Enteric glia regulate Paneth cell secretion and intestinal microbial ecology

Reviewer #1 (Public Review):

The role of enteric glial cells in regulating intestinal mucosal functions at a steady state has been a matter of debate in recent years. Enteric glial cell heterogeneity and related methodological differences likely underlie the contrasting findings obtained by different laboratories. Here, Prochera and colleagues used Plp1-CreERT2 driver mice to deplete the majority of enteric glia from the gut. They found that glial loss has very limited effects on the transcriptome of gut cells 11 days after tamoxifen treatment (used to induce DTA expression), and by extension - more specifically, has only minimal impact on cells of the intestinal mucosa. Interestingly, in the colon (where Paneth cells are not present) they did observe transcriptomic changes related to Paneth cell biology. Although no overt gene expression alterations were found in the small intestine - also not in Paneth cells - morphological, ultrastructural, and functional changes were detected in the Paneth cells of enteric glia-depleted mice. In addition, and possibly related to Paneth cell dysfunction, enteric glia-depleted mice also show alterations in intestinal microbiota composition.

In their analyses of enteric glia from existing single-cell transcriptomic data sets, it is stated that these come from 'non-diseased' humans. However, the data on the small intestine is obtained from children with functional gastrointestinal disorders (Zheng 2023). Data on colonic enteric glia was obtained from colorectal cancer patients (Lee 2020). Although here the cells were isolated from non-malignant regions, saying that the large intestines of these patients are non-diseased is probably an overstatement. Another existing dataset including human mucosal enteric glia of healthy subjects is presented in Smillie et al (2019). It would be interesting to see how the current findings relate to the data from Smillie et al.

The time between enteric glia depletion and analyses (mouse sacrifice) must be a crucial determinant of the type of effects, and the timing thereof. In the current study 11 days after tamoxifen treatment was chosen as the time point for analyses, which is consistent with earlier work by the lab using the same model (Rao et al 2017). What would happen when they wait longer than 11 days after tamoxifen treatment? Data, not necessarily for all parameters, on later time points would strengthen the manuscript significantly.

The authors found transcriptional dysregulation related to Paneth cell biology in the colon, where Paneth cells are normally not present. Given the bulk RNA sequencing approach, the cellular identity in which this shift is taking place cannot be determined. However, it would be useful if the authors could speculate on which colonic cell type they reckon this is happening in. On the other hand, enteric glia depletion was found to affect Paneth cells structurally and functionally in the small intestine, where transcriptional changes were initially not identified. Only when performing GSEA with the in silico help of cell type-specific gene profiles, differences in Paneth cell transcriptional programs in the small intestine were uncovered. A comment on this discrepancy would be helpful, especially for the non-bioinformatician readers among us.

From looking at Figure 3B it is clear that Paneth cells are not the only epithelial cell type affected (after less stringent in silico analyses) by enteric glial cell depletion. Although the authors show that this does not translate into ultrastructural or numerical changes of most of these cell types, this makes one wonder how specific the enteric glia - Paneth cell link is. Besides possible indirect crosstalk (via neurons), it is not clear if enteric glia more closely associate with Paneth cells as compared to these other cell types. Immunofluorescence stainings of some of these cells in the Plp1-GFP mice would be informative here. The authors mention IL-22 as a possible link, but do Paneth cells express receptors for transmitters commonly released by enteric glia? Maybe they can have a look at putative cell-cell interactions by mapping ligand-receptor pairs in the scRNAseq datasets they used.

Previously the authors showed that enteric glia regulation of intestinal motility is sex-dependent (Rao et al 2017). While enteric glia depletion caused dysmotility in female mice, it did not affect motility in males. For this reason, most experiments in the current study were conducted in male mice only. However, for the experiments focusing on the effect of enteric glia depletion on host-microbiome interactions and intestinal microbiota composition both male and female mice were used. In Figure 8A male and female mice are distinctly depicted but this was not done for Figure 8C. Separate characterization of the microbiome of male and female mice would have helped to figure out how much intestinal dysmotility (in females) contributes to the effect on gut microbial composition. This is an important exercise to confirm that the effect on the microbiome is indeed a consequence of altered Paneth cell function, as suggested by the authors (in the results and discussion, and in the abstract). In this context, it would also be interesting to compare the bulk sequencing data after enteric glia depletion between female and male mice.

