A PhysioMechanical Model of Esophageal Function in Eosinophilic Esophagitis.

BACKGROUND & AIMS: Eosinophilic esophagitis (EoE) is characterized by eosinophilic inflammation, but also heterogeneous presentations involving fibrostenotic esophageal remodeling and esophageal dysmotility. We aimed to define and evaluate phenotypes of EoE using functional lumen imaging probe (FLIP) panometry (ie, a PhysioMechanical classification of EoE). METHODS: Patients with EoE who completed FLIP during endoscopy were included in a cross-sectional study. FLIP studies were analyzed for distensibility plateau and compliance of the esophageal body, maximum esophagogastric junction diameter, and contractile response pattern. These FLIP features were then applied to define PhysioMechanical classifications. RESULTS: A total of 215 patients with EoE (mean [standard deviation] age 38 [12] years; 31% female) were included. Seven PhysioMechanical classifications were identified that differed by various clinical characteristics, including symptom duration (P < .001) and Endoscopic EoE Reference Scores (EREFS) (P < .001). In particular, patients with "nonreactive fibrostenosis" (n = 14), had greater symptom duration (median [interquartile range] 20 [10-30] years) and more frequently had EREFS grade 2 or 3 ring scores (14 of 14 patients) than patients with a "normal" PhysioMechanical classification (symptom duration: 3 [1-8] years; 4 of 50 [8%] had EREFS grade 2 or 3 rings). In addition, among patients off treatment at cross-sectional evaluation (n = 46), there was a difference between PhysioMechanical classifications in future proton pump inhibitor (PPI) response rates (ie, achieving peak mucosal eosinophil count <15 per high-powered field after PPI treatment); P = .009. PPI response ranged from 87% (13 of 15 patients) with "isolated esophagogastric junction outflow obstruction" to 11% (1 of 9 patients) with "spastic-reactive fibrostenosis." CONCLUSIONS: Classifying PhysioMechanical esophageal function in EoE based on FLIP panometry features may facilitate defining disease severity and directing management in EoE.

The Severity of Reduced Esophageal Distensibility Parallels Eosinophilic Esophagitis Disease Duration.

BACKGROUND & AIMS: Chronic inflammation of eosinophilic esophagitis (EoE) results in progressive, fibrostenotic remodeling of the esophageal wall. This study aimed to demonstrate objective changes in esophageal distensibility relative to duration of EoE disease using a functional lumen imaging probe (FLIP). METHODS: Adult patients with EoE who completed a 16-cm FLIP protocol during endoscopy were evaluated in a cross-sectional study. FLIP analysis focused on distensibility plateau (DP) of the esophageal body. The time from onset of symptoms to time of endoscopy with FLIP was assessed, as was time from symptom onset to EoE diagnosis (ie, diagnostic delay). RESULTS: A total of 171 patients (mean age 38 ± 12 years; 31% female) were included; the median symptom duration was 8 (interquartile range, 3-15) years and diagnostic delay was 4 (interquartile range, 1-12) years. At the time of endoscopy with FLIP, there were 54 patients (39%) in histologic remission (<15 eosinophils per high-power field [eos/hpf]). Symptom duration and diagnostic delay were negatively correlated with DP (rho = -0.326 and -0.309; P values < .001). Abnormal esophageal distensibility (DP ≤17 mm) was more prevalent with increased duration of symptoms (P < .004): 23% at <5 years to 64% at ≥25 years. When stratifying the cohort based on mucosal eosinophil density, patients with ≥15 eos/hpf had significantly lower DP with greater symptom duration (P = .004), while there was not a significant difference among patients with <15 eos/hpf (P = .060). CONCLUSIONS: Esophageal distensibility objectively measured with FLIP was reduced in EoE patients with greater symptom duration and diagnostic delay. This supports that EoE is a progressive, fibrostenotic disease and that FLIP may be a useful tool to monitor disease progression in EoE.

Composite score of physiomechanical esophageal function using functional lumen imaging probe panometry in eosinophilic esophagitis.

