Regulation of esophageal epithelial function in Eosinophilic Esophagitis
A dissertation submitted to the Graduate School of the University of Cincinnati
In partial fulfillment of the requirement of the degree of
DOCTOR OF PHILOSOPHY
In the Department of Pharmacology & Systems Physiology of the College of Medicine
2018
By
Chang Zeng B.S. Sun Yat-sen University, 2012
Committee Chair: Anjaparavanda P. Naren, Ph.D. Mentor: Simon P. Hogan, Ph.D.
Abstract
Eosinophilic Esophagitis (EoE) is an allergic inflammatory disorder with increasing prevalence in the western world. Patients with EoE demonstrate symptoms including vomiting, dysphagia and food impaction which decreased the quality of life.
One of the histopathological features of EoE is esophageal tissue remodeling, including dilated intercellular spaces (DIS) and basal zone hyperplasia (BZH). However, the underlying molecular s that drive these features is largely unknown. Here, we investigate the 1) involvement of sodium‐hydrogen exchanger 3 (NHE3) in esophageal epithelium remodeling and 2) the role of the transcription factors, signal transducer and activator of transcription (STAT), in the regulation of gene networks that control esophageal epithelial proliferation and histopathological features of EoE.
By analyzing RNA sequencing comparing transcriptome difference in esophageal biopsies from normal control (NL) and EoE patients, we identified NHE3 as the most upregulated transmembrane transporters in patients with active EoE. We found that the expression pattern of NHE3 closely correlated with the disease severity and DIS. Functional analyses demonstrated that NHE3 activity is upregulated in IL‐13 treated primary esophageal epithelial cells derived from EoE patients, as well as in IL‐13‐induced stratified squamous epithelium generated by the air‐liquid interface (EPC2‐ALI). Pharmacological
Inhibition of NHE3 activity protected from IL‐13 induced DIS in esophageal epithelium. Thus, we concluded that NHE3 plays a functional role in DIS formation and pharmacologic interventions targeting
SLC9A3 function may suppress the histopathologic manifestations in EoE
IL‐13 has previously been shown to activate STAT proteins, particularly STAT3 and STAT6 and regulate the transcriptome changes in EoE patients. Using transcription factor binding site (TFBS) analysis, we identified STAT protein binding motif is one of the most enriched transcription factor binding site (TFBS) in the dysregulated genes in both EoE biopsies and IL‐13 treated EPC2‐ALI cultures. In particular, we
ii identified the STAT3 binding site as the most enriched TFBS in IL‐13 induced upregulated genes in EPC‐
ALI. By knocking down STAT3 in EPC2 cells, we revealed a role for STAT3 in the regulation of epithelium barrier function and IL‐13‐induced proliferation. In contrast, we show that STAT6 plays a pro‐ inflammatory role regulating cytokine and chemokine production in esophageal epithelium.
Together, we identified several molecular targets in the esophageal epithelium that are important in modulating the esophageal epithelium remodeling in EoE. This dissertation, for the first time, uncovers the role of transmembrane transporters in the pathogenesis of EoE. Also, we provide valuable information on the two distinct divergent pathways regulated by STAT3 and STAT6 driving IL‐13‐ mediated transcriptome changes in EoE. Further investigations into the contribution of these mechanisms in the clinical manifestations of EoE will facilitate the evolvement of new therapeutic approaches in EoE treatment.
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Acknowledgements
First, I would like to express my utmost gratitude to my mentor Dr. Simon Hogan. I had a tough beginning for my graduate study, but it all paid off when I ended up joined Simon’s laboratory. Simon is an excellent mentor. He showed me his enthusiasm and passion for science, guided me of how to do science correctly and efficiently, and told me to explore all the possibility of life in the future. These are all valuable assets and shaped me into who I am right now.
Next, I would like to thank my thesis committee members. All of my committee members, Drs.
Anjaparavanda Naren, Hong‐Sheng Wang, Gary Shull, Marshall Montrose and Robert Rapoport have provided me invaluable support and advice, which are essential for my completion of the dissertation.
