResponse: We thank the reviewer for this important point. Our data show that IL-17A\nnot only increases Paneth cell numbers but also enhances their stem-like properties.\nBecause the organoids begin to die after five days in culture, we trypsinize them at that\nstage to study stem-like Paneth cells. As suggested, we have performed additional\nexperiments to further support a role for IL-17A in Paneth cell dedifferentiation.We have\nprovided cleared images and brightfield images in figure 5I which illustrate mature\norganoid morphology (Fig. 5I). Lastly, we then stained the IL-17A treated green\norganoids for MUC2 and ChGA. MUC2 marks terminally differentiated goblet cells while\nChGA marks terminally differentiated enteroendocrine cells. Since the staining revealed\nthat these green organoids were composed of both GFP+/MUC2+ double positive cells\nand GFP+/ChGA+ double positive cells, we can ascertain that these green organoids\narise from Paneth cells and are mature organoids composed of terminally differentiated\nepithelial cells (Fig. 5J).\nComment: The rationale behind the selection of genes analyzed in the context of\nPaneth cell dedifferentiation (Figure 6A) is not entirely clear. For example, why did the\nauthors examine Wnt9b and Wnt6 to assess Wnt signaling rather than canonical Wnt\ntarget genes such as Axin2, CyclinD1, c-Myc, or CD44? Similarly, why were Dll1 and\nDll4 measured instead of established Notch target genes such as HES or HEY to\nevaluate pathway activity? In addition, the authors did not include YAP target genes,\ndespite their own re-analysis of scRNA-seq data showing that YAP targets are\nupregulated in the same Paneth cell subcluster with the highest expression of ADAM17\nand Nox1 (Figure 6H). Likewise, in irradiated IL17RAfl/fl;Defa6-cre mice, the authors\nmeasured expression of Hif1α, Hk2, Slc2a1, and Pgk1, but only Hif1α is discussed in\nthe text. What was the rationale for including the other genes?\nResponse: We have included analysis of canonical Wnt target genes (Axin2, CyclinD1,\nc-Myc, and Cd44). We have also included established Notch target gene (Hes1).\nIn addition, we included YAP target genes (Yap and Hbegf). We did not observe a\ndifference between the expression of these genes in the ileum between irradiated\nIl17rafl/fl;Defa6-cre+/- mice (Fig. S4A). We have altered the text to include the rationale\nof including Hk2, Slc2a1, and Pgk1 (line 341). Specifically, we state: “Hif1 has been\nshown to promote Adam17 expression (46, 47). Thus, we explored if the expression of Hif1\nand its target genes (Hk2, Slc2a1, Pgk1) were also reduced in in the terminal ileum of irradiated\nIl17rafl/fl;Defa6-cre+ mice.”\nComment: The title of the manuscript suggests that Paneth cell dedifferentiation is\nregulated through the NOX1–ADAM17 axis, which is a strong claim given that the only\nevidence indicating an involvement of NOX1 is the upregulation of NOX1 mRNA upon\nirradiation and IL-17A treatment, and the downregulation of NOX1 mRNA in irradiated\nADAM17fl/fl; Defa6-Cre mice. At a minimum, effects on NOX1 expression should be\nconfirmed at the protein level, ideally by immunostaining in mouse tissues. Additionally,\ndemonstrating reduced ROS production upon Paneth cell–specific IL-17RA knockout\n(e.g., by ROS analysis in irradiated organoids using indicators such as CellROX or\nH2DCFDA) would further strengthen the proposed link between IL-17RA signaling and\nROS-mediated ADAM17 activation. Even then, the title might falsely indicate regulation\nby NOX1, as ROS might be increased by multiple other mechanisms upon irradiation.\nResponse: We stained and quantified NOX1 protein via immunohistochemistry in the\nilium of irradiated Adam17fl/fl;Defa6-Cre+/- and Il17rafl/fl;Defa6-cre+/-. Protein data matched\nexpression data as we saw reduced NOX1 in both Adam17fl/fl;Defa6-Cre+ and Il17rafl/fl;Defa6-\ncre+ as compared to their cre- counter parts (Figs. S4B & S7G). We also updated our title and\nremoved NOX1 from it.\nComment: Regarding the title, I have an additional remark: the authors do not provide\ndirect evidence that the observed effect is specifically mediated by immune cell–derived\nIL-17A. To be accurate, either immune cells should be removed from the title, or\nexperiments should be included to specifically address their involvement. Notably, IL-\n17A can be expressed by other cell types, such as fibroblasts, as reported by Lotti et al.,\n2013 (https://doi.org/10.1084/jem.20131195).\nResponse: We have updated our title to remove any mention of immune derived IL-\n17A. Furthermore, we specify the importance of IL-17RA signaling on Paneth\nprogenitors rather than IL-17A signaling.\nComment: As stated in the main text, ADAM17 is expressed in Paneth cells of both\nnaïve and irradiated mice (Fig. S5A). Based on the images, ADAM17 expression\nappears to be considerably lower in irradiated mice compared to non-irradiated controls.\nThe authors neither comment on this nor provide further analysis of differential ADAM17\nexpression between the two conditions. If confirmed, this would contradict the results\nshown in Figure 6H, where re-analysis of published scRNA-seq data from irradiated\nmice indicated increased ADAM17 expression. This discrepancy should be addressed\nand discussed.