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Background and Aims: Tesnatilimab, a monoclonal antibody targeting NKG2D, was evaluated in Crohn's disease [CD] patients who had failed or were intolerant to biologic or conventional therapy., Methods: TRIDENT was a phase 2b, two-part, randomised, double-blind, placebo-controlled, parallel-arm, multicenter study. In Part 1 [proof of concept], 145 patients who were biologic intolerant or refractory [Bio-IR] or had not failed biologic therapy [Bio-NF] were randomised in a 1:1 ratio to placebo subcutaneously [SC] or tesnatilimab 400 mg SC. In Part 2 [dose ranging], 243 Bio-IR and Bio-NF patients were randomised in a 1:1:1:1:1 ratio to placebo, tesnatilimab [50 mg, 150 mg, 400 mg], or intravenous infusion of ustekinumab ~6 mg/kg at Week 0 and 90 mg SC at Weeks 8 and 16. The primary endpoint was mean change from baseline in Crohn's Disease Activity Index [CDAI] at Week 8 [Part 1] and Week 12 [Part 2]. Clinical and endoscopic remission/response were evaluated. Efficacy analyses were also assessed by NKG2D and MICB single nucleotide polymorphism [SNP] status [SNP-positive means positive in at least one of two SNPs]. Safety events were summarised., Results: In Part 1, mean change from baseline in CDAI score was significantly greater with tesnatilimab vs placebo at Week 8 [-103.6 vs -60.0; p < 0.01]. In Part 2, no dose-response signal was detected. Mean changes from baseline in CDAI at Week 12 were -93.2, -72.2, and -84.3 for low, middle, and high doses of tesnatilimab, respectively, vs -59.2 for placebo and -148.8 for ustekinumab. Similar reductions from baseline in CDAI score were observed in patients receiving tesnatilimab, regardless of SNP status. Clinical remission rates were greater with tesnatilimab than placebo in Parts 1 and 2, whereas endoscopic response rates were greater with tesnatilimab only in Part 1. No unexpected safety events occurred., Conclusions: Tesnatilimab was well tolerated. The efficacy of tesnatilimab in patients with CD was significant for the primary endpoint in Part 1; however, no dose-response signal was detected for the primary endpoint in Part 2. Based on these inconsistent findings, tesnatilimab was not considered an effective treatment for patients with CD and no further development is planned., Clinicaltrials.gov Identifier: NCT02877134., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: This ongoing Phase-2, randomised, placebo-controlled, double-blind study evaluated the efficacy, safety and pharmacokinetics of intravenous belimumab in childhood-onset systemic lupus erythematosus (cSLE)., Methods: Patients (5 to 17 years) were randomised to belimumab 10 mg/kg intravenous or placebo every 4 weeks, plus standard SLE therapy. Primary endpoint: SLE Responder Index (SRI4) response rate (Week 52). Key major secondary endpoints: proportion of patients achieving the Paediatric Rheumatology International Trials Organisation/American College of Rheumatology (PRINTO/ACR) response using 50 and '30 alternative' definitions (Week 52), and sustained response (Weeks 44 to 52) by SRI4 and Parent Global Assessment of well-being (Parent-global). Safety and pharmacokinetics were assessed. Study not powered for statistical testing., Results: Ninety-three patients were randomised (belimumab, n=53; placebo, n=40). At Week 52, there were numerically more SRI4 responders with belimumab versus placebo (52.8% vs 43.6%; OR 1.49 (95% CI 0.64 to 3.46)). PRINTO/ACR 30 alternative (52.8% vs 27.5%; OR 2.92 (95% CI 1.19 to 7.17)) and PRINTO/ACR 50 (60.4% vs 35.0%; OR 2.74 (95% CI 1.15 to 6.54)) responses were more frequent with belimumab than placebo, as were sustained responses for SRI4 (belimumab, 43.4%; placebo, 41.0%; OR 1.08 (95% CI 0.46 to 2.52)) and Parent-global (belimumab, 59.1%; placebo, 33.3%; OR 3.49 (95% CI 1.23 to 9.91)). Serious adverse events were reported in 17.0% of belimumab patients and 35.0% of placebo patients; one death occurred (placebo). Week-52, geometric mean (95% CI) belimumab trough concentration was 56.2 (45.2 to 69.8) µg/mL., Conclusion: The belimumab intravenous pharmacokinetics and benefit-risk profile in cSLE are consistent with adult belimumab studies and the 10 mg/kg every 4 weeks dose is appropriate., Trial Registration Number: NCT01649765., Competing Interests: Competing interests: DB, KC, AH, AN, DAR, BJ and HS are employees of GSK and hold shares/options in the company; M-LW is a former employee of GSK; HIB has served the speakers bureau of GSK, Roche and Novartis, has been a consultant to Hoffman-La Roche, Novartis, Pfizer, Sanofi Aventis, Merck Serono, AbbVie, Amgen, Alter, AstraZeneca, Baxalta Biosimilars, Biogen Idec, Boehringer, BMS, Celgene, EMD Serono, Janssen, MedImmune, Novartis, Pfizer and UCB Biosciences GmbH. Payments are made to CCHMC, the employer of HIB; AM has received honoraria for consultancies (<10 000 US$ each) from