Primarily, I want to thank Dr. Naren for the guidance on experimental techniques and preparedness to assist with completion of my thesis dissertation. Also, I am grateful to Dr. Rapoport for being supportive not only as my committee member but also my graduate program director.
I would like to offer my special thanks to Dr. Ronald Millard. He helped and guided me to get through the most terrible time of my graduate study. I would not be able to join Simon’s lab and achieve what I have made now without him. I would also like to thank Nancy for her endless help during both my graduate program transition and process towards graduation.
During the past four years, I have met amazing people in Hogan Lab, and I would like to express my thankfulness individually. Taeko, thank you for sharing your experiences and stupid videos with me all the time, I always enjoyed the time we spent together. Simone, thank you for your guidance on experiments and your company to different places in Cincinnati. It is sort of sad that we are not going to drink KSFM when I graduate. Amna, we have been together to go through both foundation and numerous dreadful lab meetings, I think this would be enough to keep our friendship for a long time.
Lisa, thank you for always being like a mom and take care of me, I wish I could bring you more exotic
v food to try. Now I’m starting to run out of spaces, but I still want to thank Jazib, Yanfen, Sunil, Varsha,
Andy, Justin, Heather, Nianrong, David, Ania, and Marjan. We have shared amazing memories together, and thank you all for your friendship.
I would also like to thank the great people in Division of Allergy and Immunology at Cincinnati Children’s
Hospital Medical Center who offered me help and friendship during my training as a graduate student.
Special thanks to Julie, Nurit and Mark Rochman, who have given me useful advice on both scientific problems and career development.
Finally, I would like to thank my mom Zhi, my aunt Tun, my boyfriend Yongkun, my friend Yu and
Jiuzhou, and all my other friends and families for their unconditional support and love.
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Table of Contents
Regulation of esophageal epithelial function in Eosinophilic Esophagitis ...... i
Abstract ...... ii
Acknowledgements ...... v
Table of Contents ...... vii
List of Abbreviations ...... xi
List of Figures and Tables ...... xiii
1 Chapter I: Introduction ...... 15
1.1 Eosinophilic Esophagitis ...... 15
1.1.1 Introduction ...... 15
1.1.2 Pathogenesis of EoE ...... 15
1.1.3 Therapy ...... 25
1.1.4 Summary ...... 28
1.2 Interleukin‐13 (IL‐13) ...... 29
1.2.1 Introduction ...... 29
1.2.2 Production of IL‐13 ...... 29
1.2.3 IL‐13 signaling pathway ...... 29
1.2.4 IL‐13 function under physiological and pathological conditions ...... 32
1.2.5 IL‐13 in EoE ...... 33
1.2.6 IL‐13‐related treatment ...... 34
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1.2.7 Summary ...... 34
1.3 Ion transporters ...... 36
1.3.1 Introduction of Ion transporters ...... 36
1.3.2 Ion transporters in the esophagus ...... 37
1.3.3 Summary ...... 38
1.4 Sodium hydrogen exchangers (NHEs) ...... 39
1.4.1 Introduction ...... 39
1.4.2 NHE3 ...... 39
1.4.3 Summary ...... 41
1.5 Summary ...... 42
2 Chapter II: SLC9A3/NHE3 dysregulation and dilated intercellular spaces in eosinophilic esophagitis
43
2.1 Copyright and Student Contribution ...... 45
2.2 Abstract ...... 45
2.3 Introduction ...... 47
2.4 Material and Methods ...... 50
2.5 Results ...... 58
2.5.1 Transmembrane transporter SLC9A3/NHE3 specifically upregulated and correlated with
eosinophil count and DIS in EoE ...... 58
2.5.2 Increased NHE3 function in IL‐13–stimulated primary esophageal epithelial cells...... 59
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2.5.3 IL‐13 induces an EoE‐like transcriptome including increased transmembrane transporter
activity and SLC9A3 overexpression in an in vitro, matured esophageal epithelium model system. 60
