Intestinal epithelial cell barrier dysfunction and elevated Angiopoietin-like 4 identified in orally-susceptible peanut allergy model

To the editors:

Peanut remains the number one cause of death due to food-related anaphylaxis. Co-factors (e.g., febrile illness, vigorous exercise) that modify the severity of food allergic reaction are known to increase intestinal epithelial cell (IEC) barrier permeability (1), and patients with peanut allergy have higher serum levels of a major peanut allergen (Ara h 6) after peanut ingestion (2). Yet, we do not understand basic interactions between peanut and IECs.

Orgel, et al., identified the Collaborative Cross CC027/GeniUnc (“CC027”) mouse strain as susceptible to peanut allergy by intragastric administration without a Th2-skewing adjuvant (3). We asked whether peanut allergy in CC027 mice was associated with increased IEC barrier permeability. Peanut-sensitized and -challenged (Peanut/Peanut) CC027 mice (3) demonstrated a significant decrease in rectal temperature (Fig. 1A), a measure of reaction severity, from baseline, whereas Peanut/Peanut C3H mice showed no difference in rectal temperature from baseline (Fig. 1B). PBS-sensitized and peanut-challenged (PBS/Peanut) CC027 mice showed a significant but mild and transient temperature decrease after 15 minutes (Fig. 1A); this could be due to the direct effect of peanut on IEC tight junctions (4). We saw similar significant temperature decreases from baseline when we challenged naïve CC027 mice with egg or beef, but this was significantly less than in naïve CC027 mice challenged with peanut (Fig. 1C). Notably, comparable allergen doses are not known. A significant proportion of Peanut/Peanut CC027, compared to C3H, mice developed watery diarrhea during challenge (Fig. 1D), although no differences in intestinal motility between similarly-treated strains were seen (Fig. 1E). Serum levels of a major peanut allergen (Ara h 2) 30 minutes after peanut challenge positively correlated with maximum decrease in rectal temperature in Peanut/Peanut CC027 (Fig. 1F) but not C3H mice (Fig. 1G). After intragastric FITC-dextran 4 kDa (FD4) administration during challenge, plasma FD4 levels in Peanut/Peanut CC027 mice were significantly higher compared to that of PBS/Peanut CC027 mice at 30, 60, 120, and 240 minutes after challenge (Fig. 1H), but not in similarly treated C3H mice (Fig. 1I). Decreases in rectal temperature correlated with peak plasma FD4 concentration in CC027 (Fig. 1J) but not C3H (Fig. 1K) mice. Mast cells in CC027 mice robustly respond to IgE-dependent stimulation (9), which could contribute to the increased intestinal permeability in sensitized CC027 mice; however, the tempered effect of peanut on intestinal permeability in non-sensitized CC027 mice (Figs. 1C, H) may be IEC-intrinsic.

Figure 1. Peanut allergy in CC027 mice and pediatric patients is associated with increased IEC permeability.