BACKGROUND AND AIMS: The evaluation provided by functional lumen imaging probe (FLIP) panometry includes esophageal distensibility/compliance (mechanics) of the esophageal body and esophagogastric junction (EGJ) and esophageal motility (secondary peristalsis). We developed a composite score using these parameters to characterize physiomechanical function in patients with eosinophilic esophagitis (EoE). METHODS: Two hundred fifteen adult patients with EoE who completed FLIP panometry during sedated endoscopy with esophageal biopsy sampling were included. FLIP metrics of esophageal body Compliance, Contractile response, Distensibility plateau, and maximum EGJ Diameter (C2D2) were scored as 0 for normal versus 1 or 2 for increasing degree of abnormality. Scores were summed to calculate the composite C2D2 score. RESULTS: The C2D2 score had a significant positive correlation with mucosal eosinophil count (ρ = .241) and total Endoscopic EoE Reference Score (ρ = .467). Among 46 patients off treatment at the baseline evaluation, future proton pump inhibitor (PPI) responders (ie, achieved mucosal eosinophil count <15 per high-powered field after PPI treatment) had lower C2D2 scores than PPI nonresponders (median, 2 [interquartile range, 1-3] vs 4 [interquartile range, 2-6], respectively; P = .003). A regression model (that controlled for age, sex, and baseline eosinophil count) showed a C2D2 score ≤3 had an odds ratio of 14.5 (95% confidence interval, 2.6-85) to predict future PPI response. However, total Endoscopic EoE Reference Scores (P = .142) and baseline eosinophil count (P = .480) did not differ between PPI responders and PPI nonresponders. CONCLUSIONS: This composite score of FLIP panometry metrics, the C2D2 score, may facilitate characterizing physiomechanical function in EoE and serve as an objective outcome measure.

GITR agonistic stimulation enhances the anti-tumor immune response in a mouse model of ESCC.

Esophageal cancer is a significant health burden in the United States and worldwide and is the 8th leading cause of cancer-related death. Over 90% of esophageal cancers are squamous cell cancers (ESCC). Despite the development of new therapies, the overall 5-year survival rate remains lower than 20%. Recent clinical trials of immunotherapy approaches in ESCC have shown that blocking PD-1/PD-L1 interactions can reduce tumor burden and increase survival, but this only occurs in a fraction of patients. This emphasizes the need for additional therapeutic options to improve overall response rates, duration of response, and overall survival. Glucocorticoid-induced TNFR-related protein (GITR) stimulation has emerged as a promising immunotherapy target, as its stimulation appears to promote tumor regression. In this study, we evaluated the consequences of GITR agonistic stimulation with the DTA-1 antibody (anti-GITR agonist) on esophageal squamous cell carcinoma (ESCC) progression. Increased expression of GITR was observed in esophageal tumors from ESCC patients in comparison to normal adjacent tissue and in a mouse model of ESCC. 100% of mice treated with 4-NQO/IgG control antibody developed invasive squamous cell carcinoma. Less advanced esophageal tumors were seen in mice treated with 4-NQO/anti-GITR agonist compared to 4-NQO/IgG treatment. 4-NQO/anti-GITR agonist-treated mice demonstrated a significant increase in mucosal CTL/Treg ratios as well as decreased gene expression profiles of pathways related to esophageal squamous cell carcinogenesis. Thus, GITR agonism merits further study as a treatment strategy for ESCC patients.

GI Manifestations With a Focus on the Esophagus: Recent Progress in Understanding Pathogenesis.

PURPOSE OF REVIEW: Esophageal dysfunction is common in systemic sclerosis (SSc) patients. Limited treatment options are available for scleroderma esophageal disease. Here, we discuss recent updates on the diagnosis, treatment, and characterization that have been made in patients with scleroderma esophageal disease. RECENT FINDINGS: In the past few years, novel diagnostic tools have provided insight into esophageal dysmotility in SSc patients. New drugs are being tested and might improve symptoms and quality of life in SSc patients with esophageal dysfunction. Molecular stratification methods have facilitated the identification of molecular signatures in the esophagus of SSc patients. The Friend leukemia integration 1 (Fli1) conditional knockout mouse is the first animal model to report an esophageal phenotype with SSc features. The clinical presentation in SSc patients with esophageal dysfunction is heterogeneous, complicating diagnosis and management. The improvement of diagnostic tools for esophageal symptoms and dysfunction and the use of molecular approaches in SSc mouse models and patient biopsies offer an opportunity to improve the characterization of SSc esophageal disease, which should help improve management and treatment decisions.

