Homeostatic activation of aryl hydrocarbon receptor by dietary ligands dampens cutaneous allergic responses by controlling Langerhans cells migration

  1. Adeline Cros
  2. Alba De Juan
  3. Renaud Leclère
  4. Julio L Sampaio
  5. Mabel San Roman
  6. Mathieu Maurin
  7. Sandrine Heurtebise-Chrétien
  8. Elodie Segura

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Abstract

Dietary compounds can affect the development of inflammatory responses at distant sites. However, the mechanisms involved remain incompletely understood. Here, we addressed the influence on allergic responses of dietary agonists of aryl hydrocarbon receptor (AhR). In cutaneous papain-induced allergy, we found that lack of dietary AhR ligands exacerbates allergic responses. This phenomenon was tissue-specific as airway allergy was unaffected by the diet. In addition, lack of dietary AhR ligands worsened asthma-like allergy in a model of ‘atopic march.’ Mice deprived of dietary AhR ligands displayed impaired Langerhans cell migration, leading to exaggerated T cell responses. Mechanistically, dietary AhR ligands regulated the inflammatory profile of epidermal cells, without affecting barrier function. In particular, we evidenced TGF-β hyperproduction in the skin of mice deprived of dietary AhR ligands, explaining Langerhans cell retention. Our work identifies an essential role for homeostatic activation of AhR by dietary ligands in the dampening of cutaneous allergic responses and uncovers the importance of the gut–skin axis in the development of allergic diseases.

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  1. Version published to 10.7554/elife.86413 on eLife
  2. Review Commons

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    Reply to the reviewers

    1. General Statements

    Imbalance in gut-derived AhR ligands has been shown to be involved in inflammatory bowel disease and in neuro-inflammation. The aim of our study was to address the role of dietary AhR ligands in a context that had not been previously explored. We decided to focus on allergy because AhR has broad functions in barrier tissues homeostasis, which is directly relevant to allergy.

    2. Description of the planned revisions

    Additional experiments in response to Reviewer 2

    "The authors make a strong claim that the epidermal barrier function is not affected by AhR poor diet conditions (claim made in abstract and last paragraph of the discussion). This should be experimentally validated."

    We already performed footpad histology and observed that the stratum corneum is not affected by the diet (fig1A and figS1E). We will provide a quantitative analysis by measuring stratum corneum thickness on the images, and add this data to figure 1. To strengthen this point, we will also perform ultra-structural analysis of the epidermis in the two diet groups using electron microscopy of the skin. This will provide a deeper characterization of the epidermal structure, including cornified layers and intercellular tight junctions.

    "Injection into the footpad as a route of administration is also physiologically distinct from epicutaneous sensitization given the natural barriers are artificially breached via needle puncture. Did the authors consider epicutaneous sensitization via the skin without additional barrier disruption? Does this yield the same response?"

    We will perform skin sensitization without barrier disruption by applying papain (or vehicle) on shaved flank skin. To minimize skin abrasion, mice will be shaved the day before the application. We will analyze dendritic cells migration to the draining lymph nodes after 48h by flow cytometry, and helper T cell responses in the draining lymph nodes after 6 days by measuring cytokine secretion.

    Text edits

    Comments from Reviewer 1

    • We will add appropriate references in response to comments from reviewer 1: " in several places they cited review articles instead of original articles for key findings. Ex. For the expression of Mucin 5 and CLCA1 a review is cited." and " the role of AHR in ILC2 (PMID: 30446384) and alveolar epithelial cells (PMID: 35935956) has been documented. The authors should add these references."
    • We will modify the figures legends according to reviewer 1's suggestions: " Although the authors mentioned treatment schedule and stimulants used in the method, a short description in the figure legend will be helpful for the readers".
    • We will address other comments from reviewer 1 by modifying the text where appropriate:

    "1. In the introduction section, the authors should explain adequately why they thought that AHR signaling is important for allergy.

    1. Since IL-5, IL-13 production by skin draining lymph nodes and pulmonary lymph nodes was different, is this difference due to difference in AHR expression?
    2. In Fig.3, the authors showed that intra-nasal stimulation does not induce eosinophil migration or IL-5, IL-13 induction in I3C diet group. These data and the data shown in figure-2 are in contrast. The authors should discuss this discrepancy."

    Comments from Reviewer 2

    • "How to explain the difference between IL4 (no effect between the two diets/or absence/presence LCs in Fig. 4D) and IL5/IL13 (small effect in Fig 1D and 4D). "

    This is an interesting point. It has been shown that IL4 is produced in lymph nodes by T cells distinct from those producing IL5 and IL13 (https://doi.org/10.1038/ni.2182). In addition, IL4 expression is regulated at the transcriptional level by distinct mechanisms from IL5 and IL13 expression (https://doi.org/10.1016/S1074-7613(00)80073-4, https://doi.org/10.1038/ni.1966).
    We speculate that IL4-producing T cells are not affected by Langerhans cells presence in the lymph nodes. We will add a point in the discussion section to discuss this.

