IL-4-STAT6 axis amplifies histamine-induced vascular endothelial dysfunction and hypovolemic shock.

BACKGROUND: Mast cell (MC)-derived mediators induce vasodilatation and fluid extravasation, leading to cardiovascular failure in severe anaphylaxis. We have previously revealed a synergistic interaction between the cytokine IL-4 and the MC-derived mediator histamine in modulating vascular endothelial (VE) dysfunction and severe anaphylaxis. The mechanism by which IL-4 exacerbates histamine-induced VE dysfunction and severe anaphylaxis are unknown. OBJECTIVE: To identify the IL-4-induced molecular processes regulating the amplification of histamine-induced VE barrier dysfunction and the severity of IgE-mediated anaphylaxic reactions. METHODS: RNAseq, Western blot, Ca2+ imaging and barrier functional analyses on the vascular endothelial cell line (EA.hy926). Pharmacologic degraders (selective PROTAC (proteolysis-targeting chimera) and genetic (lentiviral shRNA) inhibitors were used to determine the roles of STAT3 and STAT6 in conjunction with in vivo model systems of histamine-induced hypovolemic shock. RESULTS: IL-4 enhancement of histamine-induced VE barrier dysfunction was associated with increased VE-Cadherin degradation, intracellular calcium flux, phospho-Src levels and required transcription and de novo protein synthesis. RNAseq analyses of IL-4 stimulated VE cells identified dysregulation of genes involved in cell proliferation, cell development, and cell growth and transcription factor motif analyses revealed a significant enrichment of differential expressed genes (DEGs) with putative STAT3 and STAT6 motif. IL-4 stimulation in EA.hy926 cells induced both STAT3Y705 and STAT3S727 phosphorylation. Genetic and pharmacologic ablation of VE STAT3 activity revealed a role for STAT3 in basal VE barrier function, however IL-4 enhancement and histamine-induced VE barrier dysfunction was predominantly STAT3-independent. In contrast, IL-4 enhancement and histamine-induced VE barrier dysfunction was STAT6-dependent. Consistent with this finding, pharmacologic knockdown of STAT6 abrogated IL-4-mediated amplification of histamine-induced hypovolemia. CONCLUSIONS: These studies unveil a novel role of the IL-4/ STAT6 signaling axis in the priming of VE cells predisposing to exacerbation of histamine-induced anaphylaxis.

IL-13-induced STAT3-dependent signaling networks regulate esophageal epithelial proliferation in eosinophilic esophagitis.

BACKGROUND: Basal zone hyperplasia (BZH) and dilated intercellular spaces (DISs) are thought to contribute to the clinical manifestations of eosinophilic esophagitis (EoE); however, the molecular pathways that drive BZH remain largely unexplored. OBJECTIVE: We sought to define the role of IL-13-induced transcriptional programs in esophageal epithelial proliferation in EoE. METHODS: We performed RNA sequencing, bioinformatics, Western blot, reverse transcriptase quantitative PCR, and histologic analyses on esophageal biopsies from healthy control and patients with EoE, primary esophageal cells derived from patients with EoE, and IL-13-stimulated esophageal epithelial keratinocytes grown at the air-liquid interface (EPC2-ALI). Genetic (shRNA) and pharmacologic (proteolysis-targeting chimera degrader) approaches and in vivo model of IL-13-induced esophageal epithelial remodeling (Krt5-rtTA x tetO-IL-13Tg) were used to define the role of signal transducer and activator of transcription 3 (STAT3) and STAT6 and secreted frizzled-related protein 1 (SFRP1) in esophageal epithelial proliferation. RESULTS: RNA-sequencing analysis of esophageal biopsies (healthy control vs EoE) and EPC2-ALI revealed 82 common differentially expressed genes that were enriched for putative STAT3 target genes. In vitro and in vivo analyses revealed a link between IL-13-induced STAT3 and STAT6 phosphorylation, SFRP1 mRNA expression, and esophageal epithelial proliferation. In vitro studies showed that IL-13-induced esophageal epithelial proliferation was STAT3-dependent and regulated by the STAT3 target SFRP1. SFRP1 mRNA is increased in esophageal biopsies from patients with active EoE compared with healthy controls or patients in remission and identifies an esophageal suprabasal epithelial cell subpopulation that uniquely expressed the core EoE proinflammatory transcriptome genes (CCL26, ALOX15, CAPN14, ANO1, and TNFAIP6). CONCLUSIONS: These studies identify SFRP1 as a key regulator of IL-13-induced and STAT3-dependent esophageal proliferation and BZH in EoE and link SFRP1+ esophageal epithelial cells with the proinflammatory and epithelial remodeling response in EoE.