Reviewer #2 (Public Review):

This is an excellent and timely study from the Rao lab investigating the interactions of enteric glia with the intestinal epithelium. Two early studies in the late 1990s and early 2000s had previously suggested that enteric glia play a pivotal role in control of the intestinal epithelial barrier, as their ablation using mouse models resulted in severe and fatal intestinal inflammation. However, it was later identified that these inflammatory effects could have been an indirect product of the transgenic mouse models used, rather than due to the depletion of enteric glia. In previous studies from this lab, the authors had identified expression of PLP1 in enteric glia, and its use in CRE driver lines to label and ablate enteric glia.

In the current paper, the authors carefully examine the role of enteric glia by first identifying that PLP1-creERT2 is the most useful driver to direct enteric glial ablation, in terms of the number of glial cells targeted, their proximity to the intestinal epithelium, and the relevance for human studies (GFAP expression is rather limited in human samples in comparison). They examined gene expression changes in different regions of the intestine using bulk RNA-seq following ablation of enteric glia by driving expression of diphtheria toxin A (PLP1-creERT2;Rosa26-DTA). Alterations in gene expression were observed in different regions of the gut, with specific effects in different regions. Interestingly, while there were gene expression changes in the epithelium, there were limited changes to the proportions of different epithelial cell types identified using immunohistochemistry in control vs glial-ablated mice. The authors then focused on the investigation of Paneth cells in the ileum, identifying changes in the ultrastructural morphology and lysozyme activity. In addition, they identified alterations in gut microbiome diversity. As Paneth cells secrete antimicrobial peptides, the authors conclude that the changes in gut microbiome are due to enteric glia-mediated impacts on Paneth cell activity.

Overall, the study is excellent and delves into the different possible mechanisms of action, including the investigation of changes in enteric cholinergic neurons innervating the intestinal crypts. The use of different CRE drivers to target enteric glial cells has led to varying results in the past, and the authors should be commended on how they address this in the Discussion.

Reviewer #3 (Public Review):

In this study, Prochera, et al. identify PLP1+ cells as the glia that most closely interact with the gut epithelium and show that genetic depletion of these PLP1+ glia in mice does not have major effects on the intestinal transcriptome or the cellular composition of the epithelium. Enteric glial loss, however, causes dysregulation of Paneth cell gene expression that is associated with morphological disruption of Paneth cells, diminished lysozyme secretion, and altered gut microbial composition. Overall, the authors need to first prove whether the Plp1CreER Rosa26DTA/+ mice system is viable. Also, most experimental systems have been evaluated by immunohistochemistry, scRNAseq, and electron microscopy, but need quantitative statistical processing. In addition, the value of the paper would be enhanced if the significance of why the phenotype appeared in the large intestine rather than the small intestine when PLP1 is deficient for Paneth cells is clarified.

Weaknesses:

Major:

(1) Supplementary Figure 2; Cannot be evaluated without quantification.

(2) Figure 2A; Is Plp1CreER Rosa26DTA/+ mice system established correctly? S100B immunohistology picture is not clear. A similar study is needed for female Plp1CreER Rosa26DTA/+ mice. What is the justification for setting 5 dpt, 11 dpt? Any consideration of changes to organs other than the intestine? Wouldn't it be clearer to introduce Organoid technology?

1. Figure 2B; Need an explanation for the 5 genes that were altered in the colon. Five genes should be evaluated by RT-qPCR. Why was there a lack of change in the duodenum and ileum?

(4) Supplementary Figure 3; Top 3 genes should be evaluated by RT-qPCR.

(5) Supplementary Figure 4B, C, and D; Why not show analysis in the small intestine?

(6) Supplementary Figure 4D; Cannot be evaluated without quantification.

(7) Figure 3D; Cannot be evaluated without quantification.

(8) Supplementary Figure 5B and C; Top 3 genes should be evaluated by RT-qPCR.

(9) Supplementary Figure 6; Top 3 genes should be evaluated by RT-qPCR.

(10) Figure 4A; Cannot be evaluated without quantification.

(11) Figure 4D; Cannot be evaluated without quantification.

