Homeostatic activation of aryl hydrocarbon receptor by dietary ligands dampens cutaneous allergic responses by controlling Langerhans cells migration
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.
This important study uncovers the role of Aryl Hydrocarbon Receptor (AhR) in tempering allergic responses. The authors present compelling data supporting the function of AhR ligands in limiting cutaneous allergic type 2 responses but not airway allergic responses, underscoring an interesting tissue-specific role of this axis. The work will be of broad interest to immunologists, including those with a special interest in mechanisms of regulation of allergy.https://doi.org/10.7554/eLife.86413.sa0
Development of immune-mediated diseases is affected by numerous environmental factors, including nutrition. Dietary compounds can modulate immune cells homeostasis and inflammatory immune responses (Wu et al., 2018), affecting in particular the susceptibility to allergy (Julia et al., 2015). However, the impact of individual nutrients and the molecular mechanisms involved remain incompletely understood. In particular, whether dietary metabolites that activate the aryl hydrocarbon receptor (AhR) influence type 2 allergic responses remains unclear.
Type 2 allergic responses result from dysregulated immune responses mediated mainly by IL4, IL5, and IL13. In the skin and lungs, allergens trigger the secretion by keratinocytes or epithelial cells of alarmins, such as thymic stromal lymphopoietin (TSLP) (Deckers et al., 2017a; Akdis et al., 2020). These soluble mediators stimulate the production of IL4, IL5, and IL13 by type 2 innate-like lymphoid cells (ILCs) and basophils, and the activation of dendritic cells (DCs) (Deckers et al., 2017a; Akdis et al., 2020). After their migration to the lymph nodes, activated DCs polarize CD4 T cells into Th2 cells that themselves secrete IL4, IL5, and IL13, thereby stimulating the production of IgE and amplifying the type 2 inflammatory response (Deckers et al., 2017a; Akdis et al., 2020). Nutritional compounds can affect multiple players of the type 2 inflammatory cascade through recognition by G-protein-coupled and nuclear receptors. For instance, mice fed on a high-fiber diet have increased levels of short-chain fatty acid propionate and decreased susceptibility to airway allergy due to impaired induction of Th2 polarization by lung DC (Trompette et al., 2014). Mice with a vitamin D-deficient diet display lung inflammation, high blood IgE levels, and increased numbers of lung Th2 cells (Vasiliou et al., 2014). Dietary interventions are therefore a promising strategy for preventing the development of allergic diseases, in particular in children (Trambusti et al., 2020; Trikamjee et al., 2020). However, a better understanding of the effects of each family of dietary compounds is needed.
AhR is a nuclear receptor-sensing metabolite produced mainly by the breakdown of food components or from tryptophan catabolism by intestinal microbiota (Rothhammer and Quintana, 2019; De Juan and Segura, 2021). Imbalance in gut-derived AhR ligands worsens inflammation in the intestine during inflammatory bowel disease (Monteleone et al., 2011; Lamas et al., 2016; Hubbard et al., 2017) and in the central nervous system during neuroinflammation (Rothhammer et al., 2018; Rothhammer et al., 2016). Whether dietary AhR ligands also play a role in other pathological contexts requires further investigation. In particular, AhR exerts broad functions in barrier tissues including skin and lung (Esser and Rannug, 2015), where AhR activation has been reported to limit inflammation (Di Meglio et al., 2014; Beamer and Shepherd, 2013). The impact of dietary AhR ligands in allergic responses at such barrier sites remains unknown.
In this study, we explored the role of dietary AhR ligands in the development of type 2 allergic responses using papain as a model allergen. We showed that lack of dietary AhR ligands exacerbates cutaneous, but not airway, papain-induced allergy. In addition, we found that lack of dietary AhR ligands during allergen cutaneous sensitization worsened asthma-like airway allergy in a recall phase, a model for ‘atopic march.’ We demonstrated that lack of dietary AhR ligands impacts the inflammatory profile of epidermal cells and increases the production of bioactive TGF-β, causing the retention of Langerhans cells in the skin, in turn leading to exaggerated Th2 responses in the lymph nodes. Our results identify a major role for dietary AhR ligands in the modulation of cutaneous allergic responses.
Lack of dietary AhR ligands exacerbates cutaneous allergic type 2 responses
To study the impact of dietary AhR agonists on allergic responses, we sought to compare mice fed with diets either poor or rich in AhR agonists. Normal mouse chow contains phytochemicals that can act as precursors of AhR agonists, in particular indole-3-carbinole (I3C) (Bjeldanes et al., 1991), which is detected in the serum of mice fed on normal chow diet (Figure 1—figure supplement 1A). To avoid confounding effects, we used a standard purified diet (AIN-93M) that is naturally poor in phytochemicals (hereafter termed ‘AhR-poor diet’) and the same diet enriched for I3C (hereafter termed ‘I3C diet’). As a proof of principle, we verified that I3C concentration in the serum of mice fed on the AhR-poor or I3C diet was significantly different (Figure 1—figure supplement 1A). To confirm that these diets induced different levels of AhR activation in vivo, we analyzed the expression in liver cells of the canonical AhR target gene Cyp1a1. Mice fed on the normal chow and I3C diets showed similar expression of Cyp1a1, while mice fed on the AhR-poor diet had almost undetectable expression of Cyp1a1 (Figure 1—figure supplement 1B), validating that the AhR-poor diet contains negligeable amounts of natural AhR agonists. To study type 2 allergic responses, we chose the model protease allergen papain (Shimura et al., 2016; Kamijo et al., 2013; Sokol et al., 2008). To validate that the I3C diet would mimic normal conditions in this model, we analyzed Th2 cells induction in mice fed on normal chow or I3C diet. After footpad injection of papain, we observed similar Th2 induction in the draining lymph nodes in both groups (Figure 1—figure supplement 1C). IL4 and IL13 were increased by papain exposure, while IL5 secretion was not significantly different from the control condition. Finally, to confirm that only AhR agonists from the diet were modulated in this setting, we measured the serum concentration of L-kynurenin (produced by host metabolism), 3,3'-diindolylmethane (DIM, generated by the degradation of I3C in the stomach), and indole-3-acetic acid (produced by microbiota metabolism from food components) (De Juan and Segura, 2021; Bjeldanes et al., 1991). DIM, but not L-kynurenin or indole-3-acetic acid, was decreased in the serum of mice fed on the AhR-poor diet (Figure 1—figure supplement 1D). Collectively, these results validate our experimental set up. For the rest of the study, we compared mice fed on the I3C and AhR-poor diets.
