Willem S. Lexmond

Involvement of the iNKT Cell Pathway Is Associated With Early-Onset Eosinophilic Esophagitis and Response to Allergen Avoidance Therapy

OBJECTIVES:Recent experimental evidence suggests that environmental microbial factors early in life determine susceptibility to allergic diseases through inappropriate chemotaxis and local activation of CD1d-restricted, invariant chain natural killer T (iNKT) cells. In this study, we analyzed the involvement of these pathways in pediatric patients with eosinophilic esophagitis (EoE) before and after dietary allergen elimination.METHODS:mRNA expression levels of components of the C-X-C motif chemokine ligand 16 (CXCL16)–iNKT–CD1d axis were compared in esophageal biopsies from EoE patients vs. normal or inflammatory controls and before and after treatment.RESULTS:CXCL16, iNKT cell–associated cell marker Vα24, and CD1d were significantly upregulated in esophageal biopsies from EoE patients and correlated with the expression of inflammatory mediators associated with allergy. Upregulation of each of these factors was significantly more pronounced in patients aged <6 years at diagnosis, and this early-onset EoE subpopulation was characterized by a more prominent food allergic disease phenotype in a cohort-wide analysis. Successful, but not unsuccessful, treatment of early-onset EoE patients with dietary elimination of instigating allergens led to reduction in infiltrating iNKT cells and complete normalization of mRNA expression levels of CXCL16 and CD1d.CONCLUSIONS:Our observations place iNKT cells at the center of allergic inflammation associated with EoE, which could have profound implications for our understanding, treatment and prevention of this and other human allergic diseases.

Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

Antigen-mediated cross-linking of Immunoglobulin E (IgE) bound to mast cells/basophils via FcɛRI, the high affinity IgE Fc-receptor, is a well-known trigger of allergy. In humans, but not mice, dendritic cells (DCs) also express FcɛRI that is constitutively occupied with IgE. In contrast to mast cells/basophils, the consequences of IgE/FcɛRI signals for DC function remain poorly understood. We show that humanized mice that express FcɛRI on DCs carry IgE like non-allergic humans and do not develop spontaneous allergies. Antigen-specific IgE/FcɛRI cross-linking fails to induce maturation or production of inflammatory mediators in human DCs and FcɛRI-humanized DCs. Furthermore, conferring expression of FcɛRI to DCs decreases the severity of food allergy and asthma in disease-relevant models suggesting anti-inflammatory IgE/FcɛRI signals. Consistent with the improved clinical parameters in vivo, antigen-specific IgE/FcɛRI cross-linking on papain or lipopolysaccharide-stimulated DCs inhibits the production of pro-inflammatory cytokines and chemokines. Migration assays confirm that the IgE-dependent decrease in cytokine production results in diminished recruitment of mast cell progenitors; providing a mechanistic explanation for the reduced mast cell-dependent allergic phenotype observed in FcɛRI-humanized mice. Our study demonstrates a novel immune regulatory function of IgE and proposes that DC-intrinsic IgE signals serve as a feedback mechanism to restrain allergic tissue inflammation.

FOXP3+ Tregs require WASP to restrain Th2-mediated food allergy

In addition to the infectious consequences of immunodeficiency, patients with Wiskott-Aldrich syndrome (WAS) often suffer from poorly understood exaggerated immune responses that result in autoimmunity and elevated levels of serum IgE. Here, we have shown that WAS patients and mice deficient in WAS protein (WASP) frequently develop IgE-mediated reactions to common food allergens. WASP-deficient animals displayed an adjuvant-free IgE-sensitization to chow antigens that was most pronounced for wheat and soy and occurred under specific pathogen-free as well as germ-free housing conditions. Conditional deletion of Was in FOXP3+ Tregs resulted in more severe Th2-type intestinal inflammation than that observed in mice with global WASP deficiency, indicating that allergic responses to food allergens are dependent upon loss of WASP expression in this immune compartment. While WASP-deficient Tregs efficiently contained Th1- and Th17-type effector differentiation in vivo, they failed to restrain Th2 effector responses that drive allergic intestinal inflammation. Loss of WASP was phenotypically associated with increased GATA3 expression in effector memory FOXP3+ Tregs, but not in naive-like FOXP3+ Tregs, an effect that occurred independently of increased IL-4 signaling. Our results reveal a Treg-specific role for WASP that is required for prevention of Th2 effector cell differentiation and allergic sensitization to dietary antigens.

