Allergic sensitization is initiated by protein and epithelia interaction, although the molecular mechanisms leading this encounter toward an allergic phenotype remain unknown. Here, we apply the two-hit hypothesis of inflammatory diseases to the study of food allergy sensitization. First, we studied the effects of long-term depilation in mice by analyzing samples at different time points. Several weeks of depilation were needed until clear immunological changes were evidenced, starting with upregulation of NLRP3 protein levels, which was followed by overexpression of Il1b and Il18 transcripts. Secondly, we assessed the effects of allergen addition (in this case, Pru p 3 in complex with its natural lipid ligand) over depilated skin. Systemic sensitization was evaluated by intraperitoneal provocation with Pru p 3 and measure of body temperature. Anaphylaxis was achieved, but only in mice sensitized with Prup3_complex and not treated with the NLRP3 inhibitor MCC950, thus demonstrating the importance of both hits (depilation + allergen addition) in the consecution of the allergic phenotype. In addition, allergen encounter (but not depilation) promoted skin remodeling, as well as CD45+ infiltration not only in the sensitized area (the skin), but across several mucosal tissues (skin, lungs, and gut), furtherly validating the systemization of the response. Finally, a low-scale study with human ILC2s is reported, where we demonstrate that Prup3_complex can induce their phenotype switch (↑CD86, ↑S1P1) when cultured in vitro, although more data is needed to understand the implications of these changes in food allergy development.
Food allergy is a chronic inflammatory condition affecting 8% of children and 5% of adults with no definitive treatment available[
The two-hit hypothesis was initially assembled to explain inflammatory mechanisms underlying some chronic pathologies[
As previously reported, cutaneous exposure to food allergens in children and mice predisposes to IgE-mediated anaphylaxis[
On the other hand, lipids have been described as adjuvants in allergy[
Over the past decades, it has been shown that allergen encounter induces the secretion of alarmins (IL25, IL33) by epithelial cells and keratinocytes, promoting pro-allergic responses mediated by dendritic cells (DCs), mast cells, eosinophils, and type 2 innate lymphoid cells (ILC2s), among others[
Although in vivo models have broadened our knowledge of skin-mediated food allergy sensitization, we still ignore the molecular mechanisms underlying this event, as well as the contribution of lipid ligands in the process. Within this context, we produced a mouse model of sensitization using the major peach allergen, the LTP Pru p 3[
To study the role of depilation as a first hit in allergic sensitization, first we compared the permeability of the skin barrier between depilated and non-depilated mice. As shown in Fig. 1A, blue toluidine uptake was greater in depilated specimens than in non-depilated ones, suggesting that depilation induces epithelial permeability in vivo. This result might suggest that lower layers from depilated skins are more exposed to ambient stimulus, including allergens, than untreated ones. This feature was not strain-dependent and was maintained through time, after 3 and 6 weeks of continuous depilation (Supplementary Fig. 1).
Graph: Figure 1 Skin depilation induces epithelial permeability and inflammasome priming in vivo. (A) Blue toluidine absorption in vivo by skins from non-depilated (n = 4) and depilated (n = 6) mice. The images show the opposite face where the staining was added. Representative images from confocal microscopy (×63 magnification) are also shown, remarking the staining uptake by the skin in the case of depilated specimens. Bar = 40 µm. (B,C) Kinetics and quantification of NLRP3 expression (red) in non- (0) and depilated (1–6) mice. 546-labelled anti-rabbit IgG was used as an isotype control. Three individual sections from each mouse (n = 5 animals/group and time point) were separately stained. NLRP3 expression in the whole tissue was then analyzed with a Zeiss LSM 880 confocal microscope, with two representative but distant regions from each section being chosen for quantification purposes. Data are presented as representative images and mean (SEM. Kruskal–Wallis test with Dunn's correction for multiple comparisons). ***P < 0.001, ****P < 0.0001. (D) RTqPCR analysis showing the kinetics of transcriptional activation of Nlrp3, Il1b, Il18 and alarmin genes (Il25, Il33) in skin biopsies from non-depilated (0) versus depilated (
Additionally, we found that keratinocytes from depilated rodents showed a significant increase in the expression of NLRP3 protein, when compared to non-depilated animals (Fig. 1B,C). Regarding the kinetics of this protein expression, we observed that NLRP3 protein detection remained unaltered during the first 2 weeks after depilation. After the third week (Fig. 1B,C t = 3), however, the mean fluorescence intensity in the epidermis associated with NLRP3 detection was significantly enhanced when compared to non-depilated (Fig. 1B,C t = 0) specimens, remaining constant over time at least up to the last week studied in this report (Fig. 1B,C t = 6 weeks).
