Correlation of increased PARP14 and CCL26 expression in biopsies from children with eosinophilic esophagitis

Abstract

PARP14 is one of 18 poly-ADP ribosyl polymerase (PARP) family members that contain a catalytic domain conferring ADP-ribosyltransferase activity, and was initially identified as a transcriptional cofactor for signal transducer and activator of transcription (STAT)6 activity.1Goenka S. Kaplan M.H. Transcriptional regulation by STAT6.Immunol Res. 2011; 50: 87-96Crossref PubMed Scopus (235) Google Scholar Cytokine-stimulated STAT6 DNA binding activates PARP14 catalytic activity, resulting in changes in target gene chromatin.2Mehrotra P. Riley J.P. Patel R. Li F. Voss L. Goenka S. PARP-14 functions as a transcriptional switch for Stat6-dependent gene activation.J Biol Chem. 2011; 286: 1767-1776Crossref PubMed Scopus (107) Google Scholar Because STAT6 plays an obligate role in the development of allergic inflammation,1Goenka S. Kaplan M.H. Transcriptional regulation by STAT6.Immunol Res. 2011; 50: 87-96Crossref PubMed Scopus (235) Google Scholar and other PARPs including PARP1 may contribute to allergic inflammation,3Rosado M.M. Bennici E. Novelli F. Pioli C. Beyond DNA repair, the immunological role of PARP-1 and its siblings.Immunology. 2013; 139: 428-437Crossref PubMed Scopus (124) Google Scholar we previously assessed the requirement for PARP14 in the development of allergic inflammation. In a model of allergic airway inflammation, mice deficient in PARP14 had diminished cellular infiltrates, increased lung function, and decreased TH2 cytokine production compared with control mice.4Mehrotra P. Hollenbeck A. Riley J.P. Li F. Patel R.J. Akhtar N. et al.Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease.J Allergy Clin Immunol. 2013; 131 (e1-12): 521-531Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Similarly, treatment of wild-type mice with PARP inhibitors during or after the development of disease resulted in decreased airway inflammation, TH2 cell development, and increased lung function compared with control mice.4Mehrotra P. Hollenbeck A. Riley J.P. Li F. Patel R.J. Akhtar N. et al.Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease.J Allergy Clin Immunol. 2013; 131 (e1-12): 521-531Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar At least part of the mechanism of PARP14 function was through direct effects of PARP14 on TH2 cytokine genes, and the TH2 transcription factor Gata3.4Mehrotra P. Hollenbeck A. Riley J.P. Li F. Patel R.J. Akhtar N. et al.Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease.J Allergy Clin Immunol. 2013; 131 (e1-12): 521-531Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar However, the requirement for PARP14 in STAT6-dependent gene expression in nonlymphoid cells, and whether PARP14 expression is altered in human disease, has not been assessed.We initially tested whether there are changes in the expression of members of the macro-PARP subfamily of PARP genes in children with eosinophilic esophagitis (EoE) compared with control samples. We obtained esophageal biopsies from children with EoE (Indiana University [IU] population; see Table E1 in this article's Online Repository at www.jacionline.org) and control samples from children who had esophageal biopsies for diagnostic purposes but did not have eosinophilic esophagitis (Table E1). RNA was isolated from biopsies, and cDNA was assessed for gene expression by using quantitative PCR. We observed a 5.95-fold average increase in PARP14 expression, a 3.1-fold average increase in PARP1 expression, and a decrease in PARP15 expression in EoE biopsies compared with controls (Fig 1, A). In contrast, there was no significant difference in the expression of PARP9 (Fig 1, A).To confirm this finding, we examined PARP14 expression in a population from Cincinnati Children's Hospital Medical Center through the use of high-throughput RNA sequencing.5Lu T.X. Sherrill J.D. Wen T. Plassard A.J. Besse J.A. Abonia J.P. et al.MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers.J Allergy Clin Immunol. 