Mark Rochman, PhD, Jared Travers, BS, Cora E. Miracle, Mary C

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Presentation transcript:

Profound loss of esophageal tissue differentiation in patients with eosinophilic esophagitis Mark Rochman, PhD, Jared Travers, BS, Cora E. Miracle, Mary C. Bedard, BS, Ting Wen, PhD, Nurit P. Azouz, PhD, Julie M. Caldwell, PhD, Kiran KC, BS, Joseph D. Sherrill, PhD, Benjamin P. Davis, MD, PhD, Jeffrey K. Rymer, BS, Kenneth M. Kaufman, PhD, Bruce J. Aronow, PhD, Marc E. Rothenberg, MD, PhD Journal of Allergy and Clinical Immunology Volume 140, Issue 3, Pages 738-749.e3 (September 2017) DOI: 10.1016/j.jaci.2016.11.042 Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Altered transcription of esophagus-specific genes in patients with EoE. A, Graph shows percentage of organ-specific genes altered in the EoE transcriptome, as assessed by means of RNA sequencing.3 B, Venn diagram shows overlap of genes specifically expressed in the esophagus according to the Human Protein Atlas (Esophagus) with genes differentially expressed in the esophageal tissue of patients with active EoE compared with control esophageal tissue (EoE). For each group of genes (see the text and the Methods section for definitions), the number of upregulated (Up) and downregulated (Down) genes is shown (not to scale). A set of 117 overlapping genes is designated as Eso-EoE genes. C, Normalized expression levels for each gene (fragments per kilobase per million reads [FPKM]) in esophageal biopsy tissue from patients with active EoE (EoE, blue rectangles, n = 10) and control subjects (Ctrl, green rectangles, n = 6) for the 12 genes most highly expressed in the homeostatic esophagus are presented as a box-and-whiskers plot. Genes upregulated in samples from patients with active EoE are in orange. The P value for each gene is shown on the right. Statistical significance was determined by using the Holm-Sidak method. D, Venn diagram shows the overlap of genes specifically expressed in the stomach according to the Human Protein Atlas with genes differentially expressed in biopsy specimens of patients with EG.14 Journal of Allergy and Clinical Immunology 2017 140, 738-749.e3DOI: (10.1016/j.jaci.2016.11.042) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Functional enrichment analysis of esophagus-specific Eso-EoE genes altered in patients with EoE. Shown are the 10 most significant terms identified for Eso-EoE genes by functional enrichment analysis in the following categories: Molecular Function (A), Biological Processes (B), Human Phenotype (C), and Protein Domain (D). Genes for the selected terms (gray bars) are listed with the corresponding fold change in expression for active EoE (EoE) compared with control (Ctrl) samples. Upregulated genes are in orange. The x-axes represent the negative log10 P value. Journal of Allergy and Clinical Immunology 2017 140, 738-749.e3DOI: (10.1016/j.jaci.2016.11.042) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Effect of differentiation on expression of esophagus-specific Eso-EoE genes. A, Schematic diagram for generating ALI cultures from EPC2 esophageal epithelial cells is shown. Cells are initially grown in submerged culture at low and then high calcium concentrations to promote initial differentiation; terminal differentiation is achieved by growing cells in ALI. Note that the presence of IL-13 during the differentiation process weakens esophageal epithelial integrity, as represented by increased spaces between cell layers. B, Venn diagram shows overlap between Eso-EoE genes and transcriptional changes during initial differentiation. A heat map represents log2 fold change in gene expression when comparing biopsy specimens from patients with EoE with normal biopsy specimens and comparing day 8 with day 3 (Initial Diff). The analysis is focused on the 90 common Eso-EoE genes. Clustering was performed by using Euclidean distance and average linkage parameters; yellow and blue colors correspond to increased or decreased expression, respectively; and black indicates a nonsignificant change compared with control values. Journal of Allergy and Clinical Immunology 2017 140, 738-749.e3DOI: (10.1016/j.jaci.2016.11.042) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Effect of IL-13 treatment on expression of esophagus-specific Eso-EoE genes at the ALI. A, Venn diagram shows overlap of Eso-EoE genes with transcriptional signature of terminally differentiated ALI in the absence or presence of IL-13. D0 represents the first day and D6 represents the last day of terminal differentiation at ALI (see Fig 3 for