Eli-Lilly, EMD Serono, Janssen, Novartis, Pfizer and AbbVie; DML has received honoraria and/or consulting fees (<10 000 US$ each) from AbbVie, Janssen and Sobi; NR has received speaker’s bureau and reimbursement of travel expenses from GSK, honoraria for consultancies (<10 000 US$ each) from Ablynx, AbbVie, AstraZeneca-MedImmune, Biogen, Boehringer, Bristol Myers Squibb, Eli-Lilly, EMD Serono, GSK, Hoffmann-La Roche, Janssen, Merck, Novartis, Pfizer, R-Pharma, Sanofi, Servier, Sinergie, Sobi and Takeda. The IRCCS Istituto Giannina Gaslini (IGG), where NR works as full-time public employee has received contributions (>10 000 US$ each) from BMS, Eli-Lilly, GSK, Hoffmann-La Roche, Janssen, Novartis, Pfizer and Sobi. This funding has been reinvested for research activities of the hospital in a fully independent manner, without any commitment with third parties.; JA has received consulting fees and/or honoraria from AbbVie, BMS, Gebro, Novartis, Pfizer, Roche and Sobi; CA-M has received honoraria for consultancies or speaker bureaus from Pfizer, Eli Lilly and Takeda; ICP has received consulting fees from AbbVie, BMS, Novartis, Pfizer, Roche and Sobi; MS has received honoraria for consultancies or speaker bureaus (<10 000 USD each) from AbbVie, Medac Pharm and Novartis., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Objective: This extension study of the Phase III, randomized, placebo-controlled Belimumab International SLE Study (BLISS)-52 and BLISS-76 studies allowed non-US patients with SLE to continue belimumab treatment, in order to evaluate its long-term safety and tolerability including organ damage accrual., Methods: In this multicentre, long-term extension study (GlaxoSmithKline Study BEL112234) patients received i.v. belimumab every 4 weeks plus standard therapy. Adverse events (AEs) were assessed monthly and safety-associated laboratory parameters were assessed at regular intervals. Organ damage (SLICC/ACR Damage Index) was assessed every 48 weeks. The study continued until belimumab was commercially available, with a subsequent 8-week follow-up period., Results: A total of 738 patients entered the extension study and 735/738 (99.6%) received one or more doses of belimumab. Annual incidence of AEs, including serious and severe AEs, remained stable or declined over time. Sixty-nine (9.4%) patients experienced an AE resulting in discontinuation of belimumab or withdrawal from the study. Eleven deaths occurred (and two during post-treatment follow-up), including one (cardiogenic shock) considered possibly related to belimumab. Laboratory parameters generally remained stable. The mean (s.d.) SLICC/ACR Damage Index score was 0.6 (1.02) at baseline (prior to the first dose of belimumab) and remained stable. At study year 8, 57/65 (87.7%) patients had no change in SLICC/ACR Damage Index score from baseline, indicating low organ damage accrual., Conclusion: Belimumab displayed a stable safety profile with no new safety signals. There was minimal organ damage progression over 8 years., Trial Registration: ClinicalTrials.gov, https://clinicaltrials.gov, NCT00424476 (BLISS-52), NCT00410384 (BLISS-76), NCT00732940 (BEL112232), NCT00712933 (BEL112234)., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Objectives: TSLP has been shown to be associated with eosinophilic esophagitis (EoE). Specifically, children with EoE often have the nucleotides AA or AG instead of GG at the single nucleotide polymorphism position RS3806932. Presently, the phenotypic characteristics in EoE children with the TSLP EoE risk allele are unknown., Methods: A retrospective analysis was performed of all children with EoE who had TSLP genotyping at The Children's Hospital of Philadelphia from 2008-2014. EoE food allergen triggers, presence of atopic features, IgE mediated food allergy and skin prick testing results were reviewed. The number and type of EoE food allergen triggers were compared with genotype using chi-square analysis. Primary cell cultures from EoE patients with or without the risk allele were stimulated with ovalbumin and TSLP secretion was measured by ELISA., Results: Fifty three of 309 patients were found to have no copies of the TSLP risk allele, whereas 256 patients were found to have one or more copies of the risk allele. There was an increase in the number of patients with three or more EoE food allergens among those who were either homozygous or heterozygous for the risk allele compared to those without the risk allele (P < 0.0001). This was independent of their atopic background. Primary cultures from patients homozygous for the risk allele had greater TSLP secretion than those isolated from heterozygous patients., Conclusions: The TSLP risk allele is associated with having multiple EoE food allergen triggers. This novel EoE genotypic-phenotypic correlation may guide future treatment for those with the TSLP risk allele.

GSK2982772 is a highly selective inhibitor of receptor-interacting protein kinase 1 (RIPK1) being developed to treat chronic inflammatory diseases. This first-in-human study evaluated safety, tolerability, pharmacokinetics (PK), and exploratory pharmacodynamics (PD) of GSK2982772 administered orally to healthy male volunteers. This was a Phase I, randomized, placebo-controlled, double-blind study. In Part A, subjects received single ascending doses of GSK2982772 (0.1-120 mg) or placebo in a crossover design during each of 4 treatment periods. In Part B, subjects received repeat doses of GSK2982772 (20 mg once daily [QD] to up to 120 mg twice daily [BID]) or placebo for 14 days. Part C was an open-label relative bioavailability study comparing 20-mg tablets vs capsules. Safety, tolerability, pharmacokinetics (PK), RIPK1 target engagement (TE), and pharmacodynamics (PD) were assessed. The most common adverse events (AEs) were contact dermatitis and headache. Most AEs were mild in intensity, and there were no deaths or serious AEs. The PK of GSK2982772 was approximately linear over the dose range studied (up to 120 mg BID). There was no evidence of drug accumulation upon repeat dosing. Greater than 90% RIPK1 TE was achieved over a 24-hour period for the 60-mg and 120-mg BID dosing regimens. Single and repeat doses of GSK2982772 were safe and well tolerated. PK profiles showed dose linearity. The high levels of RIPK1 TE support progression into Phase II clinical trials for further clinical development., (© 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Objective: The influence of eosinophilic oesophagitis (EoE)-associated inflammation upon oesophageal epithelial biology remains poorly understood. We investigated the functional role of autophagy in oesophageal epithelial cells (keratinocytes) exposed to the inflammatory EoE milieu., Design: Functional consequences of genetic or pharmacological autophagy inhibition were assessed in endoscopic oesophageal biopsies, human oesophageal keratinocytes, single cell-derived ex vivo murine oesophageal organoids as well as a murine model recapitulating EoE-like inflammation and basal cell hyperplasia. Gene expression, morphological and functional characterisation of autophagy and oxidative stress were performed by transmission electron microscopy, immunostaining, immunoblotting, live cell imaging and flow cytometry., Results: EoE-relevant inflammatory conditions promoted autophagy and basal cell hyperplasia in three independent murine EoE models and oesophageal organoids. Inhibition of autophagic flux via chloroquine treatment augmented basal cell hyperplasia in these model systems. Oesophageal keratinocytes stimulated with EoE-relevant cytokines, including tumour necrosis factor-α and interleukin-13 exhibited activation of autophagic flux in a reactive oxygen species-dependent manner. Autophagy inhibition via chloroquine treatment or depletion of Beclin-1 or ATG-7, augmented oxidative stress induced by EoE-relevant stimuli in murine EoE, oesophageal organoids and human oesophageal keratinocytes. Oesophageal epithelia of paediatric EoE patients with active inflammation displayed increased autophagic vesicle content compared with normal and EoE remission subjects. Functional flow cytometric analysis revealed autophagic flux in human oesophageal biopsies., Conclusions: Our findings reveal for the first time that autophagy may function as a cytoprotective mechanism to maintain epithelial redox balance and homeostasis under EoE inflammation-associated stress, providing mechanistic insights into the role of autophagy in EoE pathogenesis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Objectives: Eosinophilic esophagitis (EoE) is an immune-mediated allergic disease characterized by progressive esophageal dysmotility and fibrotic stricture associated with chronic esophageal fibroblast activation. It remains unknown how esophageal fibroblasts respond to EoE-relevant matrix stiffness or inflammatory cytokines., Methods: Immunofluorescence was used to evaluate α-smooth muscle actin (α-SMA) expression in endoscopic esophageal biopsies. Primary esophageal fibroblasts from adult and pediatric patients with or without EoE were exposed to transforming growth factor (TGF)β to determine gene expression, collagen-matrix contractility, and cytoskeletal organization. The influence of matrix stiffness upon fibroblast behavior was assessed on the engineered surface of polyacrylamide gels with varying stiffness. Fibroblast traction forces were measured using microfabricated-post-array-detectors., Results: EoE esophageal fibroblasts had enhanced α-SMA expression. TGFβ not only stimulated enhanced fibroblast-specific gene expression but also promoted fibroblast-mediated collagen-matrix contraction, despite disease state or age of patients as the origin of cells. Unlike conventional monolayer cell, culture conditions using plastic surface (1 GPa) that activates fibroblasts constitutively, our engineered platforms recapitulating physiologically relevant stiffness (1-20 kPa) revealed that matrix stiffness defines the extent of α-SMA expression, intracellular collagen fibril organization, SMAD3 phosphorylation, and fibroblast traction force., Conclusions: Matrix stiffness may critically influence TGFβ-mediated gene expression and functions of esophageal fibroblasts ex vivo independent of age and disease conditions. These findings provide a novel insight into the pathogenesis of fibrostenotic disease in EoE.

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl2 differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.

Background: Evidence supports a possible link between eosinophilic esophagitis (EoE) and environmental aeroallergens, which can manifest as seasonal exacerbation of esophageal eosinophilia. Few studies have examined this link in pediatric patients with EoE., Objective: To identify the proportion of patients with seasonal induced esophageal eosinophilia., Methods: A retrospective chart review was conducted of all patients diagnosed with EoE at the authors' institution. Demographic data were collected by chart review. Seasonal variation or flare was defined as a change from fewer than to at least 15 eosinophils per high-power field and a minimum of a 2-fold increase in eosinophil count between 2 consecutive biopsy specimens in different seasons without dietary or medication modifications., Results: Of the 1,180 patients with EoE, 160 (14%) were suspected of having aeroallergen-associated triggers by history. Of these 160 patients, 32 (20%) had biopsy examination-confirmed variation of EoE triggered by aeroallergens. Most of these patients were boys (84%), all had a history or examination consistent with allergic rhinitis, and most had a history of asthma (75%). Thirty-two subjects had obvious seasonal variation, 22 of whom also had known food-induced symptoms., Conclusion: Children with EoE and allergic rhinitis might have exacerbations in their esophageal eosinophilia during certain seasons depending on the specific aeroallergens to which they are sensitized. Identification of environmental allergens to sensitized patients is important and can guide therapy., (Copyright © 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Eosinophilic 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., Results: Firmicutes 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., Conclusions: In conclusion, the esophageal microbiome in EoE is distinct from that of non-EoE controls, with maximal differences observed during active allergic inflammation.

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 Barrett's 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.

Eosinophilic Esophagitis (EoE) is a chronic allergic disorder, whose pathobiology is incompletely understood. Histamine-producing cells including mast cells and basophils have been implicated in EoE. However, very little is currently known about the role of histamine and histamine receptor (HR) expression and signaling in the esophageal epithelium. Herein, we characterized HR (H1R, H2R, H3R, and H4R) expression in human esophageal biopsies and investigate the role of histamine signaling in inducible cytokine expression in human esophageal epithelial cells in vitro. HR expression was quantified in esophageal biopsies from non-EoE control (N = 23), inactive EoE (<15 eos/hpf, N = 26) and active EoE (>15 eos/hpf, N = 22) subjects using qRT-PCR and immunofluorescent localization. HR expression and histamine-mediated cytokine secretion were evaluated in human primary and telomerase-immortalized esophageal epithelial cells. H1R, H2R, and H4R expression were increased in active EoE biopsies compared to inactive EoE and controls. H2R was the most abundantly expressed receptor, and H3R expression was negligible in all 3 cohorts. Infiltrating eosinophils expressed H1R, H2R, and H4R, which contributed to the observed increase in HR in active subjects. H1R and H2R, but not H3R or H4R, were constitutively expressed by primary and immortalized cells, and epithelial histamine stimulation induced GM-CSF, TNFα, and IL-8, but not TSLP or eotaxin-3 secretion. Epithelial priming with the TLR3 ligand poly (I:C) induced H1R and H2R expression, and enhanced histamine-induced GM-CSF, TNFα, and IL-8 secretion. These effects were primarily suppressed by H1R antagonists, but unaffected by H2R antagonism. Histamine directly activates esophageal epithelial cytokine secretion in vitro in an H1R dependent fashion. However, H1R, H2R and H4R are induced in active inflammation in EoE in vivo. While systemic antihistamine (anti-H1R) therapy may not induce clinical remission in EoE, our study suggests that further study of histamine receptor signaling in EoE is warranted and that targeting of additional histamine receptors may lead to novel treatment strategies for this important disease.

Background and Aims: Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro., Methods and Results: Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production., Conclusions: Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis., (Copyright © 2014. Published by Elsevier Inc.)

Eosinophilic esophagitis (EoE) is an allergic disorder characterized by infiltration of the oesophagus 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 diseases, 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 oesophagus. The identification of five EoE loci, not only expands our aetiological understanding of the disease but may also represent new therapeutic targets to treat the most debilitating aspect of EoE, oesophageal inflammation and remodelling.

Objective: To review the understanding of the pathogenesis of eosinophilic esophagitis (EoE) and the role of the immune system in the disease process., Data Sources: Peer-reviewed articles on EoE from PubMed searching for "Eosinophilic Esophagitis and fibrosis" in the period of 1995 to 2013., Study Selections: Studies on the clinical and immunologic features, pathogenesis, and management of EoE., Results: Recent work has revealed that thymic stromal lymphopoietin and basophil have an increased role in the pathogenesis of disease. Additional understanding on the role of fibrosis in EoE is emerging., Conclusion: The incidence of EoE is increasing like most atopic disease. Similar to other allergic diseases, EoE is treated with topical steroids and/or allergen avoidance., (Copyright © 2014 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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 Children's 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., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

The mechanisms by which gastroesophageal reflux disease esophagitis develops are controversial. Although many support the notion that caustic injury leads to reflux esophagitis, others have proposed that reflux esophagitis is caused by esophageal epithelial cytokine-mediated inflammation. We previously demonstrated that Toll-like receptor 3 (TLR3) is highly expressed and functional in the nontransformed human esophageal epithelial cell line EPC2-hTERT. In addition to activation by viral double-stranded RNA, TLR3 can be activated by endogenous mRNA released by necrotic cells. In the present study, we investigated the role of esophageal epithelial TLR3 to sense danger signals released by necrotic esophageal epithelial cells in vitro. Following induction of freeze-thaw necrosis, necrotic EPC2-hTERT cell supernatants (NCS) were used to stimulate EPC2-hTERT monolayers, leading to NF-κB-dependent induction of IL-8 mRNA expression. Responses to self-derived NCS were not observed in transformed gastrointestinal epithelial cell lines, including TE-1 and Caco-2 cells, suggesting that the ability to sense endogenous danger signals is unique to nontransformed esophageal epithelial cells. To determine the immunostimulatory role of epithelial RNA, EPC2-hTERT cells were stimulated with self-derived mRNA, which significantly induced IL-8 mRNA expression. Finally, suppression of TLR3 signaling in a DN-TLR3 cell line, hTERT-ΔTIR-TLR3, led to reduced NCS-induced IL-8 induction by both NCS and mRNA stimulation. Our results demonstrate that human esophageal epithelial cells can sense endogenous danger signals, in part through TLR3 signaling. This supports the concept that epithelial injury plays an inciting role in the pathogenesis of reflux-induced esophagitis, providing important insights into the mechanisms by which epithelial injury leads to inflammation.