Esophageal Expression of Active IκB Kinase-ß in Mice Up-Regulates Tumor Necrosis Factor and Granulocyte-Macrophage Colony-Stimulating Factor, Promoting Inflammation and Angiogenesis.

BACKGROUND & AIMS: IκB kinase-ß (IKKß) mediates activation of the nuclear factor-κB, which regulates immune and inflammatory responses. Although nuclear factor-κB is activated in cells from patients with inflammatory diseases or cancer, little is known about its roles in the development and progression of esophageal diseases. We investigated whether mice that express an activated form of IKKß in the esophageal epithelia develop esophageal disorders. METHODS: We generated ED-L2-Cre/Rosa26-IKK2caSFL mice, in which the ED-L2 promoter activates expression of Cre in the esophageal epithelia, leading to expression of a constitutively active form of IKKß (IKKßca) in epithelial cells but not in inflammatory cells or the surrounding stroma (IKKßca mice). Mice lacking the Cre transgene served as controls. Some mice were given intraperitoneal injections of neutralizing antibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor (TNF), or immunoglobulin G1 (control), starting at 1 month of age. Epithelial tissues were collected and analyzed by immunofluorescence, immunohistochemical, and quantitative real-time polymerase chain reaction assays. Transgenes were overexpressed from retroviral vectors in primary human keratinocytes. RESULTS: IKKßca mice developed esophagitis and had increased numbers of blood vessels in the esophageal stroma, compared with controls. Esophageal tissues from IKKßca mice had increased levels of GM-CSF. Expression of IKKßca in primary human esophageal keratinocytes led to 11-fold overexpression of GM-CSF and 200-fold overexpression of TNF. Incubation of human umbilical vein endothelial cells with conditioned media from these keratinocytes increased endothelial cell migration by 42% and promoted formation of capillary tubes; these effects were blocked by a neutralizing antibody against GM-CSF. Injections of anti-GM-CSF reduced angiogenesis and numbers of CD31+ blood vessels in esophageal tissues of IKKßca mice, but did not alter the esophageal vasculature of control mice and did not alter recruitment of intraepithelial leukocytes to esophageal tissues of IKKßca mice. Injections of anti-TNF prevented the development of esophagitis in IKKßca mice. CONCLUSIONS: Constitutive activation of IKKß in the esophageal epithelia of mice leads to inflammation and angiogenesis, mediated by TNF and GM-CSF, respectively.

WNT10A promotes an invasive and self-renewing phenotype in esophageal squamous cell carcinoma.

Esophageal cells overexpressing epidermal growth factor receptor (EGFR) and TP53 mutation can invade into the extracellular matrix when grown in 3D-organotypic cultures (OTC) and mimic early invasion in esophageal squamous cell carcinoma (ESCC). We have performed laser capture microdissection with RNA microarray analysis on the invasive and non-invasive tumor cells of p53(R175H)-overexpressing OTC samples to determine candidate genes facilitating tumor invasion. WNT10A was found to be >4-fold upregulated in the invasive front. Since WNT10A is also prominently upregulated during placode promotion in hair follicle development, a process that requires epithelial cells to thicken and elongate, in order to allow downward growth, we hypothesized that WNT10A may be important in mediating a similar mechanism of tumor cell invasion in ESCC. We have found that WNT10A expression is significantly upregulated in human ESCC, when compared with normal adjacent tissue. Furthermore, high WNT10A expression levels correlate with poor survival. Interestingly, we observe that WNT10A is expressed early in embryogenesis, but is reduced dramatically postnatally. We demonstrate that overexpression of WNT10a promotes migration and invasion, and proliferation of transformed esophageal cells. Lastly, we show that WNT10A overexpression induces a greater CD44(High)/CD24(Low) population, which are putative markers of cancer stem cells, and increases self-renewal capability. Taken together, we propose that WNT10A acts as an oncofetal factor that is highly expressed and may promote proper development of the esophagus. During tumorigenesis, it is aberrantly overexpressed in order to promote ESCC migration and invasion, and may be linked to self-renewal of a subset of ESCC cells.

