From genetics to treatment of eosinophilic esophagitis

Abstract

Purpose of review

Eosinophilic Esophagitis (EoE) is an emerging chronic atopic disease. Recent advances in understanding its genetic and molecular biology pathogenesis may lead to a better management of the disease

Recent findings

EoE is an atopic disease. Most of the patients affected by EoE have other atopic diseases such as allergic rhinitis, asthma, IgE-mediated food allergies and/or atopic dermatitis. The local inflammation is a T helper type 2 (Th2) flogosis, which most likely is driven by a mixed IgE and n-IgE-mediated reaction to food and/or environmental allergens. Epidemiological studies show that EoE is an atopic disease with a strong genetic component. Genetic studies have shown that EoE is associated with single nucleotide polymorphism on genes, which are released by the epithelium and important in atopic inflammation such as thymic stromal lymphopoietin located (TSLP) close to the Th2 cytokine cluster [interleukin (IL)-4, IL-5, IL-13] on chromosome 5q22, Calpain 14, EMSY, and Eotaxin3. When the EoE diagnosis is made, it is imperative to control the local eosinophilic inflammation not only to give symptomatic relief to the patient, but also to prevent complications such as esophageal stricture and food impaction.

Summary

EoE is treated like many other atopic diseases with a combination of topical steroids and/or food antigen avoidance. The new understanding of EoE may lead to more specific and definitive treatments of EoE.

INTRODUCTION

Eosinophilic Esophagitis (EoE) is a chronic atopic clinical-pathologic disease affecting both children and adults [1,2]. It is defined by a significant pathological eosinophil infiltration limited to the esophagus that causes esophageal dysfunction and, if left untreated, fibrosis [1,2]. As the esophageal gastro-duodenal endoscopy has become readily available at the beginning of the 21st century, EoE has been exponentially more recognized in western countries, with a yearly incidence now estimated to be similar to Crohn’s disease [3–8].

In the last few year, using traditional epidemiology, a novel genetics study approach and traditional molecular biology, there has been great progress in understanding EoE pathogenesis [9▪]. This progress has led to establish a well defined globally accepted management of the EoE and a search for more specific treatments that will hopefully result in the cure of this puzzling and ever more prevalent disease [10–13].

EPIDEMIOLOGY

Several epidemiological studies [5–7,14▪,15,16] have revealed that EoE is a highly hereditable atopic disease that affect mainly Caucasian men regardless of their age. EoE affects children and adults from all continents [5–7,14▪,15,17]; however, the western world has a highest prevalence, with a north–south and west–east gradients similar to the one described for the incidence of atopic diseases [5–7,14▪,15,16]. In children in the Unite States, EoE has prevalence of 50.5/10000, equivalent to pediatric inflammatory bowel disease [18].

The epidemiological data suggest both atopic and genetic components in patients with EoE:

1. Patients with EoE are highly atopic [19,20] (Table 1). The most common comorbidity is the allergic rhinitis, but also the rate of IgE-mediated food allergy is 10 times higher than the general population [19–22]. Unlike classic food allergy that typically involves a limited set of foods, EoE patients are often sensitized to a myriad of foods, often including food groups not typically considered to elicit IgE-mediated food allergy [23].

2. EoE affects predominantly men with a male to female ratio of 3 : 1 in both children and adults [2,6,18].

3. Family history, especially in men, is very frequent with nearly 10% of parents of EoE patients having a history of esophageal strictures and about 8% having biopsy-proven EoE [24]. EoE also shows a sibling-risk ratio of 80, meaning that having a sibling with the disease increase 80 times the risk of developing a similar disease in other siblings. This is extremely high compared with the one for related atopic diseases such as asthma that has a sibling-risk ratio of about 2 [4,25,26].

Table 1.

