Dietary L-Tryptophan consumption determines the number of colonic regulatory T cells and susceptibility to colitis via GPR15

Environmental factors are the major contributor to the onset of immunological disorders such as ulcerative colitis. However, their identities remain unclear. Here, we discover that the amount of consumed L-Tryptophan (L-Trp), a ubiquitous dietary component, determines the transcription level of the colonic T cell homing receptor, GPR15, hence affecting the number of colonic FOXP3+ regulatory T (Treg) cells and local immune homeostasis. Ingested L-Trp is converted by host IDO1/2 enzymes, but not by gut microbiota, to compounds that induce GPR15 transcription preferentially in Treg cells via the aryl hydrocarbon receptor. Consequently, two weeks of dietary L-Trp supplementation nearly double the colonic GPR15+ Treg cells via GPR15-mediated homing and substantially reduce the future risk of colitis. In addition, humans consume 3–4 times less L-Trp per kilogram of body weight and have fewer colonic GPR15+ Treg cells than mice. Thus, we uncover a microbiota-independent mechanism linking dietary L-Trp and colonic Treg cells, that may have therapeutic potential.

gut microbiota, it would be good to confirm this finding in germ-free mice.Moreover, what is the impact of L-Trp supplementation on the gut microbiota composition?7. The identification of the AhR ligands involved in GPR15 induction is very interesting.
While the impact of L-Trp seems independent of the gut microbiota according to Fig 2, microbes derived I3-PYA, I3S and pyocyanin induce GPR15+ Treg in vitro.Can the processing of L-Trp by the gut microbiota, during L-Trp supplementation, lead to the generation of these ligands?Are they naturally found in lower proportion in the colon of germ-free mice?
The difficulty to identify the natural ligand for AhR in naïve T cells is understandable, is it possible to stimulate naïve T cells with L-trp and run a metabolomic analysis to identify which AhR ligands are generated?10.The therapeutic potential of L-Trp is discussed yet only a prophylactic approach was used in the study.What is the impact of L-trp supplementation when it is initiated the day after the C rodentium infection.Regardless of the results the findings will be very interesting.
11.The benefits of L-Trp supplementation in colitis are clear but it would be valuable to discuss the potential side effects of increasing colonic Treg particularly in the context of colorectal cancer.

Reviewer #2 (Remarks to the Author):
In this manuscript, Van and colleagues investigated the effect of L-Tryptophan on GPR15+Treg generation in vivo via AhR signaling.The authors mainly used AhR conditional KO mice and L-Tryptophan-supplemented/reduced diet to evaluate how GPR15+Treg cells developed.This is an interesting paper, which may contribute to better understanding of the function of AhR to control Treg generation and intestinal disorders such as ulcerative colitis associated with AhR deficiency.However, it has several essential problems which need to be addressed to improve the quality of the report.