(12) Additional experiments on in vivo infection systems comparing Plp1CreER Rosa26DTA/+ mice and controls would be great.

Author response:

We thank the reviewers for their thoughtful consideration of our study and are delighted they found the findings to be important. In this initial response to the overall positive reviews, we want to address common themes raised, clarify points relevant to a few specific reviewer concerns, and frame plans for the revised manuscript.

(1) Analysis of data from human tissue: Reviewer 1 notes “In their analyses of enteric glia from existing single-cell transcriptomic data sets, it is stated that these come from 'non-diseased' humans. However, the data on the small intestine is obtained from children with functional gastrointestinal disorders (Zheng 2023). Data on colonic enteric glia was obtained from colorectal cancer patients (Lee 2020). Although here the cells were isolated from non-malignant regions, saying that the large intestines of these patients are non-diseased is probably an overstatement.

In the Zheng et al. dataset, “functional GI disorders” refers to biopsies from children that do not have any histopathologic evidence of digestive disease. The children do, however, have at least one GI symptom that prompted a diagnostic endoscopy with biopsies, leading to the designation of “functional” disorder. Given that diagnostic endoscopies are invasive procedures that necessitate anesthesia, obtaining biopsies from completely healthy, asymptomatic children without any clinical indication would not be allowable per most institutional review boards, leading the authors of that study to use these samples as a control group. We thus used the “non-diseased” label to encompass these samples as well as those from the unaffected regions of large intestine from colorectal cancer patients. We recognize, however, that this label might be misleading and will revise the manuscript to more accurately reflect the information on control tissue origin.

Another existing dataset including human mucosal enteric glia of healthy subjects is presented in Smillie et al (2019). It would be interesting to see how the current findings relate to the data from Smillie et al.”

We thank the reviewer for directing us to the Smillie et al. 2019 dataset. This dataset derives from colonic mucosal biopsies from 12 healthy adults (8480 stromal cells) and 18 adults with ulcerative colitis (10,245 stromal cells from inflamed bowel segments and 13,146 from uninflamed), all between the ages of 20-77 years. Our preliminary analysis shows that the putative glial cluster in this dataset does not separate by inflammation or disease state based on the common glial genes: S100B, PLP1, and SOX10. PLP1 and S100B are broadly expressed across this cluster while GFAP is not detected in this dataset, consistent with our observations from the two other human datasets included in our manuscript. In the revised manuscript, we will include the Smillie et al. 2019 data in a supplemental figure as additional supportive evidence.

(2) Validation and further details of the Plp1CreER-DTA model for genetic depletion of enteric glia: Reviewer 1 notes “The time between enteric glia depletion and analyses (mouse sacrifice) must be a crucial determinant of the type of effects, and the timing thereof. In the current study 11 days after tamoxifen treatment was chosen as the time point for analyses, which is consistent with earlier work by the lab using the same model (Rao et al 2017). What would happen when they wait longer than 11 days after tamoxifen treatment?” Reviewer 3 asks whether “the Plp1CreER Rosa26DTA/+ mice system established correctly” and raises concern about quantitative characterization.

In previous work, we discovered that the gene Plp1 is broadly expressed by enteric glia and, within the mouse intestine, is quite specific to glial cells (PMID: 26119414). We characterized the Plp1CreER mouse line as a genetic tool in detail in this initial study. Then in a subsequent study, we used Plp1CreER-DTA mice to genetically deplete enteric glia and study the consequences on epithelial barrier integrity, crypt cell proliferation, enteric neuronal health and gastrointestinal motility (PMID: 28711628). In this second study, we performed extensive validation of the Plp1CreER-DTA mouse model including detailed quantification of glial depletion in the small and large intestines across the myenteric, intramuscular and mucosa compartments by immunohistochemical (IHC) staining of whole tissue segments to sample thousands of cells. We found that the majority of S100B+ enteric glia were depleted within 5 days in both sexes, including more than 88% loss of mucosal glia, and that this loss was stable at 3 subsequent timepoints (7, 9 and 14 days post-tamoxifen induction of Cre activity). Glial loss was further confirmed by IHC for GFAP in the myenteric plexus, and by ultrastructural analysis of the small intestine to ensure cell depletion rather than simply loss of marker expression. Our group was the first to use this model to study enteric glia, and since then similar models and our key observations have been replicated by other groups (PMID: 33282743, 34550727). Thus, we consider this model to be well established.