    • We will tune down our conclusion regarding the different effects of diet-derived and microbiota-derived AhR ligands according to the comments of the reviewer: "This part seemed far-fetched. There are many more differences between germ free and specific pathogen free mice than only the presence/absence of AhR ligands. Hence, it seemed like a very big step to compare both conditions and draw the conclusion that microbiota-derived AhR ligands activate different sets of genes. It would also make more sense if Fig. 5 would be immediately followed by Fig. 7". We also propose to move Fig6 to the supplementary data.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    4. Description of analyses that authors prefer not to carry out

    Comments from Reviewer 1

    "In Fig.4, the authors show there is no difference in total number, but difference in migration, was there a difference in expression of migratory markers?"

    We assume the reviewer is referring to the number of Langerhans cells in the epidermis in steady-state, which is not different between diets (fig4A). We actually already show in supplementary figure S3E classical cell surface markers that are upregulated upon dendritic cells migration (MHC class II and CD40). We found no difference in the expression of these markers between diet groups.

    Comments from Reviewer 2

    "Fig. 1D Cytokine production
    In AhR poor diet the spread between the individual data points is much larger and the difference between presence/absence of dietary ligands in IL5 and IL13 seems to be based merely on a few outliers (which especially in the case of IL13 appear to be completely out of range). Most other datapoints do not seem to be highly different from the ones in the AhR rich diet.
    Where does this high variation come from in AhR poor diet (and what is the reason for these high outliers)? Would the data have been significantly different without the outliers? "

    Throughout the manuscript, we have represented raw data and individual data points for transparency. We observed some variability between biological replicates, not just for cytokine secretion (fig1D) but in most assays (for instance cell counts in lymph nodes in fig1C or inflammatory cell counts in fig2A and fig3A or antibody production in fig2E), yet the reviewer focuses their comments on fig1D. In the case of fig1D, we have performed Kruskal-Wallis statistical tests to account for this variation, and the difference between diet groups was statistically significant. We do not understand how we could remove the so-called ‘outliers’ without data manipulation to perform an alternative statistical test. We also disagree with the reviewer that 4 out of 11 points can be considered ‘outliers’.

    "In general, increases of all canonical T-helper cytokine responses (except for IL4) can be noted in the LN and the difference in IL10, IL17 or IFNg production between AhR poor and rich diet appears even more pronounced than the difference in IL5/IL13 (Fig. S1F). Still the authors decide to focus the entire story on the allergic response after stating that a 'lack of dietary AhR ligands amplifies allergic responses'. Why was this choice made?"

    Imbalance in gut-derived AhR ligands has been shown to be involved in inflammatory bowel disease and in neuro-inflammation. The aim of the project was to address the role of dietary AhR ligands in a context that had not been previously explored. We decided to focus on allergy because AhR has broad functions in barrier tissues homeostasis, which is directly relevant to allergy. We will better explain this point in the introduction. In the course of the study, we analyzed IL10, IL17 and IFNg production by lymph node T cells to get a complete view of helper responses, and we provided this data in supplementary information for transparency. We believe this information might be useful for other groups studying other types of skin inflammation.

    "Would the authors expect other inflammatory models via the skin (e.g. bacterial, viral infection) to confer better/worse outcomes under an AhR poor diet?"

    This is an interesting question. Unfortunately, we do not have the means to analyze bacterial or viral skin infections for lack of adequate facilities (i.e. BSL2 animal facility) or ethics approval for this kind of experiments. We believe that our work may prompt in the future other groups to analyze the impact of dietary AhR ligands in other inflammatory skin contexts.

    "At a mechanistic level, how do LC suppress the activation of T cells in the LN, and how would this impact secretion of certain cytokines but not others?"

    "it remains a bit speculative how migration of LCs to the dLNs of the skin contributes to suppressing Th2 immunity in the airways. Several hypotheses have been put forward in the discussion. What is their thought about this and how to validate experimentally?"

    This is an important question. A regulatory role for Langerhans cells has been evidenced by other studies, but the molecular mechanisms involved remain elusive. This point is discussed in the discussion part of the manuscript. We believe that deciphering the mechanism of action of Langerhans cells is beyond the scope of the present study (and is unrelated to the direct effect of the diet), and would represent an entire project in itself.