\nResponse: We have provided higher quality images in figure S5A.\nComment: Figure S6 is intended to show a Paneth cell–specific ADAM17 knockout;\nhowever, ADAM17 expression also appears reduced in other epithelial cells. Here either\nhigher-quality images should be provided to clarify this or it should be commented on\nwhy a Paneth cell–specific knockout might lead to a broader reduction of ADAM17\nexpression.\nResponse: We have provided higher quality images in figure S6.\nComment: Figure 6K shows a reduction of lineage tracing in NAC-treated mice. Is it\npossible to assess ADAM17 activity in these mice? This would help to strengthen the\nlink between ROS production and ADAM17 activation.\nResponse: We performed ADAM17 activity assays from the tissue of these mice. We\nobserved a trend towards a decrease of ADAM17 activity in tissue from NAC treated\nmice as compared to the H2O control cohort (Fig. S4C).\nComment: Regarding Figure 7F, Kempen et al. state that there is no difference in the\ntight junction protein ZO-1 in ADAM17fl/fl;Defa6-Cre mice and conclude that ADAM17\nlimits damage in response to irradiation by promoting cellular proliferation rather than\nmodulating gut barrier proteins. However, based on the images provided in Figure 7F, it\nappears that ZO-1 is absent in the crypts of Adam17fl/fl;Defa6-cre mice. The authors\nshould either reconsider their statement or provide additional data to support their\ninterpretation.\nResponse: We have quantified this data via mean fluorescence intensity using ImageJ.\nWe observed no differences (Fig. 7H).\nMinor Comments\nComment: In Figure 1A and F, Figure 3A, Figure 5E, Figure 6M, Figure 7A, Figure S3A\nit is not described what the arrows should indicate.\nResponse: We have updated figure legends to describe what arrows should indicate.\nComment: Y-axes in Figure 1A,, 3A, 5E, 6M, 7A, 7D should start from 0.\nResponse: We have updated figures to ensure they all start from 0.\nComment: It is not described how PCNA+ cells in Figure 3D and Figure 7D were\nquantified (cells per crypt? How many crypts per mouse?)\nResponse: We have updated the y-axis of these figures to better describe how the\nimages were quantified (PCNA+ cells/crypt structure). Furthermore, all cell counts were\ndone on at least 30 crypts per mouse. This information has been added to the statistical\nanalysis section in the methods.\nComment: Authors should describe in more detail how outliers were identified and in\nwhich experiments outliers were excluded.\nResponse: Outliers were determined by using GraphPad Prism (ROUT analysis, Q=1%). This\ninformation has been included in the statistical analysis section in the methods.\nFurthermore, figures in which outliers have been removed have been noted in figure\nlegends.\nComment: It is not always clear how many times the experiments were repeated or\nhow many samples were analyzed and quantified. For example, the authors state:\n“Figures 5A, 5B, 5G, and 5I are representative of 2–3 mice. Figures 5B, 5D, 5E,\n5G, and 5H are generated from 2–3 independent experiments, and representative\nimages are shown and quantified.” This level of reporting is insufficient for a high-quality journal\nsuch as Nature Communications. The exact number of biological and technical\nreplicates, as well as the precise number of mice analyzed in each experiment, must be\nclearly specified.\nResponse: The exact number of replicates, as well as the precise number of mice\nanalyzed in each experiment, has been clearly specified in figure legends.\nComment: The manuscript refers to Figure S6A, however, Figure S6 has no A or B.\nResponse: This discrepancy has been fixed in the manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my concerns.", "reviewer_2": "Kempen et al. have performed substantial additional work and have considerably improved their manuscript. They addressed most of my previous suggestions and concerns, and the revisions have strengthened the overall quality and interpretability of the study.\nHowever, two points still need to be clarified:\n1. The age of the mice.\nI appreciate that the authors included mouse ages in the figure legends. Unfortunately, it is not clear whether Cre+ and Cre- mice were age-matched or not. This is very critical, as previously the authors reported no difference of lysozyme expression in the intestinal epithelium of 6-week-old and a reduction of Paneth cell number in 10-week-old IL17RAfl/fl;Villin-cre mice (Lin et al., 2022 PMID: 35081371) and hence the age of the mice might substantially influence the outcome of some experiments. For example, in Figure 2E where the other show a reduction of Paneth cells, the figure legend indicates: “mice were 7.9, and 14.6 weeks old”. If these mice were not age-matched the authors cannot rule out that the mouse age influenced the outcome of the experiments. The same concern applies to Figure S1J, in which 6.7, 10 and 11.6 week old mice were used, and the number of Lyz1 positive cells in Cre+ mice show noticeable variation.