Eosinophilic esophagitis: Immune mechanisms and therapeutic targets.

Eosinophilic esophagitis (EoE) is an emerging chronic inflammatory disease of the oesophagus and is clinically characterized by upper gastrointestinal (GI) symptoms including dysphagia and esophageal food impaction. Histopathologic manifestations, which include intraepithelial eosinophilic inflammation and alterations of the esophageal squamous epithelium, such as basal zone hyperplasia (BZH) and dilated intercellular spaces (DIS), are thought to contribute to esophageal dysfunction and disease symptoms. Corroborative clinical and discovery science-based studies have established that EoE is characterized by an underlying allergic inflammatory response, in part, related to the IL-13/CCL26/eosinophil axis driving dysregulation of several key epithelial barrier and proliferative regulatory genes including kallikrein (KLK) serine proteases, calpain 14 (CAPN14) and anoctamin 1 (ANO1). The contribution of these inflammatory and proliferative processes to the clinical and histological manifestations of disease are not fully elucidated. Herein, we discuss the immune molecules and cells that are thought to underlie the clinical and pathologic manifestations of EoE and the emerging therapeutics targeting these processes for the treatment of EoE.

An experimental evaluation of the efficacy of perinatal sulforaphane supplementation to decrease the incidence and severity of vinclozolin-induced hypospadias in the mouse model.

Determining the mechanisms of toxicity induced by pollutants has long been a research priority in lieu of considering the mechanisms of resilience that prevent deleterious impacts. Protective mechanisms in many taxa can be therapeutically targeted to enhance resilience to synthetic toxicants. For example, the environmental sensor, Nuclear factor (erythroid-derived 2)-like 2 (Nfe2l2 or Nrf2), a transcription factor, facilitates transcription of many protective genes. Hypospadias is a common malformation of the penis. The risk of being born with hypospadias increases with pollutant exposure. We use vinclozolin-induced hypospadias in the mouse as a model to test the hypothesis that pollutant-induced birth defects can be prevented and reduced in severity by augmenting natural mechanisms of resilience. Pregnant mice were exposed to the demasculinizing toxicant, vinclozolin, in combination with increasing doses of the NRF2 activator, sulforaphane. The sulforaphane dose that most effectively increased masculinization (anogenital distance) was identified and used to test the hypothesis that sulforaphane reduces the hypospadias-inducing potency of vinclozolin. Finally, a Nrf2 knockout study was conducted to test whether NRF2 was required for the sulforaphane-induced rescue effects. Sulforaphane supplementation to vinclozolin exposed embryos increased anogenital distance in a nonlinear fashion typical of Nrf2 activators. The most effective dose of sulforaphane (45 mg/kg) reduced the occurrence and severity of vinclozolin-induced hypospadias and corrected penis morphogenesis. The sulforaphane-induced rescue effect was dependent on the presence of Nrf2. Nrf2 plays a critical role in protecting the fetus from vinclozolin and reduces the incidence and severity of hypospadias, the most common birth defect in boys in many countries. This work lays a foundation for developing prenatal supplements that will protect the fetus from pollutant-induced hypospadias. Studying the protective mechanisms that drive resilience to toxicants will facilitate innovation of protective therapies.