However, as seen in Fig. 1D, expression of Nlrp3 transcripts was significantly upregulated when compared to non-depilated skins only after 6 weeks of depilation with the cream. This could indicate that the increase in protein detection we are observing after the third week of depilation is not due to an actual increase in the translation ratios of the protein, but rather to the formation of NLRP3 complexes, pointing to an effective priming of the pathway. Nevertheless, further studies should be conducted in order to fully validate this theory. Regarding the inflammasome products Il1b and Il18, their induction over time seems to be slightly different, with Il18 transcripts being detected earlier (after 5 weeks) during the depilation protocol, while Il1b is not detected until the sixth week. A small induction of alarmins (Il25, Il33) transcription seems to be detected after 5 weeks of depilation, although this tendency was only statistically significant for the latter. However, the strongest induction was observed again after the sixth week of the protocol. When we tried to correlate the levels of Il1b and Il33 transcripts with their corresponding protein counterparts, we were unable to detect them by means of Western blot and ELISA, respectively (data not shown).
Finally, after 6 weeks, skin depilation also significantly upregulated several genes related to the establishment of T1 and T3 immune responses (Cxcl10, Il17a, Il23a, S100a8) and oxidative stress (Dusp1, Fig. 1E), thus confirming the establishment of a proinflammatory genetic landscape when this treatment is applied long-term. Altogether, these results suggest that skin damage must be sustained over time in order to clearly induce transcriptomic changes in the tissue related to a proinflammatory background, thus validating the safety of depilatory creams when not applied regularly over healthy skins. However, the induction of protein expression due to continuous depilation over time could not be demonstrated with the studies here presented, although other depilation protocols (such as tape-stripping) have shown to induce the production of IL33 in the skin of mice[
In order to furtherly validate the results obtained in mice, we sought to study the effects of the first hit (depilation) over cells from human origin. We chose thioglycolate exposure as a model of in vitro depilation because it constitutes a universal component of depilatory cream formulations. Also, previous reports had pointed that keratinocytes in the presence of this compound express an altered morphology and significantly higher levels of the stress-related chaperone Hsp70[
Graph: Figure 2 Thioglycolate induces inflammasome activation in vitro in HaCaT cells. (A) Dose-dependent effect of thioglycolate addition over monolayer integrity of HaCaT cells cultured in 24-well plates (n = 6/group). (B) Comparison of TEER values from HaCaT cells in Transwell, when treated or not with thioglycolate (450 mM). Data are presented as mean (SEM. Mann–Whitney test). **P < 0.01. (C) Dose-dependent effect of thioglycolate addition over the production of IL18 in HaCaT cells, as quantified by ELISA. All the experiments were performed at least three times and analyzed in triplicates. (D) Immunodetection of bioactive caspase-1 (p20) in lysates from HaCaT cells treated with different concentrations of thioglycolate. The experiment was performed using a pool from three biological replicates performed independently. β-tubulin was used as a loading control.
Moreover, thioglycolate exposure prompted the release of soluble IL18 to the media, in a dose-dependent manner (Fig. 2C). Analyzing the kinetics of its production, we found that both 450 mM and 2250 mM concentrations of thioglycolate induced a small IL18 liberation 3 h after treatment; however, only with the highest thioglycolate concentration the amount of IL18 in the supernatant increased over time (Fig. 2C). Surprisingly, the addition of thioglycolate was not able to induce IL1β release to the media, with its basal concentration remaining unaffected by the stimulus after 3 h from its addition (Supplementary Fig. 2A).