2012; 129: 1064-1075.e1069Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar Compared with the IU population, this population had more severe inflammation5Lu T.X. Sherrill J.D. Wen T. Plassard A.J. Besse J.A. Abonia J.P. et al.MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers.J Allergy Clin Immunol. 2012; 129: 1064-1075.e1069Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar (Table E1). Following analysis of the RNA-sequencing data, we observed similar (4.5-fold) increases in PARP14 expression as seen in the IU population (Fig 1, B).The chemokine CCL26 (Eotaxin-3) is a critical component of the pathology of EoE. CCL26 expression is dramatically increased in biopsies from patients with EoE, and single nucleotide polymorphisms in the CCL26 gene are associated with increased disease incidence.6Blanchard C. Wang N. Stringer K.F. Mishra A. Fulkerson P.C. Abonia J.P. et al.Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis.J Clin Invest. 2006; 116: 536-547Crossref PubMed Scopus (696) Google Scholar, 7Gupta S.K. Fitzgerald J.F. Kondratyuk T. HogenEsch H. Cytokine expression in normal and inflamed esophageal mucosa: a study into the pathogenesis of allergic eosinophilic esophagitis.J Pediatr Gastroenterol Nutr. 2006; 42: 22-26Crossref PubMed Scopus (151) Google Scholar Moreover, STAT6 regulates CCL26 in esophageal cells.8Lim E.J. Lu T.X. Blanchard C. Rothenberg M.E. Epigenetic regulation of the IL-13-induced human eotaxin-3 gene by CREB-binding protein-mediated histone 3 acetylation.J Biol Chem. 2011; 286: 13193-13204Crossref PubMed Scopus (35) Google Scholar To determine whether PARP14 expression correlated with CCL26 expression, we tested the association of expression of these 2 genes in esophageal biopsies from patients with EoE and observed a strong correlation coefficient (IU population: R = 0.81; P = .0002, Cincinnati Children's Hospital Medical Center population: R = 0.61, P = .03) (Fig 1, C). In contrast, there was no significant correlation between PARP1 and CCL26 expression (R = 0.30, P = .27). There is significant heterogeneity in the expression of PARP14 in the biopsy samples, with some overlap in the control biopsy samples (Fig 1, A). This heterogeneity was lessened in samples from patients with more severe inflammation (Fig 1, B). Thus, PARP14 expression is increased and CCL26 and PARP14 gene expression is correlated in 2 populations interrogated by 2 distinct methods.Because our results suggested a relationship between PARP14 and CCL26, we tested the ability of PARP14 to regulate CCL26 directly. The esophageal cell line TE-7 was transfected with a CCL26 luciferase reporter vector and plasmids encoding STAT6 and/or PARP14 before incubation for 24 hours in the presence or absence of the STAT6-activating cytokines IL-4 and IL-13. Consistent with previous results, transfection of STAT6-expressing plasmids increased CCL26 reporter activity (Fig 2, A). Moreover, transfection of PARP14 alone increased basal and cytokine-induced reporter activity (Fig 2, A). Importantly, cotransfection of STAT6 and PARP14 significantly increased CCL26 reporter activity over cells transfected with STAT6 alone (Fig 2, A). The effects of PARP14 expression were entirely dependent on STAT6 binding because they were not observed when the plasmid was cotransfected with a CCL26 reporter that had a mutation in the STAT6 binding site (Fig 2, A). These results suggested that PARP14 might be a viable target for modulating the expression of CCL26. To test this directly, TE-7 cells were incubated with IL-4 or IL-13 in the presence or absence of the PARP activity inhibitor PJ34 before expression of the endogenous CCL26 gene was assessed. We observed that IL-4 and IL-13 increased CCL26 mRNA and that incubation with the PARP inhibitor attenuated the induction in response to