Autophagy Contributes to Homeostasis in Esophageal Epithelium Where High Autophagic Vesicle Level Marks Basal Cells With Limited Proliferation and Enhanced Self-Renewal Potential.

BACKGROUND & AIMS: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. METHODS: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. RESULTS: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. CONCLUSIONS: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.

Immune Assessment Today: Optimizing and Standardizing Efforts to Monitor Immune Responses in Cancer and Beyond.

As part of a symposium, current and former directors of Immune Monitoring cores and investigative oncologists presented insights into the past, present and future of immune assessment. Dr. Gnjatic presented a classification of immune monitoring technologies ranging from universally applicable to experimental protocols, while emphasizing the need for assay harmonization. Dr. Obeng discussed physiologic differences among CD8 T cells that align with anti-tumor responses. Dr. Lyerly presented the Soldano Ferrone lecture, commemorating the passionate tumor immunologist who inspired many, and covered a timeline of monitoring technology development and its importance to immuno-oncology. Dr. Sonabend presented recent achievements in glioblastoma treatment, accentuating the range of monitoring techniques that allowed him to refine patient selection for clinical trials. Dr. Guevara-Patiño focused on hypoxia within the tumor environment and stressed that T cell viability is not to be confused with functionality. Dr. Butterfield accentuated monitoring of dendritic cell metabolic (dys)function as a determinant for tumor vaccine success. Lectures were interspersed with select abstract presentations. To summarize the concepts, Dr. Maecker from Stanford led an informative forum discussion, pointing towards the future of immune monitoring. Immune monitoring continues to be a guiding light towards effective immunotherapeutic strategies.

Suprabasal cells retain progenitor cell identity programs in eosinophilic esophagitis-driven basal cell hyperplasia.

Eosinophilic esophagitis (EoE) is an esophageal immune-mediated disease characterized by eosinophilic inflammation and epithelial remodeling, including basal cell hyperplasia (BCH). Although BCH is known to correlate with disease severity and with persistent symptoms in patients in histological remission, the molecular processes driving BCH remain poorly defined. Here, we demonstrate that BCH is predominantly characterized by an expansion of nonproliferative suprabasal cells that are still committed to early differentiation. Furthermore, we discovered that suprabasal and superficial esophageal epithelial cells retain progenitor identity programs in EoE, evidenced by increased quiescent cell identity scoring and the enrichment of signaling pathways regulating stem cell pluripotency. Enrichment and trajectory analyses identified SOX2 and KLF5 as potential drivers of the increased quiescent identity and epithelial remodeling observed in EoE. Notably, these alterations were not observed in gastroesophageal reflux disease. These findings provide additional insights into the differentiation process in EoE and highlight the distinct characteristics of suprabasal and superficial esophageal epithelial cells in the disease.

Suprabasal cells retaining stem cell identity programs drive basal cell hyperplasia in eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an esophageal immune-mediated disease characterized by eosinophilic inflammation and epithelial remodeling, including basal cell hyperplasia (BCH) and loss of differentiation. Although BCH correlates with disease severity and with persistent symptoms in patients in histological remission, the molecular processes driving BCH remain poorly defined. Here, we demonstrate that despite the presence of BCH in all EoE patients examined, no increase in basal cell proportion was observed by scRNA-seq. Instead, EoE patients exhibited a reduced pool of KRT15+ COL17A1+ quiescent cells, a modest increase in KI67+ dividing epibasal cells, a substantial increase in KRT13+ IVL+ suprabasal cells, and a loss of differentiated identity in superficial cells. Suprabasal and superficial cell populations demonstrated increased quiescent cell identity scoring in EoE with the enrichment of signaling pathways regulating pluripotency of stem cells. However, this was not paired with increased proliferation. Enrichment and trajectory analyses identified SOX2 and KLF5 as potential drivers of the increased quiescent identity and epithelial remodeling observed in EoE. Notably, these findings were not observed in GERD. Thus, our study demonstrates that BCH in EoE results from an expansion of non-proliferative cells that retain stem-like transcriptional programs while remaining committed to early differentiation.