    “Fig. 3 - Why would the alteration of diet pose a confounding factor to the model? Did the authors determine that such diet-associated changes are only important at the sensitization phase? The footpad (Fig. 1) and pulmonary (Fig 2) models were performed with the altered diets throughout the entire length of the experiment. If anything, wouldn't changing the diet after sensitization also provide an additional variable here? Is it known what happens (e.g. inflammatory state, genetic changes) when a normal diet is resumed after a period of adaptation? This reviewer does not understand the reason for all-of-a-sudden changing the diet after the sensitization phase.”

    Our goal with this experiment was to address the effect of the dietary AhR ligands during the skin sensitization phase only. This is why diets are different only in this phase of the protocol. We want to emphasize that the IC3 diet and the AhR-poor diet only differ in the presence of one molecule, which is I3C. The composition of the food is otherwise exactly the same, therefore we do not believe that a change between AhR-poor and I3C would represent a confounding factor. This is different to the adaptation period when we place the mice on I3C or AhR-poor diets instead of normal chow diet (which has a completely different formulation). We will make this point clearer in the text.

    "Fig. 7 Role of TGFb
    At first site, it seems counterintuitive that TGFb, which is a molecule generally associated with homeostasis and dampening of inflammation, is associated here with more profound inflammation. How to reconcile? At this point the data on TGFb are merely correlative. Did the authors directly test the contribution of TGFb to LC migration? In addition, did they check whether they could restore defects in LC migration in absence of AhR ligands by blocking the formation of active TGFb? "

    We agree with the reviewer that the role of TGFb seems counter-intuitive. However, multiple studies have shown that TGFb produced by keratinocytes retains Langerhans cells in the epidermis, using a variety of experimental approaches including genetic tools (https://doi.org/10.1073/pnas.1119178109, https://doi.org/10.1038/ni.3396,

    https://doi.org/10.4049/jimmunol.1000981, https://doi.org/10.1016/j.xjidi.2021.100028). We do not have any reason to doubt the validity of these studies. Therefore, we believe that demonstrating again the role of TGFb in Langerhans cells migration is not necessary.

    Using blocking antibodies against TGFb or its receptor, as suggested by the reviewer, would most probably not allow us to address whether it restores the defect in Langerhans cells migration. Indeed, results from the literature (cited above) indicate that such blocking would increase Langerhans cells migration in both diet groups, therefore it will most likely be impossible to conclude.

    In addition, we have provided several lines of evidence that AhR activation regulates the expression of Integrin-beta8 in keratinocytes and the release of bioactive TGFb, at transcriptomic and protein levels, in both mouse and human keratinocytes (fig7). Therefore, we believe that additional experiments to support the link between AhR ligands and TGFb are not necessary within the scope of the revision.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    In this paper Cros et al describe how the absence of dietary ligands of AhR exacerbate cutaneous papain-induced allergy. This was only observed when papain was applied topically, but not intranasally. However, lack of dietary AhR ligands also worsened allergic airway inflammation after cutaneous sensitization. At a mechanistic level, the authors found that the absence of dietary AhR ligands hampered migration of Langerhans cells (LC) to the lymph nodes, where they are claimed to be needed to suppress T cell activation. Complementary models that lead to loss of LCs gave a similar phenotype. The authors performed RNA-sequencing on epidermal cells derived from mice that were either fed an AhR ligand rich or poor diet to define differences in transcriptome signature. They uncovered increased expression of the integrin Itgb8 in absence of AhR ligands, which is needed for production of active TGF, a factor known from literature to contribute to LC retention in the skin.

    In general, the study is well done, and the different experimental conditions are well controlled for. The experiments are built up in a logical fashion, and most of the times, the interpretation is appropriate (except for a few claims, see further). The paper reads very fluently, and the key points are interesting.

    Major comments:

    • Fig. 1D Cytokine production
    • In AhR poor diet the spread between the individual data points is much larger and the difference between presence/absence of dietary ligands in IL5 and IL13 seems to be based merely on a few outliers (which especially in the case of IL13 appear to be completely out of range). Most other datapoints do not seem to be highly different from the ones in the AhR rich diet.
      Where does this high variation come from in AhR poor diet (and what is the reason for these high outliers)? Would the data have been significantly different without the outliers?
      How to explain the difference between IL4 (no effect between the two diets/or absence/presence LCs in Fig. 4D) and IL5/IL13 (small effect in Fig 1D and 4D).
    • In general, increases of all canonical T-helper cytokine responses (except for IL4) can be noted in the LN and the difference in IL10, IL17 or IFNg production between AhR poor and rich diet appears even more pronounced than the difference in IL5/IL13 (Fig. S1F). Still the authors decide to focus the entire story on the allergic response after stating that a 'lack of dietary AhR ligands amplifies allergic responses'. Why was this choice made?
      Would the authors expect other inflammatory models via the skin (e.g. bacterial, viral infection) to confer better/worse outcomes under an AhR poor diet?
      At a mechanistic level, how do LC suppress the activation of T cells in the LN, and how would this impact secretion of certain cytokines but not others?