Comments:
1. Regarding the AhR CD4-cKO condition, Yoshimatsu et al., Cell Reports 2022 (Fig. 4) showed basically no significant changes in the frequency of total Treg in colonic LP.In this manuscript by Van et al, however, it seems that there was some change in total colonic Treg cells especially in Fig. 5.The authors need to show not only GPR15+Foxp3+ or GPR15−Foxp3+ classification but also the frequency of colonic Treg cells throughout the manuscript.3.In the colitis model (especially in Fig. 6), the authors used infection-induced colitis with C.Rodentium.Is it suitable as an ulcerative colitis model? 4. In Fig. 1a (and other figures), the authors indicate some change in the frequency of GPR15+Foxp3− fracfion (Tconv cells) which was reduced in AhR cKO condifion.In the whole manuscript, the author didn't analyze GPR15+ Tconv cells.Were they Th1, Th2, or Th17, for example?Do they produce Th specific cytokines ? 5.In Fig. 4, FICZ and BaP differentially functioned as T cell polarizers; i.e., FICZ actively induced GPR15+Tconv cells while BaP induced GPR15+Treg cells.Could FICZ production occur in a specific condition in the presence of some microbiota and induce GPR15+Tconv cells in vivo?Also, does p.o. administration of BaP work as well as i.p. administration they performed?6.In Fig. 6d, the authors estimated the amount of L-tryptophan consumption in mice and humans and concluded that less GPR15+Treg cells in humans could be due to the difference of L-tryptophan consumption.However, just one representative data of FACS plot of GPR15 and FOXP3 expression is shown.How many samples were analyzed?This Fig. 6d can be moved to Supplemental data.7.In Fig. 1e, the authors show that the L-Tryptophan-reduced food reduced GPR15+Treg cells in the spleen as well.This needs to be discussed.
8. In Fig. 3, the authors show that GPR15+Foxp3− fracfion (Tconv) decreased in the GF condition whereas GPR15+Treg cells had no change.This means that GPR15+Treg cells developed in a microbiota-independent manner, or, conversely GPR15+Tconv cells development is microbiota-dependent.This needs to be discussed.9.In Fig. 5b,  One main concern I have is the choice of the model to test the ability of L-Trp to ameliorate colitis -the Citro rodentium model.This is a model of EPEC/EHEC (authors refer to this as enterotoxogenic?), but is not a good model for chronic intestinal inflammation that mimics human IBD (CD/UC).The data obtained using this model are only somewhat convincing.
Several of the readout show no and minor difference between WT and GPR15 KO mice suggesting that GPR15+ endogenous Tregs are not playing a robust role in Citro infection.
Overall, this is a poor model to test the hypothesis that L-Trp supplementation can ameliorate colitis.Other colitis models should be considered and explored.Similarly, only a prevention model was tested and a treatment model should be explored in order to demonstrate whether L-Trp supplementation can prevent and/or treat chronic colitis.
Jun 28 th , 2023 Dear Reviewers: On behalf of my colleagues, I would like to thank you all for your incredibly helpful suggestions to strengthen our findings.The following is a point-by-point response to your comments on our manuscript: "Dietary L-tryptophan consumption determines the number of colonic regulatory T cells and susceptibility to colitis via GPR15."All changes to the manuscript were highlighted in yellow, either in the text or in the figure legends.
8. L-Trp decreases the Th1 response, what about Th17?This is particularly relevant as Th17 is induced by C rodentium.Showing the impact of Trp suppl on Th1 and Th17 in the colon of control vs infected mice would be a nice addition to the manuscript.9.It is unclear how Fig 9b was generated.What is the n number of mice and humans used to compare their proportion of colonic GPR15+ Treg?How was the resulting plot done?

2 .
Regarding AhR function for Treg cells, Treg specific AhR KO by utilizing the Foxp3-Cre system needs to be analyzed.The authors' experiments do not exclude the possibility of GPR15+Tconv conversion into GPR15+Treg cells in colonic LP, as a recent report has shown that some Th17 cells, called Treg-lineage committed Tconv cells, bear the potential to differentiate into Foxp3+Treg cells in a specific condition (van der Veeken et al., Immunity 2022).
3'distal Gpr15 locus contains not only AHR-binding but also AP-1-binding domains.Is AP-1 binding needed to express GPR15 in T cells including Treg and others?Reviewer #3 (Remarks to the Author): In the manuscript entitled Dietary L-Tryptophan consumption determines the number of colonic regulatory T-cells and susceptibility to colitis via GPR15 by Van et al, the author investigated the role of L-Trp on colonic Foxp3+ Treg expression of GPR15.Several key findings are presented: • The amount of dietary L-Trp correlated with colonic Foxp3+ Tregs numbers via the AhR-GPR15 axis • L-Trp increased GPR15 during CD4+ T cell activation/migration to the LI via GPR15 • The ability of L-Trp to increase GPR15+Foxp3+ Tregs was dependent upon IDO1/2 but not microbiota • AhR ligands have differential ability to induce Teff vs Tregs and AhR directly regulates GPR15 • L-Trp supplementation reduced Citro rodentium colitis While it is published that AhR regulates GPR15 expression and colonic Foxp3+ Tregs, it has been unclear which natural ligands may be regulating this pathway.The present study provides compelling evidence that L-Trp supplementation can regulate Foxp3+ Treg numbers in the colonic LP.The use of Ahr-CD4-CKO, Gpr15(gfp/gfp)Foxp3mrfp, Cd4creAhrfl/fl, HH7-2TgRag1(n/n) and Ido1/2 DKO all provided supporting in vivo evidence for the role of the L-Trp pathway through IDO1/2 to induce GPR15.The experiments are all very thorough and nicely presented.