Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

STAT5 controls the intestinal epithelial stem cell niche by regulating Wnt/β-catenin signaling, and lack of active pYSTAT5 induces Clostridium difficile infection. STAT5 may be a transcription factor that restricts niche cell differentiation.

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Full guidelines are available on our Instructions for Authors page, http://life-sciencealliance.org/authorguide We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Reviewer #1 (Comments to the Authors (Required)): This is a very interesting paper investigating the role of STAT5 in IESC homeostasis and the response to C. difficile infection. The authors have generated a series of transgenic mice whose utilization in quite elegant tracing experiments, together with ex vivo experiments in organoids, definitively established the role of STAT5 loss of function or gain of function in the control of IESC lineage specification as well as its role under conditions of colitis induced by C. difficile. Importantly they establish that the loss of STAT5 results in Paneth cell dysfunction, which makes these mice more vulnerable to the pathological effects of C. difficile. They also do the inverse set of experiments using a permanently active mutant of STAT5 that led to very interesting and consistent results. These are well performed studies that are of potential great significance and of relative high novelty.
The last part of the paper is a little bit weaker. Thus, although some experiments were performed to try to explain mechanistically how STAT5 works at a more molecular level, these more mechanistic studies lack the depth and detail that would make of this a really highly impactful paper. In any case, as it is now, the results presented, although somehow correlative at the molecular level, are of great interest and need to be reported because the mouse in vivo studies are solid and of interest to the signaling and intestinal research community.
The paper is in need of some English editing to improve the flow and make it easier to follow. There are also some panel labels that seem to have been switched that need to be fixed and in general the presentation of the figures and the text could use some more careful editing.
Reviewer #2 (Comments to the Authors (Required)): The manuscript by Liu et al. focuses on an important issue for C.difficile infection and tissue regeneration: Paneth cell maturation. Overall I find the manuscript suitable for the journal, however there are some points that need to be changed and/or answered.
Major points: -In the discussion the authors claim "Our data indicated that IESC differentiation to Paneth cell niche requires a persistent pYSTAT5 activation" yet in Figure 2a .the epithelial knockout still shows lysozyme positive Paneth cells.
-Is pYSTAT5 downregulated in chronic colitis models? -Paneth cell quantification in figure 3 would be clearer if repeated with another methodology (flow cytometry, western blot) -It would be interesting to see if the different STAT5 mouse models presented have a difference in budding formation capacity, something considered as a good output for regenerative capacity of an organoidin homeostasis and inflammation.
Minor points: -How does STAT5 activate Wnt? -The term Paneth-like cell was first coined in this manuscript " Schewe et al. 2016 Cell Stem Cell" as such this reference should be added.