In addition, we confirmed the detection of bioactive caspase-1 (p20) in cell lysates from HaCaT cells cultured with thioglycolate (2250 mM), in contrast with its absence in the same cells cultured at low concentrations (450 mM) of the stimulus (Fig. 2D). Caspase-1 is needed for both IL1β and IL18 production. Thus, the presence of the latter in cell supernatants is furtherly validated, while the absence of the first suggests an IL1β-independent inflammation mechanism. It is known that pro-IL18 is constitutively produced by epithelial cells, while translation of pro-IL1β must be activated prior to its cleavage by caspase enzymes[
In order to validate these data and its reproducibility in other human epidermis cell lines, we sought to complement the inflammasome activation study using A431 cells as another model. In this case, we observed that monolayer integrity was unaffected at concentrations used in approved formulations (450 mM), but it was compromised at higher concentrations (Supplementary Fig. 2B). Accordingly, the Il1b and Il18 expression was slightly upregulated (Supplementary Fig. 2C,D) not only at a transcriptional level but also with IL18 present in cell supernatants, in a dose-dependent manner (Supplementary Fig. 2E). In the case of IL1β, we only observed small quantities of it when cells were stimulated with 2250 mM of thioglycolate, but not at lower concentrations (data not shown). Thus, these assays confirm that the results obtained with the HaCaT model are not dependent of cell line and might be extended to other human epidermis models.
Once we have understood some of the effects derived from depilation (first hit) in skin-mediated allergic sensitization, we sought to evaluate the effects of allergen encounter (second hit) over the stressed mucosa. Pru p 3 was used as a model allergen in an adjuvant-free mouse model of anaphylaxis (Fig. 3A), based on previously published reports[
Graph: Figure 3 The skin pathway can induce anaphylaxis in a mouse model of allergy. (A) Mice were epicutaneously sensitized in the abdomen weekly (6 weeks) after depilation with Pru p 3 or Prup3_complex (n = 8/group). Sensitization with Prup3_complex was also performed in mice without functional NLRP3 (MCC950; n = 5). Untreated depilated and non-depilated animals were also included in the experimental design (n = 5/group). Challenge was performed intraperitoneally in all groups studied with 100 µg of Pru p 3 at week 7. (B) Comparison of body temperature drop post-challenge by sensitizing agent (n = 8 in Pru p 3 and Prup3_complex specimens; n = 5 in depilated-only and MCC950-treated animals). Data are presented as mean (SEM. Mann–Whitney test). *P < 0.05, **P < 0.01. (C) H&E staining of skin sections from sensitized (Prup3_complex, n = 8 mice), untreated depilated (n = 5), non-depilated (n = 5) and sensitized + MCC950-treated (n = 5) mice at ×10 and (D) ×40 magnification. Three individual sections from each mouse were separately stained and photographed with a Zeiss LSM 880 confocal microscope, with two representative but distant regions from each section being analyzed. After that, representative images were chosen for publication. D = dermis, PA = panniculus adiposus, PC = panniculus carnosus. Bar = 100 µm (×10) or 50 µm (×40). In all cases, mouse models were performed in two independent set of animals.
In order to demonstrate the importance of the first hit (depilation and NLRP3 priming) to establish the allergic phenotype, mice sensitized with Prup3_complex were treated with MCC950. This compound has been extensively used as an inhibitor of NLRP3 oligomerization[
Besides, histological characterization of Prup3_complex sensitized mice revealed remodeling of skins, with thickened dermis and striated muscle layers compared to depilated non-sensitized specimens, as well as non-depilated ones (Fig. 3C). Nevertheless, NLRP3 priming due to first hit seems crucial to allow the remodeling induced by Prup3_complex, since specimens sensitized with the allergen and treated with MCC950 presented a phenotype more similar to both depilated non-sensitized specimens, as well as non-depilated ones (Fig. 3C). Additionally, cellular infiltration into the dermis was higher in sensitized animals, suggesting that Prup3_complex exposure prompts cellular mobilization in the mucosa (Fig. 3D). All these images were taken after the mice were challenged with Pru p 3, although the possibility that the observed changes are due to the challenge is ruled out, since untreated and non-depilated specimens did also receive the same challenge. So, these results might point that Prup3_complex addition after depilation aggravates the skin inflammation previously observed in depilated only mice (Fig. 1).
