Mark Rochman

IL-33 Markedly Activates Murine Eosinophils by an NF-κB–Dependent Mechanism Differentially Dependent upon an IL-4–Driven Autoinflammatory Loop

Eosinophils are major effector cells in type 2 inflammatory responses and become activated in response to IL-4 and IL-33, yet the molecular mechanisms and cooperative interaction between these cytokines remain unclear. Our objective was to investigate the molecular mechanism and cooperation of IL-4 and IL-33 in eosinophil activation. Eosinophils derived from bone marrow or isolated from Il5-transgenic mice were activated in the presence of IL-4 or IL-33 for 1 or 4 h, and the transcriptome was analyzed by RNA sequencing. The candidate genes were validated by quantitative PCR and ELISA. We demonstrated that murine-cultured eosinophils respond to IL-4 and IL-33 by phosphorylation of STAT-6 and NF-κB, respectively. RNA sequence analysis of murine-cultured eosinophils indicated that IL-33 induced 519 genes, whereas IL-4 induced only 28 genes, including 19 IL-33–regulated genes. Interestingly, IL-33 induced eosinophil activation via two distinct mechanisms, IL-4 independent and IL-4 secretion/autostimulation dependent. Anti–IL-4 or anti–IL-4Rα Ab-treated cultured and mature eosinophils, as well as Il4- or Stat6-deficient cultured eosinophils, had attenuated protein secretion of a subset of IL-33–induced genes, including Retnla and Ccl17. Additionally, IL-33 induced the rapid release of preformed IL-4 protein from eosinophils by a NF-κB–dependent mechanism. However, the induction of most IL-33–regulated transcripts (e.g., Il6 and Il13) was IL-4 independent and blocked by NF-κB inhibition. In conclusion, we have identified a novel activation pathway in murine eosinophils that is induced by IL-33 and differentially dependent upon an IL-4 auto-amplification loop.

Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation

Although interleukin (IL)-13 and neurotrophins are functionally important for the pathogenesis of immune responses, the interaction of these pathways has not been explored. Herein, by interrogating IL-13-induced responses in human epithelial cells we show that neurotrophic tyrosine kinase receptor, type 1 (NTRK1), a cognate, high-affinity receptor for nerve growth factor (NGF), is an early transcriptional IL-13 target. Induction of NTRK1 was accompanied by accumulation of activating epigenetic marks in the promoter; transcriptional and epigenetic changes were signal transducer and activator of transcription 6 dependent. Using eosinophilic esophagitis as a model for human allergic inflammation, we found that NTRK1 was increased in inflamed tissue and dynamically expressed as a function of disease activity and that the downstream mediator of NTRK1 signaling early growth response 1 protein was elevated in allergic inflammatory tissue compared with control tissue. Unlike NTRK1, its ligand NGF was constitutively expressed in control and disease states, indicating that IL-13-stimulated NTRK1 induction is a limiting factor in pathway activation. In epithelial cells, NGF and IL-13 synergistically induced several target genes, including chemokine (C-C motif) ligand 26 (eotaxin-3). In summary, we have demonstrated that IL-13 confers epithelial cell responsiveness to NGF by regulating NTRK1 levels by a transcriptional and epigenetic mechanism and that this process likely contributes to allergic inflammation.

