Desmoplakin and periplakin genetically and functionally contribute to eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease with a complex underlying genetic etiology. Herein, we conduct whole-exome sequencing of a multigeneration EoE pedigree (discovery set) and 61 additional multiplex families with EoE (replication set). A series of rare, heterozygous, missense variants are identified in the genes encoding the desmosome-associated proteins DSP and PPL in 21% of the multiplex families. Esophageal biopsies from patients with these variants retain dilated intercellular spaces and decrease DSP and PPL expression even during disease remission. These variants affect barrier integrity, cell motility and RhoGTPase activity in esophageal epithelial cells and have increased susceptibility to calpain-14–mediated degradation. An acquired loss of esophageal DSP and PPL is present in non-familial EoE. Taken together, herein, we uncover a pathogenic role for desmosomal dysfunction in EoE, providing a deeper mechanistic understanding of tissue-specific allergic responses.

A novel herpes-like virus inducing branchial lesions in a tiger shark (Galeocerdo cuvier)

A juvenile, male tiger shark ( Galeocerdo cuvier) developed illness after capture in Florida waters and was euthanized. Gross lesions included mild skin abrasions, hepatic atrophy, and coelomic fluid. Histologically, gills contained multifocal lamellar epithelial cell necrosis and thromboses. Scattered gill and esophageal epithelial cells had large, basophilic, intracytoplasmic, and intranuclear inclusions. Ultrastructurally, lamellar epithelial cells contained arrays of intracytoplasmic viral particles and scattered intranuclear nucleocapsids. Capsulated virions were 148 ± 11 nm with an 84 ± 8 nm icosahedral nucleocapsid and an electron-dense core. Next-generation sequencing, quantitative polymerase chain reaction, and in situ hybridization performed on formalin-fixed tissue confirmed a herpes-like viral infection. The viral polymerase shared 24% to 31% protein homology with other alloherpesviruses of fish, indicating a divergent virus. This report documents the pathologic findings associated with a molecularly confirmed novel herpes-like virus in an elasmobranch.

Coexpression Network Analysis of lncRNA Associated with Overexpression of DNMT1 in Esophageal Epithelial Cells

Screening and preliminary identification of high DNMT1 expression-related lncRNA, which is involved in various interrelated signaling pathways, has led to the development of a theoretical basis for various types of disease mechanisms. Differential expression profiles of lncRNA and mRNA were identified in a microarray. Ten lncRNAs with high levels of variation were identified by qRT-PCR. KEGG and GO analyses were used to identify differentially expressed mRNAs. Six signaling pathways were selected based on the KEGG results of the lncRNA-mRNA expression network analysis. From the microarrays in the experimental and control groups, we found a total of 6987 differentially expressed lncRNAs, and 7421 differentially expressed mRNAs were obtained ( P < 0.05 ; fold change > 2.0 x ). GO analysis and KEGG pathway analysis showed high expression of DNMT1 in esophageal epithelial cells. Nine pathways were involved in mRNA upregulation, including natural killer cell-mediated cytotoxicity and many other prominent biochemical pathways. Forty-six pathways were associated with downregulated mRNAs and ribosomes involving multiple biological pathways. Coexpression network analysis showed that 8 mRNAs and 16 lncRNAs were linked to the p53 signaling pathway. In Helicobacter pylori infections, interactions occurred between 22 lncRNAs and 11 mRNAs in the ErbB signaling pathway and between 19 lncRNAs and 8 mRNAs in epithelial cell signal transduction. Interactions were present between 19 lncRNAs and 5 mRNAs in the sphingolipid signaling pathway, along with interactions between 21 lncRNAs and 12 mRNAs in the PI3K-Akt signaling pathway. Cytotoxicity interactions occurred between 22 lncRNAs and 9 mRNAs in natural killer cells.

Inhibition of NHE-1 Increases Smoke-Induced Proliferative Activity of Barrett’s Esophageal Cell Line

Several clinical studies indicate that smoking predisposes its consumers to esophageal inflammatory and malignant diseases, but the cellular mechanism is not clear. Ion transporters protect esophageal epithelial cells by maintaining intracellular pH at normal levels. In this study, we hypothesized that smoking affects the function of ion transporters, thus playing a role in the development of smoking-induced esophageal diseases. Esophageal cell lines were treated with cigarettesmoke extract (CSE), and the viability and proliferation of the cells, as well as the activity, mRNA and protein expression of the Na+/H+ exchanger-1 (NHE-1), were studied. NHE-1 expression was also investigated in human samples. For chronic treatment, guinea pigs were exposed to tobacco smoke, and NHE-1 activity was measured. Silencing of NHE-1 was performed by using specific siRNA. CSE treatment increased the activity and protein expression of NHE-1 in the metaplastic cells and decreased the rate of proliferation in a NHE-1-dependent manner. In contrast, CSE increased the proliferation of dysplastic cells independently of NHE-1. In the normal cells, the expression and activity of NHE-1 decreased due to in vitro and in vivo smoke exposure. Smoking enhances the function of NHE-1 in Barrett’s esophagus, and this is presumably a compensatory mechanism against this toxic agent.