Incidence of other atopic diseases in eosinophilic esophagitis

	Number of patients with EoE	Atopy	Asthma	Allergic rhinitis	Atopic dermatitis	IgE specific for foods	Anaphylaxis to foods
General population	NA	30%	8.5%	25%	10%	10%	0.2%
Spergel et al., Philadelphia	620	NA	50%	61%	21%	50	10%
Assa’ad et al., Cincinnati	89	79%	39%	30%	19%	75%	NA
Sugnanam et al., Australia	45	NA	66%	93%	55%	NA	24%
Guajardo et al., World registry	39	80%	38%	64%	26%	62%	23%

EoE, eosinophilic esophagitis; NA, not available.

All together, the epidemiological data suggest a strong atopic and genetic component in EoE development.

GENETICS BASIS OF EOSINOPHILIC ESOPHAGITIS

The epidemiological data show that EoE is an atopic, multifactorial, and complex disease with a strong genetic component, whose inheritance does not follow the classical Mendelian patterns [26,27,28▪▪, 29,30]. It is believed that multiple interacting genes, some having a protective effect and other a causative one, act together with each gene having its own tendency to be influenced by the environment [26,27,28▪▪,29,30]. Two study designs are commonly used to determine the genetic contributions in complex diseases candidate gene association studies and genome-wide association studies (GWAS) [30]. Genome-wide linkage study design can be also used in large families affected by complex multifactorial diseases, but it will not be discussed as this approach has not been used to study EoE.

The candidate gene association studies allow the identification of genes and pathways suspected of contributing to the disease based on its biological plausibility, and the incidence of small gene variations such as single nucleotide polymorphysim (SNP) in the suspected gene or sets of genes are compared between a population affected by the disease (cases) and a group of controls [30]. The main limitations of such a design are its inability to identify novel or unsuspected genes and pathways contributing to the pathogenesis of a disorder [30].

The availability of microarray technology has made possible the high-throughput genotyping of hundreds of thousands SNPs on the entire genome and has allowed the development of like GWAS [30]. In this design many SNPs are compared across the entire genome between cases and controls. As shown in the candidate gene association study [30], the larger the number of cases and controls for analysis, the better the statistical power. GWAS allows also performing a hypothesis-free search for gene variants associated with a certain disease and this has been a powerful tool to unveil new targets for researchers [30]. Moreover, independent replication of genes identified through GWAS is much more common than those identified with the single gene approach association [30].

The first candidate gene for EoE identified was CCL-26 (Eotaxin 3), after a genome-wide profiling revealed that CCL-26 was overexpressed about 50-fold compared with normal controls or patients with gastroesophageal acid reflux [31]. This study highlighted the important role of CCL-26 in EoE pathogenesis. A potential proposed mechanism is that T helper type 2 (Th2) cell activation leads to overexpression of CCL-26 from the esophageal epithelial cells, especially if genetically predisposed, and to the migration of eosinophils to the esophagus [29,31–40].

Aceves et al. [41–43] examined how SNP could influence the response to topical corticosteroids in patients with EoE. The patients who responded to the topical therapy were more likely to have a CC genotype at the −509 position in the TGF-β promoter than were nonresponders. These data suggest that response to therapy may be influenced by the genetic background.

More recently, GWAS analysis has identified three genes as important candidate gene in the pathogenesis of EoE: the thymic stromal lymphopoietin (TSLP) gene, at 5q22, the Calpain 14 one on chr2p23.1, and the c11orf30/EMSY gene one on chr11q13.5 [27,28▪▪,44▪▪,45] (Table 2).

Table 2.

Eosinophilic esophagitis genetic risk description of single nucleotide polymorphysims found in eosinophilic esophagitis genome-wide association studies

Variant (effect allele)	chr: pos hg19	Gene	Location	Function	Effect allele frequency	EoE OR	P EoE GWAS	SE EoE GWAS
rs1438673 (T)	chr5: 110467499	TSLP	Intergenic	TSLP eSNP/   ENCODE   transcription   factor binding site	0.496	0.626	2.74 × 10−12	0.063
rs55646091 (A)	chr11: 76299431	c11orf30	Intergenic	ENCODE   transcription   factor binding site	0.044	2.219	5.38 × 0−10	0.157
rs74732520 (G)	chr2: 31396392	CAPN14	3’ UTR	Transcribed and   high LD with three   variants   (rs77997242,   rs113412973,   rs78464756) in   ENCODE   transcription   factor binding   sites	0.067	1.782	1.69 × 10−8	0.131

EoE, eosinophilic esophagitis; CAPN14, Calpain 14; ENCODE, encyclopedia of DNA elements; GWAS, genome-wide association studies; LD, linkage disequilibrium; SNP, single nucleotide polymorphysim; TSLP, thymic stromal lymphopoietin.