Reviewer 1 raises an excellent question about examining epithelial health beyond 11 days post-tamoxifen (11dpt) in this model. Particularly given the longer-lived nature of Paneth cells relative to other epithelial cell types, this would be very interesting to explore. Through 11dpt, Cre+ mice are well-appearing and indistinguishable from their Cre-negative control littermates. Unfortunately, a limitation of the Plp1CreER-DTA model is that beyond 11dpt, Cre+ mice become anorexic, lose body weight, and have signs of neurologic debility such as hindlimb weakness and uncoordinated gait that are prominent by 14dpt. These phenotypes are likely the consequence of targeting Plp1+ glia outside the gut, such as Schwann cells and oligodendrocytes (as described in another study which used a similar model to study demyelination in the central nervous system, PMID: 20851998). Given these CNS effects and that starvation is well known to affect Paneth cell phenotypes (PMIDs: 1167179, 21986443), we elected not to examine timepoints beyond 11dpt. Technological advances that enable more selective cell depletion would allow study of more chronic effects of enteric glial loss.

(3) Sex differences in the microbiome data: All 3 reviewers queried whether there were sex differences in the microbiome data with Reviewer 1 explaining “Previously the authors showed that enteric glia regulation of intestinal motility is sex-dependent (Rao et al 2017). While enteric glia depletion caused dysmotility in female mice, it did not affect motility in males. For this reason, most experiments in the current study were conducted in male mice only. However, for the experiments focusing on the effect of enteric glia depletion on host-microbiome interactions and intestinal microbiota composition both male and female mice were used. In Figure 8A male and female mice are distinctly depicted but this was not done for Figure 8C. Separate characterization of the microbiome of male and female mice would have helped to figure out how much intestinal dysmotility (in females) contributes to the effect on gut microbial composition. This is an important exercise to confirm that the effect on the microbiome is indeed a consequence of altered Paneth cell function…”

In our microbiome analysis, we initially analyzed males and females separately but did not observe significant differences between the two sexes. Thus, we merged the data to increase the statistical power of the genotype comparisons. It was an oversight on our part to not label the female and male datapoints in Figure 8C as we did for the other data in the manuscript. We will update this graph and related supplemental figures in the revised version. Per Reviewer 2’s suggestion, we will also address this further in the Results and Discussion.

(4) Reconciling RNA-Seq identification of transcriptional changes in the colon, but not the small intestine, while the GSEA and downstream tissue level morphological and functional analyses detected phenotypes in the small intestine. Reviewers 1 and 3 raised this question with Reviewer 1 noting “…enteric glia depletion was found to affect Paneth cells structurally and functionally in the small intestine, where transcriptional changes were initially not identified. Only when performing GSEA with the in silico help of cell type-specific gene profiles, differences in Paneth cell transcriptional programs in the small intestine were uncovered. A comment on this discrepancy would be helpful, especially for the non-bioinformatician readers among us.”

Standard differential gene expression analysis (DEG) of the effects of glial loss revealed significant differences only in the colon, and even there only a handful of genes were changed. These changes were not accompanied by corresponding changes at the protein level, at least as detectable by IHC. In the small intestine, there were no significant differences by standard DEG thresholds. Unlike DEG, gene set enrichment analyses (GSEA), provides a significance value based on whether there is a higher than chance number of genes that are changing in a uniform direction without consideration for the significance of the magnitude of change. Therefore, the GSEA detected that a significant number of genes in the curated Paneth cell gene list exhibited a positive fold change difference in the bulk RNA sequencing data. This prompted us to examine Paneth cells and other epithelial cell types in more detail by IHC, functional and ultrastructural analyses, which all converged on the observation that Paneth cells were relatively selectively disrupted in the epithelium of glial depleted mice.

(5) Other: We will address all remaining comments in our detailed author response that will accompany our revised manuscript. We thank Reviewer 2 for the very positive feedback overall and highlighting opportunities to better label findings in some of the figures. We will make these suggested changes in our revised manuscript.