\n2. Interpretion of Figure S3C:\nIn Figure S3C, the authors examine whether treatment with IL-25 or IL-17C induces the formation of green Defa6-cre;mTmG mice organoids. For IL-25 they show a reduction from 7% to 0% composition between untreated and treated organoids. For IL-17C they show a rather drastic increase from 0% to more than 20%, which is comparable to the effect of IL-17A. Despite this the authors write:”Our results showed no significant increase in green organoid development from passaged Defa6-cre;mT/mG mice post-irradiation after IL-25 or IL-17C treatment (Fig. S3C)”. Which seems to me like a misinterpretation of the experiments. Additionally, the figure legend states: “There were 3 mice in each treatment group in figures S3C”. Yet, only one data point per group is shown in the graph. Can the authors elaborate on this discrepancy?\nMinor point:\nIn Figure 6A, the authors show differences in secreted factors regulating Wnt and Notch ligands. They then state:\n“Interestingly, Ly6a but not Ly6d, Anxa1, Anxa3, Anxa5, Yap, and Hbegf expression was increased in the small intestine of Cre+ mice as compared to Cre– mice (Fig. 6A & S4A).”\nA) The authors should include the Wnt targets in the text.\nB) They should also mention that the increased ligand expression does not appear to translate into a noticeable effect on downstream targets.\nAdditional typos:\n• Line 1133: Should this be “3 experiments”?\n• Figure S3A: “Defe6-cre” should be corrected to “Defa6-cre.”"}, "author_response": "Dear Reviewers,\nWe would like to thank the reviewers for their consideration of our manuscript and for\nproviding insightful comments. In addition, we appreciate the opportunity to revise and\nresubmit this manuscript. Below, we provide each of the reviewers’ comments followed\nby a detailed point-by-point response.\nReviewer 1:\nAll concerns have been addressed.\nReviewer 2:\nSummary and Significance: Kempen et al. have performed substantial additional work\nand have considerably improved their manuscript. They addressed most of my previous\nsuggestions and concerns, and the revisions have strengthened the overall quality and\ninterpretability of the study.\nHowever, two points still need to be clarified:\nMajor Comments\nComment: I appreciate that the authors included mouse ages in the figure legends. This\nis very critical, as previously the authors reported no difference of lysozyme expression\nin the intestinal epithelium of 6-week-old and a reduction of Paneth cell number in 10-\nweek-old IL17RAfl/fl;Villin-cre mice (Lin et al., 2022 PMID: 35081371) and hence the\nage of the mice might substantially influence the outcome of some experiments. For\nexample, in Figure 2E where the other show a reduction of Paneth cells, the figure\nlegend indicates: “mice were 7.9, and 14.6 weeks old”. If these mice were not age-\nmatched the authors cannot rule out that the mouse age influenced the outcome of the\nexperiments. The same concern applies to Figure S1J, in which 6.7, 10 and 11.6 week\nold mice were used, and the number of Lyz1 positive cells in Cre+ mice show noticeable\nvariation.\nResponse: We would like to clarify this important point. We have used multiple litters\nfor each experiment, but in each litter we have cre- and cre+ mice which are age\nmatched. We have clarified this in the methods section as well and stated: “Littermate\ncontrols were used, thus cre- and cre+ mice were age matched.” (Lines 519-520)\nComment: In Figure S3C, the authors examine whether treatment with IL-25 or IL-17C\ninduces the formation of green Defa6-cre;mTmG mice organoids. For IL-25 they show a\nreduction from 7% to 0% composition between untreated and treated organoids. For IL-\n17C they show a rather drastic increase from 0% to more than 20%, which is\ncomparable to the effect of IL-17A. Despite this the authors write: ”Our results showed\nno significant increase in green organoid development from passaged Defa6-\ncre;mT/mG mice post-irradiation after IL-25 or IL-17C treatment (Fig. S3C)”. Which\nseems to me like a misinterpretation of the experiments. Additionally, the figure legend\nstates: “There were 3 mice in each treatment group in figures S3C”. Yet, only one data\npoint per group is shown in the graph. Can the authors elaborate on this discrepancy?\nResponse: In figure S3C we use 3 mice. Only one data point is visible because the\nother two mice had 0% composition of green organoids with and without IL-17C\ntreatment, thus the data points are superimposed on each other at 0. We have provided\nall raw results as supplementary information in accordance with Nature\nCommunications reporting guidelines, and thus the numerical values for figure S3C can\nbe found there.\nMinor Comments\nComment: In Figure 6A, the authors show differences in secreted factors regulating\nWnt and Notch ligands. They then state: “Interestingly, Ly6a but not Ly6d, Anxa1,\nAnxa3, Anxa5, Yap, and Hbegf expression was increased in the small intestine of Cre+\nmice as compared to Cre– mice (Fig. 6A & S4A).”\nResponse: We state in the prior sentence before the one the reviewer has highlighted\nthe following: “We showed decreased expression of Adam17, Dll1, and Wnt9b in\nIl17rafl/fl;Defa6-cre+ mice after radiation injury compared to cre- mice.” (Lines 327-328).\nWe have also further clarified that the increase in Ly6a does not seem to result in down\nstream effect as suggested. We specifically state: “The increased ligand expression of\nLy6a does not appear to result in noticeable down stream effects.” (Lines 332-333).\nAll typos have been fixed."}]

Decision letter