We first analyzed cutaneous allergic responses to papain challenge in the footpad. Histological analysis showed epidermal hyperplasia upon papain exposure only in the foot of mice fed on the AhR-poor diet (Figure 1A and B and Figure 1—figure supplement 1E). To assess the induction of Th2 responses, we analyzed draining lymph nodes after 6 d. The number of CD4 T cells and B cells was similar in mice fed on the IC3 or AhR-poor diets (Figure 1C), suggesting that the diet had no impact on CD4 T cells or B cells proliferation. We restimulated normalized numbers of lymph nodes T cells ex vivo and measured cytokine secretion. While IL4 secretion was induced similarly in both groups of mice, the production by T cells of Th2 cytokines IL5 and IL13 was exacerbated in mice fed on the AhR-poor diet (Figure 1D). To confirm these results in a setting of epicutaneous sensitization, we applied papain topically on the skin, without any abrasion, and restimulated T cells from the draining lymph node after 6 d (Figure 1E). Similarly, we found that mice fed on the AhR-poor diet showed higher secretion of IL5 and IL13 upon papain exposure. The secretion of IL10, IFN-γ, and IL17A upon papain challenge was also increased in mice fed on the AhR-poor diet in the footpad setting (Figure 1—figure supplement 1F), but not with topical application (Figure 1—figure supplement 1G). We concluded that lack of dietary AhR ligands amplifies allergic responses after cutaneous allergen exposure.
Lack of dietary AhR ligands does not impact airway allergic type 2 inflammation
To address whether this phenomenon occurred with other exposure routes, we used a model of asthma-like airway allergy induced by repetitive intranasal exposure to papain. Papain challenge induced airway inflammation with infiltration of eosinophils, monocytes, and CD4 T cells as assessed in the bronchoalveolar space (Figure 2A and Figure 2—figure supplement 1). There was no difference in cell numbers between mice fed on the I3C or AhR-poor diets (Figure 2A). We could also detect IL13 and IL5 in the bronchoalveolar lavage, but cytokine secretion was not increased by the AhR-poor diet compared to I3C diet (Figure 2B). The production of Th2 cytokines by T cells from the pulmonary lymph nodes was also similar between diets (Figure 2C). We also assessed airway hyperreactivity by measuring in lung tissues the expression of the genes coding for mucus protein Mucin 5a (Muc5ac) (Young et al., 2007) and CLCA1 (Gob5), a molecule produced by goblet cells during hyperplasia (Leverkoehne and Gruber, 2002; Nakanishi et al., 2001). Papain challenge increased the expression of Muc5ac and Gob5 in the lungs, to a similar extent in mice fed on I3C or AhR-poor diets (Figure 2D). Finally, we measured the concentration of IgE in the blood and found comparable levels in both groups of papain-exposed mice (Figure 2E). Collectively, these results indicate that the lack of dietary AhR ligands does not affect airway type 2 allergic responses.
Lack of dietary AhR ligands worsens airway allergy after skin sensitization
Cutaneous allergen exposure can lead to airway allergy to the same allergen via the induction of Th2 memory CD4 T cells by skin DC (Deckers et al., 2017a), a phenomenon referred to as ‘atopic march’ (Bantz et al., 2014). Therefore, we addressed whether dietary AhR ligands impact asthma-like airway allergy after skin sensitization. To this aim, we fed mice on the I3C or AhR-poor diet only during the sensitization phase and placed all experimental groups on I3C diet 7 d after cutaneous exposure to papain or vehicle via footpad injection. Then, all groups were exposed to papain intranasally. While the number of alveolar macrophages was unaffected by the diet, lack of dietary AhR ligands during the sensitization phase increased the infiltration in the bronchoalveolar space of eosinophils, monocytes, and CD4 T cells (Figure 3A). IL13 secretion was higher in the bronchoalveolar lavage of mice fed on the AhR-poor diet during the sensitization phase, and IL5 concentration showed an increased tendency that was not statistically significant (Figure 3B). Finally, the expression of Muc5ac and of Gob5 in lung tissues was significantly increased in mice deprived of dietary AhR ligands during the sensitization phase (Figure 3C). These results show that the lack of dietary AhR ligands during cutaneous allergen exposure worsens subsequent airway allergy.
Lack of dietary AhR ligands impairs Langerhans cells migration that increases Th2 responses in the lymph nodes
The contrasting results obtained in cutaneous versus airway papain-induced allergy suggest tissue-specific effects. Because of their central role in Th2 cells induction, we analyzed DC populations. One difference between skin and lung tissues is the presence of Langerhans cells, residing in the epidermis and absent from the lungs. AhR is expressed by Langerhans cells and has been proposed to regulate their numbers in the epidermis (Hong et al., 2020). To address whether lack of dietary AhR ligands affects Langerhans cells maintenance in the epidermis, we quantified epidermal Langerhans cells from mice fed on the I3C or AhR-poor diet. We first imaged the epidermis of mice expressing green fluorescent protein (GFP) under the promoter of Cd207/Langerin gene (Kissenpfennig et al., 2005; Figure 4A). We found no significant difference between diets in Langerhans cells density. To confirm this observation using another approach, we analyzed footpad epidermal cells by flow cytometry (Figure 4—figure supplement 1A and B) and found no significant difference between diets in Langerhans cells numbers. We then analyzed skin DC migration to the skin-draining lymph nodes. We assessed DC numbers 24 hr and 48 hr after papain footpad injection compared with vehicle (Figure 4—figure supplement 1C and D). Langerhans cells numbers in the lymph nodes were significantly lower upon papain exposure in mice fed on the AhR-poor diet (Figure 4B). By contrast, papain exposure increased the number of migratory skin cDC1 and cDC2 independently of the diet (Figure 4B). In particular, migratory PDL2+ cDC2 play an essential role in the induction of allergic Th2 responses (Gao et al., 2013; Kumamoto et al., 2013), and their number was similar in papain-treated mice from both diet groups (Figure 4B). In addition, the number of lymph node resident DC was similar in both diet groups (Figure 4B). The expression of MHC class II molecules or co-stimulatory molecule CD40 was not affected by the diet (Figure 4—figure supplement 1E). To confirm these results in the epicutaneous sensitization model, we analyzed draining lymph nodes 48 hr after papain topical application (Figure 4—figure supplement 1F). The numbers of resident and migratory cDC1 and cDC2 were increased in both diet groups upon papain exposure compared to vehicle treatment, while Langerhans cells number was significantly reduced in papain-treated mice fed on the AhR-poor diet. Collectively, our results show that lack of dietary AhR ligands impairs Langerhans cells migration upon papain challenge.
To confirm the role of AhR in this phenomenon, we examined DC migration after footpad papain challenge upon pharmacological inhibition of AhR. We used CH-223191, a potent AhR antagonist (Zhao et al., 2010), to treat mice fed on the I3C diet. Langerhans cells migration, but not that of cDC1 or cDC2, was significantly reduced in mice treated with the AhR inhibitor (Figure 4C). This result suggests that impaired Langerhans cells migration in mice fed with the AhR-poor diet is due to reduced AhR activation.