TSLP is a cytokine that belongs to the interleukin (IL)-7 family, it is secreted by epithelial cells in response danger stimuli (i.e. infectious agent, stimulation of toll-like receptor, allergens) and it is elevated in EoE patients [27,46,47]. TSLP is a major driver of atopic inflammation and tissue remodeling, as it strongly promotes Th2 inflammation by: activating professional antigen-presenting cells (APC to prime naive T-cell to initiate Th2-type allergic responses; inhibiting Tregs; directly promoting Th2-cytokine secretion from T cells, basophils, eosinophils, mast cells, and invariant natural killer cells [46,48–59]. The first ever GWAS conducted in EoE showed an association between a SNP in TSLP gene and risk for EoE in a relative small population of 500 EoE children, confirming the strong influence of genetics in EoE [27]. Those who were homozygous for the EoE risk allele (AA) had increased TSLP expression and basophil infiltration in the esophageal epithelium compared with those carrying heterozygous (AG) risk allele and homozygous (GG) protective minor alleles [27]. In addition, Sherrill et al. [45] not only confirmed TSLP protective SNP but also found that male patients with EoE had more often a SNP in the TSLP receptor (TSLPR) gene, which is encoded by a pseudoautosomal region on Xp22.3 and Yp11.3 [45]. This finding could explain the epidemiological data that EoE is more common in men by a 3 : 1 ratio [15].

Subsequent molecular biology studies [52,53,60] conducted by our group with collaboration with Dr Artis’s group showed that TSLP may promote Th2 inflammation in EoE through basophils. Basophils are known to secrete histamine and Th2 cytokine (IL-4 and IL-13) if stimulated through their high affinity receptor for immunoglobulin (Ig)E (FcεRI) [61]. However, Siracusa et al. [53] have described that TSLP may act as an independent growth factor for a subpopulation of basophils, which is overexpressed in EoE patients and is able to produce significant Th2 cytokines (IL-4, IL-6, CCL3, CCL4, and CCL12). Even more interestingly Noti et al. [52] recently described a novel mouse model of EoE in which the development of EoE-like features was dependent upon both TSLP and basophils, but independent of IgE responses. In his mouse model, blocking of TSLP or basophil by monoclonal antibodies prevented the development of EoE.

Together, these studies show how a GWAS identified in TSLP and subsequent studies confirmed its role in EoE making it a promising therapeutic target.

Th2 cytokines are responsible of the inflammation observed in EoE [9▪]. The Th2 cytokines appear to exert redundant functions, as they have many similar and overlapping effects; therefore, treatment strategies that are based on abolishing a single cytokine are probably not going to be sufficient to treat EoE. Indeed, anti-IL-5 administration in patients with EoE has resulted to partial reduction of esophageal inflammation and minimal symptomatic relief [62,63]. But, targets against TSLP would may be a better option, as they would abolish more globally the Th2 response.

Building on the success of our first GWAS, we performed an expanded GWAS 936 cases and 4312 controls to identify additional targets worthy of further biological study. In addition to confirming the relevance of our previously reported TSLP locus [27], we discovered three novel loci on c110rf30, Calpain 14 (CAPN14), [28▪▪] (Table 2). A summary of the findings is shown in Table 2.

Variants at the c11orf30 locus (EMSY) have been associated with seasonal allergic rhinitis [64], ulcerative colitis [65], Crohn’s disease [66], atopic dermatitis [67,68], asthma [69], and allergic sensitization [70], albeit with much lower odds ratios (range 1.09 in asthma to 1.22 in atopic dermatitis). EMSY has been identified as a central component in a novel Akt-dependent mechanism by which interferon and other growth factors regulate the expression of interferon-stimulated genes (ISGs) [71]. Interferon and ISGs play a central role in Th1 inflammation and Th2 suppression; consequently, a dysregulation in EMSY expression could lead to allergic diseases [71–73].