Mouse organoid culture is a suitable model to study esophageal ion transport mechanisms

Altered esophageal ion transport mechanisms play a key role in inflammatory and cancerous diseases of the esophagus, but epithelial ion processes have been less studied in the esophagus because of the lack of a suitable experimental model. In this study, we generated 3D esophageal organoids (EOs) from two different mouse strains and characterized the ion transport processes of the EOs. EOs form a cell-filled structure with a diameter of 250-300 µm and generated from epithelial stem cells as shown by FACS analysis. Using conventional PCR and immunostaining, the presence of Slc26a6 Cl−/HCO3− anion exchanger (AE), Na+/H+ exchanger (NHE), Na+/HCO3- cotransporter (NBC), cystic fibrosis transmembrane conductance regulator (CFTR) and anoctamin 1 Cl− channels were detected in EOs. Microfluorimetric techniques revealed high NHE, AE, and NBC activities, whereas that of CFTR was relatively low. In addition, inhibition of CFTR led to functional interactions between the major acid-base transporters and CFTR. We conclude that EOs provide a relevant and suitable model system for studying the ion transport mechanisms of esophageal epithelial cells, and they can be also used as preclinical tools to assess the effectiveness of novel therapeutic compounds in esophageal diseases associated with altered ion transport processes.

Multi-omics of the esophageal microenvironment identifies signatures associated with progression of Barrett’s esophagus

Background The enrichment of Gram-negative bacteria of oral origin in the esophageal microbiome has been associated with the development of metaplasia. However, to date, no study has comprehensively assessed the relationships between the esophageal microbiome and the host. Methods Here, we examine the esophageal microenvironment in gastro-esophageal reflux disease and metaplasia using multi-omics strategies targeting the microbiome and host transcriptome, followed by targeted culture, comparative genomics, and host-microbial interaction studies of bacterial signatures of interest. Results Profiling of the host transcriptome from esophageal mucosal biopsies revealed profound changes during metaplasia. Importantly, five biomarkers showed consistent longitudinal changes with disease progression from reflux disease to metaplasia. We showed for the first time that the esophageal microbiome is distinct from the salivary microbiome and the enrichment of Campylobacter species as a consistent signature in disease across two independent cohorts. Shape fitting and matrix correlation identified associations between the microbiome and host transcriptome profiles, with a novel co-exclusion relationship found between Campylobacter and napsin B aspartic peptidase. Targeted culture of Campylobacter species from the same cohort revealed a subset of isolates to have a higher capacity to survive within primary human macrophages. Comparative genomic analyses showed these isolates could be differentiated by specific genomic features, one of which was validated to be associated with intracellular fitness. Screening for these Campylobacter strain-specific signatures in shotgun metagenomics data from another cohort showed an increase in prevalence with disease progression. Comparative transcriptomic analyses of primary esophageal epithelial cells exposed to the Campylobacter isolates revealed expression changes within those infected with strains with high intracellular fitness that could explain the increased likelihood of disease progression. Conclusions We provide a comprehensive assessment of the esophageal microenvironment, identifying bacterial strain-specific signatures with high relevance to progression of metaplasia.

Loss of grand histone H3 lysine 27 trimethylation domains mediated transcriptional activation in esophageal squamous cell carcinoma

Trimethylation of histone H3 lysine 27 trimethylation (H3K27me3) may be recruited by repressive Polycomb complexes to mediate gene silencing, which is critical for maintaining embryonic stem cell pluripotency and differentiation. However, the roles of aberrant H3K27me3 patterns in tumorigenesis are not fully understood. Here, we discovered that grand silencer domains (breadth > 50 kb) for H3K27me3 were significantly associated with epithelial cell differentiation and exhibited high gene essentiality and conservation in human esophageal epithelial cells. These grand H3K27me3 domains exhibited high modification signals involved in gene silencing, and preferentially occupied the entirety of topologically associating domains and interact with each other. We found that widespread loss of the grand H3K27me3 domains in of esophageal squamous cell carcinomas (ESCCs) were enriched in genes involved in epithelium and endothelium differentiation, which were significantly associated with overexpression with increase of active modifications of H3K4me3, H3K4me1, and H3K27ac marks, as well as DNA hypermethylation in the gene bodies. A total of 208 activated genes with loss of grand H3K27me3 domains in ESCC were identified, where the higher expression and mutation of T-box transcription factor 20 (TBX20) were associated with worse patients’ outcomes. Our results showed that knockdown of TBX20 may have led to a striking defect in esophageal cancer cell growth and carcinogenesis-related pathway, including cell cycle and homologous recombination. Together, our results reveal that loss of grand H3K27me3 domains represent a catalog of remarkable activating regulators involved in carcinogenesis.