Langerhans cells play an essential role in dampening T cell responses in skin-draining lymph nodes (Igyarto et al., 2009; Kaplan et al., 2005; Gomez de Agüero et al., 2012). We hypothesized that reduced Langerhans cells presence in the lymph nodes could cause the observed exacerbated Th2 responses. To address this, we used Cd207(Langerin)-DTR mice, in which Langerhans cells can be depleted by Diphtheria Toxin injection (Kissenpfennig et al., 2005). We confirmed efficient Langerhans cells depletion in the footpad epidermis (Figure 4—figure supplement 1G). We injected papain or vehicle in the footpad of mice depleted of Langerhans cells or WT littermates (treated similarly with Diphtheria Toxin). To assess Th2 cells induction, we analyzed draining lymph nodes after 6 d. While IL4 secretion was similar in both groups, IL5 and IL13 secretion was significantly increased by Langerhans cells depletion (Figure 4D). These results mirror the observations made in mice fed with the AhR-poor diet (Figure 1D). We concluded that exacerbated Th2 responses in the absence of dietary AhR ligands are caused by impaired Langerhans cells migration to the lymph nodes.
Dietary AhR ligands regulate the inflammatory profile of epidermal cells
To address how dietary AhR ligands control Langerhans cells migration, we analyzed the transcriptomic profile of epidermal cells in mice fed on the I3C or AhR-poor diet, in basal conditions (vehicle treatment) or upon allergen challenge (6 hr after papain treatment). Papain exposure induced a common transcriptomic program (Figure 5—figure supplement 1A), enriched for type I interferon pathway, cytokine-mediated signaling, matrix remodeling, and oncostatin M pathway (a regulator of keratinocyte activation) (Boniface et al., 2007; Figure 5—figure supplement 1B). Consistent with pathway enrichment results, both papain-treated groups had significantly increased expression of inflammatory mediators genes (such as Csf1, Ccl20, and Tslp), interferon-stimulated genes (including Stat1 and Oasl2) (Figure 5—figure supplement 1C), and genes involved in tissue repair and remodeling (such as Areg, Il24, Mmp13, Osmr, Tgfa, and Vegfa) (Figure 5—figure supplement 1D).
Differentially expressed genes between diets were overexpressed mostly in the epidermis of mice fed on the AhR-poor diet (Figure 5A), both in basal conditions and upon papain challenge (Figure 5—figure supplement 1E). Diet-modulated genes were enriched for regulation of extracellular matrix, cytokine signaling, and inflammatory responses, including the leptin pathway (Figure 5B and C). Consistent with this, mice fed on the AhR-poor diet had in papain-treated epidermis significantly higher expression of matrix components (such as Col1a1, Col4a1, Col5a1) and cell adhesion molecules (including Itgb2 and Itgb7) (Figure 5—figure supplement 1F), and inflammatory genes including cytokine Il1b (Figure 5D) and chemokines Cxcl1, Cxcl3, Cxcl5, Ccl2, and Ccl3 (Figure 5D). To confirm these results at the protein level, we analyzed the secretion of chemokines using skin explants. CCL2, CCL3, and CXCL1 were more released upon papain treatment in the skin of mice deprived of dietary AhR ligands (Figure 5E), consistent with mRNA expression. Collectively, these results show that homeostatic activation of AhR via dietary ligands down-modulates inflammatory pathways in epidermal cells.
Diet-derived AhR ligands do not affect keratinocyte barrier
AhR is involved in keratinocyte differentiation and maintenance of skin barrier integrity (Haas et al., 2016; van den Bogaard et al., 2015). AhR ligands produced by commensal microbiota have been shown to be essential in this process (Uberoi et al., 2021). To address whether microbiota-derived and diet-derived AhR agonists have similar impact on epidermal genes, we reanalyzed public transcriptomic data from the epidermis of germ-free and specific pathogen-free (SPF) mice (Uberoi et al., 2021). The GO signature for keratinocyte differentiation was enriched in the epidermis of SPF mice (Figure 5—figure supplement 2A), consistent with previous findings that microbiota-derived AhR ligands control the expression of genes involved in barrier function such as Cdsn, Cldn1, Dsc1, Dsg1a, Flg, Ivl, and Tjp3, and of keratine molecules including Krt10 (Uberoi et al., 2021; Figure 5—figure supplement 2A). By contrast, the genes overexpressed in the epidermis of mice fed on the AhR-poor diet were not enriched in any group (Figure 5—figure supplement 2B). In particular, the expression of chemokine genes or Itgb8 was comparable between SPF and germ-free mice (Figure 5—figure supplement 2B). In addition, we found that lack of dietary AhR ligands had no impact on, or even increased, the expression of keratinocyte barrier genes (Figure 5—figure supplement 2C). Consistent with this, we did not observe by histology any diet-dependent defect in stratum corneum, the outer layer of epidermis formed by cornified keratinocytes (Figure 1—figure supplement 1E and Figure 5—figure supplement 3A). In addition, the ultra-structure of the epidermis was similar in both diet groups (Figure 5—figure supplement 3B). These observations suggest that the epidermal barrier function was not compromised in mice deprived of dietary AhR ligands and that diet-derived AhR ligands do not impact keratinocyte barrier.
AhR activation regulates keratinocyte production of TGF-β in mouse and human
Langerhans cells migration is regulated by Tgf-β, which retains Langerhans cells in the epidermis (Kel et al., 2010; Bobr et al., 2012; Mohammed et al., 2016). Bioactive Tgf-β can be produced from the latent form upon cleavage by integrin-β8 expressed on keratinocytes (Mohammed et al., 2016; Cambier et al., 2005). One of the pathways enriched in the epidermis of mice fed on the AhR-poor diet was related to Tgf-β regulation (Figure 5B). Indeed, expression of Itgb8 was significantly higher in the epidermis of mice fed on the AhR-poor diet, with or without papain treatment (Figure 6A), suggesting increased release of bioactive Tgf-β in the skin of mice deprived of dietary AhR ligands. To directly test this, we measured total and bioactive Tgf-β release in skin explants. The concentration of bioactive Tgf-β, but not that of total Tgf-β, was significantly higher in the skin of mice fed on the AhR-poor diet (Figure 6B).
To address the relevance of these results in human, we first reanalyzed public transcriptomic data from human skin exposed ex vivo to an AhR antagonist (Stemregenin-1, SR1) or agonist (6-formylindolo(3,2-b)carbazole, FICZ) (Di Meglio et al., 2014). FICZ is an endogenous AhR ligand produced from the photo-oxidation of tryptophan (Oberg et al., 2005; Wincent et al., 2009). As expected, canonical AhR target genes were upregulated in the FICZ-treated samples (Figure 6C). By contrast, chemokine genes (CCL2, CCL3, CXCL1, CXCL3, CXCL5) and ITGB8 were more expressed in SR1-treated skin (Figure 6C). These results are consistent with our transcriptomic data from mouse epidermis (Figure 5D and Figure 6A). To confirm that AhR activation regulates bioactive Tgf-β release by human keratinocytes, we used a human keratinocyte cell line (HaCaT cells). We cultured differentiated human keratinocytes in the presence of an AhR antagonist (SR1) or two different physiological AhR agonists, FICZ (produced by photo-oxidation) or DIM (produced from phytonutrients). AhR activation increased the expression of canonical target genes CYP1A1 and CYP1B1, but decreased the expression of ITGB8 (Figure 6D). To directly address the impact of AhR activation on Tgf-β secretion, we measured total and bioactive Tgf-β in the supernatant. While total Tgf-β concentration was similar between treatments, bioactive Tgf-β was significantly more released by SR1-treated keratinocytes (Figure 6E). Taken together, these results indicate that lack of AhR activation increases the production of bioactive Tgf-β in the skin in both mouse and human, thereby inhibiting Langerhans cells migration.
