Esophageal IgG4 levels correlate with histopathologic and transcriptomic features in eosinophilic esophagitis

Abstract

Background: Recent data associate eosinophilic esophagitis (EoE) with IgG4 rather than IgE, but its significance and function have not been determined. Our aims were to measure esophageal IgG4 levels and to determine functional correlations as assessed by histologic and transcriptome analyses.

Methods: This case-control study included pediatric subjects with EoE (≥15 eosinophils/HPF) and non-EoE controls. Protein lysates were analyzed for IgA, IgM, and IgG1-IgG4 using the Luminex 100 system; IgE was quantified by ELISA. Esophageal biopsies were scored using the EoE histology scoring system. Transcripts were probed by the EoE diagnostic panel, designed to examine the expression of 96 esophageal transcripts.

Results: Esophageal IgG subclasses, IgA, and IgM, but not IgE, were increased in subjects with EoE relative to controls. The greatest change between groups was seen in IgG4 (4.2 mg/g protein [interquartile range: 1.0-13.1 mg/g protein] vs 0.2 mg/g protein [0.1-0.9]; P < .0001). Tissue IgG4 levels correlated with esophageal eosinophil counts (P = .0006); histologic grade (P = .0011) and stage (P = .0112) scores; and IL4, IL10, IL13, but not TGFB1, expression and had strong associations with a subset of the EoE transcriptome. Esophageal IgG4 transcript expression was increased and correlated with IgG4 protein levels and IL10 expression.

Conclusion: These findings extend prior studies on IgG4 in adult EoE to the pediatric population and provide deeper understanding of the potential significance and regulation of IgG4, demonstrating that IgG4 is a relevant feature of the disease; is closely related to esophageal eosinophil levels, type 2 immunity and T regulatory cytokines; and is likely produced locally.

Keywords: IgG4; eosinophilic esophagitis; eosinophilic oesophagitis; histology; pediatric; transcriptome.

Figure 1

Esophageal tissue IgG4 content. The data are expressed as milligrams of IgG4 per gram of total protein (A) and as a fraction of total IgG (B). Each data point represents the mean IgG4 measurement from an individual distal biopsy from 17 biologically independent subjects. The lines represent the median and interquartile range for each group.

Figure 2

Esophageal tissue IgG4 content and histologic features of EoE. (A) Correlation between esophageal peak eosinophil count (peak eosinophil count/HPF) and esophageal tissue homogenate IgG4 content (mg IgG4 per gram of total protein) in subjects with EoE. Each data point represents the mean IgG4 measurement from an individual distal biopsy from 17 biologically independent subjects. The lines represent the standard deviation for each data point. (B) Correlation between histologic grade (as a fraction of the total histologic score) and esophageal tissue homogenate IgG4 content (mg IgG4 per gram of total protein) in subjects with EoE. (C) Correlation between histologic stage (as a fraction of the total histologic score) and esophageal tissue homogenate IgG4 content (mg IgG4 per gram of total protein) in subjects with EoE. (D) A heat map was generated demonstrating the degree of correlation between esophageal tissue IgG4 content and each of the histologic features by grade and stage. Red indicates a higher degree of correlation (closer to Spearman ρ of 1.0) and blue represents a lower degree of correlation (closer to a Spearman ρ of zero). The magnitude of the color change is proportional to the correlation. Eosinophil inflammation (EI) was evaluated using peak eosinophil counts obtained by counting eosinophils in the most densely inflamed HPF. Additional features were basal zone hyperplasia (BZH): >15% of the total epithelial thickness; eosinophil abscess (EA): solid mass of intraepithelial eosinophils; eosinophil surface layering (SL): linear alignment of eosinophils parallel to the epithelial surface; dilated intercellular spaces (DIS): spaces around squamous epithelial cells that exhibit intercellular bridges; surface epithelial alteration (SEA): surface epithelial cells that exhibit altered tinctorial properties, manifest as dark red staining, with or without intraepithelial eosinophils; dyskeratotic epithelial cells (DEC): individual cells with deeply eosinophilic cytoplasm and hyperchromatic nuclei; lamina propria fibers (LPF): thickened connective tissue fibers in the lamina propria.

Figure 3

Esophageal tissue IgG4 content and cytokine mRNA expression in the esophageal mucosa. Correlations between mRNA expression of (A) IL4, (B) IL13, (C) IL10, and (D) TGFB1 and esophageal IgG4 content (mg IgG4 per gram of total protein) in esophageal biopsies from subjects with EoE.

Figure 4

Expression of IgG4 heavy chain mRNA in the esophageal mucosa. (A) Correlation between IGHG4 mRNA expression and IgG4 protein content (mg IgG4 per gram of total protein) in esophageal biopsies from subjects with EoE. (B) Correlation between IL10 mRNA expression and IGHG4 mRNA expression in esophageal biopsies from subjects with EoE.

Figure 5

Associations between esophageal tissue IgG4 content and a diagnostic subset of the EoE transcriptome. The x-axis represents the negative log10 P value of the Spearman correlation between tissue IgG4 levels and a diagnostic subset of genes from the EoE transcriptome (EDP). Genes shown on the y-axis are organized within functional groupings. Red shading denotes upregulated genes, and blue shading denotes downregulated genes. The dashed line indicates FDR-corrected P < 0.05.