Given these results, we sought to analyze if allergen encounter could induce modifications in the immunological state of depilated skins. As shown in Fig. 4A,B, we found that Prup3_complex addition induced a significant CD45+ infiltration in the skin of depilated mice. Interestingly, this recruitment of leukocytes was also observed in distant mucosal locations, such as lungs, duodena, and ilea (Fig. 4C–H). To frame the contribution of depilation or Pru p 3 (without its ligand) in the recruitment of immune cells, we also analyzed non-depilated mice, as well as depilated Pru p 3-sensitized animals. As shown in Supplementary Fig. 4, non-depilated and depilated mice present similar levels of cell infiltration in the dermis. Also, Pru p 3 presents slightly greater levels of CD45+ cells in the region, although Prup3_complex is the only treatment presenting significant differences compared to non- and depilated animals. So, immune cell recruitment results from allergen encounter (second hit) during sensitization, with the ligand of Pru p 3 playing an important role in this process. As in the case of Fig. 3C,D, all these images were taken after the mice were challenged with Pru p 3, but the possibility that the observed changes are due to challenge is ruled out, since non-depilated, depilated and Pru p 3-sensitized specimens did also receive the same challenge, but their infiltration is still below Prup3_complex-sensitized mice's levels.
Graph: Figure 4 CD45+ infiltration in mucosa after Prup3_complex cutaneous treatment. Representative images and quantification of (A,B) skins, (C,D) lungs, (E,F) duodena and (G,H) ilea hybridized with anti-CD45 antibody (green). Nuclei (blue) stained with DAPI. Quantification of CD45+ cell infiltration was calculated as the ratio between CD45+ and DAPI+ cells per image (n = 5/group; at least 3 sections were separately stained from each mouse at distal depths of the tissue and 3–5 images were taken per section). Data are presented as mean (SEM. Mann–Whitney test). **P < 0.01. Bar = 100 µm. In all cases, mouse models correspond to samples from two independent sets of animals.
To characterize the nature of the allergen-induced infiltration, different cell types were stained with specific cell markers in skin biopsies of Prup3_complex sensitized mice. We observed that the CD45+ infiltration included mainly dermal CD207+ (Fig. 5A,B), CD3+ (Fig. 5C,D) and CD127+CD3− cells (Fig. 5E,F; zoomed in Supplementary Fig. 5), the later ones resembling an ILC-like phenotype[
Graph: Figure 5 Prup3_complex prompts inflammatory phenotype in the skin. Representative images and quantification of immune infiltration in the skin of depilated sensitized (Prup3_complex; n = 5) and untreated (Untreated; n = 5) mice. Nuclei (blue) stained with DAPI. (A,B) CD207+ (DCs), (C,D) CD3+ (T cells) and (E,F) CD45+CD127+CD3− (ILCs) cells. Quantification of DCs and ILCs is referred to dermal areas where these cells were grouped forming clusters (at least 4 sections were obtained from each mouse at distal depths of the tissue and 3–5 images were taken per section), while T cells were homogeneously distributed through the whole tissue. Data are presented as mean (SEM. Mann–Whitney test). *P < 0.05, **P < 0.01. Bar = 50 µm. (G) Heat map of RTqPCR analyses from skin biopsies from depilated + Prup3_complex (n = 2) vs non-depilated (n = 4) mice. Each sample was analyzed in two technical replicates. Data are presented as mean (SEM. Mann–Whitney test). *P < 0.05, **P < 0.01. In all cases, mouse models correspond to samples from two independent sets of animals.
Given the relevance that ILC2s have gained over the last decades in the study of allergic pathologies[
Graph: Figure 6 Prup3_complex induces phenotype changes in peripheral human ILC2s in vitro. (A) Quantification of ILC2s in the bloodstream of allergic (LTP+ ; n = 9) and healthy (n = 9) donors. Total cell counts were determined with a hemocytometer. Data are presented as mean (SEM. Mann–Whitney test). *P < 0.05. (B,C) Flow cytometry phenotyping of circulating ILC2s incubated for 96 h with Pru p 3, Pru p 3's ligand or Prup3_complex. The experiment was repeated 5 independent times in duplicates. Data are presented as a mean of ∆ILC2 = (% Positive ILC2s)sample – (% Positive ILC2s)control (SEM. Kruskal–Wallis test with Dunn's correction for multiple comparisons). *P < 0.05, **P < 0.01. (D) Work diagram from the 3D in vitro skin model. (E,F) Flow cytometry as in (B,C).