Downregulation of miR-135b-5p Suppresses Progression of Esophageal Cancer and Contributes to the Effect of Cisplatin

Esophageal cancer (EC) is one of the commonest human cancers, which accompany high morbidity. MicroRNAs (miRNAs) play a pivotal role in various cancers, including EC. Our research aimed to reveal the function and mechanism of miR-135b-5p. Our research identified that miR-135b-5p was elevated in EC samples from TCGA database. Correspondingly real-time PCR assay also showed the miR-135b-5p is also higher expressed in Eca109, EC9706, KYSE150 cells than normal esophageal epithelial cells (Het-1A). CCK8, Edu, wound healing, Transwell assay, and western blot demonstrated miR-135b-5p inhibition suppresses proliferation, invasion, migration and promoted the apoptosis in Eca109 and EC9706 cells. Moreover, the miR-135b-5p inhibition also inhibited xenograft lump growth. We then predicted the complementary gene of miR-135b-5p using miRTarBase, TargetScan, and DIANA-microT. TXNIP was estimated as a complementary gene for miR-135b-5p. Luciferase report assay verified the direct binding site for miR-135b-5p and TXNIP. Real-time PCR and western blot assays showed that the inhibition of miR-135b-5p remarkably enhanced the levels of TXNIP in Eca109 and EC9706 cells. Furthermore, cisplatin (cis-diamminedichloroplatinum II, DDP) decreased miR-135b-5p expression and increased TXNIP expression. Enhanced expression of miR-135b-5p attenuated the inhibitory ability of cisplatin (cis-diamminedichloroplatinum II, DDP) in Eca109 cells, accompanied by TXNIP downregulation. In conclusion, the downregulation of miR-135b-5p suppresses the progression of EC through targeting TXNIP. MiR-135b-5p/TXNIP pathway contributes to the anti-tumor effect of DDP. These findings may provide new insight into the treatment of EC.

N-3 Polyunsaturated Fatty Acids Protect Esophageal Epithelial Cell From Acid Exposure

Objectives Our previous study has found that n-3 polyunsaturated fatty acids (PUFAs) inhibited inflammation in rats with esophagitis. This study was aimed to observe the protective effect of n-3 PUFAs on esophageal epithelial cells against acid damage and to explore its mechanism. Methods Human esophageal epithelial cells (Het-1A) were repeatedly treated with acidified medium (pH 5.0) and PUFAs with different ratios of n-6/n-3 (9:1, 3:1, 1:1 and 1:3, respectively), and Nrf2 agonists or inhibitors were used to assess the role of Nrf2 in mechanism of n-3 PUFAs. Western Blot and Quantitative Real-time PCR (qPCR) were used to assess the expression of Nrf2, NLRP3 and caspase-1. IL-1β, IL-18 and LDH levels in cell culture supernatants were detected by enzyme linked immunosorbent assay (ELISA), while pyroptosis was assessed by caspase-1 and TUNEL double staining. Results Compared with the normal group, the level of MDA was increased after acid intervention, while that of SOD was decreased (P &lt; 0.05). In groups with different ratios of n-6/n-3 PUFAs, the expressions of Nrf2 and SOD were increased along with the increasing of n-3 PUFAS ratio, which was reached the highest at 1:1 (P &lt; 0.05) and was followed by a decrease. Compared with the 9:1 group, the expression of NLRP3, caspase-1 and the pyroptosis rate of 1:1 group were decreased (P &lt; 0.05). IL-1β, IL-18, and LDH levels in the cell culture supernatant were also decreased in 1:1 group (P &lt; 0.05). Among Nrf2 agonist group, inhibitor group, control group and solvent group, the expression of Nrf2 was highest while the expressions of NLRP3, caspase-1 and pyroptosis rate were lowest in the agonist group (P &lt; 0.05). In the inhibitor group, the expression of Nrf2 was lowest and pyroptosis related protein as well as pyroptosis rate were highest (P &lt; 0.05). There was no significant difference in all indexes between control and solvent group (P &gt; 0.05). Conclusions N-3 PUFAs protected esophageal epithelial cells from acid damage through upregulating Nrf2, which therefore disrupted oxidative stress and NLRP3 inflammasome activation, and then resulted in pyroptosis inhibition. Funding Sources This study was supported by the Science Foundation of the Fujian Province, China(Grant No. 2019J01447).