Mice received intragastric peanut or PBS once weekly for 4 weeks, followed by challenge with peanut or PBS in Week 5. A-B: Change in pre-challenge rectal temperature was measured in PBS/Peanut (closed circle/dashed line, n=12 CC027 mice), Peanut/PBS (open shape/solid line, n=8 CC027 and 5 C3H mice), and Peanut/Peanut (closed shape/solid line, n=18, 10) CC027 (A) and C3H/HeJ (B) mice. One sample t test compared to baseline, 0; **p<0.005; ***p<0.001; ****p<0.0001. (C) Non-sensitized CC027 mice were challenged with food allergens and change from baseline rectal temperature measured (n=3, 18, 18, 12 for respective groups PBS, Peanut, Egg, and Beef). One sample t test compared to baseline, 0; ****p<0.0001 for Peanut at 15, 30, and 45 minutes; **p<0.005 for Egg at 15 minutes; **p<0.005 for Beef at 15 and 45 minutes; and *p<0.05 for Beef at 30 minutes. For comparisons to Peanut: multiple unpaired t tests with Welch correction and Holm-Šídák’s multiple comparisons test; a: adj p<0.05 versus PBS and Egg; b: adj p<0.05 versus PBS and Beef; c: adj p<0.0005 versus Egg; d: adj p<0.05 versus PBS; e: adj p<0.005 versus Egg. (D) The absence or presence of watery stool was noted in CC027 and C3H mice during peanut challenge. Fisher’s exact test; ****p<0.0001. (E) Ten minutes following challenge with PBS or peanut, mice were given carmine red solution by oral gavage. Twenty minutes later mice were sacrificed and small intestines isolated. Motility was measured as the length from the pyloric junction to the carmine dye front, as a percentage of the total length of the small intestines. Ordinary one-way ANOVA with Tukey’s multiple comparisons test; ***adj p<0.0005. F-G: Serum Ara h 2 levels 30 minutes after challenge were plotted against maximum decrease in rectal temperature. Pearson correlation coefficient r was calculated. (F) CC027 mice (n=9), Pearson r=0.78, p=0.01. (G) C3H mice (n=9), Pearson r=0.49, p=0.18. H-I: Plasma FD4 was measured over time after peanut challenge of PBS- (n=4 CC027 and 5 C3H mice) and peanut-sensitized (n=13, 10) CC027 (H) and C3H (I) mice. Multiple unpaired t tests with Welch correction and Holm-Šídák’s multiple comparisons test; *adj p<0.05. J-K: Peak plasma FD4 concentration versus maximum decrease in rectal temperature for each Peanut/Peanut mouse was used to calculate Pearson r coefficient. (J) CC027 (n=26), Pearson r=0.75, p<0.0001; and (K) C3H (n=7), Pearson r=−0.2, p=0.6. L-M: Jejunal IECs isolated from CC027 mice were grown on collagen-coated Transwell^® inserts. Transepithelial electrical resistance (TEER) was measured across the monolayer on each day of culture, and experiments were performed on cultures at their plateau TEER level (mature monolayer). Jejunal IECs were isolated from PBS/PBS (n=4 CC027 and 2 C3H mice) and Peanut/Peanut (n=6, 4) CC027 and C3H mice. Monolayers were treated with peanut (100 ¼g/mL) in the apical compartment and TEER (L) and apical-to-basolateral FD4 (M) measured over time. Transit of FD4 across the IEC monolayer is expressed as the percentage of basolateral FD4 that had moved across collagen-coated Transwells^® without cells. Experimental duplicates were averaged. Ordinary one-way ANOVA with Šídák’s multiple comparisons test comparing PBS/PBS and Peanut/Peanut within and between strains; *adj p<0.05; ***adj p<0.0005. (N) Food challenge-confirmed peanut allergic patients and age-matched non-peanut-allergic patients were enrolled. Age, gender, and serum peanut-specific IgE were collected. ^AMann-Whitney test; ^BFisher’s exact test; ^COne sample Wilcoxon test. O-Q: Serum was collected from food challenge-confirmed peanut-allergic pediatric patients and age-matched non-peanut-allergic control patients. (O) Serum LPS-binding protein (LBP) levels were measured by ELISA; red bars represent means. One-tailed, unpaired t test with Welch’s correction; p=0.05. (P) Peanut-allergic patients were stratified based on their highest successfully consumed dose during enrollment food challenge (≤25 mg and >25 mg) and serum LBP levels plotted for each group; red bars represent means. Two-tailed, unpaired t test; *p<0.05. (Q) Serum LBP levels were graphed against highest successfully consumed dose to calculate the Pearson correlation coefficient; r=−0.3, p=0.03. R-U: Peanut-sensitized CC027 (R,S) and C3H (T,U) mice were challenged with peanut (mg): 0 (n=10 CC027 and 5 C3H mice), 1 (n=4, 4), 5 (n=4, 4), 10 (n=8, 4), or 12 (n=12, 4). (R,T) Change from pre-challenge rectal temperature was measured. One sample t test compared to baseline, 0; *p<0.05, **p<0.005, ***p<0.0005. (S,U) Post-challenge plasma FD4 levels were measured. Ordinary one-way ANOVA with Tukey’s multiple comparisons test (S) or Welch’s ANOVA with Dunnett’s T3 multiple comparisons test (U), versus 0 mg; *adj p<0.05. V-W: Plasma FD4 levels were measured 30 minutes after challenge with PBS (n=7 CC027 and 5 C3H mice), egg (n=5, 4), walnut (n=5, 4), or milk (n=4, 3) in respectively orally-exposed/sensitized CC027 (V) and C3H (W) mice. Welch’s ANOVA test with Dunnett’s T3 multiple comparisons test versus PBS; **adj p<0.005.