Calpain-14 and its association with eosinophilic esophagitis

Calpains are a family of intracellular, calcium-dependent cysteine proteases involved in a variety of regulatory processes, including cytoskeletal dynamics, cell-cycle progression, signal transduction, gene expression, and apoptosis. These enzymes have been implicated in a number of disease processes, notably for this review involving eosinophilic tissue inflammation, such as eosinophilic esophagitis (EoE), a chronic inflammatory disorder triggered by allergic hypersensitivity to food and associated with genetic variants in calpain 14 (CAPN14). Herein we review the genetic, structural, and biochemical properties of CAPN14 and its gene product CAPN14, and its emerging role in patients with EoE. The CAPN14 gene is localized at chromosome 2p23.1-p21 and is most homologous to CAPN13 (36% sequence identity), which is located 365 kb downstream of CAPN14. Structurally, CAPN14 has classical calpain motifs, including a cysteine protease core. In comparison with other human calpains, CAPN14 has a unique expression pattern, with the highest levels in the upper gastrointestinal tract, particularly in the squamous epithelium of the esophagus. The CAPN14 gene is positioned in an epigenetic hotspot regulated by IL-13, a TH2 cytokine with increased levels in patients with EoE that has been shown to be a mediator of the disease. CAPN14 induces disruptive effects on the esophageal epithelium by impairing epithelial barrier function in association with loss of desmoglein-1 expression and has a regulatory role in repairing epithelial changes induced by IL-13. Thus CAPN14 is a unique protease with distinct tissue-specific expression and function in patients with EoE and is a potential therapeutic target for EoE and related eosinophilic and allergic diseases.

Profound loss of esophageal tissue differentiation in patients with eosinophilic esophagitis

Background A key question in the allergy field is to understand how tissue‐specific disease is manifested. Eosinophilic esophagitis (EoE) is an emerging tissue‐specific allergic disease with an unclear pathogenesis. Objective Herein we tested the hypothesis that a defect in tissue‐specific esophageal genes is an integral part of EoE pathogenesis. Methods We interrogated the pattern of expression of esophagus‐specific signature genes derived from the Human Protein Atlas in the EoE transcriptome and in EPC2 esophageal epithelial cells. Western blotting and immunofluorescence were used for evaluating expression of esophageal proteins in biopsy specimens from control subjects and patients with active EoE. Whole‐exome sequencing was performed to identify mutations in esophagus‐specific genes. Results We found that approximately 39% of the esophagus‐specific transcripts were altered in patients with EoE, with approximately 90% being downregulated. The majority of transcriptional changes observed in esophagus‐specific genes were reproduced in vitro in esophageal epithelial cells differentiated in the presence of IL‐13. Functional enrichment analysis revealed keratinization and differentiation as the most affected biological processes and identified IL‐1 cytokines and serine peptidase inhibitors as the most dysregulated esophagus‐specific protein families in patients with EoE. Accordingly, biopsy specimens from patients with EoE evidenced a profound loss of tissue differentiation, decreased expression of keratin 4 (KRT4) and cornulin (CRNN), and increased expression of KRT5 and KRT14. Whole‐exome sequencing of 33 unrelated patients with EoE revealed 39 rare mutations in 18 esophagus‐specific differentially expressed genes. Conclusions A tissue‐centered analysis has revealed a profound loss of esophageal tissue differentiation (identity) as an integral and specific part of the pathophysiology of EoE and implicated protease‐ and IL‐1–related activities as putative central pathways in disease pathogenesis.

Cadherin 26 is an alpha integrin-binding epithelial receptor regulated during allergic inflammation

Cadherins (CDH) mediate diverse processes critical in inflammation, including cell adhesion, migration, and differentiation. Herein, we report that the uncharacterized cadherin 26 (CDH26) is highly expressed by epithelial cells in human allergic gastrointestinal tissue. In vitro, CDH26 promotes calcium-dependent cellular adhesion of cells lacking endogenous CDHs by a mechanism involving homotypic binding and interaction with catenin family members (alpha, beta, and p120), as assessed by biochemical assays. Additionally, CDH26 enhances cellular adhesion to recombinant integrin α4β7 in vitro; conversely, recombinant CDH26 binds αE and α4 integrins in biochemical and cellular functional assays, respectively. Interestingly, CDH26-Fc inhibits activation of human CD4+ T cells in vitro including secretion of IL-2. Taken together, we have identified a novel functional CDH regulated during allergic responses with unique immunomodulatory properties, as it binds α4 and αE integrins and regulates leukocyte adhesion and activation, and may thus represent a novel checkpoint for immune regulation and therapy via CDH26-Fc.