2.5.4 IL‐13–induced NHE3 expression and function in differentiated esophageal epithelial cells.
61
2.5.5 Increased SLC9A3 expression and activity is linked with DIS formation...... 62
2.6 Discussion ...... 64
2.7 Figures ...... 69
3 Chapter III: IL‐13 activated STAT3 and STAT6 pathways in Eosinophilic Esophagitis ...... 104
3.1 Copyright and Student Contribution ...... 105
3.2 Abstract ...... 105
3.3 Introduction ...... 107
3.4 Materials and methods ...... 110
3.5 Results ...... 115
3.5.1 STAT binding site is one of the most enriched TFBS in EoE dysregulated genes ...... 115
3.5.2 STAT binding site is the most enriched in both dysregulated genes and transcriptionally
active genes following 4 hours incubation with IL‐13 in EPC2‐ALI ...... 115
3.5.3 STAT3 is the most enriched TFBS in IL‐13‐induced upregulated genes...... 116
3.5.4 STAT3 and STAT3‐related genes regulate barrier integrity of the esophageal epithelium
117
3.5.5 IL‐13 induced STAT3‐dependent genes are involved in IL‐13 induced cell proliferation . 118
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3.5.6 IL‐13 induced STAT6‐dependent genes are involved in cytokine production but not IL‐13
induced cell proliferation ...... 119
3.6 Discussion ...... 121
3.7 Figures ...... 125
4 Chapter IV: General Discussion and Summary ...... 144
4.1 Alteration of transmembrane transporters expression in EoE ...... 145
4.2 SLC9A3/NHE3 dysregulation in esophageal epithelium ...... 149
4.3 STAT involvement in EoE transcriptome ...... 153
4.4 STAT3 regulates tissue remodeling in esophageal epithelium ...... 155
4.5 STAT6 regulates cytokine production in esophageal epithelium ...... 157
4.6 Summary ...... 158
4.7 Figures ...... 160
5 References ...... 166
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List of Abbreviations
Abbreviation Full Name AD‐HIES Autosomal‐dominant hyper‐IgE syndrome ALI air‐liquid interface AR allergic rhinitis BE Barrett’s esophagus BrdU 5‐Bromo‐2’‐deoxyuridine BZH basal zone hyperplasia CAPN14 Calpain 14 CCL26 Chemokine ligand 26 CRAC calcium release‐activated calcium CRLF2 Cytokine receptor‐like factor 2 DIS dilated intercellular spaces DSG1 Desmoglein‐1 EA Esophageal adenocarcinoma ECP eosinophil cationic protein EDN Eosinophil‐derived neurotoxin EDP EoE diagnostic panel EMT epithelial‐mesenchymal transition ENaC epithelium sodium channel EoE Eosinophilic Esophagitis ERK Extracellular signal‐regulated kinase FLG Filaggrin GERD gastroesophageal reflux disease GO gene ontology GWAS genome‐wide association study H&E hematoxylin and eosin H3K4me3 trimethylation of lysine 4 on histone H3 IF immunofluorescence ILC2 Group 2 Innate lymphoid cells iNKT invariant natural killer T cell JAK Janus kinase KD knockdown KLK kallikrein LRRC31 leucine‐rich repeat‐containing protein 31 MBP major basic protein NHE sodium‐hydrogen exchanger NL Normal healthy control pHi intracellular pH PPI Proton pump inhibitor proton pump inhibitor‐ responsive eosinophilic PPI‐REE esophagitis
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RNAseq RNA sequencing SFED Six‐food elimination diet SLC Solute carrier family SNP single nucleotide polymorphisms STAT Signal transduction and activator of transcription TEER Trans‐epithelial electrical resistance TFBS transcription factors binding site TGFB1 Transforming growth factor, beta 1 TSLP Thymic stromal lymphopoietin
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List of Figures and Tables
Figure 2‐1. SLC9A3 is the most upregulated transmembrane transporter activity gene in the EoE transcriptome, and levels correlate with EoE severity and DIS ...... 69
Figure 2‐2. Increased SLC9A3/NHE3 expression and activity in primary esophageal epithelial cells derived from EoE biopsy in response to IL‐13...... 72
Figure 2‐3. Mature stratified squamous esophageal epithelium model using EPC‐ALI culture system. .... 74
Figure 2‐4. Increased SLC9A3/NHE3 expression and activity in IL‐13–stimulated EPC2‐ALI...... 76
Figure 2‐5. Blockade of NHE3 protected EPC2‐ALI from IL‐13–induced dilated intercellular spaces (DIS).
...... 78
Figure 2‐6. Demographics of patient cohorts examined in RNAseq analysis or qPCR...... 80
Figure 2‐7. GO analysis of dysregulated genes in EoE patients ...... 82
Figure 2‐8. IL‐13–induced proliferation in EPC2‐ALI is attenuated by NHE3 inhibitor EIPA...... 84
Table 2‐1. Expression level of 572 genes dysregulated in mature EPC2‐ALI by IL‐13...... 86
Table 2‐2. GO analysis of IL‐13–induced dysregulated genes in mature EPC2‐ALI...... 101
Figure 3‐1. STAT binding site is one of the most enriched TFBS in EoE dysregulated genes ...... 125
Figure 3‐2. STAT binding site is the most enriched in both dysregulated genes and transcriptionally active genes following 4hr incubation with IL‐13 in EPC2‐ALI ...... 128
Figure 3‐3. STAT3 is the most upregulated STAT protein in 4hr IL‐13 treated EPC2‐ALI. And STAT3 binding site is the most enriched TFBS in IL‐13‐induced upregulated genes...... 130
Figure 3‐4. STAT3 and STAT3‐related genes regulate barrier integrity of esophageal epithelium ...... 132
Figure 3‐5. IL‐13 induced STAT3‐dependent genes are involved in IL‐13 induced cell proliferation ...... 135
Figure 3‐6. IL‐13 induced STAT6‐dependent genes are involved in cytokine production but not IL‐13 induced cell proliferation ...... 138
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Figure 3‐7. Proposed mechanism of IL‐13 driven STAT3 and STAT6 dependent transcriptome alteration in esophageal epithelium ...... 142
Figure 4‐1. Acid protection mechanism in esophagus ...... 160
Figure 4‐2. JAK/STAT signaling pathway ...... 162
Figure 4‐3. Proposed Mechanism of IL‐13 induced NHE3‐dependent DIS formation in EoE ...... 164
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1 Chapter I: Introduction
1.1 Eosinophilic Esophagitis
1.1.1 Introduction
In the past 20 years, Eosinophilic Esophagitis (EoE) has been increasing in prevalence worldwide. Its prevalence reached 0.5‐1 in 1000 in 20141, which is similar to diseases including the inflammatory bowel diseases, Ulcerative colitis and Crohn’s Disease. Observed in both pediatrics and adults, EoE is characterized by esophageal eosinophilia, leading to symptoms such as dysphagia, food impaction, and abdominal pain2. In 2015, patients with EoE cost an estimated $1.4 billion per year in United States3.
However, with such high health‐care cost, the current therapeutic options for EoE is still not optimal.
EoE patients who are on a food elimination diet still suffered from the low quality of life, with chances of disease remission. As a result, better therapeutic strategies need to be developed. The following section will focus on the current knowledge of the pathogenesis of EoE and the available therapeutic options.
1.1.2 Pathogenesis of EoE
1.1.2.1 Food allergens
Food is considered as one of the major triggers for EoE4. Different than usual food allergy, EoE patients are usually hypersensitized with multiple food allergens5,6. However, a case study on 30 children identified close association with food allergies with EoE occurrences7. Also, direct food allergen extract injection in the esophagus in EoE patients caused acute EoE symptoms including luminal obstruction and mucosal blanching8. The causative role of food allergens in EoE is further supported by the success of food elimination diet in the treatment of EoE9. Multiple clinical studies showed that elimination of positive foods identified in skin prick or patch testing led to resolution or partial improvement of
15 symptom in confirmed in EoE patients while introducing these foods back to diet caused the re‐ emergence of esophageal eosinophilia10‐13.