To analyze a model closer to physiological conditions, we developed an in vitro skin model based on keratinocytes (HaCaT cells) and fibroblasts cultured in a Transwell format. ILC2s from healthy volunteers were seeded onto the basolateral chamber. Pru p 3 or Prup3_complex was added to the apical side in the presence or absence of thioglycolate, to replicate an in vitro sensitization process (Fig. 6D). Expression of CD86 and S1P1 in ILC2s was analyzed by flow cytometry, showing CD86 was overexpressed after incubation with both Pru p 3 and Prup3_complex, independently of thioglycolate addition (Fig. 6E). In contrast, S1P1 expression in ILC2s was only significantly induced by Prup3_complex after thioglycolate addition (Fig. 6F) suggesting that overexpression of S1P1 in ILC2s is dependent of tight junction disruption in the skin monolayer due to thioglycolate treatment (as shown in Fig. 2B), resulting in a permeability increase in the tissue and a greater availability of the allergen in the basolateral chamber after that. Nonetheless, these results should not be seen as a reflection of what is happening in murine skins, since experimental conditions from both assays differ greatly. For example, based on our experimental data, we cannot discriminate if ILCs detected in murine skins (Fig. 5E,F) are resident ILCs or migratory peripheral ones that were recruited to the tissue after allergen exposure, while in the case of human ILC2s (Fig. 6) they have been isolated in all cases from peripheral circulation. For future publications, it would be interesting to isolate the dermal ILC-like subset detected in Prup3_complex-sensitized mice, to analyze if its phenotype has also been switched (when compared to non-sensitized controls) in a similar fashion of human ILC2s do when they get in contact with the allergen.
Based on the two-hits hypothesis to explain the immune mechanism of several chronic inflammatory diseases, allergy could be the result of: first, skin depilation, which primes the inflammasome pathway; and second, allergen encounter, which activates cell infiltration and systematizes the response. In this work, we have deepened in the mechanisms by which depilated skin can prime allergic sensitization to food allergens. We produced an epicutaneously-sensitized murine model of allergy that showed significant anaphylactic symptoms after allergen challenge, accompanied by skin remodeling, which included higher levels of cellular infiltration in the dermal layer, a histopathological feature frequently reported in atopic dermatitis[
Even though skin has been classically described as a tolerogenic tissue[
Additionally, depilation prompted the upregulation of Il25 and Il33 transcripts in vivo, suggesting a possible relationship with the initiation of T2 responses in the tissue, although detection of these proteins in murine skins cannot be confirmed. The importance of NLRP3 priming to the promotion of allergic sensitization is supported by the fact that mice treated with MCC950 (i.e., lacking a functional NLRP3 route) are unable to develop a strong anaphylactic response against Prup3_complex (Fig. 3B). This result is in accordance with recent reports in the literature which demonstrate that inflammasome activation due to respiratory syncytial virus infection can lead to asthma development in BALB/c mice[
Regarding the effects of the "second hit" (allergen encounter), we found that cutaneous exposure to Prup3_complex induces an immune response based on the recruitment of CD45+ cells to the exposed tissue. This immune infiltration is exclusively a result of allergen presence, since depilated untreated animals present CD45+ infiltration levels analogous to non-depilated control mice. Moreover, we also have shown that this infiltration is not limited to the skin but spread through different mucosal locations. Systemization of immune reactions initiated locally in the skin has been reported not only in food allergy[
On the other hand, when we isolated ILC2s from peripheral blood of patients allergic to Pru p 3, we observed that they present significantly higher levels of these cells in circulation than healthy control donors. This observation is in accordance with previous reports of peripheral ILC2s growing in number after intranasal challenge and during pollinic season in cat and Phleum pratense allergic patients, respectively[
Moreover, in an impaired barrier by thioglycolate addition, Prup3_complex is able to induce the overexpression of the receptor S1P1 in the surface of ILC2s. Given the structural resemblance between Pru p 3 ligand and this immune mediator[
Considering all these observations, the following model is proposed to explain a possible timeline of allergic sensitization. As shown in Fig. 7, allergen sensitization through the skin would require two necessary hits: (
Graph: Figure 7 Proposed model for skin-mediated allergy sensitization and observations made with human ILC2s.