Elevated levels of leukotriene C4 synthase mRNA distinguish a subpopulation of eosinophilic oesophagitis patients

Cysteinyl leukotrienes contribute to Th2‐type inflammatory immune responses. Their levels in oesophageal tissue, however, do not distinguish patients with eosinophilic oesophagitis (EoE) from controls.

Accuracy of digital mRNA profiling of oesophageal biopsies as a novel diagnostic approach to eosinophilic oesophagitis

Quantification of tissue eosinophils remains the golden standard in diagnosing eosinophilic oesophagitis (EoE), but this approach suffers from poor specificity. It has been recognized that histopathological changes that occur in patients with EoE are associated with a disease‐specific tissue transcriptome.

Eosinophilic esophagitis: published evidences for disease subtypes, indications for patient subpopulations, and how to translate patient observations to murine experimental models

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder of the esophagus and commonly classified as a Th2-type allergy. Major advances in our understanding of the EoE pathophysiology have recently been made, but clinicians struggle with highly unpredictable therapy responses indicative of phenotypic diversity within the patient population. Here, we summarize evidences for the existence of EoE subpopulations based on diverse inflammatory characteristics of the esophageal tissue in EoE. Additionally, clinical characteristics of EoE patients support the concept of disease subtypes. We conclude that clinical and experimental evidences indicate that EoE is an umbrella term for conditions that are unified by esophageal eosinophilia but that several disease subgroups with various inflammatory esophageal patterns and/or different clinical features exist. We further discuss strategies to study the pathophysiologic differences as observed in EoE patients in murine experimental EoE. Going forward, models of EoE that faithfully mimic EoE subentities as defined in humans will be essential because mechanistic studies on triggers which regulate the onset of diverse EoE subpopulations are not feasible in patients. Understanding how and why different EoE phenotypes develop will be a first and fundamental step to establish strategies that integrate individual variations of the EoE pathology into personalized therapy.

Spontaneous food allergy in Was−/− mice occurs independent of FcεRI‐mediated mast cell activation

Food allergies are a growing health problem, and the development of therapies that prevent disease onset is limited by the lack of adjuvant‐free experimental animal models. We compared allergic sensitization in patients with food allergy or Wiskott‐Aldrich syndrome (WAS) and defined whether spontaneous disease in Was−/− mice recapitulates the pathology of a conventional disease model and/or human food allergy.

An algorithm for the classification of mRNA patterns in eosinophilic esophagitis: Integration of machine learning

Background: Diagnostic evaluation of eosinophilic esophagitis (EoE) remains difficult, particularly the assessment of the patients allergic status. Objective: This study sought to establish an automated medical algorithm to assist in the evaluation of EoE. Methods: Machine learning techniques were used to establish a diagnostic probability score for EoE, p(EoE), based on esophageal mRNA transcript patterns from biopsies of patients with EoE, gastroesophageal reflux disease and controls. Dimensionality reduction in the training set established weighted factors, which were confirmed by immunohistochemistry. Following weighted factor analysis, p(EoE) was determined by random forest classification. Accuracy was tested in an external test set, and predictive power was assessed with equivocal patients. Esophageal IgE production was quantified with epsilon germ line (IGHE) transcripts and correlated with serum IgE and the Th2‐type mRNA profile to establish an IGHE score for tissue allergy. Results: In the primary analysis, a 3‐class statistical model generated a p(EoE) score based on common characteristics of the inflammatory EoE profile. A p(EoE) ≥ 25 successfully identified EoE with high accuracy (sensitivity: 90.9%, specificity: 93.2%, area under the curve: 0.985) and improved diagnosis of equivocal cases by 84.6%. The p(EoE) changed in response to therapy. A secondary analysis loop in EoE patients defined an IGHE score of ≥37.5 for a patient subpopulation with increased esophageal allergic inflammation. Conclusions: The development of intelligent data analysis from a machine learning perspective provides exciting opportunities to improve diagnostic precision and improve patient care in EoE. The p(EoE) and the IGHE score are steps toward the development of decision trees to define EoE subpopulations and, consequently, will facilitate individualized therapy. GRAPHICAL ABSTRACT Figure. No caption available.

