The allergy-promoting capacity of the selected barrier disruptors was assessed in a food allergy mouse model where mice were repeatedly exposed to the barrier disruptors together with a food allergen. Two short-term mouse experiments were also carried out, to investigate the early effects on the gut barrier after a single exposure to the same barrier disruptors with (exp. 1) or without (exp. 2) the allergen.
The levels of the alarmins TSLP, IL-25 and IL-33 and the IL-33 receptor ST2 were determined in intestinal tissue homogenate supernatants (see below). ST2 was also analyzed in serum, as a measure of altered barrier function (40, 41). As markers of altered intestinal barrier permeability, serum concentrations of the stable peanut allergen Ara h2 and FABP2 were examined (42). As markers of lymphoid tissue immune responses, the cytokines TNF-α, IFNγ, IL-1β, -2, -4, -5, -6, -10, -13, and -17 were assessed after ex vivo stimulation of cells from the mesenteric lymph nodes (MLNs), Peyer’s patches (PP) or spleen. Cells were stimulated with ConA to activate T cells or LPS to activate B cells and monocytes.
Test solutions
We tested the environmental contaminants with known or suspected barrier disrupting capacities, cholera toxin (CT), deoxyvalenol (DON) and house dust mite (HDM) (9, 10, 21). We defined CT, DON and HDM as known barrier disruptors, and the pesticide Glyphosate (GLY) as a suspected barrier disruptor. The doses for CT (EMD Biosciences Inc., San Diego, CA, USA), DON (Sigma Aldrich, St. Louis, MO, USA), HDM (Stallergenes Greer, London, UK), and GLY (SUPELCO, Bellefonte, PA, USA), were all based on literature where exposure to these doses gave biological effects (doses and references are stated in Table 1). Phosphate buffered saline (PBS, 10 mM 0.9% NaCl pH 7.4) was given to groups as negative control and was used as vehicle. Peanut extract (PE, Stallergenes Greer, London, UK) was used as the allergen, as peanut is one of the most potent food allergens and a widely used ingredient in foods.
Table 1. Test solutions, doses (all oral exposures) and references used to choose dose levels
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Test compounds and dose per animal (in 250 µL PBS)
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Effects demonstrated at this dose level in earlier studies
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exp. 1*
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exp. 2*
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Food allergy model#
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PBS: vehicle ctr.
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PBS
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PBS
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PBS
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-
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PE wo adjuvant: neg. ctr.
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1 mg PE
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1 mg PE
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1 mg PE
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Peanut food allergy (with CT)
(7)
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CT:
pos. adjuvant ctr.
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15 µg CT
1 mg PE
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15 µg CT
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15 µg CT
1 mg PE
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Increased food allergy (9)
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DON
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100 µg DON
1 mg PE
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100 µg DON
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100 µg DON
1 mg PE
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Increased food allergy (9)
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HDM
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20 µg HDM
1 mg PE
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20 µg HDM
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20 µg HDM
1 mg PE
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Increased colonic permeability (19)
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GLY
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20 µg GLY
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20 µg GLY
1 mg PE
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Effects on gut microbiome (43)
Effects of Roundup® (44)
Effects on behavior (24, 45)
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CT wo allergen: neg. ctr.
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15 µg CT
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Neg. ctr.
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*single exposure
# repeated exposures
§Roundup® herbicide contains approximately 40% GLY (Monsanto Company, St. Louis, MO, USA).
Abbreviations: Pos positive, Neg negative, ctr control, wo without
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Animals
Female C3H/HeOuJ mice were purchased from Charles River Laboratories (Sulzfeld, Germany), and Jackson Laboratory (Bar Harbor, ME, USA) for exp. 2 due to supply issues. The mice were 4 to 5 weeks old at arrival and randomly allocated in groups and housed on Nestpack bedding (Datesand Ltd, Manchester, UK) at room temperature of 21±2°C, humidity of 55±10 % and exposed to 12 hours light/dark cycle. The mice were given pelleted feed (RM1, SDS, Essex, UK) and tap water ad libitum, and were acclimatized for at least one week before dosing. The experiments with live animals were performed in conformity with the laws and regulations in Norway and were approved by the Experimental Animal Board under the Ministry of Agriculture in Norway (FOTS ID 11121).