Recombinant Pru p 3 was produced in Pichia pastoris as previously described[
For the skin permeability assay, 24-week-old female C57BL/6J mice (Charles River, L'Arbresle, France) were anesthetized by inhalation of isoflurane and abdominal fur was removed just one time with depilatory cream (Depilated) or scissors, without damaging the skin (Non-depilated). Immediately after, the abdomen was briefly washed with warm water and 40 µL of blue toluidine solution 0.1% (Sigma) were added in small droplets in the region. After 5 min, the staining was removed with a dry gauze and the animals were euthanized using a CO
For the effects of depilation over inflammasome priming, 6–8-week-old female BALB/c mice (Charles River) were weekly depilated once per week (for 6 weeks) in the abdomen with depilatory cream and euthanized with CO
For the effects of allergen encounter over cellular infiltration and RTqPCR analyses, 6–8-week-old female BALB/c mice were weekly sensitized once per week (for 6 weeks) epicutaneously with the allergen (100 µg of Pru p 3 or Prup3_complex; Fig. 3A) after depilation. The depilation protocol we followed was analogous to the one described in the prior paragraph. One week after the last sensitization, mice were provoked with an intraperitoneal injection of 100 µg of Pru p 3. Body temperature was measured before and 15 min after challenge by rectal thermometer (VWR). Mice were euthanized in a CO
In order to evaluate the relevance of NLRP3 priming towards the induction of allergic sensitization, we repeated the sensitization with Prup3_complex in BALB/c mice, but 1 week prior to the first sensitization protocol (days − 7 to 0) we started to treat them intraperitoneally as previously reported[
All animals were randomly assigned and separated into each group immediately after its arrival to our facilities. They were fed ad libitum with a Pru p 3-free diet (Labdiet Eurodent Diet 22% pellet for rodents). All the procedures were carried at the IIS- Fundación Jiménez Díaz (FJD, Madrid, Spain) with the permission of the Institutional Animal Care and Use Committee from the Community of Madrid (Ref. PROEX 392/15), under the current legislation (European Union Directive 2010/63/EU) and in compliance to ARRIVE guidelines.
Sections (7 µm) of the paraffin-embedded organs were cut using a microtome (Leica RM1235, Wetzlar, Germany). After deparaffination and rehydration, hematoxylin/eosin (H&E) staining was performed following provider's instructions (Sigma-Aldrich, St. Louis, MO, USA) and samples were mounted using DPX (Sigma-Aldrich). Immunofluorescence (IF) assays were performed as previously described[
mRNA was isolated from frozen skin samples using the PureLink RNA Micro Kit (ThermoFisher). mRNA samples from non-depilated (n = 4), depilated (n = 4) and depilated + sensitized (n = 2) animals were isolated, and cDNA was obtained with the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher), following provider's instructions. qPCR was performed from each individual sample using Bio-Rad's PrimePCR Pathways (Atopic Dermatitis M384; Hercules, CA, USA), following provider's instructions. Each assay was repeated twice. Additionally, gene expression from individually selected genes was analyzed using the FastStart Universal SYBR Green (ROX) (Roche) as previously described[
A431 cells (ATCC CRL-1555; human epidermal keratinocytes) or HaCaT cells (CVCL 0038; human epidermal keratinocytes) were seeded into 24-well plates (Costar, New York, NY, USA) and grown in supplemented DMEM. Once confluence was reached, cells were incubated with growing concentrations of sodium thioglycolate (
Additionally, 6 h after the last thioglycolate addition cells were lysed and homogenized with GIT extraction buffer (pH 7; 4 M guanidine isothiocyanate, Sigma-Aldrich; 25 mM sodium citrate, Sigma-Aldrich; 0.5% sarcosyl, Sigma-Aldrich; 0.1 M 2-mercaptoethanol, Carl Roth, Germany). RNA was purified by phenol:chloroform extraction and precipitated with ethanol. cDNA was obtained and qPCR was performed as previously described in the "Gene expression by qPCR" section. Gapdh was used as an endogenous gene to normalize the results. Alternatively, intracellular protein content was isolated by incubation for 10 min (4 °C) with 0.5 mL of RIPA buffer (20 mM Tris–HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.1% SDS) and EDTA-free protease inhibitor cocktail (Roche). After that, samples were centrifuged for 10 min (4 °C) at 10,000g and supernatants were collected. Equal amounts of protein were loaded into 15% SDS-PAGE gels and transferred to PVDF membranes. After blocking O/N with Casein Blocking Buffer (Sigma), Western blot was performed with an anti-caspase-1 (1:500; 22915-1-AP) antibody O/N, followed by extensive washing with PBS-Tween (0.05%) and 1 h incubation with anti-rabbit-HRP (Sigma, 1:30000). The reaction was revealed using Thermo Scientific's Super Signal West Pico PLUS Chemiluminescent Substrate for 4 min (RT) and GE Healthcare's Amersham Hyperfilm ECL chemiluminescence films. Uncropped images can be found in Supplementary Fig. 7.