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., (Copyright © 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

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.

In the intestinal epithelium, activation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT pathways, via growth factor-mediated signaling, has been shown to regulate cell proliferation and inhibit apoptosis. An immune-activated receptor critical for Th2 immune responses, IL-4Ralpha can also activate PI3-kinase via insulin receptor substrate (IRS)-dependent signaling. Here, using the intestinal goblet cell-specific gene RELMbeta, we investigated the effect of PI3-kinase activation via Th2 immune responses on the goblet cell phenotype. IL-13 stimulation activated PI3-kinase and AKT signal transduction in LS174T cells. Not only did pharmacological inhibition of PI3-kinase and AKT1/2 inhibit RELMbeta induction by IL-13, but AKT inhibition also significantly reduced constitutive basal expression of RELMbeta, a response reproduced by the simultaneous pharmacological inhibition of both epidermal growth factor receptor and IGF-I receptor signaling. In vivo, the disruption of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), an inhibitor of PI3-kinase activation, led to the activation of RELMbeta expression in the small intestine. Furthermore, induction of an intestinal Th2 immune response by infection with a small intestinal nematode parasite, Heligmosomoides polygyrus, led to enhanced epithelial cell proliferation, activation of AKT as demonstrated by the loss of Foxo1 nuclear localization, and robust induction of RELMbeta expression in wild-type, but not IL-4Ralpha knockout, mice. These results demonstrate that Th2 immune responses can regulate goblet cell responses by activation of PI3-kinase and AKT pathways via IL-4Ralpha.

SAMP1/Fc mice develop spontaneous ileitis that shares many features with human Crohn's disease. One of the earliest features of ileitis in SAMP1/Fc mice is an increase in the number of ileal goblet and intermediate cells. Resistin-like molecule beta (RELMbeta) is a goblet cell-specific, cysteine-rich peptide previously shown to function as part of the innate immune response. In this study, we examined the role of expression of RELMbeta in the initiation of ileal inflammation in SAMP1/Fc mice. RELMbeta was highly induced in the ilea of SAMP1/Fc mice beginning at age 5 wk, coincident with the histological appearance of inflammation. RELMbeta was found in ileal goblet cells and some intermediate and Paneth cells. Surprisingly, RELMbeta mRNA levels were significantly increased in the ilea of 80% of germ-free SAMP1/Fc mice examined compared with specific pathogen-free AKR control mice of similar age. Ileitis was observed in germfree SAMP1/Fc mice, although it was attenuated relative to specific pathogen-free SAMP1/Fc mice. These data suggest that neither the early induction of RELMbeta expression nor ileal inflammation requires the presence of viable intestinal flora. Neither was the induction of RELMbeta dependent on the major Th1 or Th2 cytokines. However, RELMbeta stimulated naive bone marrow-derived macrophages to secrete significant amounts of TNF-alpha, IL-6, and RANTES. Our data suggest that RELMbeta is involved in the initiation of ileitis in SAMP1/Fc mice and may act through the induction of proinflammatory cytokines from resident immune cells within the mucosa.