details). Group 1 shows 32 genes altered in biopsy specimens and in the ALI in the presence and absence of IL-13. Group 2 shows 8 genes with altered expression in biopsy specimens and in the presence of IL-13 during ALI (D6 vs D6 + IL-13). Group 3 shows 28 genes with altered expression in biopsy specimens and by ALI differentiation but was not affected by IL-13 (D6 vs D0). B, Heat map shows a log2 fold change in expression of genes from the IL-1 and SERPINB gene families in biopsy specimens and ALI culture from patients with EoE at the indicated conditions. Orange color represents increased expression of SERPIN genes in biopsy specimens from patients with EoE. For Fig 4, A and B, the heat maps show the log2 fold change in expression of genes from indicated groups. For all heat maps, the EoE biopsy specimen represents the expression level of the indicated genes in the EoE transcriptome compared with that in unaffected subjects. Clustering was performed by using Euclidean distance and average linkage parameters. Yellow and blue colors correspond to increased or decreased expression, respectively, and black color indicates a nonsignificant change compared with control subjects. C, Expression level of indicated Eso-EoE genes after the EoE diagnostic panel analysis23 was assessed by using RT-PCR in control subjects (Ctrl), patients with active EoE (EoE), and patients with gastroesophageal reflux disease (GERD). Each dot represents an individual biopsy specimen, and expression was normalized to GAPDH. Error bars represent the SEM of the mean relative expression. *P < .05, **P < .01, and ***P < .001, 1-way ANOVA. ns, Not significant. Journal of Allergy and Clinical Immunology 2017 140, 738-749.e3DOI: (10.1016/j.jaci.2016.11.042) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Protein expression, protease activity, and expression of differentiation markers in esophageal biopsy specimens of patients with EoE. A, Representative Western blot for the indicated proteins in control (Ctrl) and active EoE (EoE) samples (n = 3 biopsy specimens). Red and green arrows point to the bands corresponding to analyzed proteins; molecular weight markers are shown on the right. B, Graphs show relative expression of the indicated proteins normalized to the loading control (HSP90) by using Western blotting (n = 5-10 samples for each group). Error bars represent the SEM of the mean relative expression. *P < .05 and **P < .01, Mann-Whitney test. C-F, Immunofluorescence of distal esophageal biopsy specimens from control subjects (Ctrl, top row) or patients with active EoE (EoE Inflamed, middle row) or proximal esophageal biopsy specimens from regions that do not have active inflammation from patients with active EoE (EoE Non-inflamed, bottom row). Staining with the nuclear dye DAPI or with an antibody directed against the indicated differentiation marker (KRT5, KRT14, KRT4, or CRNN) are indicated by blue or green, respectively. White broken line indicates basal membrane. Scale bar = 100 μm. Journal of Allergy and Clinical Immunology 2017 140, 738-749.e3DOI: (10.1016/j.jaci.2016.11.042) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Model of esophagus-specific allergic inflammation. Under healthy conditions, the esophagus expresses tissue-specific markers of terminal epithelial differentiation (represented by KRT4 and CRNN). Profound loss of esophageal differentiation, either acquired or genetically inherited (represented by loss of KRT5 and KRT14), predisposes and/or propagates EoE. Loss of differentiation is triggered by imbalanced proteolytic activity in the esophagus, as indicated by the ratio between proteases and protease inhibitors (note that protease and protease inhibitors can be either upregulated or downregulated in patients with EoE). Journal of Allergy and Clinical Immunology 2017 140, 738-749.e3DOI: (10.1016/j.jaci.2016.11.042) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Effect of IL-1α replenishment on ALI. A, Relative transepithelial electrical resistance (TEER) in EPC2 ALI cultures treated with IL-13 and IL-1α either alone or in combination. IL-13 treatment was started when ALI was induced at day 8, and IL-1α was added for the last 2 days of culture. TEER measurements were normalized to average TEER in the untreated (Ctrl) cultures at day 4. B, Expression level of CCL26 and SERPINB4 was assessed by using RT-PCR. Expression was normalized to GAPDH. Combined data for 2 independent experiments are shown. Journal of Allergy and Clinical Immunology 2017 140, 738-749.e3DOI: (10.1016/j.jaci.2016.11.042) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions