Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder associated with allergic hypersensitivity to food. We interrogated >1.5 million genetic variants in European EoE cases and subsequently in a multi-site cohort with local and out-of-study control subjects. In addition to replication of the 5q22 locus (meta-analysis p = 1.9×10−16), we identified association at 2p23 (encoding CAPN14, p = 2.5×10−10). CAPN14 was specifically expressed in the esophagus, dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to IL-13, and located in an epigenetic hotspot modified by IL-13. There was enriched esophageal expression for the genes neighboring the top 208 EoE sequence variants. Multiple allergic sensitization loci were associated with EoE susceptibility (4.8×10−2 < p < 5.1×10−11). We propose a model that elucidates the tissue specific nature of EoE that involves the interplay of allergic sensitization with an EoE-specific, IL-13–inducible esophageal response involving CAPN14.
Background & Aims
Gene expression profiling provides an opportunity for definitive diagnosis but has not yet been well applied to inflammatory diseases. Herein, we describe an approach for diagnosis of an emerging form of esophagitis, eosinophilic esophagitis (EoE), currently diagnosed by histology and clinical symptoms.
We developed an EoE diagnostic panel (EDP), comprising a 96-gene quantitative PCR array and an associated dual-algorithm that uses cluster analysis and dimensionality reduction, using a cohort of randomly selected esophageal biopsy samples from pediatric patients with EoE (n = 15) or without EoE (non-EoE controls, n = 14), subsequently vetted using a separate cohort of 194 pediatric and adult patient samples derived from both fresh or formalin-fixed paraffin embedded (FFPE) tissue: active EoE (n = 91), control (non-EoE and EoE remission, n = 57), histologically ambiguous (n = 34), and reflux (n = 12) samples.
The EDP identified adult and pediatric patients with EoE with ~96% sensitivity and ~98% specificity, and distinguished patients with EoE in remission from controls, as well as identified patients exposed to swallowed glucorticoids. The EDP could be used with FFPE tissue RNA and distinguished patients with EoE from those with reflux esophagitis, identified by pH-impedance testing. Preliminary evidence showed that the EDP could identify patients likely to have disease relapse following treatment.
We developed a molecular diagnostic test (referred as the EDP) that identifies patients with esophagitis in a fast, objective, and mechanistic manner, offering an opportunity to improve diagnosis and treatment, and a platform approach for other inflammatory diseases.
EoE; GERD; NERD; diagnostic panel; signature; eosinophil; EoE transcriptome; fluidic card; genomics; transcriptome; esophagus
Eosinophils are major effector cells in type 2 inflammatory responses and become activated in response to IL-4 and IL-33, yet the molecular mechanisms and cooperative interaction between these cytokines remain unclear. Our objective was to investigate the molecular mechanism and cooperation of IL-4 and IL-33 in eosinophil activation. Eosinophils derived from bone marrow or isolated from Il5-transgenic mice were activated in the presence of IL-4 or IL-33 for 1 or 4h and the transcriptome was analysed by RNA-sequencing. The candidate genes were validated by qPCR and ELISA. We first demonstrated that murine cultured eosinophils respond to IL-4 and IL-33 by phosphorylation of STAT-6 and NFκB, respectively. RNA sequence analysis of murine cultured eosinophils indicated that IL-33 induced 519 genes; whereas, IL-4 induced only 28 genes, including 19 IL-33-regulated genes. Interestingly, IL-33 induced eosinophil activation via two distinct mechanisms, IL-4 independent and IL-4 secretion/auto-stimulation dependent. Anti-IL-4 or anti-IL-4Rα antibody-treated cultured and mature eosinophils, as well as Il4- or Stat6-deficient cultured eosinophils, had attenuated protein secretion of a subset of IL-33-induced genes, including Retnla and Ccl17. Additionally, IL-33 induced the rapid release of pre-formed IL-4 protein from eosinophils by an NFκB-dependent mechanism. However, the induction of most IL-33-regulated transcripts (e.g. Il6 and Il13) was IL-4 independent and blocked by NFκB inhibition. In conclusion, we have identified a novel activation pathway in murine eosinophils that is induced by IL-33 and differentially dependent upon an IL-4 auto-amplification loop.
IL-13-induced epithelial gene and protein expression changes are central to the pathogenesis of multiple allergic diseases. Herein, using human esophageal squamous and bronchial columnar epithelial cells, we identified miRNAs that were differentially regulated after IL-13 stimulation. Among the IL-13-regulated miRNAs, miR-375 showed a conserved pattern of down-regulation. Furthermore, miR-375 was down regulated in the lung of IL-13 lung transgenic mice. We subsequently analyzed miR-375 levels in a human disease characterized by IL-13 overproduction – the allergic disorder eosinophilic esophagitis (EE), and observed down-regulation of miR-375 in EE patient samples compared to control patients. MiR-375 expression levels reflected disease activity, normalized with remission, and inversely correlated to the degree of allergic inflammation. Using a lentiviral strategy and whole-transcriptome analysis in epithelial cells, miR-375 over-expression was sufficient to markedly modify IL-13-associated immunoinflammatory pathways in epithelial cells in vitro, further substantiating interactions between miR-375 and IL-13. Taken together, our results support a key role of miRNAs, particularly miR-375, in regulating and fine-tuning IL-13 mediated responses.
Surfactant protein D (SP-D) has been proposed to be protective in allergic airway responses.
We aimed to determine the effect of SP-D deficiency on murine and human airway allergy.
Immunological responses of SP-D gene deficient mice (Sftpd−/−) at baseline and following four Aspergillus fumigatus exposures were assessed. In addition, the significance of a single nucleotide polymorphism (Met11Thr) in the human SP-D gene (known to decrease SP-D function) on asthma susceptibility was investigated.
Macrophage BALF levels and lung CD-4+ T-cells were increased in naive Sftpd−/− mice in association with increased lung CCL17 levels. Th2-associated antibody levels (IgG1 and IgE) were significantly lower in 4–6 week old Sftpd−/− mice (p<0.05). Accordingly, naive Sftpd−/− splenocytes released significantly less IL-4 and IL-13 upon anti-CD3/CD28 stimulation (p<0.01). Following intranasal allergen exposures, a modest decrease in BALF eosinophilia was observed in Sftpd−/− mice compared to wild type mice (p<0.01). Translational studies in a pediatric population of Caucasians with asthma revealed that a single nucleotide polymorphism in the SFTPD gene, affecting SP-D levels and pathogen binding, was associated with lower asthma susceptibility (p<0.05).
Sftpd−/− mice have an impaired systemic Th2 response at baseline and modestly reduced pulmonary eosinophilia following allergen exposure. Translational studies revealed that a mutation in the SFTPD gene was associated with lower asthma susceptibility in Caucasians. Taken together, these results support the hypothesis that SP-D-dependent innate immunity influences atopy and asthma.
SP-D deficiency results in increased pulmonary inflammation and decreased susceptibility to asthma, perhaps related to impaired endotoxin and pathogen clearance.
allergy; lung; surfactant protein D; polymorphism; eosinophil; IL-13; Aspergillus; endotoxin
Intestinal anaphylaxis (manifested by acute diarrhea) is dependent on IgE and mast cells.
We aimed to define the respective roles of IL-4 and IL-13 and their receptors in disease pathogenesis.
Wild-type mice and mice deficient in IL-4, IL-13, and IL-13Rα1 (part of the type 2 IL-4R) were sensitized with ovalbumin (OVA)/alum and subsequently given repeated intragastric OVA exposures. IL-4Rα chain was targeted with anti-IL-4Rα mAb prior to or after intragastric OVA exposures.
IL-4−/− (and IL-4/13−/−) mice produced almost no IgE and were highly resistant to OVA-induced diarrhea, whereas allergic diarrhea was only partially impaired in IL-13−/− and IL-13Rα1−/− mice. IL-13Rα1-deficient mice developed decreased IgE despite having normal baseline IL-4 levels. Intestinal mast cell accumulation and activation also depended mainly on IL-4 and to a lesser extent on IL-13. Prophylactic anti-IL-4Rα mAb treatment, which blocks all IL-4 and IL-13 signaling, suppressed development of allergic diarrhea. However, treatment with anti-IL-4Rα mAb for 7 days only partially suppressed IgE and did not prevent intestinal diarrhea.
Endogenously-produced IL-13 supplements the ability of IL-4 to induce allergic diarrhea by promoting oral allergen sensitization rather than the effector phase of intestinal anaphylaxis.
allergy; anaphylaxis; IL-4; IL-13; IL-13Ralpha1; intestine; mast cell
The gastrointestinal (GI) tract is in constant “negotiation” with the microbial flora present in the lumen. Resident hematopoietic cells (i.e. lymphocytes, mast cells and eosinophils) are part of this ongoing and silent homeostatic battle. Eosinophilic GI diseases (EGID) are characterized by an increased number of eosinophil infiltrates with no identified cause. In this review, we describe the past and present knowledge regarding the chemotactic factors involved in GI eosinophilia.
Eosinophil; gastrointestinal; chemokine; cytokine; pathogenesis
Eosinophilic esophagitis (EoE) is an emerging chronic inflammatory disease mediated by immune hypersensitization to multiple foods and strongly associated with atopy and esophageal remodeling.
We provide clinical and molecular evidence indicating a high prevalence of EoE in patients with inherited connective tissue disorders (CTDs).
We examined the rate of EoE among patients with CTDs and subsequently analyzed esophageal mRNA transcript profiles in patients with EoE with or without CTD features.
We report a cohort of 42 patients with EoE with a CTD-like syndrome, representing 0.8% of patients with CTDs and 1.3% of patients with EoE within our hospital-wide electronic medical record database and our EoE research registry, respectively. An 8-fold risk of EoE in patients with CTDs (relative risk, 8.1; 95% confidence limit, 5.1-12.9; χ21 = 112.0; P < 10−3) was present compared with the general population. Esophageal transcript profiling identified a distinct subset of genes, including COL8A2, in patients with EoE and CTDs.
There is a remarkable association of EoE with CTDs and evidence for a differential expression of genes involved in connective tissue repair in this cohort. Thus, we propose stratification of patients with EoE and CTDs into a subset referred to as EoE-CTD.
Eosinophilic esophagitis; eosinophilic gastrointestinal disease; eosinophil; connective tissue disorders; Ehlers-Danlos syndrome; Marfan syndrome; hypermobility syndrome
Eosinophilic Esophagitis (EE) is now a commonly encountered disorder that was rarely diagnosed a decade ago.
We aimed to determine the epidemiologic and histologic features of retrospective pediatric esophageal eosinophilia before the first case of EE at our institution was recognized.
Esophageal biopsies obtained between 1982–1999 with reflux esophagitis were re-examined and re-organized into 2 groups based on peak esophageal eosinophil number (<15 eos/hpf, ≥ 15 eos/hpf). The epidemiology and histology of the entire cohort and a population based cohort were evaluated.
807 biopsies from 666 patients were re-examined; 198 patients had ≥ 15 eos/hpf. Among a population-based cohort of patients with ≥ 15 eos/hpf, there was a modest increase in incidence (p < 0.001; IRR 1.18; CI 1.09–1.28). After correcting for a 40-fold increase in the number of endoscopies during this time period, the proportion of biopsies with ≥ 15 eos/hpf did not change (0.08 in 1982 vs. 0.08 in 1996 (peak), (p = 0.9; IRR 1.02; CI 0.73 –1.44). Patients who had as few as 5 eos/hpf were more likely to have persistent esophageal eosinophilia on repeat EGD, evidence of basal layer hyperplasia and lamina propria fibrosis compared to patients with < 5 eos/hpf (p <0.001).
Esophageal Eosinophilia, at levels consistent with EE, was present among 30% of patients diagnosed with reflux esophagitis and the incidence of esophageal eosinophilia did not change over time. Patients with ≥ 5 eos/hpf had evidence of other histologic abnormalities and were likely to have persistent esophageal eosinophilia.
eosinophilic esophagitis; incidence; diagnosis; chronic esophagitis; eosinophil; esophagitis; epidemiology; retrospective
In this review, we aim to put in perspective the biology of a multifunctional leukocyte, the eosinophil, by placing it in the context of innate and adaptive immune responses. Eosinophils have a unique contribution in initiating inflammatory and adaptive responses due to their bidirectional interactions with dendritic cells and T cells, as well as their large panel of secreted cytokines and soluble mediators. The mechanisms and consequences of eosinophil responses in experimental inflammatory models are discussed.
Eosinophil; chemokine; cytokine; pathogenesis; gastrointestinal; asthma
Allergic inflammation is accompanied by the coordinated expression of a myriad of genes and proteins that initiate, sustain, and propagate immune responses and tissue remodeling. MicroRNAs (miRNAs) are a class of short single-stranded RNA molecules that post-transcriptionally silence gene expression and have been shown to fine-tune gene transcriptional networks as single miRNAs can target hundreds of genes. 7 Considerable attention has focused on the key role of miRNAs in regulating homeostatic immune architecture and acquired immunity. Recent studies have identified miRNA profiles in multiple allergic inflammatory diseases including asthma, eosinophilic esophagitis, allergic rhinitis, and atopic dermatitis. Specific miRNAs have been found to have critical roles in regulating key pathogenic mechanisms in allergic inflammation including polarization of adaptive immune responses and activation of T cells (e.g. miR-21 and miR-146), regulation of eosinophil development (e.g. miR-21 and miR-223), and modulation of interleukin (IL)-13-driven epithelial responses (e.g. miR-375). This review discusses recent advances in our understanding of the expression and function of miRNAs in allergic inflammation, their role as disease biomarkers, and perspectives for future investigation and clinical utility.
Allergy; microRNA; miRNA; non-coding RNA; asthma; eosinophilic esophagitis; atopic dermatitis; allergic rhinitis; eosinophils; inflammation; biomarkers
Recently, microRNAs (miRNAs) have been shown to be involved in hematopoietic cell development but their role in eosinophilopoeisis has not yet been described. Here we show that miR-223 is up-regulated during eosinophil differentiation in an ex vivo bone marrow derived eosinophil culture system. Targeted ablation of miR-223 leads to an increased proliferation of eosinophil progenitors. We found up-regulation of a miR-223 target gene – IGF1R in the eosinophil progenitor cultures derived from miR-223-/- mice compared to miR-223+/+ littermate controls. The increased proliferation of miR-223-/- eosinophil progenitors was reversed by treatment with the IGF1R inhibitor (picropodophyllin). Whole genome microarray analysis of differentially regulated genes between miR-223+/+ and miR-223-/- eosinophil progenitor cultures identified a specific enrichment in genes that regulate hematologic cell development. Indeed, miR-223-/- eosinophil progenitors had a delay in differentiation. Our results demonstrate that miRNAs regulate the development of eosinophils by influencing eosinophil progenitor growth and differentiation and identify a contributory role for miR-223 in this process.
Eosinophils; Esophagitis; Symptoms; Therapy
DNA demethylation has been primarily studied in the context of development biology, cell fate, and cancer, with less attention on inflammation. Herein, we investigate the association between DNA methylation and production of the chemoattractant cytokine eotaxin-3 in the tissue of patients with allergic disease. Regions of the human eotaxin-3 promoter were found to be hypomethylated in primary epithelial cells obtained from allergic tissue compared with normal control tissue (CTL). The demethylation of a specific CpG site (designated CpG 2), which is juxtaposed to a key cyclic AMP-responsive element (CRE) site, was significantly demethylated in patient-derived compared to CTL-derived epithelial cells. Levels of methylation at CpG 2 inversely correlated with basal and IL-13–induced eotaxin-3 gene expression. Conversely, global inhibition of methylation with 5-azacytidine (5-AzaC) promoted eotaxin-3 production in association with decreasing CpG 2 methylation. In addition, the basal and IL-13-induced eotaxin-3 transcriptional activity was suppressed by promoter methylation using a methylation-free in vitro system. Further, electrophoretic mobility shift assays (EMSA) demonstrated that the attachment of CREB binding protein (CBP) and activating transcription factor 2 (ATF-2) to the CRE site was methylation dependent. Taken together, these data identify a contributory role for DNA methylation in regulating eotaxin-3 production in human allergic inflammation.
Allergic airway inflammation is characterized by marked in situ changes in gene and protein expression, yet the role of microRNAs (miRNAs), a new family of key mRNA regulatory molecules, in this process has not yet been reported. Using a highly sensitive microarray based approach, we identified 21 miRNAs with differential expression between doxycycline-induced lung-specific IL-13 transgenic mice (with allergic airway inflammation) and control mice. In particular, we observed over-expression of miR-21 and under-expression of miR-1 in the induced IL-13 transgenic mice compared to control mice. These findings were validated in two independent models of allergen-induced allergic airway inflammation and in IL-4 lung transgenic mice. While IL-13 induced miR-21 expression was IL-13 receptor alpha 1 dependent, allergen induced miR-21 expression was mediated mainly independent of IL-13 receptor alpha 1 and STAT6. Notably, predictive algorithms identified potential direct miR-21 targets among IL-13-regulated lung transcripts such as IL-12p35 mRNA that was decreased in IL-13 transgenic mice. Introduction of pre-miR-21 dose-dependently inhibited cellular expression of a reporter vector harboring the 3’UTR of IL-12p35. Moreover, mutating miR-21 binding sites in IL-12p35 3’UTR abrogated miR-21 mediated repression. In summary, we have identified a miRNA signature in allergic airway inflammation, which includes miR-21 that modulates IL-12, a molecule germane to T helper cell polarization.
Allergy; lung; inflammation; gene regulation
Eosinophilic esophagitis is a chronic, immune-mediated inflammatory disorder that responds to dietary therapy; however, data evaluating the effectiveness of dietary therapeutic strategies is limited.
This study compared the effectiveness of three frequently prescribed dietary therapies [elemental, six-food elimination, and skin prick and atopy patch-directed elimination] and assessed the remission predictability of skin tests and their utility in directing dietary planning.
A retrospective cohort of proton-pump inhibitor-unresponsive, non-glucocorticoid-treated eosinophilic esophagitis patients who had two consecutive endoscopic biopsies associated with dietary intervention was identified. Biopsy histology and remissions (< 15 eosinophils/high-power field) following dietary therapy and food reintroductions were evaluated.
Ninety-eight of 513 patients met eligibility criteria. Of these 98, 50% (49), 27% (26), and 23% (23) received elemental, six-food elimination, and directed diets, respectively. Remission occurred in 96%, 81%, and 65% of patients on elemental, six-food elimination, and directed diets, respectively. The odds of post-diet remission vs. non-remission were 5.6-fold higher (P=0.05) on elemental vs. six-food elimination, 12.5-fold higher (P=0.003) on elemental vs. directed, and were not significantly different (P=0.22) on six-food elimination vs. directed diets. Following 116 single-food reintroductions, the negative predictive value of skin testing for remission was 40%–67% (milk 40%, egg 56%, soy 64%, and wheat 67%).
All three dietary therapies are effective; however, an elemental diet is superior at inducing histologic remission compared with six-food elimination and skin test-directed diets. Notably, an empiric six-food elimination diet is as effective as a skin test-directed diet. The negative predictive values of foods most commonly reintroduced in single-food challenges are not sufficient to support the development of dietary advancement plans solely based on skin tests.
Eosinophilic esophagitis; eosinophils; histologic remission; pediatric; dietary therapy; food allergy; negative predictive values; elemental diet; six-food elimination diet; skin test-directed elimination diet
The role of microRNAs (miRNAs), a key class of regulators of mRNA expression and translation, in patients with eosinophilic esophagitis (EoE) has not been explored.
We aimed to identify miRNAs dysregulated in patients with EoE and assess the potential of these miRNAs as disease biomarkers.
Esophageal miRNA expression was profiled in patients with active EoE and those with glucocorticoid-induced disease remission. Expression profiles were compared with those of healthy control subjects and patients with chronic (noneosinophilic) esophagitis. Expression levels of the top differentially expressed miRNAs from the plasma of patients with active EoE and patients with EoE remission were compared with those of healthy control subjects.
EoE was associated with 32 differentially regulated miRNAs and was distinguished from noneosinophilic forms of esophagitis. The expression levels of the most upregulated miRNAs (miR-21 and miR-223) and the most downregulated miRNA (miR-375) strongly correlated with esophageal eosinophil levels. Bioinformatic analysis predicted interplay of miR-21 and miR-223 with key roles in the polarization of adaptive immunity and regulation of eosinophilia, and indeed, these miRNAs correlated with key elements of the EoE transcriptome. The differentially expressed miRNAs were largely reversible in patients who responded to glucocorticoid treatment. EoE remission induced a single miRNA (miR-675) likely to be involved in DNA methylation. Plasma analysis of the most upregulated esophageal miRNAs identified miR-146a, miR-146b, and miR-223 as the most differentially expressed miRNAs in the plasma.
We have identified a marked dysregulated expression of a select group of miRNAs in patients with EoE and defined their reversibility with glucocorticoid treatment and their potential value as invasive and noninvasive biomarkers.
Eosinophilic esophagitis; microRNA; glucocorticoid; biomarkers
IL-13 is expressed in lesions of atopic dermatitis (AD) and has been associated with increased disease severity. IL-13 has two cognate receptors: IL-13Rα1 and IL-13Rα2. Although IL-13Rα2 expression is known to be induced in response to IL-13 in keratinocytes, its function in AD has never been evaluated. We characterized the loss of skin barrier function and the development of cutaneous inflammation in IL-13Rα2–null versus wild-type BALB/c mice following an epicutaneous allergen-sensitization/challenge model that shares similarities with human AD. Mice lacking IL-13Rα2 had significantly increased transepidermal water loss, cutaneous inflammation, peripheral eosinophilia, and IgG1 and IgE levels compared with wild-type mice. The rate of resolution of the cutaneous inflammation was not significantly altered in the IL-13Rα2–null mice. IL-13 induced expression of IL-13Rα2 in keratinocyte cell lines and primary human keratinocytes. Depletion of IL-13Rα2 in a keratinocyte cell line resulted in increased STAT6 signaling in response to IL-13. In conclusion, IL-13Rα2 serves a protective role in the pathogenesis of allergic inflammation and loss of skin barrier function in a mouse model of AD, suggesting that it may be an important endogenous regulator of IL-13–induced cutaneous inflammation in humans.
CD22 is currently recognized as a B cell-specific Siglec and has been exploited therapeutically with humanized anti-CD22 monoclonal antibody having been used against B cell leukemia. Herein, tissue-specific eosinophil mRNA microarray analysis identified that CD22 transcript levels of murine gastrointestinal (GI) eosinophils are 10-fold higher than those of lung eosinophils. In order to confirm the mRNA data at the protein level, we developed a FACS-based protocol designed to phenotype live GI eosinophils isolated from the murine lamina propria. Indeed, we found that jejunum eosinophils expressed remarkably high levels of surface CD22, similar to levels found in B cells across multiple mouse strains. In contrast, CD22 was undetectable on eosinophils from the colon, blood, thymus, spleen, uterus, peritoneal cavity and allergen-challenged lung. Eosinophils isolated from newborn mice did not express CD22 but subsequently upregulated CD22 expression to adult levels within the first 10 days after birth. The GI lamina propria from CD22 gene-targeted mice harbored more eosinophils than wild-type control mice, while the GI eosinophil turnover rate was unaltered in the absence of CD22. Our findings identify a novel expression pattern and tissue eosinophilia-regulating function for the “B cell-specific” inhibitory molecule CD22 on GI eosinophils.
Eosinophils; mucosa; Siglec; CD22
Objective: We report the first pediatric specific Phenome-Wide Association Study (PheWAS) using electronic medical records (EMRs). Given the early success of PheWAS in adult populations, we investigated the feasibility of this approach in pediatric cohorts in which associations between a previously known genetic variant and a wide range of clinical or physiological traits were evaluated. Although computationally intensive, this approach has potential to reveal disease mechanistic relationships between a variant and a network of phenotypes.
Method: Data on 5049 samples of European ancestry were obtained from the EMRs of two large academic centers in five different genotyped cohorts. Recently, these samples have undergone whole genome imputation. After standard quality controls, removing missing data and outliers based on principal components analyses (PCA), 4268 samples were used for the PheWAS study. We scanned for associations between 2476 single-nucleotide polymorphisms (SNP) with available genotyping data from previously published GWAS studies and 539 EMR-derived phenotypes. The false discovery rate was calculated and, for any new PheWAS findings, a permutation approach (with up to 1,000,000 trials) was implemented.
Results: This PheWAS found a variety of common variants (MAF > 10%) with prior GWAS associations in our pediatric cohorts including Juvenile Rheumatoid Arthritis (JRA), Asthma, Autism and Pervasive Developmental Disorder (PDD) and Type 1 Diabetes with a false discovery rate < 0.05 and power of study above 80%. In addition, several new PheWAS findings were identified including a cluster of association near the NDFIP1 gene for mental retardation (best SNP rs10057309, p = 4.33 × 10−7, OR = 1.70, 95%CI = 1.38 − 2.09); association near PLCL1 gene for developmental delays and speech disorder [best SNP rs1595825, p = 1.13 × 10−8, OR = 0.65(0.57 − 0.76)]; a cluster of associations in the IL5-IL13 region with Eosinophilic Esophagitis (EoE) [best at rs12653750, p = 3.03 × 10−9, OR = 1.73 95%CI = (1.44 − 2.07)], previously implicated in asthma, allergy, and eosinophilia; and association of variants in GCKR and JAZF1 with allergic rhinitis in our pediatric cohorts [best SNP rs780093, p = 2.18 × 10−5, OR = 1.39, 95%CI = (1.19 − 1.61)], previously demonstrated in metabolic disease and diabetes in adults.
Conclusion: The PheWAS approach with re-mapping ICD-9 structured codes for our European-origin pediatric cohorts, as with the previous adult studies, finds many previously reported associations as well as presents the discovery of associations with potentially important clinical implications.
PheWAS; ICD-9 code; genetic polymorphism
Interleukin (IL)-13 and IL-4 are hallmark cytokines of Th2-associated diseases including asthma. Recent studies revealed that IL-13Rα1 regulates asthma pathogenesis by mediating both IL-4 and IL-13-mediated responses. Nonetheless, the relative contribution of each cytokine in response to aeroallergen challenge and the degree of functional dichotomy between IL-4 and IL-13 in asthma remains unclear. Consistent with prior publications, we demonstrate that IL-13Rα1 regulates aeroallergen-induced airway resistance and mucus production but not IgE and Th2 cytokine production. We demonstrate that aeroallergen-induced eosinophil recruitment and chemokine production were largely dependent of IL-13Rα1 following Aspergillus (Asp) but not house dust mite (HDM) challenges. Notably, Asp-challenged mice displayed increased IL-13Rα1-dependent accumulation of dendritic cell subsets into lung draining lymph nodes in comparison with HDM. Comparison of IL-4 and IL-13 levels in the different experimental models revealed increased IL-4:IL-13 ratios following HDM challenge, likely explaining the IL-13Rα1-independent eosinophilia and chemokine production. Consistently, eosinophil adoptive-transfer experiments revealed near ablation of lung eosinophilia in response to Asp in Il13ra1−/− mice, suggesting that Asp-induced lung eosinophil recruitment is regulated by IL-13-induced chemokine production, rather than altered IL-13 signaling in eosinophils. Furthermore, the near complete protection observed in Il13ra1−/− mice in response to Asp-challenge was dependent on mucosal sensitization since Alum/Asp-sensitized mice that were re-challenged with Asp developed IL-13Rα1-independent eosinophilia although other asthma parameters remained IL-13Rα1-dependent. These results establish that IL-13Rα1 is required for aeroallergen-induced airway resistance and that allergen-induced chemokine production and consequent eosinophilia is dictated by the balance between IL-4 and IL-13 production in situ.