363 Human Hematopoietic Stem Cells With a Defined Immunodeficiency and Enteropathy Transfer Clinical Phenotype to a Novel Humanized Mouse Strain

Investigation into the pathogenesis of IBD has been limited by the absence of humanized murine IBD models. The development of immunodeficient mouse strains has facilitated the study of human immune reconstitution in murine hosts. However, adaptive immune responses and antibody class switching are generally weak in models that lack autologous fetal thymic grafts, possibly due to human CD4+ T cell selection in the mouse thymus occurring on murine major histocompatibility complex II (MHCII). To address this, we modified the widely utilized NOD.Prkdc.Il2rγ (NSG) immunocompromised strain to be deficient for murine MHCII (NSGAbo) and instead expressed human leukocyte antigen-DR1 under the control of the murine MHCII promoter (NSGAboDR1). We tested whether CD4+ T cell development and adaptive immune responses are improved following reconstitution using human HLA-matched CD34+ hematopoietic stem cells (HSCs) and whether immune reconstitution using HSCs from a patient with IPEX (immunodeficiency, polyendocrinopathy, enteropathy, X-linked), a well-defined immunodeficiency, that is caused by a mutation in the transcription factor FOXP3, would transfer the clinical phenotype to NSGAboDR1 mice. Results: At 20 weeks following HSC injection into radiation-conditioned neonates, we found that the development of human T cells and dendritic cells were increased in NSGAboDR1 mice and that these mice displayed delayed-type hypersensitivity response to a secondary recall antigen challenge. There was an increase in T cell clonotype diversity as determined by CDR3 sequencing of the TCRβ gene in CD4+ T cells in NSGAboDR1 mice compared to NSG mice. In addition, NSGAboDR1 mice had an increase in mature B cells and antibody class switching compared to NSG and NSGAbo mice. We then reconstituted NSGAboDR1 mice using HSCs from a patient with IPEX that is associated with functional defects in Treg and assessed if the clinical phenotype would be transferred to NSGAboDR1 mice. All mice reconstituted with IPEX CD34+ HSCs were deceased by 18 weeks while all control mice receiving healthy bone marrow CD34+ cells remained viable. IPEX mice developed lymphocytic infiltrate in lung, portal tract and bile ducts of the liver, and slight accumulation of lymphocytes in the small intestine similar to scurfy mice, which also harbor a mutation in Foxp3. CD4+ T cell in IPEX mice had an expanded effector memory T cell population and concomitant decrease in Naive CD4+ T cells. Conclusions: We demonstrate that human HSCs from a patient with a defined genetic immunodeficiency and enteropathy can transfer the disease phenoytpe in a novel humanized mouse strain. This strain does not require grafts of human fetal tissue nor is it restricted to autologous fetal HSCs allowing for reconstitution using human HSCs with defined genetics to study human immune cells and IBD pathogenesis in vivo.

Electrophysiological Studies into the Safety of the Anti-diarrheal Drug Clotrimazole during Oral Rehydration Therapy

Background and Aims Morbidity and mortality from acute diarrheal disease remains high, particularly in developing countries and in cases of natural or man-made disasters. Previous work has shown that the small molecule clotrimazole inhibits intestinal Cl- secretion by blocking both cyclic nucleotide- and Ca2+-gated K+ channels, implicating its use in the treatment of diarrhea of diverse etiologies. Clotrimazole, however, might also inhibit transporters that mediate the inwardly directed electrochemical potential for Na+-dependent solute absorption, which would undermine its clinical application. Here we test this possibility by examining the effects of clotrimazole on Na+-coupled glucose uptake. Materials and Methods Short-circuit currents (Isc) following administration of glucose and secretagogues were studied in clotrimazole-treated jejunal sections of mouse intestine mounted in Ussing chambers. Results Treatment of small intestinal tissue with clotrimazole inhibited the Cl- secretory currents that resulted from challenge with the cAMP-agonist vasoactive intestinal peptide (VIP) or Ca2+-agonist carbachol in a dose-dependent fashion. A dose of 30 μM was effective in significantly reducing the Isc response to VIP and carbachol by 50% and 72%, respectively. At this dose, uptake of glucose was only marginally affected (decreased by 14%, p = 0.37). There was no measurable effect on SGLT1-mediated sugar transport, as uptake of SGLT1-restricted 3-O-methyl glucose was equivalent between clotrimazole-treated and untreated tissue (98% vs. 100%, p = 0.90). Conclusion Treatment of intestinal tissue with clotrimazole significantly reduced secretory responses caused by both cAMP- and Ca2+-dependent agonists as expected, but did not affect Na+-coupled glucose absorption. Clotrimazole could thus be used in conjunction with oral rehydration solution as a low-cost, auxiliary treatment of acute secretory diarrheas.