We cultured jejunal IEC “enteroids” derived from CC027 mice in 2-D monolayers. Peanut protein (100 μg/mL) and FD4 were added to the apical side and permeability measured. Monolayer transepithelial electrical resistance (TEER) increases when paracellular permeability decreases. Peanut/Peanut CC027 mouse-derived IECs, but not those from respective C3H mice, showed a significant decrease in TEER (Fig. 1L) and increase in apical-to-basolateral FD4 (Fig. 1M) compared to PBS/PBS CC027 mouse-derived jejunal IECs. Increased IEC permeability can correlate with intestinal mast (5) cell burden; Peanut/Peanut CC027 mice have increased intestinal mast cells (3). However, our culture data suggest an immune cell-independent mechanism by which peanut affects IEC permeability, for instance, through increased secretory cell antigen passages (6), although this hasn’t been explored in our model.

We next sought to determine intestinal permeability in peanut-allergic subjects with challenge-confirmed peanut allergy from the Sublingual Immunotherapy for Peanut Allergy and Induction of Tolerance study (ClinicalTrials.org ID NCT01373242) and in age-matched control patients with undetectable serum peanut-specific and aero-allergen-specific IgE levels (Fig. 1N). During double-blind, placebo-controlled, food challenge, patients ingested increasing doses of peanut protein until they developed challenge-limiting symptoms. The highest successfully consumed dose estimated patient reactivity as a measure of severity. There were no age or gender differences between the groups. We measured serum LPS-binding protein (LBP), a serum marker of IEC permeability. Serum LBP levels were significantly higher in peanut-allergic patients (Fig. 1O). We stratified peanut-allergic patients by their highest successfully consumed dose of peanut; patients who were unable to tolerate more than 25 mg peanut protein (approximately 1/12^th of a kernel) had significantly higher serum LBP levels than patients who were able to tolerate more than 25 mg peanut (Fig. 1P). Serum LBP significantly negatively correlated with the highest successfully consumed dose (Fig. 1Q).

To define the eliciting dose of peanut for a systemic reaction in our murine peanut allergy model, we challenged peanut-sensitized mice with 1, 5, 10 or 12 mg peanut or PBS as a control (“0” time-point). Peanut/Peanut CC027 mouse rectal temperatures and plasma FD4 levels 30 minutes after challenge with 12 mg were significantly decreased from baseline (Fig. 1R) and increased compared to mice challenged with 0 mg (Fig. 1S), respectively; there were increases from baseline rectal temperature in respective C3H mice (Fig. 1T). There was a significant but small increase in plasma FD4 in C3H mice given 12 mg peanut compared to the PBS, 1 mg, and 5 mg groups, which could represent the ability of peanut to alter tight junction proteins (Fig. 1U). Plasma FD4 levels showed wide variation in the mice that received 12 mg peanut, suggesting that other factors may contribute to IEC paracellular permeability during peanut allergy.

To determine the effect of other common food allergens on IEC barrier permeability in CC027 mice, we sensitized mice to proteins from egg, walnut, or milk without adjuvant (7). Thirty minutes after challenge with the respective non-peanut allergens, CC027 mice challenged with egg and milk, compared to PBS, had significantly lower plasma FD4 levels (Fig. 1V). In C3H mice, challenge with none of the non-peanut allergens produced significant changes in plasma FD4 concentration (Fig. 1W). The eliciting dose of different allergens in our model are unknown and affected by both features of the mouse strain and the food’s intrinsic allergenicity. Peanut was the most potent allergen in this model for exploring allergen-associated increases in IEC barrier permeability.