either cytokine (Fig 2, B). Similar results were observed in the TE-1 esophageal cell line. Thus, PARP14, and PARP activity, contribute to the regulation of CCL26 in esophageal cells. These results do not exclude the possibility that PARP14 is expressed by, and functions in, additional cell types that contribute to EoE.Fig 2PARP14 activates the CCL26 gene. A, CCL26 promoter reporter activity with cotransfection of STAT6- and/or PARP14-expressing plasmids into TE-7 esophageal cells. *P < .05; **P < .001, compared with control plasmid transfection; ‡P < .05 compared with STAT6 transfection. B, CCL26 expression in cytokine-stimulated TE-7 esophageal cells incubated with the PARP inhibitor PJ34. Results are the average of at least 3 experiments. *P < .05 and **P < .001. P values determined by using the Student t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although we are only beginning to understand the in vivo functions of PARP14, this report, coupled with our previous work,4Mehrotra P. Hollenbeck A. Riley J.P. Li F. Patel R.J. Akhtar N. et al.Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease.J Allergy Clin Immunol. 2013; 131 (e1-12): 521-531Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar suggests that PARP14 has a significant role in the development of allergic inflammation. It likely works in multiple cell types, including in T cells, where it results in increased TH2 and TH9 development,4Mehrotra P. Hollenbeck A. Riley J.P. Li F. Patel R.J. Akhtar N. et al.Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease.J Allergy Clin Immunol. 2013; 131 (e1-12): 521-531Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 9Goswami R. Jabeen R. Yagi R. Pham D. Zhu J. Goenka S. et al.STAT6-dependent regulation of Th9 development.J Immunol. 2012; 188: 968-975Crossref PubMed Scopus (168) Google Scholar and in target organ epithelial cells, enhancing the production of proallergic chemokines. Our results raise the possibility that targeting PARP14, or even PARP activity in general, might be an effective therapy for allergic diseases including EoE. PARP14 is one of 18 poly-ADP ribosyl polymerase (PARP) family members that contain a catalytic domain conferring ADP-ribosyltransferase activity, and was initially identified as a transcriptional cofactor for signal transducer and activator of transcription (STAT)6 activity.1Goenka S. Kaplan M.H. Transcriptional regulation by STAT6.Immunol Res. 2011; 50: 87-96Crossref PubMed Scopus (235) Google Scholar Cytokine-stimulated STAT6 DNA binding activates PARP14 catalytic activity, resulting in changes in target gene chromatin.2Mehrotra P. Riley J.P. Patel R. Li F. Voss L. Goenka S. PARP-14 functions as a transcriptional switch for Stat6-dependent gene activation.J Biol Chem. 2011; 286: 1767-1776Crossref PubMed Scopus (107) Google Scholar Because STAT6 plays an obligate role in the development of allergic inflammation,1Goenka S. Kaplan M.H. Transcriptional regulation by STAT6.Immunol Res. 2011; 50: 87-96Crossref PubMed Scopus (235) Google Scholar and other PARPs including PARP1 may contribute to allergic inflammation,3Rosado M.M. Bennici E. Novelli F. Pioli C. Beyond DNA repair, the immunological role of PARP-1 and its siblings.Immunology. 2013; 139: 428-437Crossref PubMed Scopus (124) Google Scholar we previously assessed the requirement for PARP14 in the development of allergic inflammation. In a model of allergic airway inflammation, mice deficient in PARP14 had diminished cellular infiltrates, increased lung function, and decreased TH2 cytokine production compared with control mice.4Mehrotra P. Hollenbeck A. Riley J.P. Li F. Patel R.J. Akhtar N. et al.Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease.J Allergy Clin Immunol. 2013; 131 (e1-12): 521-531Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Similarly, treatment of wild-type mice with PARP inhibitors during or after the development of disease resulted in decreased airway inflammation, TH2 cell development, and increased lung function compared with control mice.4Mehrotra P. Hollenbeck A. Riley J.P. Li F. Patel R.J. Akhtar N. et al.Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease.J Allergy Clin Immunol. 2013; 131 (e1-12): 521-531Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar At least part of the mechanism of PARP14 function was through direct effects of PARP14 on TH2 cytokine genes, and the TH2 transcription factor Gata3.4Mehrotra P. Hollenbeck A. Riley J.P. Li F. Patel R.J. Akhtar N. et al.Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease.J Allergy Clin Immunol. 2013; 131 (e1-12): 521-531Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar However, the requirement for PARP14 in STAT6-dependent gene expression in nonlymphoid cells, and whether PARP14 expression is altered in human disease, has not been assessed. We initially tested whether there are changes in the expression of members of the macro-PARP subfamily of PARP genes in children with eosinophilic esophagitis (EoE) compared with control samples. We obtained esophageal biopsies from children with EoE (Indiana University [IU] population; see Table E1 in this article's Online Repository at www.jacionline.org) and control samples from children who had esophageal biopsies for diagnostic purposes but did not have eosinophilic esophagitis (Table E1). RNA was isolated from biopsies, and cDNA was assessed for gene expression by using quantitative PCR. We observed a 5.95-fold average increase in PARP14 expression, a 3.1-fold average increase in PARP1 expression, and a decrease in PARP15 expression in EoE biopsies compared with controls (Fig 1, A). In contrast, there was no significant difference in the expression of PARP9 (Fig 1, A). To confirm this finding, we examined PARP14 expression in a population from Cincinnati Children's Hospital Medical Center through the use of high-throughput RNA sequencing.5Lu T.X. Sherrill J.D. Wen T. Plassard A.J. Besse J.A. Abonia J.P. et al.MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers.J Allergy Clin Immunol. 2012; 129: 1064-1075.e1069Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar Compared with the IU population, this population had more severe inflammation5Lu T.X. Sherrill J.D. Wen T. Plassard A.J. Besse J.A. Abonia J.P. et al.MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers.J Allergy Clin Immunol. 2012; 129: 1064-1075.e1069Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar (Table E1). Following analysis of the RNA-sequencing data, we observed similar (4.5-fold) increases in PARP14 expression as seen in the IU population (Fig 1, B). The chemokine CCL26 (Eotaxin-3) is a critical component of the pathology of EoE. CCL26 expression is dramatically increased in biopsies from patients with EoE, and single nucleotide polymorphisms in the CCL26 gene are associated with increased disease incidence.6Blanchard C. Wang N. Stringer K.F. Mishra A. Fulkerson P.C. Abonia J.P. et al.Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis.J Clin Invest. 2006; 116: 536-547Crossref PubMed Scopus (696) Google Scholar, 7Gupta S.K. Fitzgerald J.F. Kondratyuk T. HogenEsch H. Cytokine expression in normal and inflamed esophageal mucosa: a study into the pathogenesis of allergic eosinophilic esophagitis.J Pediatr Gastroenterol Nutr. 2006; 42: 22-26Crossref PubMed Scopus (151) Google Scholar Moreover, STAT6 regulates CCL26 in esophageal cells.8Lim E.J. Lu T.X. Blanchard C. Rothenberg M.E. Epigenetic regulation of the IL-13-induced human eotaxin-3 gene by CREB-binding protein-mediated histone 3 acetylation.J Biol Chem. 2011; 286: 13193-13204Crossref PubMed Scopus (35) Google Scholar To determine whether PARP14 expression correlated with CCL26 expression, we tested the association of expression of these 2 genes in esophageal biopsies from patients with EoE and observed a strong correlation coefficient (IU population: R = 0.81; P = .0002, Cincinnati Children's Hospital Medical Center population: R = 0.61, P = .03) (Fig 1, C). In contrast, there was no significant correlation between PARP1 and CCL26 expression (R = 0.30, P = .27). There is significant heterogeneity in the expression of PARP14 in the biopsy samples, with some overlap in the control biopsy samples (Fig 1, A). This heterogeneity was lessened in samples from patients with more severe inflammation (Fig 1, B). Thus, PARP14 expression is increased and CCL26 and PARP14 gene expression is correlated in 2 populations interrogated by 2 distinct methods. Because our results suggested a relationship between PARP14 and CCL26, we tested the ability of PARP14 to regulate CCL26 directly. The esophageal cell line TE-7 was transfected with a CCL26 luciferase reporter vector and plasmids encoding STAT6 and/or PARP14 before incubation for 24 hours in the presence or absence of the STAT6-activating cytokines IL-4 and IL-13. Consistent with previous results, transfection of STAT6-expressing plasmids increased CCL26 reporter activity (Fig 2, A). Moreover, transfection of PARP14 alone increased basal and cytokine-induced reporter activity (Fig 2, A). Importantly, cotransfection of STAT6 and PARP14 significantly increased CCL26 reporter activity over cells transfected with STAT6 alone (Fig 2, A). The effects of PARP14 expression were entirely dependent on STAT6 binding because they were not observed when the plasmid was cotransfected with a CCL26 reporter that had a mutation in the STAT6 binding site (Fig 2, A). These results suggested that PARP14 might be a viable target for modulating the expression of CCL26. To test this directly, TE-7 cells were incubated with IL-4 or IL-13 in the presence or absence of the PARP activity inhibitor PJ34 before expression of the endogenous CCL26 gene was assessed. We observed that IL-4 and IL-13 increased CCL26 mRNA and that incubation with the PARP inhibitor attenuated the induction in response to either cytokine (Fig 2, B). Similar results were observed in the TE-1 esophageal cell line. Thus, PARP14, and PARP activity, contribute to the regulation of CCL26 in esophageal cells. These results do not exclude the possibility that PARP14 is expressed by, and functions in, additional cell types that contribute to EoE. Although we are only beginning to understand the in vivo functions of PARP14, this report, coupled with our previous work,4Mehrotra P. Hollenbeck A. Riley J.P. Li F. Patel R.J. Akhtar N. et al.Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease.J Allergy Clin Immunol. 2013; 131 (e1-12): 521-531Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar suggests that PARP14 has a significant role in the development of allergic inflammation. It likely works in multiple cell types, including in T cells, where it results in increased TH2 and TH9 development,4Mehrotra P. Hollenbeck A. Riley J.P. Li F. Patel R.J. Akhtar N. et al.Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease.J Allergy Clin Immunol. 2013; 131 (e1-12): 521-531Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 9Goswami R. Jabeen R. Yagi R. Pham D. Zhu J. Goenka S. et al.STAT6-dependent regulation of Th9 development.J Immunol. 2012; 188: 968-975Crossref PubMed Scopus (168) Google Scholar and in target organ epithelial cells, enhancing the production of proallergic chemokines. Our results raise the possibility that targeting PARP14, or even PARP activity in general, might be an effective therapy for allergic diseases including EoE. We thank Pierre Hainaut and Jean-Yves Scoazec for supplying esophageal cell lines and the CCL26 reporter plasmid. MethodsGene expressionRNA was isolated from the esophageal biopsies (IU population), and gene expression was assessed for the indicated genes by using quantitative PCR. The mRNA expression of CCL26 was determined by using the ΔCt method. RNA isolated from the Cincinnati Children's Hospital Medical Center population was sequenced at the Cincinnati Children's Hospital Medical Center Gene Discovery and Genetic Variation Core as previously described.5Lu T.X. Sherrill J.D. Wen T. Plassard A.J. Besse J.A. Abonia J.P. et al.MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers.J Allergy Clin Immunol. 