Patients with a clear clinical history of peach allergy and healthy donors were voluntarily recruited and peripheral blood was donated at the FJD Hospital (Madrid, Spain). All individuals were included in the research after providing informed consent. All experimental protocols were conducted in accordance with the latest revision of the Declaration of Helsinki after being approved by the ethical committee from Universidad Politécnica de Madrid (LILIPAL_BIO2017-84548-R).
Peripheral blood mononuclear cells (PBMCs) from each subject were isolated using a Lymphoprep density gradient and ILC2s were purified using STEMCELL's EasySe Human ILC2 Isolation Kit (STEMCELL Technologies, Grenoble, France), following provider's instructions.
ILC2s were seeded into 96-well plates (Costar) at a concentration of 4000 cells/mL and cultured for 96 h in supplemented RPMI medium in the presence of Prup3_complex (5 ng/µL) or only Pru p 3 (5 ng/µL) or Pru p 3 ligand (0.5 ng/µL), respectively. After, cells were incubated for 1 h at RT with specific antibodies against sphingosine-1-phosphate receptor type 1 (S1P1; 1:50; FAB7089P PE-conjugated; R&D Systems) and CD86 (1:50; BU63 APC-conjugated; ImmunoTools). Samples were analyzed using a BD Accuri cytometer. Unstained samples were used as background control.
To simulate the two-hit allergic sensitization hypothesis in vitro, we developed a 3D skin model based on 24-well Transwell plates as previously reported, with slight modifications[
Statistically significant differences were analyzed by GraphPad6 (GraphPad Software Inc., La Jolla, CA, USA) using Mann–Whitney test, except where noted. In all cases, P values < 0.05 were considered significant.
This research was funded by the Spanish Government (MINECO, grant BIO2017-84548- R); Instituto de Salud Carlos III (ISCIII) co-founded by FEDER Thematic Networks and Cooperative Research Centers: ARADYAL (RD16/0006/0003; RD16/0006/0013) and the Community of Madrid (FOODAL-CM_S2018/BAAA-4574). DPC was funded by Universidad Politécnica de Madrid and Banco Santander with a predoctoral Programa Propio grant. ARS was funded from the Community of Madrid included in the project FOOD-AL (FOODAL-CM_S2018/BAAA-4574). GHR was funded by European Comission (H2020-NMBP-X-KET-2017-768641—AllerScreening). JTA was funded from Instituto de Salud Carlos III (ISCIII) co-founded by FEDER Thematic Networks and Cooperative Research Centers: ARADYAL (RD16/0006/0003).
A.D.P., J.T.A., M.G.A. and D.P.C. designed the study. D.P.C., Z.G.K., A.Y.M., G.H.R. and J.T.A. performed experimental assays. Data was analysed by D.P.C., Z.G.K., J.T.A., V.E., and A.D.P. Manuscript was written and revised by D.P.C., J.T.A., A.D.P. and A.R.S. All authors reviewed and approved the manuscript.
The authors declare no competing interests.
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By Diego Pazos-Castro; Zulema Gonzalez-Klein; Alma Yuste Montalvo; Guadalupe Hernandez-Ramirez; Alejandro Romero-Sahagun; Vanesa Esteban; Maria Garrido-Arandia; Jaime Tome-Amat and Araceli Diaz-Perales
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