March 23, 2019
We are appreciative of the insightful comments of Editorial Board Members and the Reviewers. We have uploaded the revised manuscript and provide you with a point-by-point response where we have addressed each of the major and minor concerns. Changes in the text body have been underlined for ease of review. We believe that with additional new data focused on comments we received, the revised manuscript is substantially improved. We hope that our revised manuscript and responses to critiques can be further considered for a publication by the reviewers and the Editorial Board of Life Science Alliance. A point-by-point response is included below.
Reviewer #1 (Comments to the Authors (Required)): 1. This is a very interesting paper investigating the role of STAT5 in IESC homeostasis and the response to C. difficile infection. The authors have generated a series of transgenic mice whose utilization in quite elegant tracing experiments, together with ex vivo experiments in organoids, definitively established the role of STAT5 loss of function or gain of function in the control of IESC lineage specification as well as its role under conditions of colitis induced by C. difficile. Importantly they establish that the loss of STAT5 results in Paneth cell dysfunction, which makes these mice more vulnerable to the pathological effects of C. difficile. They also do the inverse set of experiments using a permanently active mutant of STAT5 that led to very interesting and consistent results. These are well performed studies that are of potential great significance and of relative high novelty.
Author response: Thank so much for an overall positive comment on our research significance. I have revised our Summary Blurb as "Constitutively active pYSTAT5 restricts intestinal stem cells to give rise to pYSTAT5 + niche cells by regulating Wnt/β-Catenin. Lack of pYSTAT5 decreases Paneth cells to exaggerate C. difficile colitis", where we reiterate our finding. We conclude that STAT5 is an essential protein to specify niche cell differentiation.
2. The last part of the paper is a little bit weaker. Thus, although some experiments were performed to try to explain mechanistically how STAT5 works at a more molecular level, these more mechanistic studies lack the depth and detail that would make of this a really highly impactful paper. In any case, as it is now, the results presented, although somehow correlative at the molecular level, are of great interest and need to be reported because the mouse in vivo studies are solid and of interest to the signaling and intestinal research community.
Author response: Very appreciate your critiques. Our on-going study is focused on "Utilize ChIP-seq assay to determine the target genes by which Ca-pYSTAT5 differentially regulates secretory cell lineage commitment in intestines or colon", part of which will involve a tetrameric STAT5 augments EZH2 activity, subsequently stimulating epigenetic markers to induce or repress lineage-specific genes. We have included the limitation of our current research, and future research targets in the discussion in line 13 on Page 23.
Author response: 3. The paper is in need of some English editing to improve the flow and make it easier to follow. There are also some panel labels that seem to have been switched that need to be fixed and in general the presentation of the figures and the text could use some more careful editing.

The manuscript by Liu et al. focuses on an important issue for C. difficile infection and tissue regeneration: Paneth cell maturation.
Overall, I find the manuscript suitable for the journal, however there are some points that need to be changed and/or answered.
Author response: We appreciate all comments from reviewer 2. We respond to them with new data point by point. The revised parts are underlined.

Major points:
1. In the discussion the authors claim "Our data indicated that IESC differentiation to Paneth cell niche requires a persistent pYSTAT5 activation" yet in Figure 2A. the epithelial knockout still shows lysozyme positive Paneth cells.

Author response:
We reported that STAT5 acts as an intrinsic factor to regulate IESC proliferation (Gilbert et al, 2015). In our revised paper, we supplement a 14 day-lineage tracing data using Lgr5-LacZ;Stat5 mice and a single dose Tam-induction (Supplementary Fig S4A). Our new data show that loss of STAT5 in Lgr5 + IESCs leads to absence or significantly reduced LacZ staining in the jejunal, ileal and colonic crypt cells, indicating that STAT5 signaling regulates IESC stemness as an intrinsic signaling (Supplementary Fig  S4C-D). The mouse model with a constitutively active pYSTAT5 (Ca-pYSTAT5) showed an increased numbers of Paneth cells at the crypt bases and villi ( Figure 2E). Thus, STAT5 may regulate Paneth cells through specifying Paneth cell fate or controlling Paneth cell maturation. Based on the lineage tracing data from STAT5 loss function in mice, we focused on if Ca-pYSTAT5 can directly induce IESC differentiation to increase the number of Paneth cells. Our results showed that Ca-pYSTAT5 increased IESC lineage tracing at the crypt bases in the Lgr5-Rs26tdtomato;icS5 mice ( Figure 3B, and Supplementary Fig S3B and C) and led to de novo Paneth cell differentiation in the HIO xenotransplantation (Fig 6H). Together, these data indicate that Ca-pYSTAT5 in IESCs increase Paneth cell differentiation. Therefore, our experiment provides a new transcription factor that could restrict Lgr5 IESC differentiation to Paneth cells. Considering that our result displayed the possible binary effects of pYSTAT5 on Paneth cell maturation and the differentiation of Lgr5 low Ki67 + into pYSTAT5 + Lgr5 -CD24 + Lyso + niche cells, we toned done our explanation of results and revised it into: "a persistent pYSTAT5 activation can restrict IESC differentiation to Paneth cell niche", in line 2 on page 25. We will cross a Defensin 6a promoter-driven Cre transgenic mouse line and icS5 floxed mice to test the Paneth cell phenotypes affected by Ca-pYSTAT5 in the future. We have included it in the discussion in line 19 on page 25.
Using two markers of mature Paneth cells (Lyso and CD24), we observed that Stat5-deficient mice exhibited a significant reduction of Lyso + Paneth cell numbers at the crypt bottom shown in Figure 2A, which is consistent with a reduced level of Lyso and Defensin expression at the Stat5-deficient crypts shown in Figure 2B and a diminished number of CD24 + crypt Paneth cells in the Stat5-deficient mice determined by FACS analysis in Figure 4A. Knock-out of STAT5 reduced the number of Paneth cells, but neither Lyso + nor CD24 + Paneth cells are completely disappearing. STAT5 signaling could cooperate with other pathways to control Paneth cell differentiation. These data suggest that compensatory pathways independent of STAT5 also regulate IESCs towards Paneth cell differentiation. Thus, depletion of STAT5 might may lead to alternative mechanisms or other pathway activation or inhibition, such as interferon (Farin et al, 2014), MAPK (Heuberger et al, 2014) or Notch signaling (Rothenberg et al, 2012), which were reported to change Paneth cell differentiation or function. We have included some discussion in line 8 on page 7 To address these potential mechanisms will require more work beyond the scope of this manuscript, we will testify them in the future.

