Mei-Lun Wang

Thymic stromal lymphopoietin–elicited basophil responses promote eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a food allergy–associated inflammatory disease characterized by esophageal eosinophilia. Current management strategies for EoE are nonspecific, and thus there is a need to identify specific immunological pathways that could be targeted to treat this disease. EoE is associated with polymorphisms in the gene that encodes thymic stromal lymphopoietin (TSLP), a cytokine that promotes allergic inflammation, but how TSLP might contribute to EoE disease pathogenesis has been unclear. Here, we describe a new mouse model of EoE-like disease that developed independently of IgE, but was dependent on TSLP and basophils, as targeting TSLP or basophils during the sensitization phase limited disease. Notably, therapeutic TSLP neutralization or basophil depletion also ameliorated established EoE-like disease. In human subjects with EoE, we observed elevated TSLP expression and exaggerated basophil responses in esophageal biopsies, and a gain-of-function TSLP polymorphism was associated with increased basophil responses in patients with EoE. Together, these data suggest that the TSLP-basophil axis contributes to the pathogenesis of EoE and could be therapeutically targeted to treat this disease.

Identification of causative foods in children with eosinophilic esophagitis treated with an elimination diet.

BACKGROUND Eosinophilic esophagitis (EoE) is a chronic inflammatory disease with isolated eosinophils in the esophagus predominantly triggered by foods. The optimal testing to identify inciting foods remains unclear. OBJECTIVES We sought to determine the effectiveness of allergy testing-directed diets in patients with EoE. METHODS A retrospective analysis of all children with EoE seen at the Childrens Hospital of Philadelphia between 2000 and 2011 identified 941 patients with EoE. Skin prick tests (SPTs) and atopy patch tests (APTs) were conducted, and predictive values were calculated. IgE-mediated food reactions were also identified. A food was considered to cause EoE if its elimination led to resolution of esophageal eosinophilia or reintroduction led to reoccurrence of EoE. The effectiveness of the various elimination diets was compared with targeted food antigen elimination. RESULTS Definitive foods causing EoE were identified, with milk, egg, wheat, and soy as the most common foods in 319 patients. IgE-mediated reactions (urticaria and anaphylaxis) were seen in 15%. The negative predictive value for the combination of SPTs and APTs averaged 92%, with the exception of milk at 44%, and the positive predictive value averaged 44%. An empiric 6-food elimination diet or removal of positive foods on allergy testing (SPTs/APTs) both had a histologic success rate of 53%. Removal of foods identified on SPTs/APTs plus empiric elimination of milk leads to resolution in 77% of patients. CONCLUSION An elimination diet based on SPT/APT results leads to resolution of esophageal eosinophilia in a similar proportion of patients as empiric removal of foods but required that fewer foods be removed. These observations suggest that both methods are acceptable options.

GWAS identifies four novel eosinophilic esophagitis loci

Eosinophilic esophagitis (EoE) is an allergic disorder characterized by infiltration of the esophagus with eosinophils. We had previously reported association of the TSLP/WDR36 locus with EoE. Here we report genome-wide significant associations at four additional loci; c11orf30 and STAT6, which have been previously associated with both atopic and autoimmune disease, and two EoE-specific loci, ANKRD27 that regulates the trafficking of melanogenic enzymes to epidermal melanocytes and CAPN14, that encodes a calpain whose expression is highly enriched in the esophagus. The identification of five EoE loci, not only expands our etiological understanding of the disease but may also represent new therapeutic targets to treat the most debilitating aspect of EoE, esophageal inflammation and remodeling.

Invariant natural killer T cells in children with eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an atopic disease characterized by eosinophilic inflammation in which dietary antigens (in particular, milk) play a major role. EoE is most likely a mixed IgE and non‐IgE food‐mediated reaction in which overexpression of Th2 cytokines, particularly IL‐13, play a major role; however, the cells responsible for IL‐13 overexpression remain elusive. Th2‐cytokines are secreted following the ligation of invariant natural killer T cell receptors to sphingolipids (SLs). Sphingolipids (SLs) are presented via the CD1d molecule on the INKTs surface. Cows milk‐derived SL has been shown to activate iNKTs from children with IgE‐mediated food allergies to milk (FA‐MA) to produce Th2 cytokines. The role of iNKTs and milk‐SL in EoE pathogenesis is currently unknown.

Thymic stromal lymphopoietin-mediated extramedullary hematopoiesis promotes allergic inflammation.

Extramedullary hematopoiesis (EMH) refers to the differentiation of hematopoietic stem cells (HSCs) into effector cells that occurs in compartments outside of the bone marrow. Previous studies linked pattern-recognition receptor (PRR)-expressing HSCs, EMH, and immune responses to microbial stimuli. However, whether EMH operates in broader immune contexts remains unknown. Here, we demonstrate a previously unrecognized role for thymic stromal lymphopoietin (TSLP) in promoting the population expansion of progenitor cells in the periphery and identify that TSLP-elicited progenitors differentiated into effector cells including macrophages, dendritic cells, and granulocytes and that these cells contributed to type 2 cytokine responses. The frequency of circulating progenitor cells was also increased in allergic patients with a gain-of-function polymorphism in TSLP, suggesting the TSLP-EMH pathway might operate in human disease. These data identify that TSLP-induced EMH contributes to the development of allergic inflammation and indicate that EMH is a conserved mechanism of innate immunity.

BMP-driven NRF2 activation in esophageal basal cell differentiation and eosinophilic esophagitis

Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells, especially in tissues that are constantly replenished like the esophagus. Disruption of this balance is associated with pathological conditions, including eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic upon proinflammatory stimulation. However, how basal cells respond to the inflammatory environment at the molecular level remains undetermined. We previously reported that the bone morphogenetic protein (BMP) signaling pathway is critical for epithelial morphogenesis in the embryonic esophagus. Here, we address how this pathway regulates tissue homeostasis and EoE development in the adult esophagus. BMP signaling was specifically activated in differentiated squamous epithelium, but not in basal progenitor cells, which express the BMP antagonist follistatin. Previous reports indicate that increased BMP activity promotes Barretts intestinal differentiation; however, in mice, basal progenitor cell-specific expression of constitutively active BMP promoted squamous differentiation. Moreover, BMP activation increased intracellular ROS levels, initiating an NRF2-mediated oxidative response during basal progenitor cell differentiation. In both a mouse EoE model and human biopsies, reduced squamous differentiation was associated with high levels of follistatin and disrupted BMP/NRF2 pathways. We therefore propose a model in which normal squamous differentiation of basal progenitor cells is mediated by BMP-driven NRF2 activation and basal cell hyperplasia is promoted by disruption of BMP signaling in EoE.

Inflammation-associated microbiota in pediatric eosinophilic esophagitis

BackgroundEosinophilic esophagitis (EoE) is an allergic disorder characterized by eosinophil-predominant esophageal inflammation, which can be ameliorated by food antigen restriction. Though recent studies suggest that changes in dietary composition may alter the distal gut microbiome, little is currently known about the impact of a restricted diet upon microbial communities of the oral and esophageal microenvironments in the context of EoE. We hypothesize that the oral and esophageal microbiomes of EoE patients are distinct from non-EoE controls, that these differences correspond to changes in esophageal inflammation, and that targeted therapeutic dietary intervention may influence community structure. Using 16S rRNA gene sequencing, we characterized the bacterial composition of the oral and esophageal microenvironments using oral swabs and esophageal biopsies from 35 non-EoE pediatric controls and compared this cohort to samples from 33 pediatric EoE subjects studied in a longitudinal fashion before and after defined dietary changes.ResultsFirmicutes were more abundant in esophageal samples compared to oral. Proportions of bacterial communities were significantly different comparing all EoE esophageal microbiota to non-EoE controls, with enrichment of Proteobacteria, including Neisseria and Corynebacterium in the EoE cohort, and predominance of the Firmicutes in non-EoE control subjects. We detected a statistically significant difference between actively inflamed EoE biopsies and non-EoE controls. Overall, though targeted dietary intervention did not lead to significant differences in either oral or esophageal microbiota, reintroduction of highly allergenic foods led to enrichment in Ganulicatella and Campylobacter genera in the esophagus.ConclusionsIn conclusion, the esophageal microbiome in EoE is distinct from that of non-EoE controls, with maximal differences observed during active allergic inflammation.

TLR3-mediated NF-κB signaling in human esophageal epithelial cells

Despite its position at the front line against ingested pathogens, very little is presently known about the role of the esophageal epithelium in host innate immune defense. As a key player in the innate immune response, Toll-like receptor (TLR) signaling has not been well characterized in human esophageal epithelial cells. In the present study, we investigated the inflammatory response and signaling pathways activated by TLR stimulation of human esophageal cells in vitro. Using quantitative RT-PCR, we profiled the expression pattern of human TLRs 1-10 in primary esophageal keratinocytes (EPC2), immortalized nontransformed esophageal keratinocytes (EPC2-hTERT), and normal human esophageal mucosal biopsies and found that TLRs 1, 2, 3, and 5 were expressed both in vivo and in vitro. Using the cytokine IL-8 as a physiological read out of the inflammatory response, we found that TLR3 is the most functional of the expressed TLRs in both primary and immortalized esophageal epithelial cell lines in response to its synthetic ligand polyinosinic polycytidylic acid [poly(I:C)]. Through reporter gene studies, we show that poly(I:C)-induced NF-kappaB activation is critical for the transactivation of the IL-8 promoter in vitro and that nuclear translocation of NF-kappaB occurs at an early time point following poly(I:C) stimulation of esophageal epithelial cells. Importantly, we also show that poly(I:C) stimulation induces the NF-kappaB-dependent esophageal epithelial expression of TLR2, leading to enhanced epithelial responsiveness of EPC2-hTERT cells to TLR2 ligand stimulation, suggesting an important regulatory role for TLR3-mediated NF-kappaB signaling in the innate immune response of esophageal epithelial cells. Our findings demonstrate for the first time that TLR3 is highly functional in the human esophageal epithelium and that TLR3-mediated NF-kappaB signaling may play an important regulatory role in esophageal epithelial homeostasis.

Esophageal epithelial and mesenchymal cross-talk leads to features of epithelial to mesenchymal transition in vitro

BACKGROUND Esophageal fibrosis is a complication of eosinophilic esophagitis (EoE) which has been attributed to both subepithelial fibrosis and to epithelial to mesenchymal transition (EMT), a process by which epithelial cells acquire mesenchymal features. Common to both causes of EoE-fibrosis is the notion that granulocyte-derived TGF-β, induces myofibroblast differentiation of the target cell. To date, the role of esophageal epithelial cells as effector cells in esophageal fibrosis has never been explored. Herein, we investigated consequences of cross-talk between esophageal epithelial cells and fibroblasts, and identified profibrotic cytokines which influence the development of EMT in vitro. METHODS AND RESULTS Stimulation of primary fetal esophageal fibroblasts (FEF3) with conditioned media (CEM) from esophageal epithelial cells (EPC2-hTERT), primed FEF3 cells to secrete IL-1β and TNFα, but not TGFβ. To determine whether these cytokines signaled in a paracrine fashion to esophageal epithelial cells, FEF3 cells were stimulated with CEM, followed by transfer of this fibroblast conditioned media (FCM) to EPC2-hTERT cells. Epithelial FCM stimulation increased expression of mesenchymal markers and reduced E-cadherin expression, features of EMT which were TNFα and IL-1β-dependent. Using organotypic culture models, primary EoE epithelial cells exhibited features of EMT compared to non-EoE cells, corresponding to patterns of EMT in native biopsies. CONCLUSIONS Esophageal epithelial cell and fibroblast cross-talk contributes to esophageal fibrosis. Our results suggest that features of EMT can develop independent of TGF-β and granulocytes, which may have important implications in treatment of EoE.

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.