Autophagy contributes to homeostasis in esophageal epithelium where high autophagic vesicle content marks basal cells with limited proliferation and enhanced self-renewal potential.

Background & Aims: Autophagy has been demonstrated to play roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelium under homeostatic conditions. Methods: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histological and biochemical analyses. We FACS sorted esophageal basal cells based upon fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID, then subjected these cells to transmission electron microscopy, image flow cytometry, 3D organoid assays, RNA-Sequencing (RNA-Seq), and cell cycle analysis. 3D organoids were subjected to passaging, single cell (sc) RNA-Seq, cell cycle analysis, and immunostaining. Results: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells. Esophageal basal cells with high AV level (Cyto-ID High ) displayed limited organoid formation capability upon initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-ID Low ). RNA-Seq suggested increased autophagy in Cyto- ID High esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-ID Low and Cyto- ID High cells identified expansion of 3 cell populations, enrichment of G2/M-associated genes, and aberrant localization of cell cycle-associated genes beyond basal cell populations in the Cyto- ID High group. Ki67 expression was also increased in organoids generated by Cyto-ID High cells, including in cells beyond the basal cell layer. Squamous epithelial-specific autophagy inhibition induced significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Conclusions: High AV level identifies esophageal epithelium with limited proliferation and enhanced self-renewal capacity that contributes to maintenance of the esophageal proliferation- differentiation gradient in vivo .

RNA-sequencing reveals molecular and regional differences in the esophageal mucosa of achalasia patients.

Achalasia is an esophageal motility disorder characterized by the functional loss of myenteric plexus ganglion cells in the distal esophagus and lower esophageal sphincter. Histological changes have been reported in the esophageal mucosa of achalasia, suggesting its involvement in disease pathogenesis. Despite recent advances in diagnosis, our understanding of achalasia pathogenesis at the molecular level is very limited and gene expression profiling has not been performed. We performed bulk RNA-sequencing on esophageal mucosa from 14 achalasia and 8 healthy subjects. 65 differentially expressed genes (DEGs) were found in the distal esophageal mucosa of achalasia subjects and 120 DEGs were identified in proximal esophagus. Gene expression analysis identified genes common or exclusive to proximal and distal esophagus, highlighting regional differences in the disease. Enrichment of signaling pathways related to cytokine response and viral defense were observed. Increased infiltration of CD45+ intraepithelial leukocytes were seen in the mucosa of 38 achalasia patients compared to 12 controls. Novel insights into the molecular changes occurring in achalasia were generated in this transcriptomic study. Some gene changes observed in the mucosa of achalasia may be associated with esophagitis. Differences in DEGs between distal and proximal esophagus highlight the importance of better understanding regional differences in achalasia.

Heterogeneity of primary and secondary peristalsis in systemic sclerosis: A new model of "scleroderma esophagus".