In this work, we showed that lack of dietary AhR ligands exacerbates cutaneous allergic Th2 responses and airway allergy after skin sensitization. We found that homeostatic activation of AhR by diet-derived agonists down-modulates inflammation pathways in epidermal cells. In particular, mice deprived of dietary AhR ligands displayed hyperproduction of bioactive Tgf-β in the skin, impairing Langerhans cells migration and their action as down-modulators of Th2 cells induction in the lymph nodes.
Previous work has reported a role for Langerhans cells in suppressing T cell responses in various models. In hapten-induced contact hypersensitivity, which is mediated by CD8 T cells, Langerhans cells are essential for tolerance to haptens (Kaplan et al., 2005), and to down-modulate T cell responses by producing IL10 (Igyarto et al., 2009) and by inducing regulatory CD4 T cells (Gomez de Agüero et al., 2012). In models of cutaneous sensitization to ovalbumin, Langerhans cells depletion increased Th2 cutaneous responses (Marschall et al., 2021; Luo et al., 2019) and T follicular helper cells (Marschall et al., 2021), as well as airway inflammation after intranasal recall (Marschall et al., 2021). Langerhans cells depletion also exacerbated lung Th2 responses after epicutaneous sensitization to house dust mite (Deckers et al., 2017b). It has been proposed that Langerhans cells produce IL10 upon exposure to allergens (Luo et al., 2019), but the mechanisms by which Langerhans cells regulate Th2 responses, and whether regulatory T cells are involved, remain to be better characterized. Consistent with these observations, we propose a model whereby dietary AhR ligands regulate the severity of cutaneous Th2 responses via the control of Langerhans cells migration, with decreased numbers of Langerhans cells in skin-draining lymph nodes leading to exacerbated Th2 cytokine production.
We found that reduced Langerhans cells numbers in lymph nodes increased T cell secretion of IL5 and IL13, but not that of IL4. Similar observations were made in a model of house dust mite allergy (Deckers et al., 2017b). These results are consistent with previous work showing that IL4 and IL13 are produced in lymph nodes by distinct populations of T cells during helminth infection (Liang et al., 2012), and that IL4 expression is regulated in vivo by distinct transcriptional mechanisms from IL5 and IL13 expression (Kim et al., 1999; Tanaka et al., 2011; Bao and Reinhardt, 2015). We speculate that Langerhans cells modulate IL5- and IL13-producing T cells, but not IL4-producing T cells.
Previous studies have proposed a role for AhR in regulating airway inflammation. In a model of ovalbumin-induced airway allergy, AhR-deficient mice developed more severe allergic responses due to the increased ability of AhR-deficient T cells to proliferate and the higher activation of AhR-deficient lung DC (Thatcher et al., 2016). In another study using ovalbumin-induced airway allergy, systemic injection of high doses of the AhR agonist FICZ reduced eosinophilia and Th2 cytokines in the lung and blood IgE levels (Jeong et al., 2012). In addition, in a model of cockroach allergen-induced allergy, mice deficient for AhR in type II alveolar epithelial cells had increased airway hyperreactivity including eosinophilia and elevated Th2 cytokine secretion due to dysregulated autophagy (Wang et al., 2022). By contrast, we found that airway allergic responses induced by papain exposure were not affected by the lack of dietary AhR ligands. Our results suggests that dietary AhR ligands do not play a role in the induction by lung DC of Th2 polarization per se, in cytokine secretion by lung ILC2 or in the inflammatory response of lung epithelial cells. This discrepancy could be explained by ligand-specific effects or by redundant effects of AhR agonists from different sources, the lack of dietary AhR ligand being compensated in the lung by endogenous or microbiota-derived ligands.
AhR can be activated by xenobiotics, diet-derived molecules, metabolites produced by microbiota metabolism, as well as endogenous ligands produced by cellular metabolism (Rothhammer and Quintana, 2019). Here, we specifically focused on natural diet-derived AhR agonists, delivered by a physiological route, that is, food absorption. In epidermal cells, we found that dietary AhR agonists regulate inflammatory pathways, but not keratinocyte barrier genes. By contrast, AhR ligands produced by skin commensal microbiota have been shown to control genes involved in keratinocyte differentiation and function (Uberoi et al., 2021). These results could be explained by ligand-specific effects. In a model of MC903-induced atopic dermatitis, topical application or oral administration of microbiota-derived AhR ligand indole-3-aldehyde (IAId) reduced skin inflammation and type 2 responses, which was dependent on AhR (Yu et al., 2019). However, application of other AhR agonists such as kynurenin (an endogenous ligand) or indole-3-acetic acid (produced by microbiota) had no impact on disease symptoms (Yu et al., 2019). Mechanistically, it was shown that AhR activation by IAId regulated TSLP production by keratinocytes (Yu et al., 2019). By contrast, here we did not find any impact of dietary AhR ligands on TSLP production during papain-induced allergy. In a model of imiquimod-induced psoriasis, AhR-deficient mice had exacerbated skin inflammation (Di Meglio et al., 2014), while topical application of the AhR agonists FICZ (Di Meglio et al., 2014) or Tapinarof (Smith et al., 2017) ameliorated disease symptoms. Moreover, AhR-deficient keratinocytes were hyperresponsive to inflammatory stimuli ex vivo and overexpressed inflammatory mediators such as CXCL1 and CXCL5 (Di Meglio et al., 2014), consistent with our findings. Collectively, these observations suggest ligand-specific effects of AhR activation in keratinocytes.
In addition, intestinal type 3 ILC and lymphoid follicles are reduced in mice fed on an AhR-poor diet compared to I3C diet (Kiss et al., 2011), but are normal in germ-free mice (Lee et al., 2012), suggesting distinct effects of diet-derived and microbiota-derived AhR ligands on type 3 ILC and lymphoid follicles. The underlying mechanisms of such ligand-specific effects remain unclear (Rothhammer and Quintana, 2019).
In conclusion, we show that diet-derived ligands are a major source of homeostatic stimulation of AhR in keratinocytes, dampening their response to inflammation and regulating the release of bioactive Tgf-β, but not the barrier function. These results provide novel insight into the gut–skin axis and could pave the way for optimizing diet interventions to reduce the development of cutaneous allergic diseases.