SNPs in CAPN14 has been shown as a risk factor for EoE independently by Kottyan et al. [44▪▪], hence it is the second locus that has been replicated linked to EoE to date. CAPN14 belongs to the calpain family of intracellular Ca²⁺-regulated cysteine proteases known to function in diverse biological processes including the cell cycle, tight junction protein, cytokine regulation, and human fibroblast biology [74–78]. Calpains include both ubiquitous and tissue- specific members. The Genotype-Tissue Expression project [79], the Human Protein Atlas [80], and Kottyan et al. [44▪▪] demonstrated that CAPN14 is highly and selectively expressed in the esophagus. Moreover, we and others also demonstrated that CAPN14 is overexpressed in EoE esophageal epithelial cells compared with controls [28▪▪,44▪▪] and is dynamically upregulated after exposure of epithelial cells to Th2 cytokines (i.e. IL-13 [44▪▪] and IL-4 [81]).

The role of CAPN14 in esophageal biology or in EoE is not known but is a gene selective expressed in the epihelium and like TSLP points to an epithelial dysfunction as a driving force of local immune Th2 response in genetically predisposed patients. Other molecular biology studies have also shown an impaired epithelial barrier function, using gene expression profiling [82] and immunolocalization studies [83], which may contribute enhanced permeability of the epithelium to local food and environmental allergens and subsequent local inflammation [84].

POTENTIAL PATHWAYS FOR TREATMENT

The current understanding of the pathobiology of EoE is incomplete, but evolving it seems to point to three factors leading to the development of EoE: atopy, genetic predisposition, and a local Th2 inflammation driven by a dysfunctional esophageal epithelium.

Current EoE management is based on current knowledge of the disease and is mainly based on two main clinically accepted clinical treatment strategies like any other atopic disease: allergen avoidance and corticosteroid treatment (Table 3).

Table 3.

Eosinophilic esophagitis therapy

	Description	Use
Elemental diet	Diet is based on elemental formula	Currently used for short-term treatment of EoE to induce rapid resolution of EoE in almost all patients (adult and children) to establish food allergy
Six food elimination diet (SFED)	Most allergenic foods (milk, soy, egg, wheat,   treenuts, peanuts, fish, shellfish) are   eliminated	Effective in about 70% of adult an pediatric   patients
Targeted diet with allergy tests	Food eliminated based on skin tests and milk   (often only 1–2 foods are eliminated)	Effective in 80% of children, limited studies in   adults
Proton pump inhibitors (PPI)	1 mg/kg (20–40mg max dose) 1–2 times a   day	Given to everybody 8 weeks prior to diagnostic   EGD May work as a stand alone therapy in PPI   responsive EoE
Oral steroids	1 mg/kg twice a day for 10–15 days	Very effective short-term treatment used for   emergency therapy of food impaction or   debilitating symptoms (failure to thrive,   protracted vomiting)
Swallowed steroids	Fluticasone 110–220 mcg two puffs twice a   day, Budesonide 0.5–2mg a day	Effective 50–70% of patients
Anti-IL5	Reslizumab Mepolizumab	Significantly reduce eosinophilic infiltration in the   esophagus, no effects on symptoms
Anti-IgE	omalizumab	Not effective
Anti-tumor necrosis factor (TNF)α	Infliximab	Not effective
Anti-CRTH2	OC000459	Partially effective
Anti-IL-13	QAX576	Partially effective
Anti-TSLP	AMG 157	Future for EoE effective on asthma
Food allergy immunotherapy	EPIT	Future for EoE effective for IgE-mediated food   allergy (peanut)

EGD, esophageal gastroduodenal endoscopy; EoE, eosinophilic esophagitis; EPIT, epicutaneous immunotherapy; TSLP, thymic stromal lymphopoietin.