Food Allergy Model
Immunization, challenge and anaphylaxis assessment
The present peanut food allergy model was based on the mouse model for lupine allergy (Vinje, Larsen (8), and the mouse model for peanut anaphylaxis developed by Li, Serebrisky (7). Twelve mice in each of the 7 groups were immunized i.g. at day 0, 1, 2, 7, 21, 28 and 35 with PE alone or mixed with the different barrier disruptors, including a group given CT alone. CT with PE was used as a positive control for food allergy development as it has strong adjuvant effects (46), while negative control groups were the vehicle-exposed (PBS) and non-sensitized mice (PE or CT alone).
A challenge of a high dose of PE was given at the end of the experiment to induce anaphylaxis and thereby to confirm clinically relevant signs of food allergy. Due to practical considerations, half of the mice in each group were challenged at day 42 and 43. Prior to challenge, the mice were weighed, 100 µl blood sampled from the vena saphena in Microvette tubes without heparin (Sarstedt, Nümbrecht, Germany) and serum stored at -20°C for detection of Ara h2 specific IgE (see below). The mice were challenged with an intraperitoneal (i.p.) injection of 5 mg PE in 250 µL of PBS (7). Due to dilution errors on one of the days, challenge with 2.5 mg PE in 250 µL PBS was given to approximately half of each groups of mice. Temperature measurements (at 0, 15 and 30 minutes) and clinical anaphylaxis score were recorded and reported as previously described (35). After 30 minutes, all challenged mice were exsanguinated, and serum was collected. We did not manage to collect serum from four and two mice in the the PE+CT and GLY+PE groups, respectively. Serum was prepared and stored at -20°C until detection of total IgE and the anaphylaxis marker mMCP-1 (see below). Spleens were collected and kept in HBSS containing 2 % fetal bovine serum and 1 % penicillin/streptomycin on ice.
In blood collected after challenge and exsanguination, levels of mMCP-1 were determined using mouse CPT-1 (mMCP-1) ELISA kit according to the manufacturer’s protocol (ELISA Ready-SET-Go! ® Invitrogen by Thermo Fisher).
Spleen cell preparation, PE stimulation and detection of released cytokines
Spleen yields an abundant cell number compared to LNs and were chosen to assess expression of cytokines reflecting systemic changes to the immune system reported in food allergy models (7, 8). Cells counts are presented as the total cell number (106) per animal. Cell-suspensions were adjusted to 3 x 106 cells/mL in culture medium (RPMI 1640, Gibco ®, Thermo Fisher Scientific) containing 10% FBS and 1% penicillin/streptomycin). In a total volume of 200 µl per well in Microtest plates (Microtest Plate 96 Well, C, Sarstedt). Spleen cells were stimulated for 5 days in vitro with PE at a final concentration of 1 mg/m to assess the cytokine responses mainly in allergen-specific cells. After incubation at 37 °C in 5% CO2 for 5 days, the plates were centrifuged at 180 × g for 5 min at RT and the supernatants were collected and stored at −80 °C.
After thawing, supernatants were collected and stored, and TNF-α, INF-γ, IL-1β, -2, -6, -10, -13, and -17 cytokine concentrations were detected using BD Cytometric bead array (CBA) Mouse Soluble Protein Flex Sets, measured on a BD LSR II flow cytometer. The cytokines were analyzed using the FCAP Array software (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol.
Measures of peanut sensitization – total IgE and Ara h2-specific IgE
As described for other food allergen extracts (35), development of ELISAs for PE-specific IgE was challenging (unpublished data) due to high background signals from the PE extract and costs if combining all recombinant peanut allergens. Thus, as predictors of peanut allergy sensitization, the levels of the peanut component Ara h2 specific IgE and total IgE in serum were analyzed. IgE specific to the most important peanut allergen Ara h2 is considered a reliable marker of severe peanut allergy in humans (47, 48). Total IgE are reflecting specific IgE in animal models with controlled allergen exposures (35, 49).
