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. 2023 Feb;53(2):210-215.
doi: 10.1111/cea.14248. Epub 2022 Nov 6.

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

Erin C Steinbach  1   2   3 , Johanna M Smeekens  3 , Satyaki Roy  2   4 , Takahiko Toyonaga  2 , Caleb Cornaby  5 , Layna B Perini  2   6 , Ana E Berglind  2   4 , Michael D Kulis  3 , Edwin H Kim  3 , Martin T Ferris  4 , Terrence S Furey  2   4   6 , Arvil Wesley Burks  3 , Shehzad Z Sheikh  2   4   6
Affiliations

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

Erin C Steinbach et al. Clin Exp Allergy. 2023 Feb.
No abstract available

Keywords: allergy severity; angiopoietin-like 4; angptl4; barrier permeability; food allergy; intestinal epithelial cell; intestinal permeability; peanut allergy.

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Conflict of interest statement

Conflict of Interest

The authors have declared that no conflicts of interest exist.

Figures

Figure 1.
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.
Figure 2.
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.
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