The new genetic studies that are pointing to the esophageal dysfunction are shaping the treatment for tomorrow that will be based on antigen tolerance induction and specific biological treatments.

Steroid treatment

Steroids are a very effective treatment of EoE. Oral steroids are very effective, but not recommended as a long-term therapy for the well known side-effects [85] (Table 3). Swallowed inhaled corticosteroids are effective in 50–80% of patients and can be considered as first-line therapies for initial and maintenance management of EoE, as they have low bioavailability and fewer systemic side-effects. Fluticasone is administered by spraying in the mouth with a metered dose inhaler without a spacer, and swallowed twice daily [86,87]. Budesonide is used as an oral viscous slurry once or twice daily [88,89]. Swallowed inhaled corticosteroids appear to be well tolerated when used in the short term, but they can lead to increased risk of localized yeast infections and have potential long-term side-effects, including growth suppression and osteopenia (low-bone density) [87,89]. Although steroids are effective for treatment, clinical, and histologic features of EoE return upon discontinuation [1,2]. Steroids may reverse the esophageal fibrosis in children [90–92] but not in adults [93] with EoE.

Dietary intervention

The majority of patients with EoE are allergic to food allergens and aeroallergens [1,2] (Table 3). Dietary elimination therapy should be considered in all children and motivated adults diagnosed with EoE. Dietary elimination approaches include a strictly elemental diet, specific antigen avoidance based on allergy testing, and empiric food elimination based on the most common food antigens [1,2]. All three methods have been proven to be effective with improved clinical symptoms and pathology [85,94,95] and the regimen chosen should be based on the individual patient [96]. Treatment with food avoidance is highly successful, with rates close to 100% with elemental diets (amino acid formulas) and up to 80% for elimination diet. However, amino acid formulas are unpalatable and lead to low quality of life. Elimination diet on the basis of allergy testing or empirical elimination may be sustainable in the long-term albeit with some difficulties, and many patients refuse to continue them. For additional details of dietary management, please see recent review [97].

Esophageal dilation

Esophageal fibrosis and esophageal strictures are known complications of EoE. Endoscopic stricture dilation is sometimes necessary for short-term symptomatic relief but should be considered as a treatment option only if patients have failed dietary and medical therapy [1,2].

Other biological treatment

Other treatments that have been investigated include anti-IL-5 [62,63] and chemoattractant homologous receptor expressed on Th2 cells (CRTH2) antagonist (Table 3). Both strategies have shown limited or no efficacy in controlling the disease, suggesting that a broader inhibition of Th2 inflammation may be needed due to the redundancy of its mediators.

Antiinterleukin-5

IL-5 is the major survival factor for eosinophils and is indispensable for the differentiation, recruitment, and activation of the eosinophils [98]. Therefore, humanized monoclonal antibodies against IL-5 Mepolizumab (SB240563) and Reslizumab (Sch55700) have been used in clinical trials for EoE treatment [62,63]. Both antibodies in pediatrics and adult trials have failed to show symptomatic improvement beyond the placebo effect. However, both Reslizumab and Mepolizumab had a good safety profile and significantly decreased eosinophils numbers in the esophageal biopsies in adults and children with EoE, even if only few patients achieved normal biopsies. These data confirm that eosinophils are likely only one of the players in EoE inflammation and the importance of other cells and mediators in the pathogenesis of EoE that can be a target for immunological therapy.

Other biological treatment such as anti-TNFα (Infliximab) [99], anti-IgE (omalizumab) [100] have been tried in small groups of patients without showing any efficacy.

A recent study has been published on the efficacy of anti-CRTH in treatment of EoE and showed promising albeit small results. OC000459 is a potent, selective, and orally bioavailable CRTH2 antagonist, which blocks prostaglandin 2-mediated chemotaxis and activation of CRTH2-expressing cells. In a small group of patients with severe EoE dependent or resistant to corticosteroids anti-CRTH2 had a modest, but significant, antibeneficial clinical effect [101].