BACKGROUND: Although esophageal dysmotility is common in systemic sclerosis (SSc)/scleroderma, little is known regarding the pathophysiology of motor abnormalities driving reflux severity and dysphagia. This study aimed to assess primary and secondary peristalsis in SSc using a comprehensive esophageal motility assessment applying high-resolution manometry (HRM) and functional luminal imaging probe (FLIP) Panometry. METHODS: A total of 32 patients with scleroderma (28 female; ages 38-77; 20 limited SSc, 12 diffuse SSc) completed FLIP Panometry and HRM. Secondary peristalsis, i.e., contractile responses (CR), was classified on FLIP Panometry by pattern of contractility as normal (NCR), borderline (BCR), impaired/disordered (IDCR), or absent (ACR). Primary peristalsis on HRM was assessed according to the Chicago classification. RESULTS: The manometric diagnoses were 56% (n = 18) absent contractility, 22% (n = 7) ineffective esophageal motility (IEM), and 22% (n = 7) normal motility. Secondary peristalsis (CRs) included 38% (n = 12) ACR, 38% (n = 12) IDCR, 19% (n = 6) BCR, and 15% (n = 5) NCR. The median (IQR) esophagogastric junction (EGJ) distensibility index (DI) was 5.8 mm2 /mmHg (4.8-10.1) mm2 /mmHg; EGJ-DI was >8.0 mm2 /mmHg in 31%, and >2.0 mm2 /mmHg in 100% of patients. Among 18 patients with absent contractility on HRM, 11 had ACR, 5 had IDCR, and 2 had BCR. Among 7 patients with IEM, 1 had ACR, 5 had IDCR, and 1 NCR. All of the patients with normal peristalsis had NCR or BCR. CONCLUSIONS: This was the first study assessing combined HRM and FLIP Panometry in a cohort of SSc patients, which demonstrated heterogeneity in primary and secondary peristalsis. This complementary approach facilitates characterizing esophageal function in SSc, although future study to examine clinical outcomes remains necessary.

Single cell transcriptomic analysis reveals cellular diversity of murine esophageal epithelium.

Although morphologic progression coupled with expression of specific molecular markers has been characterized along the esophageal squamous differentiation gradient, the molecular heterogeneity within cell types along this trajectory has yet to be classified at the single cell level. To address this knowledge gap, we perform single cell RNA-sequencing of 44,679 murine esophageal epithelial, to identify 11 distinct cell populations as well as pathways alterations along the basal-superficial axis and in each individual population. We evaluate the impact of aging upon esophageal epithelial cell populations and demonstrate age-associated mitochondrial dysfunction. We compare single cell transcriptomic profiles in 3D murine organoids and human esophageal biopsies with that of murine esophageal epithelium. Finally, we employ pseudotemporal trajectory analysis to develop a working model of cell fate determination in murine esophageal epithelium. These studies provide comprehensive molecular perspective on the cellular heterogeneity of murine esophageal epithelium in the context of homeostasis and aging.

Aging is associated with a systemic length-associated transcriptome imbalance.

Aging is among the most important risk factors for morbidity and mortality. To contribute toward a molecular understanding of aging, we analyzed age-resolved transcriptomic data from multiple studies. Here, we show that transcript length alone explains most transcriptional changes observed with aging in mice and humans. We present three lines of evidence supporting the biological importance of the uncovered transcriptome imbalance. First, in vertebrates the length association primarily displays a lower relative abundance of long transcripts in aging. Second, eight antiaging interventions of the Interventions Testing Program of the National Institute on Aging can counter this length association. Third, we find that in humans and mice the genes with the longest transcripts enrich for genes reported to extend lifespan, whereas those with the shortest transcripts enrich for genes reported to shorten lifespan. Our study opens fundamental questions on aging and the organization of transcriptomes.

Klf4 overexpression activates epithelial cytokines and inflammation-mediated esophageal squamous cell cancer in mice.

BACKGROUND & AIMS: Esophageal squamous cell cancer accounts for more than 90% of cases of esophageal cancers. Its pathogenesis involves chronic epithelial irritation, although the factors involved in the inflammatory process and the mechanisms of carcinogenesis are unknown. We sought to develop a mouse model of this cancer. METHODS: We used the ED-L2 promoter of Epstein-Barr virus to overexpress the transcriptional regulator Krüppel-like factor 4 (Klf4) in esophageal epithelia of mice; we used mouse primary esophageal keratinocytes to examine the mechanisms by which KLF4 induces cytokine production. RESULTS: KLF4 was an epithelial-specific mediator of inflammation; we developed a new mouse model of esophageal squamous dysplasia and inflammation-mediated squamous cell cancer. KLF4 activated a number of proinflammatory cytokines, including TNF-α, CXCL5, G-CSF and IL-1α, within keratinocytes in an NF-κB-dependent manner. KLF4 was not detected in proliferating or cancer cells, indicating a non-cell autonomous effect of KLF4 on proliferation and carcinogenesis. CONCLUSIONS: KLF4 has distinct functions in carcinogenesis; upregulation of Klf4 specifically in esophageal epithelial cells induces inflammation. This mouse model might be used to determine the molecular mechanisms of esophageal squamous cell cancer and inflammation-mediated carcinogenesis.