Materials and methods
C57/B6J mice were obtained from Charles River (France). Mice were maintained on a purified diet (‘AhR-poor diet,’ AIN-93M, Safe diets) supplemented or not in indole-3-carbinol (I3C, 200 ppm, Sigma). In some experiments, mice were fed on a normal chow diet (4RF25 SV-PF 1609, Le comptoir des sciures). For Langerhans cells depletion, Cd207(Langerin)-eGFP-DTR mice (Kissenpfennig et al., 2005) were used with DTR-/- littermates as controls and injected intraperitoneally with 500 ng of Diphtheria Toxin (Sigma) 3 d prior to allergen treatment. For AhR in vivo inhibition, mice were treated by intraperitoneal injection of 100 μg of AhR inhibitor CH-223191 (Invivogen) on three consecutive days prior to allergen treatment. Only female mice were used, except for the Langerhans cells depletion experiments. Mice were placed on the specific diet at 5 wk of age for a period of adaptation of 3 wk, before being used for any experiment. Because mice fed on the same diet had to be housed in the same cage, no randomization was performed. Mice were maintained under specific pathogen-free conditions at the animal facility of Institut Curie in accordance with institutional guidelines. Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE) for the care and use of laboratory animals. Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (authorization APAFiS#24554-2020030818559195v1 given by National Authority) in compliance with the international guidelines. For all animal experiments, the experimental unit is a single animal. Sample size was not calculated a priori. No animal was excluded from analysis. Blinding was performed during outcome assessment.
Allergy modelsRequest a detailed protocol
For cutaneous allergy, mice were injected in the footpad with 50 μl of phosphate buffered saline (PBS, vehicle) containing or not 50 μg of papain (Sigma). Footpad skin was collected after 6 hr or 24 hr, popliteal lymph nodes were collected after 24 hr, 48 hr, or 6 d.
For epicutaneous sensitization, mice were first shaved on the flank. The next day, 500 μg of papain, or the same volume of PBS (vehicle), was applied on a 0.5 cm2 piece of sterile gauze that was then placed topically on the shaved skin. The gauze was protected by Tegaderm film (3 M), then removed after 2 hr. Inguinal lymph nodes were collected after 48 hr or 6 d.
For airway allergy, mice were anesthetized using isofluorane and exposed intranasally to 20 μl of PBS (vehicle) containing or not 10 μg of papain, at days 0, 1, 13, and 20. Blood was collected at day 20. Bronchoalveolar lavage, lungs and lymph nodes were collected at day 21.
For airway allergy after skin sensitization, mice were injected in the footpad with 50 μl of PBS (vehicle) containing or not 50 μg of papain (Sigma). At day 7, all mice were placed on the I3C diet. Mice were exposed intranasally to 20 μl of PBS (vehicle) containing or not 10 μg of papain at days 14, 15, 27, and 35. Bronchoalveolar lavage and lungs were collected at day 36.
IgE measurementRequest a detailed protocol
Blood was collected and left at room temperature for 3 hr to coagulate. After centrifugation (450 × g, 10 min), serum was collected for analysis and kept at –20°C. IgE concentration was measured using ELISA (Invitrogen). The limit of detection was 140 pg/ml.
HistologyRequest a detailed protocol
Whole feet from treated mice were collected and fixed in formalin. Samples were decalcified in RDO (Eurobio Scientific) for 6 hr at 37°C before paraffin embedding. Samples were cut into 3 µm thin sections, deparaffinized and stained with hematoxylin (Dako), eosin (RAL Diagnostics), and Safran (RAL Diagnostics) (HES). Slides were scanned with Philips ULTRA FAST scanner 1.6 RA. Images were analyzed using QuPath (v.0.3.1) (Bankhead et al., 2017).
Electron microscopyRequest a detailed protocol
Ear skin was collected with a 2 μm diameter punch and was fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 hr, post-fixed for 1 hr with 2% buffered osmium tetroxide, dehydrated in a graded series of ethanol solution, and then embedded in epoxy resin. Images were acquired with a digital camera Quemesa (SIS) mounted on a Tecnai Spirit transmission electron microscope (FEI Company) operated at 80kV.
Indole measurement in mouse bloodRequest a detailed protocol
Blood samples were collected in EDTA-coated tubes. Samples were centrifuged for 15 min at 1200 × g to obtain serum. Indoles levels were measured in serum using liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) (Lefèvre et al., 2019). Briefly, 50–100 µl of serum were added to 800 μl of methanol and samples were vortexed for 5 min in a thermomixer at 4°C followed by an incubation at −20 °C for 30 min. After centrifugation at 13,300 × g for 10 min, the supernatant was collected and concentrated using a SpeedVac vacuum concentrator (Thermo Scientific). Samples were resuspended in 100 µl of 10% methanol solution, vortexed in a thermomixer for 5 min, and centrifuged at 13,300 × g for 10 min. 80 µl of each sample was transferred to a liquid chromatography vial and 10 µl injected in the HPLC-HRMS system. Chromatography was carried out with a Phenomenex Kinetex 1.7 µm XB – C18 (150 mm × 2.10 mm) and 100 Å HPLC column maintained at 55 °C. The solvent system comprised mobile phase A (0.5% [vol/vol] formic acid in water), and mobile phase B (0.5% [vol/vol] formic acid in methanol). The gradient was set-up as follows: 0–2 min, 0% B; 2–7 min, 0–50% B; 7–15 min, 50–100% B; 15–18 min, 100% B, 18–18.5 min 100–0% B and 18.5–21.5 min 0%B. HRMS analyses were performed on a HPLC Vantage Flex (Thermo Fisher Scientific) coupled to a Q-Exactive Focus mass spectrometer (Thermo Fisher Scientific) that was operated in positive (ESI+). The HPLC autosampler temperature was set at 4°C. The heated electrospray ionization source was set with a spray voltage of 4.5 kV, a capillary temperature of 250°C, a heater temperature of 475°C, a sheath gas flow of 35 arbitrary units (AU), an auxiliary gas flow of 10 AU, a spare gas flow of 1 AU, and a tube lens voltage of 100 V. During the HRMS acquisition, the scan range was set to m/z = 100–500 Da, the instrument operated at 70,000 resolution (m/z = 200), with an automatic gain control (AGC) target of 1 × 106 and a maximum injection time (IT) set to automatic. Instrumental chromatography stability was evaluated by injection of a synthetic standard mixture with all metabolites of interest and quality control (QC) samples quality control obtained from a pool of the leftover of all samples analyzed. This QC sample was reinjected once at the beginning of the analysis, every 10 sample injections, and at the end of the run. Ionization and retention times were validated with pure standards and are summarized in the following table:
Peak area was used as read out for relative quantification of the metabolites across the samples and normalized by serum volume.
Skin explant analysisRequest a detailed protocol
Skin from the footpad was harvested 24 hr after vehicle or papain treatment. Skin explants from both footpads were pooled and incubated for 24 hr in 1 ml of RPMI (Gibco) medium containing 10% fetal calf serum (FCS, Biosera). After centrifugation (450 × g, 5 min), conditioned medium was collected for analysis and kept at –20°C. TGF-β concentration was measured using ELISA (Total TGF-β1 Legend MAX and Free Active TGF-β1 Legend MAX, BioLegend). The limit of detection was 8 pg/ml for TGF-β1. CCL2, CCL3, and CXCL1 concentration was measured using CBA (BD Biosciences). The limit of detection was 10 pg/ml.