Is pYSTAT5 downregulated in chronic colitis models?
Author response: As the reviewer suggested, we have provided a set of new data showing colonic mucosal pYSTAT5 during chronic DSS colitis. We created a chronic DSS colitis model by orally giving WT mice with three cycles of 7-day 3% DSS treatment with an interval of 5-day water recovery between each cycle of DSS (Supplementary Fig S1C) (Wirtz et al, 2017). We found an increased pYSTAT5 immune-staining in the nascent crypts pointed by the arrow heads, demonstrating the importance of pYSTAT5 in the colonic IEC regeneration during chronic colitis. We have included these data in the Supplementary Fig S1C and result description from line 14 to 20 on Page 8. Supplementary Fig S4 A. Lgr5CreER mice were crossed with Rs26LacZ mice to generate a mouse line (Lgr5-LacZ), then crossed with Stat5 floxed mice (Lgr5-LacZ;Stat5). Lgr5-LacZ;Stat5 mice were generated to study the effect of Stat5 on IESC self-renewal upon a single dose of Tam. B, C. Jejunal and ileal lineage tracing was analyzed 14 days after a single dose of Tam (25 mg/kg), n = 3 per group. D. Whole colon fragments were isolated from Lgr5-LacZ and Lgr5-LacZ;Stat5 mice and then stained with Whole-Mount X-Gal Staining. These stained colonic fragments were sectioned and counter-stained with HE. The LacZ + colonic crypts were counted, n = 3 mice per group.