Esophageal squamous cell dysplasia and delayed differentiation with deletion of krüppel-like factor 4 in murine esophagus.

BACKGROUND & AIMS: Krüppel-like factor 4 (Klf; previously known a gut-enriched Krüppel-like factor) is a DNA-binding transcriptional regulator highly expressed in skin and gastrointestinal epithelia, specifically in regions of cellular differentiation. Homozygous null mice for Klf4 die shortly after birth from skin defects, precluding their analysis at later stages. The aim of this study was to analyze the function of Klf4 in keratinocyte biology and epithelial homeostasis in the adult by focusing on the squamous lined esophagus. METHODS: By using the ED-L2 promoter of Epstein-Barr virus to drive Cre, we obtained tissue-specific ablation of Klf4 in the squamous epithelia of the tongue, esophagus, and forestomach. RESULTS: Mice with loss of Klf4 in esophageal epithelia survived to adulthood, bypassing the early lethality. Tissue-specific Klf4 knockout mice had increased basal cell proliferation and a delay in cellular maturation; these mice developed epithelial hypertrophy and subsequent dysplasia by 6 months of age. Moreover, loss of Klf4 in vivo was associated with increased expression of the pro-proliferative Klf5, and Klf4 down-regulated Klf5 both transcriptionally and posttranscriptionally. By using gene expression profiling, we also showed decreased expression of critical late-stage differentiation factors and identified alterations of several genes important in cellular differentiation. CONCLUSIONS: Klf4 is essential for squamous epithelial differentiation in vivo and interacts with Klf5 to maintain normal epithelial homeostasis.

Polycystin-1 is a microtubule-driven desmosome-associated component in polarized epithelial cells.

In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell-cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the beta-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell-cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.

IκB Kinase-ß Regulates Neutrophil Recruitment Through Activation of STAT3 Signaling in the Esophagus.

BACKGROUND & AIMS: The epithelial barrier is the host's first line of defense against damage to the underlying tissue. Upon injury, the epithelium plays a critical role in inflammation. The IκB kinase ß (IKKß)/nuclear factor-κB pathway was shown to be active in the esophageal epithelium of patients with esophageal disease. However, the complex mechanisms by which IKKß signaling regulates esophageal disease pathogenesis remain unknown. Our prior work has shown that expression of a constitutively active form of IKKß specifically in esophageal epithelia of mice (IkkßcaEsophageal Epithelial Cell-Knockin (EEC-KI)) is sufficient to cause esophagitis. METHODS: We generated ED-L2/Cre;Rosa26-Ikkßca+/L;Stat3L/L (IkkßcaEEC-KI;Stat3Esophageal Epithelial Cell Knockout (EEC-KO)) mice, in which the ED-L2 promoter activates Cre recombinase in the esophageal epithelium, leading to constitutive activation of IKKß and loss of Stat3. Esophageal epithelial tissues were collected and analyzed by immunostaining, RNA sequencing, quantitative real-time polymerase chain reaction assays, flow cytometry, and Western blot. IkkßcaEEC-KI mice were treated with neutralizing antibodies against interleukin (IL)23p19 and IL12p40. RESULTS: Here, we report that IkkßcaEEC-KI mice have increased activation of epithelial Janus kinase 2/STAT3 signaling. Stat3 deletion in IkkßcaEEC-KI mice attenuated the neutrophil infiltration observed in IkkßcaEEC-KI mice and resulted in decreased expression of genes related to immune cell recruitment and activity. Blocking experiments in IkkßcaEEC-KI mice showed that STAT3 activation and subsequent neutrophil recruitment are dependent on IL23 secretion. CONCLUSIONS: Our study establishes a novel interplay between IKKß and STAT3 signaling in epithelial cells of the esophagus, where IKKß/IL23/STAT3 signaling controls neutrophil recruitment during the onset of inflammation. GEO accession number: GSE154129.