Linking impaired skin barrier function to esophageal allergic inflammation via IL-33

Eosinophilic esophagitis (EoE) is an emerging chronic allergic inflammatory disorder that selectively affects the esophagus and is characterized clinically by symptoms of esophageal dysfunction, including vomiting and dysphagia.1 Molecularly, EoE is characterized by a TH2 immune response based on increased expression of the cytokines IL-4, IL-5, and IL-13.2 IL-13 promotes immune cell infiltration by inducing production of chemokines, such as CCL26 (eotaxin-3),3 and by impairing epithelial barrier function through reduced expression of the epithelial adhesion molecule desmoglein 14 and overexpression of the protease calpain 14,5 which is encoded by the CAPN14 gene at chromosome 2p23 and is a major susceptibility genetic locus for EoE.6,7 Patients with EoE often present with comorbid conditions, including asthma and atopic dermatitis,1 which also involve dysregulated TH2 responses and impaired barrier function. The similarity of the pathogeneses of these disorders is evidenced by the significant degree of shared genetic associations that exists among patients with these allergic disorders, including the overabundance of genetic variants in the thymic stromal lymphopoietin (TSLP)8,9 and filaggrin (FLG) loci.10

IL-33 is induced in undifferentiated, non-dividing esophageal epithelial cells in eosinophilic esophagitis

The molecular and cellular etiology of eosinophilic esophagitis (EoE), an emerging tissue-specific allergic disease, involves dysregulated gene expression in esophageal epithelial cells. Herein, we assessed the esophageal expression of IL-33, an epithelium-derived alarmin cytokine, in patients with EoE. IL-33 protein was markedly overexpressed within the nuclei of a subpopulation of basal layer esophageal epithelial cells in patients with active EoE compared to control individuals. IL-33 exhibited dynamic expression as levels normalized upon EoE remission. IL-33–positive basal epithelial cells expressed E-cadherin and the undifferentiated epithelial cell markers keratin 5 and 14 but not the differentiation marker keratin 4. Moreover, the IL-33–positive epithelial cells expressed the epithelial progenitor markers p75 and p63 and lacked the proliferation markers Ki67 and phospho-histone H3. Additionally, the IL-33–positive cells had low expression of PCNA. IL-33 expression was detected in ex vivo–cultured primary esophageal epithelial cells in a subpopulation of cells lacking expression of proliferation markers. Collectively, we report that IL-33 expression is induced in an undifferentiated, non-dividing esophageal epithelial cell population in patients with active EoE.

Solute carrier family 9, subfamily A, member 3 (SLC9A3)/sodium-hydrogen exchanger member 3 (NHE3) dysregulation and dilated intercellular spaces in patients with eosinophilic esophagitis

Background: Eosinophilic esophagitis (EoE) is characterized by histopathologic modifications of esophageal tissue, including eosinophil‐rich inflammation, basal zone hyperplasia, and dilated intercellular spaces (DIS). The underlying molecular processes that drive the histopathologic features of EoE remain largely unexplored. Objective: We sought to investigate the involvement of solute carrier family 9, subfamily A, member 3 (SLC9A3) in esophageal epithelial intracellular pH (pHi) and DIS formation and the histopathologic features of EoE. Methods: We examined expression of esophageal epithelial gene networks associated with regulation of pHi in the EoE transcriptome of primary esophageal epithelial cells and an in vitro esophageal epithelial 3‐dimensional model system (EPC2‐ALI). Molecular and cellular analyses and ion transport assays were used to evaluate the expression and function of SLC9A3. Results: We identified altered expression of gene networks associated with regulation of pHi and acid‐protective mechanisms in esophageal biopsy specimens from pediatric patients with EoE (healthy subjects, n = 6; patients with EoE, n = 10). The most dysregulated gene central to regulating pHi was SLC9A3. SLC9A3 expression was increased within the basal layer of esophageal biopsy specimens from patients with EoE, and expression positively correlated with disease severity (eosinophils/high‐power field) and DIS (healthy subjects, n = 10; patients with EoE, n = 10). Analyses of esophageal epithelial cells revealed IL‐13–induced, signal transducer and activator of transcription 6–dependent SLC9A3 expression and Na+‐dependent proton secretion and that SLC9A3 activity correlated positively with DIS formation. Finally, we showed that IL‐13–mediated, Na+‐dependent proton secretion was the primary intracellular acid‐protective mechanism within the esophageal epithelium and that blockade of SLC9A3 transport abrogated IL‐13–induced DIS formation. Conclusions: SLC9A3 plays a functional role in DIS formation, and pharmacologic interventions targeting SLC9A3 function may suppress the histopathologic manifestations in patients with EoE. GRAPHICAL ABSTRACT Figure. No caption available.