    Fig. 3 - Why would the alteration of diet pose a confounding factor to the model? Did the authors determine that such diet-associated changes are only important at the sensitization phase? The footpad (Fig. 1) and pulmonary (Fig 2) models were performed with the altered diets throughout the entire length of the experiment. If anything, wouldn't changing the diet after sensitization also provide an additional variable here? Is it known what happens (e.g. inflammatory state, genetic changes) when a normal diet is resumed after a period of adaptation? This reviewer does not understand the reason for all-of-a-sudden changing the diet after the sensitization phase.

    • Fig. 6: Microbiota-derived and diet-derived AhR ligands modulate different sets of epidermal genes.
      This part seemed far-fetched. There are many more differences between germ free and specific pathogen free mice than only the presence/absence of AhR ligands. Hence, it seemed like a very big step to compare both conditions and draw the conclusion that microbiota-derived AhR ligands activate different sets of genes.
      It would also make more sense if Fig. 5 would be immediately followed by Fig. 7
    • The authors make a strong claim that the epidermal barrier function is not affected by AhR poor diet conditions (claim made in abstract and last paragraph of the discussion). This should be experimentally validated. Injection into the footpad as a route of administration is also physiologically distinct from epicutaneous sensitization given the natural barriers are artificially breached via needle puncture. Did the authors consider epicutaneous sensitization via the skin without additional barrier disruption? Does this yield the same response?

    Fig. 7 Role of TGFb

    • At first site, it seems counterintuitive that TGFb, which is a molecule generally associated with homeostasis and dampening of inflammation, is associated here with more profound inflammation. How to reconcile? At this point the data on TGFb are merely correlative. Did the authors directly test the contribution of TGFb to LC migration? In addition, did they check whether they could restore defects in LC migration in absence of AhR ligands by blocking the formation of active TGFb?

    Finally, also other steps of the proposed model by the authors are based on literature rather than direct experiments. In this regard, it remains a bit speculative how migration of LCs to the dLNs of the skin contributes to suppressing Th2 immunity in the airways. Several hypotheses have been put forward in the discussion. What is their thought about this and how to validate experimentally?

    Significance

    The major strength of the paper (and the most interesting finding) is the explanation of why the effect of the diet is only detectable after cutaneous but not intranasal sensitisation and the causal link to the LCs present in the skin.

    The major limitations of the paper is that many parts of the proposed model are not experimentally validated but based on literature suggestions (eg the claim that TGFb would prevent LC migration to LN, that LC would suppress T cell responses in the LN, that the suppression of T cell cytokines (with very limited effects on IL5 and IL13 but no effect on IL4) would be sufficient to explain improved allergy symptoms in the lung...). It is also unclear why the authors studied allergic symptoms while effects on other T cell cytokines appeared more prominent. There are a few questions on the change in model from figure 1-2 to figure 3.

    The key findings are interesting and the paper is nice to read.
    The findings will be interesting to specialised audience: LC biology, allergy and Th2 immunity people
    Own research field, dendritic cell biology and papain-induced models of allergy

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript is well written. The authors mostly cited appropriate papers but in several places they cited review articles instead of original articles for key findings. Ex. For the expression of Mucin 5 and CLCA1 a review is cited.

    General comments

    1. the role of AHR in ILC2 (PMID: 30446384) and alveolar epithelial cells (PMID: 35935956) has been documented. The authors should add these references.
    2. Although the authors mentioned treatment schedule and stimulants used in the method, a short description in the figure legend will be helpful for the readers.

    Specific comments

    1. In the introduction section, the authors should explain adequately why they thought that AHR signaling is important for allergy.
    2. Since IL-5, IL-13 production by skin draining lymph nodes and pulmonary lymph nodes was different, is this difference due to difference in AHR expression?
    3. In Fig.3, the authors showed that intra-nasal stimulation does not induce eosinophil migration or IL-5, IL-13 induction in I3C diet group. These data and the data shown in figure-2 are in contrast. The authors should discuss this discrepancy.
    4. In Fig.4, the authors show there is no difference in total number, but difference in migration, was there a difference in expression of migratory markers?

    Minor points

    1. TGF-β, TCR-β and cytokine names should be written consistently across the manuscript.
    2. The authors should use "β" instead of beta

    Significance

    The work is significant and will impact the field

  5. Version published to 10.1101/2023.01.24.525336v1 on bioRxiv