To explore the effects of peanut allergy on CC027 mouse IEC function, RNA from PBS- and peanut-sensitized CC027 mouse-derived jejunal IECs before and after challenge with peanut (30, 60, and 120 minutes) was isolated for RNA-seq (Fig. 2A). Principal components analysis revealed that jejunal IEC transcriptomes from peanut-sensitized mice without challenge and 30- and 120-minutes post-challenge had closer intra- versus inter-group similarity (Fig. 2B). We found 13 differentially expressed genes (DEGs) in jejunal IECs after sensitization (FDR < 0.1), suggesting that sensitization alone does not significantly alter the jejunal IEC transcriptome. We identified 183 (110 down- and 73 up-regulated), 266 (114 down- and 152 up-regulated), and 519 (217 down- and 303 up-regulated) DEGs 30, 60, and 120-minutes post-challenge, respectively (FDR < 0.1) (Fig. 2C). Gene enrichment analysis of the three time points using Ingenuity Pathway Analysis (IPA, Qiagen) showed late enrichment in genes involved in cholesterol biosynthesis but few enriched pathways early in the time course (Fig. 2D).

Figure 2. CC027-derived jejunal IEC transcriptomes reveal angiopoietin-like 4 as a putative feature in peanut allergy.

A-D: RNA-seq was performed with jejunal IECs from PBS- or (A) peanut-sensitized & unchallenged or challenged CC027 mice after 30, 60, or 120 minutes. n=3 per group. (B) PCA plot shows expression profiles of jejunal IECs from peanut-sensitized & unchallenged (black), or challenged mice after 30 (teal), 60 (magenta), or 120 (yellow) minutes. (C) Expression log₂FC of selected genes from individual mice at each time point were compared to peanut-sensitized & unchallenged CC027 mice. n=3 per group. FDR < 0.1. (D) Gene enrichment analysis (Ingenuity Pathway Analysis, Qiagen) showed strong upregulation of cholesterol biosynthesis pathways late after challenge. (E) Time series analysis was performed by assigning expression profiles to each differentially expressed gene over the three time points then clustering based on similarity. (F) Expression profiles based on log₂FC by time point (left) and enrichment analyses of one cluster identified through time series analysis (right) are shown. (G) Whole jejunal tissue expression was confirmed by qRT-PCR for mouse strain and sensitization. Dotted line shows y=1 and all expression values are relative to PBS/PBS CC027 mouse-derived jejunum. n=5 per group. Ordinary one-way ANOVA with Tukey’s multiple comparisons test; *adj p<0.05; **adj p<0.005; ***adj p<0.0005; ****adj p<0.0001. (H) IECs and lamina propria mononuclear cells were isolated from the same peanut-sensitized CC027 mice, and Angptl4 expression was measured by qRT-PCR. Two-tailed, unpaired t test; **p<0.005. (I) RNA was extracted from whole small intestinal tissue segments and Angptl4 expression measured by qRT-PCR. Dotted line shows y=1 and all values are relative to that from PBS/PBS CC027 mouse-derived duodenum. n=5 per group. Two-way ANOVA with Šídák’s multiple comparisons test; *adj p<0.05; **adj p<0.005; ***adj p<0.0005. (J) Representative immunofluorescence of CC027 (right) and C3H (middle) jejuna with anti-Angptl4 (green) and DAPI (blue) nuclear staining. Negative control (left) had no 1° antibody. (K) Serum Angptl4 was measured by ELISA in Peanut/Peanut CC027 (n=8) and C3H (n=7) mice; red bars represent means. Two-tailed, unpaired t test; ****p<0.0001. (L) Serum ANGPTL4 was measured by ELISA in pediatric patients with (n=50) and without (n=24) peanut allergy; red bars represent means. Two-tailed, unpaired t test; **p<0.005.