To assess sensitization, Ara h2-specific IgE was assessed in serum samples collected immediately prior to challenge, since specific IgE passes from the blood to the tissues during anaphylaxis. The ELISA assay was developed in our lab. 100 µL of monoclonal rat anti-mouse IgE (2 µg/mL, Experimental Immunology unit, University of Louvain, Brussels, Belgium) in 0.05 M bicarbonate buffer (pH 9.6) was added in each well plate (Nunc MaxiSorpTM flat-bottom 96 well plate, Thermo Fisher Scientific) and incubated 1 hour at 21 °C following an over-night incubation at 4°C. The plates were then washed five times with TBS/Tween (50 mM Tris/HCL-buffer pH 8.0 with 0.05% Tween 20) using an automatic plate washer (405 LS microplate washer, Biotek), blocked with 300 µL of 4% BSA (Bovine Serum Albumin A7930-100G, Sigma Aldrich) in PBS (BSA/PBS) and incubated for 1 hour at 21°C. Plates were washed as previously described. 100 µL of diluted Ara h2 specific IgE from a mouse serum pool containing anti-Ara h2 from PE-immunized mice previously prepared in the lab (The Norwegian Institute of Public Health) was added as doublets for a standard curve. Dilutions were 1:1, following a 2-fold titration to 1:64 in 4% BSA/PBS. 4% BSA/PBS in doublets were added as blanks. Serum samples were diluted 1:5 with 4% BSA/PBS. 100 µL of diluted serum sample was added per well and incubated at 4°C over-night. Plates were then washed as earlier described and 100 µL of biotin-labeled Ara h2 (3 µg/mL, Biotin Natural Ara h2, B1-AH2-1, Indoor Biotechnologies) in 4% BSA in PBS was added. Plates were incubated 1 hour at 21 °C and then washed. Poly-HRP-strepatividin (Poly-HRP-strepatividin N200, Thermo Fisher Scientific) was diluted 1:40 000 in 4% BSA/PBS and 100 µL added to each well. Plates were incubated for 1 hour at 21 °C, washed, before addition of 100 µL of TMB solution (TMB Stabilized Chromogen SB01, Life Technologies Europe B.V., Bleiswijk, Netherlands) and incubated at RT for 15 minutes. The reaction was stopped by 50 µL of sulfuric acid 95-97% (Merck Milipore, Burlington, MA, USA) in each well. Plates were read at 450 nm in a BioTek microplate-reader.
Serum levels of the allergic sensitization marker total IgE was determined using IgE Mouse uncoated ELISA kit according to the manufacturer’s protocol (Invitrogen by Thermo Fisher Scientific). This was assessed in serum collected after challenge and exsanguination due to limited amounts of serum from the sample collected before challenge.
Short-term experiments
Experiment 1: early effects 4, 24 and 48 hours after a single exposure
Five groups of mice, 5 in each group, were exposed once by intragastric (i.g.) gavage to 250 µL of test solutions according to table 1. At 4, 24 and 48 hours after exposure, the mice were anesthetized with isoflurane gas administered as a 3.5% mixture with medical O2 in a coaxially ventilated open mask and exsanguinated by heart puncture before cervical dislocation. The collected blood samples were kept for 1-3 hours at RT before the samples were prepared and stored at – 80 °C until further use.
The mesenteric lymph nodes (MLNs) were excised and kept on ice in 5 mL Meinecker tubes with 1 mL HBSS (Hanks’ Balanced Salt Solution, Gibco® by Thermo Fisher Scientific, Waltham, MA, USA) containing 2% fetal bovine serum (FBS superior, Biochrom, Cambridge, UK) and 1% penicillin/streptomycin (10 000 units/mL penicillin, 10 mg/mL streptavidin, PAA Laboratories Inc, Etobicoke, ON, Canada), to ensure cell viability.
The small intestine was excised, and the duodenum and ileum were placed on cold metal plates to minimize degradation of proteins. The duodenum was defined as the top 7 cm of the small intestine closest to the stomach and the ileum as the lower 7 cm closest to the caecum. Duodenum and ileum were flushed thoroughly with cold PBS using a syringe, and visceral adipose tissue was removed. Two 1 cm sections from each of the 7 cm segments were snap frozen in 1.8 ml cryo tubes in liquid nitrogen and stored at – 80 °C until homogenization.