Flow cytometryRequest a detailed protocol
Cells were stained with indicated antibody cocktails supplemented with Fc block (BD Biosciences) in FACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) for 30–45 min on ice. After washing with FACS buffer, cells were resuspended in FACS buffer containing DAPI (Fisher Scientific, 100 ng/ml). Cells were acquired on a ZE5 (Bio-Rad) or FACSVerse (BD Biosciences) instrument. Supervised analysis was performed using FlowJo software v10 (FlowJo LLC).
Bronchoalveolar lavage analysisRequest a detailed protocol
Bronchoalveolar lavage was collected by injection of 1 ml of PBS in the bronchoalveolar space. Suspensions were filtered using 40 μm cell strainers. After centrifugation (450 × g, 5 min), the lavage was collected for analysis of soluble mediators and kept at –20°C. IL5 and IL13 concentration was measured using Enhanced Sensitivity CBA (BD Biosciences). The limit of detection was 274 fg/ml.
Cells were stained with anti-TCRβ BUV737 (BD Biosciences, clone H57-597), anti-CD11c PerCpCy5.5 (BD Biosciences, clone HL3), anti-SiglecF BV480 (BD Biosciences, clone E50-2440), anti-CD45 FITC (BioLegend, clone 30-F11), anti-CD11b Pe-CF594 (BD Biosciences, clone M1/70), anti-MHCII BV786 (BioLegend, clone M5/114.15.2), anti-Ly6G BV605 (BioLegend, clone 1A8), anti-CD4 BV650 (BioLegend, clone RM4-5), anti-Ly6C AF700 (BioLegend, clone HK1.4).
Lymph node cells analysisRequest a detailed protocol
For Th2 polarization analysis, lymph nodes were collected and dissociated by forcing through a 40 μm cell strainer. Cell suspensions were analyzed by flow cytometry after staining with anti-TCRβ APC (BioLegend, clone H57-597), anti-CD11b PerCpCy5.5 (BD Biosciences, clone M1/70), anti-CD4 FITC (BD Biosciences, clone RM4-5), and anti-CD19 APC-Cy7 (BD Biosciences, clone 1D3). Normalized cell numbers (2 × 105 cells/well) were cultured for 24 hr in 100 μl of RPMI medium containing 10% FCS in the presence of 5 μl of anti-CD3/CD28 beads (Thermo Fisher). After centrifugation (450 × g, 5 min), supernatant was collected for analysis and kept at –20°C. IL4, IL5, IL10, IFN-γ, IL17A, and IL13 concentration was measured using Enhanced Sensitivity CBA (BD Biosciences). The limit of detection was 274 fg/ml.
For flow cytometry of DC, lymph nodes were cut into small pieces and incubated for 30 min at 37°C in digestion mix: RPMI containing 0.5 mg/ml DNAse I (Sigma) and 0.5 mg/ml collagenase D (Roche). Cell suspensions were then filtered using 40 μm cell strainers. Antibodies used were anti-CD172a BUV737 (BD Biosciences, clone P84), anti-CD19 BV480 (BD Biosciences, clone 1D3), anti-CD3 BV480 (BD Biosciences, clone 500A2), anti-XCR1 BV510 (BioLegend, clone ZET), anti-CD11c BV785 (BioLegend, clone N418), anti-CD86 FITC (BD Biosciences, clone GL1), anti-CD26 PE (BioLegend, clone H194-112), anti-CD40 PerCP-efluor710 (eBioscience, clone 1C10), anti-PDL2 APC (BioLegend, clone TY25), anti-MHC II BV650 (BioLegend, clone M5/114.15.2), and anti-EpCAM APCFire750 (BioLegend, clone G8.8).
Epidermal cells analysisRequest a detailed protocol
Skin from the footpad was harvested using scalpels. The epidermis and dermis layers were separated after incubation at 37°C for 1 hr in 0.4 mg/ml dispase II (Roche). The epidermis was collected and then cut into small pieces using scalpels and incubated for 30 min with agitation at 37°C in RPMI containing 10% FCS and 0.5 mg/ml DNAse I. Suspensions were filtered using 40 μm cell strainers.
For Langerhans cells analysis, after centrifugation (450 × g, 5 min) cells were stained with anti-CD45 FITC (BioLegend, clone 30-F11), anti-EpCAM APCFire750 (BioLegend, clone G8.8) and anti-CD11b PerCPCy5.5 (BD Biosciences, clone M1/70).
For RNA-seq analysis, after centrifugation (150 × g, 5 min), dead cells were removed using EasySep Dead cell removal kit (StemCell). After this step, viability was around 70% as assessed by flow cytometry. Epidermal cells were composed of 95% keratinocytes (CD45- cells) as assessed by flow cytometry.
Imaging of epidermisRequest a detailed protocol
For imaging, Langerin-eGFP-DTR mice were used. Mice were placed on the AhR-poor or I3C diet for 3 wk. Epidermis was prepared from ear skin after hair removal using a depilating cream (Veet). The epidermis and dermis layers were separated after incubation at 4°C for 16 hr in 0.2 mg/ml dispase II (Roche). Epidermal layers were fixed in 4% paraformaldehyde for 20 min at room temperature. After washing in PBS, epidermal layers were placed on coverslips and preserved using Fluoromount-G mounting medium (SouthernBiotech).
eGFP fluorescence was imaged on an inverted laser scanning confocal microscope (Leica DMI8 with a sp8 scanning unit) equipped with an oil immersion objective (×40, NA = 1.35). The 488 nm laser was used for excitation and eGFP signal was collected on an Hybrid detector. A pixel size of 0.28 μm was chosen and Z stacks of three planes were acquired (Z step = 1 micron).
Image analysis was performed using Fiji software (Schindelin et al., 2012). To analyze Langerin+ cells density, a homemade macro was used. After projection of the three planes, a mask of the total eGFP was obtained. In a second step, images were blurred using a large radius (3.4 microns) to distinguish cellular stroma and count the number of cells. Finally, the number of cells was normalized to the tissue area to compute cell density.
RNA-seq library preparationRequest a detailed protocol
Epidermal cells were isolated 6 hr after vehicle or papain treatment. Cells were lysed in RLT buffer (QIAGEN). Total RNA was extracted using the RNAeasy minikit (QIAGEN) including on-column DNase digestion according to the manufacturer’s protocol. The integrity of the RNA was confirmed in BioAnalyzer using RNA 6000 Nano kit (Agilent Technologies) (8.8 < RIN < 10). Libraries were prepared according to Illumina’s instructions accompanying the TruSeq Stranded mRNA Library Prep Kit (Illumina). 500 ng of RNA was used for each sample. Library length profiles were controlled with the LabChip GXTouchHT system (Perkin Elmer). Sequencing was performed in three sequencing unit of NovaSeq 6000 (Illumina) (100-nt-length reads, paired end) with an average depth of 40 millions of clusters per sample.