To determine whether functionally-related genes showed similar expression changes across timepoints, we generated a novel time series analysis. We ranked the top 500 unique DEG (ranked by log₂(Fold-Change) (log₂FC), FDR < 0.1) among the 3 time-points and assigned each gene a profile based on the lines connecting the log₂FC between time-points (Fig. 2E). Cluster analysis identified three clusters with >100 genes that showed similar changes in expression across time-points. Gene Enrichment Analysis using IPA showed predicted activation of cell survival and proliferation pathways early, and predicted activation of cholesterol biosynthesis pathways late post-challenge (Fig. 2F).

To generate a targeted gene list, we identified unique genes from the IPA enriched pathways that were predicted to be activated or suppressed (|activation z-score| > 1). From these unique genes, we identified 16 with high expression and log₂FC compared to unchallenged mice, which were confirmed by quantitative RT-PCR from PBS/PBS and Peanut/Peanut CC027 and C3H mouse-derived jejunum (Fig. 2G). The locations of four of the 16 genes (Angptl4, Spdef, Fkbp5, and Plat) lie within genomic regions where CC027 has highly divergent haplotypes compared to C3H, suggesting these regions contribute to susceptibility to peanut sensitization by the oral route in the CC027 mice. Expression of angiopoietin-like 4 (Angptl4) was 1000-fold higher in CC027-derived, compared to C3H-derived, jejuna, suggesting it was a putative relevant feature of peanut allergy in this model.

Angptl4 is a secreted glycoprotein regulated by microbiota, fasting, fatty acids, and hypoxia (8). It is proteolytically cleaved extracellularly into two products: the N-terminal coiled-coiled domain (nAngptl4) and C-terminal fibrinogen-like domain (cAngptl4). Full-length Angptl4 and nAngptl4 bind and inhibit lipoprotein lipase, altering lipid metabolism. cAngptl4 mediates vascular permeability and wound healing. Angptl4 is primarily produced in the liver, adipose tissues, and to a lesser extent in the intestinal epithelium; its role in the intestines is poorly understood. Angptl4 expression was significantly higher in IECs compared to lamina propria mononuclear cells from the same peanut-sensitized CC027 mice (Fig. 2I, left) and in jejuna from PBS- and peanut-sensitized CC027 and C3H mice (Fig. 2I, right). Immunofluorescence of jejuna from peanut-sensitized CC027 mice revealed higher expression of Angptl4 protein compared to that from C3H mice (Fig. 2J).

We found that serum Angptl4 was significantly higher in Peanut/Peanut CC027, compared to C3H, mice (Fig. 2K). Likewise, serum ANGPTL4 was significantly higher in pediatric patients with challenge-confirmed peanut allergy compared to age-matched control patients (Fig. 2L). Our findings in the CC027 mouse model and peanut allergic patients reveals unique mechanisms of peanut allergy.

Additional information about study methods and findings are available in the following repository: https://doi.org/10.1101/2021.07.14.452416.

Key Messages:

• The genetically-susceptible CC027/GeniUnc mouse and peanut-allergic pediatric patients demonstrate increased intestinal epithelial cell (IEC) permeability.

• RNA-seq of jejunal IECs from peanut-allergic CC027 mice highlights cell survival and metabolic pathway changes.

• Angiopoietin-like 4 was identified as a putative feature of peanut allergy susceptibility in CC027 mice.

Abbreviations

Angptl4

angiopoietin-like 4

C3H

C3H/HeJ mouse strain

cAngptl4

c-terminal fibrinogen-like domain of Angptl4

CC027

CC027/GeniUnc mouse strain

DEGs

differentially expressed genes

FD4

FITC-dextran 4 kDa

IPA

Ingenuity Pathway Analysis (software, Qiagen)

IECs

intestinal epithelial cells

log₂FC

log₂(Fold-Change)

LBP

LPS-binding protein

nAngptl4

n-terminal coiled-coiled domain of Angptl4

PBS/PBS

PBS-sensitized and PBS-challenged

PBS/Peanut

PBS-sensitized and peanut-challenged

Peanut/PBS

peanut-sensitized and PBS-challenged

Peanut/Peanut

peanut-sensitized and peanut-challenged

TEER

transepithelial electrical resistance