Genetic, inflammatory, and tissue-specific factors control expression of human calpain-14

Eosinophilic esophagitis (EoE) is a chronic, food-driven allergic disease resulting in eosinophilic esophageal inflammation. We recently found that EoE susceptibility is associated with genetic variants in the promoter of CAPN14, a gene with reported esophagus-specific expression. CAPN14 is dynamically up-regulated as a function of EoE disease activity and after exposure of epithelial cells to interleukin-13 (IL-13). Herein, we aimed to explore molecular modulation of CAPN14 expression. We identified three putative binding sites for the IL-13-activated transcription factor STAT6 in the promoter and first intron of CAPN14. Luciferase reporter assays revealed that the two most distal STAT6 elements were required for the ~10-fold increase in promoter activity subsequent to stimulation with IL-13 or IL-4, and also for the genotype-dependent reduction in IL-13-induced promoter activity. One of the STAT6 elements in the promoter was necessary for IL-13-mediated induction of CAPN14 promoter activity while the other STAT6 promoter element was necessary for full induction. Chromatin immunoprecipitation in IL-13 stimulated esophageal epithelial cells was used to further support STAT6 binding to the promoter of CAPN14 at these STAT6 binding sites. The highest CAPN14 and calpain-14 expression occurred with IL-13 or IL-4 stimulation of esophageal epithelial cells under culture conditions that allow the cells to differentiate into a stratified epithelium. This work corroborates a candidate molecular mechanism for EoE disease etiology in which the risk variant at 2p23 dampens mediated CAPN14 expression in differentiated esophageal epithelial cells following IL-13/STAT6 induction of CAPN14 promoter activity.

Epithelial origin of eosinophilic esophagitis

&NA; Eosinophilic esophagitis (EoE) is a chronic, allergen‐driven inflammatory disease of the esophagus characterized predominantly by eosinophilic inflammation, leading to esophageal dysfunction. Converging data have placed the esophageal epithelium at the center of disease pathogenesis. In particular, the main EoE disease susceptibility loci at 2p23 and 5p22 encode for gene products that are produced by the esophageal epithelium: the intracellular protease calpain 14 and thymic stromal lymphopoietin, respectively. Furthermore, genetic and functional data establish a primary role for impaired epithelial barrier function in disease susceptibility and pathoetiology. Additionally, the EoE transcriptome, a set of genes dysregulated in the esophagi of patients with EoE, is enriched in genes that encode for proteins involved in esophageal epithelial cell differentiation. This transcriptome has a high proportion of esophagus‐specific epithelial genes that are notable for the unexpected enrichment in genes encoding for proteases and protease inhibitors, as well as in IL‐1 family genes, demonstrating a previously unappreciated role for innate immunity responses in the esophagus under homeostatic conditions. Among these pathways, basal production of the serine protease inhibitor, Kazal‐type 7 (SPINK7) has been demonstrated to be part of the normal differentiation program of esophageal epithelium. Profound lost expression of SPINK7 occurs in patients with EoE and is sufficient for unleashing increased proteolytic activity (including urokinase plasminogen activator), impaired barrier function, and production of large quantities of proinflammatory and proallergic cytokines, including thymic stromal lymphopoietin. Collectively, we put forth a model in which the esophagus is normally equipped as an anti‐inflammatory sensing organ and that defects in this pathway, mediated by epithelial protease/protease inhibitor imbalances, unleash inflammatory responses resulting in disorders, such as EoE.