RNA-seq data analysisRequest a detailed protocol
Genome assembly was based on the Genome Reference Consortium (mm10). Quality of RNA-seq data was assessed using FastQC. Reads were aligned to the transcriptome using STAR (Dobin et al., 2013). Differential gene expression analysis was performed using DESeq2 (v1.22.2) (Love et al., 2014). Genes with low number of counts (<10) were filtered out. Differentially expressed genes between ‘vehicle’ and ‘papain’ treatment for each diet, or between ‘AhR-poor diet’ and ‘I3C diet’ conditions for each treatment, were calculated using the design ‘group.’ Differentially expressed genes were identified based on adjusted p-value<0.01 and Log2 FoldChange>1. Complete gene lists are included in Figure 5—source data 1. Heatmaps of log2-scaled expression were generated with ComplexHeatmap. Pathway enrichment was analyzed using EnrichR (Kuleshov et al., 2016). Sequencing data has been deposited in GEO (accession number GSE198368).
Analysis of public transcriptomic dataRequest a detailed protocol
Data was downloaded from GEO. Raw count matrix was normalized using DESeq2. For human skin explant (GSE47944) (Di Meglio et al., 2014), data from non-lesional skin exposed to SR1 or FICZ was used. For mouse epidermis (GSE162925) (Uberoi et al., 2021), data from germ-free and specific pathogen-free mice were used. Heatmaps of log2-scaled expression were generated with ComplexHeatmap.
Gene set enrichment analysisRequest a detailed protocol
GSEA (Subramanian et al., 2005) was performed using the GSEA software (version 4.0.3) with the default parameters, except for the number of permutations that we fixed at n = 1000. Results are considered significant when NES > 1 and FDR < 0.25. RNA-seq data from the epidermis of germ-free and specific pathogen-free mice (GSE162925) was normalized using DESeq2. Gene signature for keratinocyte differentiation (GO:0045616) was downloaded from MSigDB (v7.5.1) (Liberzon et al., 2015). Gene signature for epidermal cells in AhR-poor diet was obtained by using the top 500 differentially expressed genes upregulated in ‘AhR-poor diet’ versus ‘I3C diet’ for vehicle treatment, based on log2 fold change.
Human keratinocyte cultureRequest a detailed protocol
Human HaCaT keratinocytes were cultured with DMEM medium without glutamine or calcium (Gibco) supplemented with antibiotics (penicillin and streptomycin), 10% FCS treated with Chelex (Sigma) to removed endogenous calcium, and 0.03 mM calcium chloride (low-calcium growth medium). Cells were kept at 80% confluence in low-calcium growth medium in order to keep a basal undifferentiated phenotype (Wilson, 2014). For differentiation, cells were switched to the same medium containing 2.8 mM calcium chloride (high-calcium growth medium). Cells were mycoplasma-free.
HaCaT cells were exposed for 24 hr to 8 μM SR1 (Cayman Chemicals), 5 μg/ml diindolylmethane (DIM, Sigma), or 60 nM 6-formylindolo[3,2-b]carbazole (FICZ, Enzo Life Sciences) in low-calcium growth medium. Medium was then replaced by high-calcium growth medium, and cells were further cultured for 24 hr. Cells were lysed in RLT buffer and supernatants were collected for analysis using ELISA (Total TGF-β1 Legend MAX and Free Active TGF-β1 Legend MAX, BioLegend). The limit of detection was 8 pg/ml for TGF-β1.
qPCRRequest a detailed protocol
Cells were harvested and lysed in RLT buffer (QIAGEN). RNA extraction was carried out using the RNAeasy micro kit (QIAGEN) according to the manufacturer’s instructions. Total RNA was retro-transcribed using the superscript II polymerase (Invitrogen), in combination with random hexamers, oligo dT and dNTPs (Promega). Transcripts were quantified by real-time PCR on a 480 LightCycler instrument (Roche). Reactions were carried out in 10 μl using a master mix (Eurogentec), with the following TaqMan Assays primers (Merck): Cyp1a1 (Mm00487218_m1), Muc5ac (Mm01276705_g1), Gob5 (Mm01320697_m1), Gapdh (Mm99999915_g1), B2m (Mm00437762_m1), Polr2a (Mm00839502_m1), CYP1B1 (Hs00164383_m1), CYP1A1 (Hs01054796_g1), ITGB8 (Hs00174456_m1), B2M (HS00187842_m1), HPRT (Hs02800695_m1), and RPL34 (Hs00241560_m1). The second derivative method was used to determine each Cp and the expression of genes of interest relative to the housekeeping genes (Gapdh, B2m, Polr2a for mouse and B2M, HPRT, RPL34 for human) was quantified.
Statistical analysisRequest a detailed protocol
Statistical tests were performed using Prism v9 (GraphPad Software). Statistical details for each experiment can be found in the corresponding figure legend. N corresponds to the number of biological replicates. Absence of asterisk indicates ‘nonsignificant.’ ANOVA was performed with Tukey’s multiple-comparisons test.
Sequencing data has been deposited in GEO (accession number GSE198368). Figure 5 - Source Data 1 contains the list of differentially expressed genes.
NCBI Gene Expression OmnibusID GSE198368. RNA-seq analysis of epidermal cells from mice fed with a diet deprived or not in AHR ligands.
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Carla V RothlinSenior and Reviewing Editor; Yale University, United States
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
[Editors' note: this paper was reviewed by Review Commons.]https://doi.org/10.7554/eLife.86413.sa1
Additional experiments in response to Reviewer 2’s comments
"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 had already performed footpad histology and observed that the stratum corneum was not affected by the diet (Figure 1A and Figure 1—figure supplement 1E). We now provide a quantitative analysis by measuring stratum corneum thickness on the images and we added this data to new supplementary figure (Figure 5—figure supplement 4). To strengthen this point, we also performed ultra-structural analysis of the epidermis using electron microscopy of the ear skin, and found no difference between diet groups (Figure 5—figure supplement 4).
"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?"
To strengthen our results, we performed epicutaneous sensitization without barrier disruption by applying papain on shaved flank skin (without any skin abrasion). We analyzed T cell responses in the draining lymph nodes after 6 days and found similar results as with footpad injection, i.e increased IL5 and IL13 secretion in mice fed on the AhR-poor diet (added as fig1E). We also analyzed dendritic cells migration to the draining lymph nodes after 48h and found that papain exposure induced cDC1 and cDC2 migration similarly in both diet groups, but Langerhans cells were reduced in mice fed on AhR-poor diet (added in Figure 4—figure supplement 1F).
Text edits in response to reviewers’ comments
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 added references to original research regarding Mucin 5 and CLCA1 : (Leverkoehne and Gruber, 2002; Nakanishi et al., 2001; Young et al., 2007). We also added references to alveolar epithelial cells in the Discussion section : "In addition, in a model of cockroach allergen-induced allergy, mice deficient for AhR in type II alveolar epithelial cells had increased airway hyperreactivity including eosinophilia and elevated Th2 cytokine secretion, due to dysregulated autophagy (Wang et al., 2022).".