Host immune responses to commensal flora and enteric pathogens are known to influence gene expression in the intestinal epithelium. Although the Cdx family of caudal-related transcription factors represents critical regulators of gene expression in the intestinal epithelium, the effect of intestinal immune responses on Cdx expression and function has not been determined. We have shown that bacterial colonization and Th2 immune stimulation by intestinal nematode infection induce expression of the intestinal goblet cell-specific gene RELM beta. In this study, we investigated the transcriptional regulation of resistin-like molecule/found in inflammatory zone (RELM/FIZZ, RELM beta) and its isoforms RELM alpha and RELM gamma to ascertain the role of Cdx in modifying intestinal gene expression associated with innate and adaptive immune responses. Analysis of the RELM beta promoter showed that Cdx2 plays a critical role in basal gene activation in vitro. This was confirmed in vivo using transgenic mice, where ectopic gastric and hepatic expression of Cdx2 induces expression of RELM beta, but not RELM alpha or RELM gamma, exclusively in the stomach. Although there was no quantitative change in colonic Cdx2 mRNA expression, protein distribution, or phosphorylation of Cdx2, bacterial colonization induced expression of RELM beta, but not RELM alpha or RELM gamma. In contrast, parasitic nematode infections activated colonic expression of all three RELM isoforms without alteration in Cdx2 expression. These results demonstrated that Cdx2 participates in directing intestine-specific expression of RELM beta in the presence of commensal bacteria and that adaptive Th2 immune responses to intestinal nematode infections can activate intestinal goblet cell-specific gene expression independent of Cdx2.

Gastrointestinal (GI) nematode infections are an important public health and economic concern. Experimental studies have shown that resistance to infection requires CD4(+) T helper type 2 (Th2) cytokine responses characterized by the production of IL-4 and IL-13. However, despite >30 years of research, it is unclear how the immune system mediates the expulsion of worms from the GI tract. Here, we demonstrate that a recently described intestinal goblet cell-specific protein, RELMbeta/FIZZ2, is induced after exposure to three phylogenetically distinct GI nematode pathogens. Maximal expression of RELMbeta was coincident with the production of Th2 cytokines and host protective immunity, whereas production of the Th1 cytokine, IFN-gamma, inhibited RELMbeta expression and led to chronic infection. Furthermore, whereas induction of RELMbeta was equivalent in nematode-infected wild-type and IL-4-deficient mice, IL-4 receptor-deficient mice showed minimal RELMbeta induction and developed persistent infections, demonstrating a direct role for IL-13 in optimal expression of RELMbeta. Finally, we show that RELMbeta binds to components of the nematode chemosensory apparatus and inhibits chemotaxic function of a parasitic nematode in vitro. Together, these results suggest that intestinal goblet cell-derived RELMbeta may be a novel Th2 cytokine-induced immune-effector molecule in resistance to GI nematode infection.

Background & Aims: Goblet cells are highly polarized exocrine cells found throughout the small and large intestine that have a characteristic morphology due to the accumulation of apical secretory granules. These granules contain proteins that play important physiologic roles in cellular protection, barrier function, and proliferation. A limited number of intestinal goblet cell-specific proteins have been identified. In this study, we investigate the expression and regulation of RELMbeta, a novel colon-specific gene., Methods: The regulation of RELMbeta messenger RNA expression was determined in LS174T, Caco-2, and HT-29 cell lines in response to stimulation with interleukin 13 and lipopolysaccharide. Quantitative reverse-transcription polymerase chain reaction, immunoblots, and immunohistochemistry were used to examine the expression of RELMbeta in BALB/c and C.B17.SCID mice housed in conventional, germ-free, and gnotobiotic environments., Results: Messenger RNA for RELMbeta is restricted to the undifferentiated, proliferating colonic epithelium. Immunohistochemistry shows that this protein is expressed in goblet cells located primarily in the distal half of the colon and cecum with lower levels detectable in the proximal colon. High levels of RELMbeta can be detected in the stool of mice and humans, where it exists as a homodimer under nonreducing conditions. Interestingly, the secretion of RELMbeta is dramatically reduced in germ-free mice. Furthermore, introduction of germ-free mice into a conventional environment results in enhanced expression and robust secretion of RELMbeta within 48 hours., Conclusions: These studies define a new goblet cell-specific protein and provide the first evidence that colon-specific gene expression can be regulated by colonization with normal enteric bacteria.