Allergy; Lung; Cytokines; Chemokines; Cytokine receptors; Eosinophils
An altered balance between Th1 and Th2 cytokines is responsible for a variety of immuno-inflammatory disorders such as asthma, yet the role of post-transcriptional mechanisms, such as those mediated by microRNAs, in adjusting the relative magnitude and balance of Th cytokine expression have been largely unexplored. Here we show that miR-21 has a central role in setting a balance between Th1 and Th2 responses to antigens. Targeted ablation of miR-21 in mice led to reduced lung eosinophilia after allergen challenge, with a broadly reprogrammed immunoactivation transcriptome, and significantly increased levels of the Th1 cytokine IFNγ. Biological network-based transcriptome analysis of OVA-challenged miR-21-/-mice identified an unexpected prominent dysregulation of IL-12/IFNγ pathways as the most significantly affected in the lungs with a key role for miR-21 in IFNγ signaling and T-cell polarization, consistent with a functional miR-21 binding site in IL-12p35. In support of these hypotheses, miR-21 deficiency led dendritic cells to produce more IL-12 after LPS stimulation, and OVA-challenged CD4+ T lymphocytes to produce increased IFNγ and decreased IL-4. Further, loss of miR-21 significantly enhanced the Th1-associated delayed-type hypersensitivity cutaneous responses. Thus, our results define miR-21 as a major regulator of Th1 vs. Th2 responses, defining a new mechanism for regulating polarized immuno-inflammatory responses.
Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder of the esophagus that is compounded by both genetic predisposition and aberrant responses to environmental antigens, particularly those that are food-derived. Data have indicated a unique transcriptional response in vivo that defines EoE and which is partially attributable to the Th2 cytokine interleukin 13 (IL-13). Moreover, a number of genetic risk variants in pro-inflammatory and epithelial cell genes associate with EoE susceptibility, demonstrating novel heritable mechanisms that contribute to disease risk. Here, we discuss recent advances in our understanding of the intrinsic (genetic) and extrinsic (environmental) components that illustrate the complex nature of EoE.
Eosinophilic esophagitis; genetics; candidate gene; genome-wide association; polymorphism
Pediatric eosinophilic esophagitis (EoE) is a newly recognized antigen-induced form of chronic esophagitis.
Characterization of long-term clinical outcomes in pediatric EoE patients is needed.
From histologic review of 3,817 pediatric esophageal biopsies from 1982–1999, we conducted a nested case-control study of patients with retrospectively-identified histologic eosinophilic esophagitis (rEoE) and chronic esophagitis (CE), as well as an age-matched control cohort. Participants were asked to complete validated health-related outcome questionnaires.
After an average of 15 years following initial endoscopy, both cohorts, 42/198 rEoE and 67/468 CE patients (as well as 100 age-matched controls), completed questionnaires. Compared to control patients, quality of life was significantly decreased among rEoE patients (P<0.001) and CE patients (P<0.001). Rates of dysphagia (rEoE 49%; CE 37%; control 6%) and food impaction (rEoE 40%; CE 14%; control 3%) were significantly increased in the rEoE cohort compared to controls (P<0.001, P<0.001 respectively). Increased esophageal eosinophil counts (OR 1.6; 95% CI 1.1–2.5; P<0.05) during childhood were predictive of dysphagia during early adulthood. Food allergy (OR 2.7; CI 1.2, 6.0; P<0.01), allergic rhinitis (OR 3.5; CI 1.8, 6.8; P<0.001), and asthma (OR 2.1; CI 1.04, 4.3; P=0.04) were associated with dysphagia. Food impaction was more common among patients with reported food allergy than those without (OR 3.1; CI 1.2, 7.8; P=0.02).
Esophageal eosinophilia is associated with reduced quality of life and persistent symptoms 15 years after presentation. Elevated esophageal eosinophil counts and the occurrence of food allergy and atopy in childhood increase the rate of dysphagia in young adulthood.
eosinophilic esophagitis; pediatric; patient-reported outcomes; natural history; eosinophil