Regarding ILC2, because it was shown in the mentioned article that only gut ILC2 express AhR, and in particular not lung or skin ILC2, we believe that a reference to this work is not relevant in the context of our study, and therefore we did not add this reference.
–" 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 have modified all figure legends to better describe the treatment schedules.
– "1. In the introduction section, the authors should explain adequately why they thought that AHR signaling is important for allergy. "
We now better explain this point in the introduction : " In particular, AhR exerts broad functions in barrier tissues including skin and lung (Esser and Rannug, 2015), where AhR activation has been reported to limit inflammation (DiMeglio et al., 2014; Beamer and Shepherd, 2013). The impact of dietary AhR ligands in allergic responses at such barrier sites remains unknown."
Comments from Reviewer 2
–"How to explain the difference between IL4 (no effect between the two diets/or absence/presence LCs in Figure 4D) and IL5/IL13 (small effect in Figure 1D and 4D). "
This is an interesting point. It has been shown that IL4 can be produced in lymph nodes by T cells distinct from those producing IL5 and IL13 (Liang et al., 2011). Consistent with this, IL4 expression is regulated in vivo by distinct mechanisms from IL5 and IL13 expression (Kim et al., 1999; Tanaka et al., 2010; Bao and Reinhardt, 2015).
We speculate that IL4-producing T cells are not affected by Langerhans cells presence. We added a point in the Discussion section to discuss this.
– "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 Figure 5 would be immediately followed by Figure 7".
We have tuned down our conclusion regarding the different effect of diet-derived and microbiotaderived AhR ligands according to the comments of the reviewer. We now conclude: “Diet-derived AhR ligands do not affect keratinocyte barrier”. We have also moved Fig6 to the supplementary data (now Figure 5—figure supplement 3). Finally, we have modified the discussion: “In epidermal cells, we found that dietary AhR agonists regulate inflammatory pathways, but not keratinocyte barrier genes. By contrast, AhR ligands produced by skin commensal microbiota have been shown to control genes involved in keratinocyte differentiation and function (Uberoi et al., 2021). These results could be explained by ligand-specific effects.”
Other additional data
During the review of the manuscript, we had the opportunity to analyze the expression of Muc5ac in lungs in the ‘atopic march’ model. We also added a few biological replicates for this experiment. We have updated Figure 3C accordingly.
Response to other comments
Comments from Reviewer 1
"In Figure 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 steadystate, which is not different between diets (Figure 4A). We actually already show in supplementary figure (now Figure 4—figure supplement 1E) some 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.
“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?”
We believe that the differences in Th2 cytokine secretion in lymph nodes are due to the difference in models. In the footpad model, we analyzed an acute reaction (day 6 after papain treatment) while in the airway model, we analyzed chronic exposure (4 intra-nasal applications over 21 days).
“In Figure 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."
In figure 3, eosinophils are actually recruited to the lungs upon papain exposure with the I3C diet (median around 5000-10000 cells in figure 3A, compared to around 100 eosinophils found with vehicle treatment in figure 2A). The reviewer’s comment shows that the representation was misleading, therefore we changed it to a log10 scale, similar to figure 2A.
Concerning cytokine detection in BAL, IL5 levels are quite similar for the I3C diet between figure 2B and figure 3B (ranging between 0 and 80 pg/mL, with a median around 20 pg/mL). Similarly for IL13, the range is quite similar between models, although the median is lower in figure 3B. This could be due to variability between experiments. Because this is a minor discrepancy, we do not believe it is necessary to add a discussion on this point in the manuscript.
Comments from Reviewer 2
"Figure 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 Figure 1C or inflammatory cell counts in Figure 2A and Figure 3A 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 addition, we made similar observations in the topical application setting (added in Fig1E).
"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 IFNγ production between AhR poor and rich diet appears even more pronounced than the difference in IL5/IL13 (Figure 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 better explained this point in the introduction: " In particular, AhR exerts broad functions in barrier tissues including skin and lung (Esser and Rannug, 2015), where AhR activation has been reported to limit inflammation (DiMeglio et al., 2014; Beamer and Shepherd, 2013). The impact of dietary AhR ligands in allergic responses at such barrier sites remains unknown."
In the course of the study, we analyzed IL10, IL17A and IFNγ 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 outside the scope of the present study (and is unrelated to the diet), and would represent an entire project in itself.
Figure 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 (Figure 1) and pulmonary (Figure 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 made this point clearer in the text: " To this aim, we fed mice on the I3C or AhR-poor diet only during the sensitization phase, and placed all experimental groups on I3C diet 7 days after cutaneous exposure to papain or vehicle."
"Figure 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 (Bobr et al., 2012; Mohammed et al., 2016; de La Cruz Diaz et al., 2021; Kel et al., 2010). 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 (now figure 6). Therefore, we believe that additional experiments to support the link between AhR ligands and TGFb are not necessary within the scope of the revision.
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Article and author information
Institut National de la Santé et de la Recherche Médicale (core funding)
- Elodie Segura
Institut Curie (core funding)
- Adeline Cros
- Elodie Segura
Institut National Du Cancer (2018-1-PLBIO-01-ICR1)
- Elodie Segura
Agence Nationale de la Recherche (ANR-10-LABX-0043)
- Mabel San Roman
- Mathieu Maurin
- Elodie Segura
Agence Nationale de la Recherche (ANR-17-CE15-0011-01)
- Elodie Segura
Agence Nationale de la Recherche (ANR-10-IDEX-0001-02 PSL)
- Elodie Segura
European Research Council (ERC Horizon 2020-Marie Sklodowska-Curie Actions (No 842535))
- Alba De Juan
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
This work was funded by INSERM, Institut Curie, Cancéropôle Ile-de-France, Institut National du Cancer (2018-1-PLBIO-01-ICR1) and Agence Nationale de la Recherche (ANR-10-LABX-0043, ANR-10-IDEX-0001-02 PSL, ANR-17-CE15-0011-01). The authors wish to thank the Flow Cytometry Core, the Pathex Platform, the NGS Platform, the Metabolomics and Lipidomics Platform, and the In Vivo Experiments Platform of Institut Curie. The authors thank S Henri for helpful advice and M Vocanson for providing founder Cd207(Langerin)-eGFP-DTR mice.
Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE) for the care and use of laboratory animals. Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (Authorization APAFiS#24554-2020030818559195-v1 given by National Authority) in compliance with the international guidelines.
Senior and Reviewing Editor
- Carla V Rothlin, Yale University, United States
- Preprint posted: January 25, 2023 (view preprint)
- Received: January 25, 2023
- Accepted: April 24, 2023
- Version of Record published: May 16, 2023 (version 1)
- Version of Record updated: May 16, 2023 (version 2)
© 2023, Cros et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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