2012; 129: 1064-1075.e1069Abstract Full Text Full Text PDF PubMed Scopus (125) Google ScholarTE-7 esophageal epithelial cells were incubated in the presence or absence of IL-4 (2.5 ng/mL) or IL-13 (20 ng/mL), with or without the PARP inhibitor PJ34 (25 μM). CCL26 mRNA expression was assessed by using quantitative PCR.Luciferase assayTable E1Patient demographicsIU populationCCHMC populationControlEoEControlEoENo.1716610Age (y), mean (range)8.5 (1.5-17.3)10.7 (2.2-17.2)12.72 (1.6-17.1)12.7 (2.9-34.8)Eosinophils/hpf046.4 ± 7.60164 ± 29% Male50535060% on PPI23.56.333.350% on H2 blocker11.8000% on corticosteroid025020% Dicyclomine5.96.3NANA% Metoclopromide5.90NANA% PPI and H2 blocker5.9000% PPI and corticosteroid00010% PPI and dicyclomine5.90NANA% PPI and metoclopromide5.90NANA% H2 blocker and metoclopromide5.90NANACCHMC, Cincinnati Children's Hospital Medical Center; NA, data not available because medications in this treatment group were not tracked; PPI, proton pump inhibitor. Open table in a new tab Gene expressionRNA was isolated from the esophageal biopsies (IU population), and gene expression was assessed for the indicated genes by using quantitative PCR. The mRNA expression of CCL26 was determined by using the ΔCt method. RNA isolated from the Cincinnati Children's Hospital Medical Center population was sequenced at the Cincinnati Children's Hospital Medical Center Gene Discovery and Genetic Variation Core as previously described.5Lu T.X. Sherrill J.D. Wen T. Plassard A.J. Besse J.A. Abonia J.P. et al.MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers.J Allergy Clin Immunol. 2012; 129: 1064-1075.e1069Abstract Full Text Full Text PDF PubMed Scopus (125) Google ScholarTE-7 esophageal epithelial cells were incubated in the presence or absence of IL-4 (2.5 ng/mL) or IL-13 (20 ng/mL), with or without the PARP inhibitor PJ34 (25 μM). CCL26 mRNA expression was assessed by using quantitative PCR. RNA was isolated from the esophageal biopsies (IU population), and gene expression was assessed for the indicated genes by using quantitative PCR. The mRNA expression of CCL26 was determined by using the ΔCt method. RNA isolated from the Cincinnati Children's Hospital Medical Center population was sequenced at the Cincinnati Children's Hospital Medical Center Gene Discovery and Genetic Variation Core as previously described.5Lu T.X. Sherrill J.D. Wen T. Plassard A.J. Besse J.A. Abonia J.P. et al.MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers.J Allergy Clin Immunol. 2012; 129: 1064-1075.e1069Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar TE-7 esophageal epithelial cells were incubated in the presence or absence of IL-4 (2.5 ng/mL) or IL-13 (20 ng/mL), with or without the PARP inhibitor PJ34 (25 μM). CCL26 mRNA expression was assessed by using quantitative PCR. Luciferase assayTable E1Patient demographicsIU populationCCHMC populationControlEoEControlEoENo.1716610Age (y), mean (range)8.5 (1.5-17.3)10.7 (2.2-17.2)12.72 (1.6-17.1)12.7 (2.9-34.8)Eosinophils/hpf046.4 ± 7.60164 ± 29% Male50535060% on PPI23.56.333.350% on H2 blocker11.8000% on corticosteroid025020% Dicyclomine5.96.3NANA% Metoclopromide5.90NANA% PPI and H2 blocker5.9000% PPI and corticosteroid00010% PPI and dicyclomine5.90NANA% PPI and metoclopromide5.90NANA% H2 blocker and metoclopromide5.90NANACCHMC, Cincinnati Children's Hospital Medical Center; NA, data not available because medications in this treatment group were not tracked; PPI, proton pump inhibitor. Open table in a new tab CCHMC, Cincinnati Children's Hospital Medical Center; NA, data not available because medications in this treatment group were not tracked; PPI, proton pump inhibitor.