Anti-IL-13 antibodies have also been tried in asthma and more recently in the treatment of EoE [102–107]. Given the importance of Th2 inflammation and IL-13 in particular in EoE pathogenesis [102–105] Rothenberg et al. [108] tried anti-IL-13 (QAX576) in a double-blind placebo-controlled clinical trial for EoE treatment. The treated group had a significant reduction of esophageal eosinophilia and other markers of Th2 inflammation and epithelial dysfunction compared with those treated with placebo. However, even if there was a trend for improved symptoms, particularly dysphagia, this did not reach statistical significance. QAX576 was well tolerated. Therefore, the authors concluded that QAX576 significantly improved intraepithelial esophageal eosinophil counts and dysregulated esophageal disease-related transcripts in adults with EoE but the clinical significance of such treatment remains to be established.

Antithymic stromal lymphopoietin

The recent identification of TSLP and its receptor as key components in the EoE pathogenesis suggests that blockage of the TSLP–TSLPR activation could provide an attractive approach to treating the cause of EoE [109]. This has been confirmed in animal studies wherein blockage of Noti et al. [52] recently described a novel mouse model of EoE in which the development of EoE-like features was dependent upon TSLP. In such a model TSLP-blocking antibodies ameliorated the EoE-like disease including the development of food impaction, when administered after the onset of disease. This is not surprising as anti-TSLP antibodies have been shown to be beneficial in various murine models of atopy [110–114].

A humanized anti-TSLP monoclonal antibody (AMG 157) that specifically binds human TSLP and prevents its interaction with TSLPR has been tested in adult asthmatic patients in a double-blind, placebo-controlled study. In the group receiving the antibody there were: attenuated allergen-induced bronchoconstriction in both early and late asthmatic responses; reduced markers of systemic and airway inflammation. Although this was only a proof-of-concept study [115▪], which did not determine whether anti-TSLP therapeutics will have clinical impact, these findings confirm that TSLP has a key role in allergic asthma. TSLP antibodies have not been tested in EoE yet.

Food immunotherapy

We and others have reported that in both adults and children, milk is the most common trigger of EoE [116–119], initiating inflammation in a genetically susceptible individual via a disrupted epithelial barrier [27,83]. Epicutaneous immunotherapy (EPIT) has recently been proposed as a way to bypass the dysfunctional and TSLP-producing esophageal epithelium, and induce lasting food tolerance in EoE [120]. In murine models of EoE, EPIT induces a persistent resolution of esophageal eosinophilia [121]. In humans, EPIT has been shown to be a well tolerated method to desensitize IgE food allergic patients [122]. EPIT, therefore, could be a promising strategy to cure food allergy and the consequent development of EoE in children with EoE due to food allergy.

CONCLUSION

In summary, to date, there is no valid primary prevention strategy nor pharmacological cure for EoE. Long-term immunotherapeutic approaches to EoE are clearly needed. Recent advances in our understanding of genetics and pathophyisology of EoE will hopefully lead to such new treatments in a not distant future.

KEY POINTS.

• Eosinophilic Esophagitis (EoE) is a clinical-pathologic disease characterized by symptoms of esophageal dysfunction and eosinophilia limited the esophagus, in absence of gastroesophageal acid reflux.

• Epidemiological studies have shown that EoE is an atopic diseases that most likely due to an interaction between environmental and genetic predisposition factors.

• Specific genetic association have been found between EoE and epithelial-related genes such as thymic stromal lymphopoietin (TSLP), CAPN14, Eotaxin 3, and other genes involved in T helper type 2 (Th2) inflammation such as TGF-β and EMSY. Recent advances in research have shown that TSLP may play a pivotal role in driving Th2 inflammation typical of EoE.

• EoE management: EoE is known to be a food antigen-driven, chronic allergic disease. There are two main clinically accepted clinical treatment strategies for EoE: dietary elimination and corticosteroid treatment.

• With the increase in our understanding of EoE pathogenesis, it is logical to anticipate that in the future there will be more specific treatment options for this rapidly increasing disease. In particular, TSLP appears to be a very promising Epicutaneous immunotherapy that may become a viable treatment if there will be studies to prove that this an effective strategy to induce food tolerance in EoE.