PIR-B Regulates CD4+ IL17a+ T-Cell Survival and Restricts T-Cell-Dependent Intestinal Inflammatory Responses.

BACKGROUND & AIMS: CD4+ T cells are regulated by activating and inhibitory cues, and dysregulation of these proper regulatory inputs predisposes these cells to aberrant inflammation and exacerbation of disease. We investigated the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of the CD4+ T-cell inflammatory response and exacerbation of the colitic phenotype. METHODS: We used Il10-/- spontaneous and CD4+CD45RBhi T-cell transfer models of colitis with PIR-B-deficient (Pirb-/-) mice. Flow cytometry, Western blot, and RNA sequencing analysis was performed on wild-type and Pirb-/- CD4+ T cells. In silico analyses were performed on RNA sequencing data set of ileal biopsy samples from pediatric CD and non-inflammatory bowel disease patients and sorted human memory CD4+ T cells. RESULTS: We identified PIR-B expression on memory CD4+ interleukin (IL)17a+ cells. We show that PIR-B regulates CD4+ T-helper 17 cell (Th17)-dependent chronic intestinal inflammatory responses and the development of colitis. Mechanistically, we show that the PIR-B- Src-homology region 2 domain-containing phosphatase-1/2 axis tempers mammalian target of rapamycin complex 1 signaling and mammalian target of rapamycin complex 1-dependent caspase-3/7 apoptosis, resulting in CD4+ IL17a+ cell survival. In silico analyses showed enrichment of transcriptional signatures for Th17 cells (RORC, RORA, and IL17A) and tissue resident memory (HOBIT, IL7R, and BLIMP1) networks in PIR-B+ murine CD4+ T cells and human CD4+ T cells that express the human homologue leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3). High levels of LILRB3 expression were associated strongly with mucosal injury and a proinflammatory Th17 signature, and this signature was restricted to a treatment-naïve, severe pediatric CD population. CONCLUSIONS: Our findings show an intrinsic role for PIR-B/LILRB3 in the regulation of CD4+ IL17a+ T-cell pathogenic memory responses.

Identification of anoctamin 1 (ANO1) as a key driver of esophageal epithelial proliferation in eosinophilic esophagitis.

BACKGROUND: The pathology of eosinophilic esophagitis (EoE) is characterized by eosinophil-rich inflammation, basal zone hyperplasia (BZH), and dilated intercellular spaces, and the underlying processes that drive the pathologic manifestations of the disease remain largely unexplored. OBJECTIVE: We sought to investigate the involvement of the calcium-activated chloride channel anoctamin 1 (ANO1) in esophageal proliferation and the histopathologic features of EoE. METHODS: We examined mRNA and protein expression of ANO1 in esophageal biopsy samples from patients with EoE and in mice with EoE. We performed molecular and cellular analyses and ion transport assays on an in vitro esophageal epithelial 3-dimensional model system (EPC2-ALI) and murine models of EoE to define the relationship between expression and function of ANO1 and esophageal epithelial proliferation in patients with EoE. RESULTS: We observed increased ANO1 expression in esophageal biopsy samples from patients with EoE and in mice with EoE. ANO1 was expressed within the esophageal basal zone, and expression correlated positively with disease severity (eosinophils/high-power field) and BZH. Using an in vitro esophageal epithelial 3-dimensional model system revealed that ANO1 undergoes chromatin modification and rapid upregulation of expression after IL-13 stimulation, that ANO1 is the primary apical IL-13-induced Cl- transport mechanism within the esophageal epithelium, and that loss of ANO1-dependent Cl- transport abrogated esophageal epithelial proliferation. Mechanistically, ANO1-dependent regulation of basal cell proliferation was associated with modulation of TP63 expression and phosphorylated cyclin-dependent kinase 2 levels. CONCLUSIONS: These data identify a functional role for ANO1 in esophageal cell proliferation and BZH in patients with EoE and provide a rationale for pharmacologic intervention of ANO1 function in patients with EoE.