Paneth cell quantification in figure 3 would be clearer if repeated with another methodology (flow cytometry, western blot).
Author response: Given that the amount of protein from a single organoid is too low to be detected by immunoblotting, and the immunostaining is not quantitated, we chose the real-time PCR and found an upregulated Lyz1 expression (Upper right panel of Figure 3E), which is consistent with the result showed in Figure 1B. In addition, to demonstrate the upregulated Lyz1 through increased Paneth cell differentiation, we measured Sox9 expression with real-time PCR. It shows the upregulated Sox9 expression in the same icS5-induced enteroids, which is consistent with increase Sox9 protein staining in the supplementary Fig S4E. These new data are respectively included in Figure 3E and Supplementary Fig S4F and in line 4 page 12 in the revised manuscript.
4. It would be interesting to see if the different STAT5 mouse models presented have a difference in budding formation capacity, something considered as a good output for regenerative capacity of an organoid in homeostasis and inflammation. Supplementary Fig S1C. STAT5 +/+ mice were orally given with three cycles of 7-day 3% DSS treatment with an interval of 5-day water recovery between each cycle of DSS. Mucosal histology was evaluated as a total score including intestinal epithelial damage (0 -3), ulceration (0 -3) and transmural lesion (0 -3). Colonic tissues were isolated and then immune-stained with pYSTAT5 pointed by arrow heads. Representative images are shown. Results are expressed as mean ± SEM, n = 4 to 5 mice per group. Author response: According to Dr. Fodde group study (Schewe et al, 2016), we have supplemented the data showing organoid forming capacity in the presence or absence of TNF-α by counting final organoid survival and initial grown organoids to calculate the percentage of regenerated organoids. Considering the length of Figure 5A, we supplement a graph of organoid forming capacity as a supplementary Fig S6A  and B to show the organoid forming capacity in the STAT5 IEC+/-, STAT5 +/+ and STAT5 IEC+++ mice. We also move the number of Org 1~3 and the size of spheroid of the controls from the original Supplementary Figure 6A to Figure 3A, which shows enteroid multiplicity at homeostasis and during inflammation. Accordingly, these data showed that LOF of STAT5 reduced the organoid forming capacity, characterized by the reduced budding numbers and enlarged spheroid size (supplementary Fig S6B) and reduced percentage of survival organoid numbers (supplementary Fig S6B), the phenotypes of which were significantly exaggerated by the presence of TNF-α. These data confirm our previous reports and extend the role of STAT5 in IESCs during inflammation, illuminating STAT5 engagement in a 3D in vitro primary organoid system with different genetic mutation. We have included these results from line 3 to 8 and line 11 on Page 16.

How does STAT5 activate Wnt?
Author response: In the Figure 7C and Supplementary Fig S7C, we showed that STAT5 activation (pYSTAT5) resulted in an increased nuclear -Catenin in the IESCs, demonstrating that pYSTAT5 amplified Wnt/β-catenin signaling to enhance IESC self-renewal and expand the IESC compartment ( Figure 2E). We also found a higher level of Sox9 expression and increased Paneth cell numbers found in the enteroids with pYSTAT5 ( Figure 7D and Supplementary Fig S4F). These data suggest the pYSTAT5-induced higher level of Wnt promotes the Lgr5 + IESCs towards Paneth cell differentiation. Using TOPflash Wnt reporter-transduced iPSCs or colon cancer cell lines that have a constitutive Wnt activation (Fuerer & Nusse, 2010), we will address in the next step if STAT5 can bind β-Catenin/Tcf4 gene locus to directly activate Wnt signaling. We have included this design in the future study and stated it in the discussion from line 13 to 20 on page 23.
Interestingly, we found that a persistent pYSTAT5 (Ca-pYSTAT5) induced by icS5-ER led a lower level of nuclear -Catenin while higher level of Lyso in the iPSC-derived HIOs compare to their expression in the transient pYSTAT5 induced by STAT5A-ER (Supplementary Fig S7C). Since Ca-pYSTAT5 has chromatin remodeling power and computation analysis shows there are many TCCN(2~5)GAA GAS motifs on the β-Catenin (Wingelhofer et al, 2018), we postulate that Ca-pYSTAT5 epigenetically controls Wnt target gene expression. For instance, Ca-pYSTAT5 may induce a formation of STAT5 tetramers on β-Catenin gene, which may lead to chromatin remodeling and epigenetically induce the repression of Wnt target genes controlling stemness while the activation of genes inducing Paneth cell differentiation. We will use CHIP-seq, ATAC-seq and RNA-seq analyses to determine the possible mechanisms by which tetrameric STAT5 stimulates epigenetic markers to induce or repress lineage-specific genes. However, it is beyond the scope of this manuscript. We have included it in the future direction from line 13 to 20 on page 23.