Homogenization of intestinal tissue
Immediately after thawing the intestinal samples, the 1 cm pieces of the 2 cm intestinal segments were weighed in 1.5 mL tubes (Micro Tubes, Brand Scientific Equipment Pvt. Ltd., Wertheim, Germany). The weights of 1 cm duodenum and ileum were noted and 0.6 mL of RIPA lysis buffer with 0.04% protease-inhibitor was added to the tubes to avoid the tissue from drying and to reduce protein degradation (9).
The tissue samples were transferred to 2 mL CK28-R tubes containing ceramic beads (Bertin Instruments) and homogenized using Precellys 24 tissue homogenizer (Bertin Instruments) at 6000 rpm (2x30 seconds, 30 seconds pause). After homogenization, the tubes were left to cool on ice for up to 30 minutes to diminish frothing. Samples were centrifuged at 1970 x g for 5 minutes, and the supernatants collected and stored at −80 °C until analysis.
ELISA analysis
Samples from serum or tissue homogenate supernatants were analyzed using ELISA kits according to the manufacturer's protocol; TSLP, IL-25, IL-33, and the IL-33 receptor ST2 (Invitrogen, Thermo Fisher Scientific), FABP2 (MyBioSource, San Diego, CA, USA) and Ara h2 (Indoor biotechnologies, Charlottesville, VA, USA). The Ara h2 concentration was determined using an anti-peroxidase-conjugated Fab fragment for detection (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). Optical density (OD) values were measured using a Microplate Reader (Elx808 Absorbance Reader, BioTek, Shoreline, WA, USA) with software Gen5TM (BioTek). Protein concentrations were determined based on a standard curve generated on each microwell plate provided in the kits. For results from the intestinal homogenates, all concentrations were normalized by dividing by the respective tissue weight.
Lymph node cell preparation, stimulation and determination of cytokine release
Preparation of MLNs was performed as described for spleen cells. To assess the cytokine responses, MLN cells were stimulated for 48 hours with either 5 µg/mL ConA (Concanavalin A from Canavalia ensiformis, Sigma-Aldrich), or 10 µg/mL LPS (lipopolysaccharides from Escherichia coli 026: B6, Sigma-Aldrich), or culture medium alone. Supernatants were collected and stored, and TNF-α, IFNγ, IL-1β, -5, -6, -10, -13 and -17 cytokine concentrations were determined as described in the food allergy model.
Experiment 2: effects 4 and 24 hours after a single exposure
In exp. 2, mice were exposed to the same test-solutions as in exp. 1, as well as the pesticide GLY, but without co-exposure to PE (Table 1). All adjustments and changes in exp. 2 were based on results from exp. 1. Since few exposure-related changes were observed at the time-point 48 hours, only the two earliest time-points 4 and 24 hours were chosen. The number of mice was increased to 8 per group at each time-point.
Furthermore, the whole length (7 cm) of intestinal tissue was used for homogenization. In addition to MLNs, up to seven PPs, primarily sampled from the ileum were collected, but also from the jejunum and duodenum if needed. PPs from each group were pooled, processed and prepared as described for MLNs. Cytokine release from stimulated MLNs and PPs were determined as described for exp. 1. However, since the levels of the cytokines IL-17, -10 and -5 were below the detection limit in exp. 1, these were excluded in exp. 2, while assessment of IL-2 and the Th2 cytokine IL-4 was included.
Due to low intestinal levels of FABP2 and Ara h2 and low levels of IL-25 in both intestines and serum samples in exp.1, these analyzes were excluded in exp. 2. All ELISA analyzes in exp. 2 were incubated overnight on microwell plates at +4°C to increase the sensitivity.
Statistical analysis
For most of the endpoints, the assumptions of equal variance and a normality distribution in our data sets were violated, thus non-parametric statistics were used: A Kruskal-Wallis test was initially performed, and if a p-value ≤ 0.05 was detected, we conducted a Dunn’s multiple comparison test to identify which groups were different. Correlation of anaphylactic score and body temperature drop in the food allergy model was performed by Spearman rank-order correlation test. All analyzes were performed in GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, CA, USA).