1.1.2.2 Environmental factors
Multiple clinical investigations have reported that a high percentage of EoE patients are sensitized to both food allergens and aeroallergens5,14, suggesting the involvement of environmental factors like aeroallergen as risk factors to induce EoE. To further support for this argument, a case study reported an
EoE patient hypersensitized to an aeroallergen but did not have food allergy15. In addition, three other patients reported the onset of EoE following exposure to a large volume of aeroallergen16, suggesting potential that environmental antigens can induce EoE. Consistent with this argument, repetitive challenge of mice with Aspergillus. Fumigatus intranasally17 or epicutaneously18 could prime pathological changes including eosinophils infiltration and epithelial hyperplasia in the esophagus, similar to the histological phenotype of EoE patients19.
Aside from aeroallergens, other environmental factors including insects, season and early life exposure have also been shown to correlate with the incidence rate with EoE. Furthermore, epidemiology studies have reported that EoE occurrence is related to geographical regions20 and climate zone20, indicating EoE prevalence being significantly higher in urban and suburban areas within the cold climate zone.
1.1.2.3 Genetic Predisposition
Although a non‐Mendelian pattern of heritance in EoE patients, epidemiology studies have reported a high occurrence of EoE in European descent, male individuals suggesting a potential genetic predisposition21,22. A clinical study investigating five families of EoE patients found that 40% of patient’s first‐generation offspring showed EoE or EoE‐like symptoms23. Furthermore, twin and family analysis within 914 families with genetic risk showed the heritability of EoE within first‐degree relative is as high as 72%, and the recurrence risk ratios are higher in male relatives24.
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One of the explanations for family inheritability of EoE is the genetic susceptibility. By performing genome‐wide association (GWAS) studies comparing EoE patients and healthy controls, genome associated single nucleotide polymorphisms (SNPs) have identified in several EoE‐related genes25.
Variants of both inflammatory‐related genes (Thymic stromal lymphopoietin (TSLP)26,27, Cytokine receptor‐like factor 2 (CRLF2)27, Chemokine ligand 26 (CCL26)28 and Transforming growth factor, beta 1
(TGFB1)29) and barrier‐forming gene (Filaggrin (FLG)30 and Calpain 14 (CAPN14)31) are found genome‐ wide associated with EoE patient cohorts. Other studies such as Phenome‐wide association study identified more SNPs in EoE patients32. However, the involvement of these SNPs in EoE need further confirmation32.
1.1.2.4 Immunological pathways
Although being induced by food allergens, different from most common food allergic disorders, EoE is a
Type‐2 T‐cell‐mediated, eosinophils‐predominant immunological disorder33. Also, one crucial histological observation in EoE is more than 15 eosinophils infiltration per high power field (Eos/HPF) in esophageal biopsies, suggesting the contribution of immunological responses in the pathogenesis of EoE34.
Immunoglobulin
Different than 80% of food allergic disorders, EoE is considered as a non‐IgE‐mediated hypersensitivity35.
Experimental studies in mouse model systems have revealed that allergen‐induced EoE phenotypes such as esophageal eosinophilia do not require B cells36 and IgE37. Also, a clinical study reported no alteration of eosinophil counts on patients receiving anti‐IgE therapy, while discovered increased serum level of
IgG4 against food allergen38. Furthermore, a recent study found increased IgG4 plasma cell density in pediatric EoE patients’ esophageal biopsies compared to normal healthy control39. Together with the finding that food‐specific IgG4 is decreased in EoE patients after receiving diet elimination therapy40,
IgG4 is considered associated with the development of EoE.
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Immune cells
Eosinophils
Under physiological condition, eosinophils are absent in esophagus. While in EoE patients, eosinophils present in both the proximal and distal esophagus41 and often associated with morphologic abnormalities42. Activation of eosinophils leads to the release of granule contents including major basic protein (MBP)， Eosinophil‐derived neurotoxin (EDN), and eosinophil cationic protein (ECP), which have proinflammatory and cytotoxic effects43. Increased EDN deposition is observed in adult EoE patients44, and its activation effect on dendritic cells may lead to the production of immunological mediators45.
Also, activated eosinophils can generate inflammatory cytokines (including IL‐5, IL‐13, TGF‐beta 1)43, which modulate the polarization, recruitment, and homeostasis of lymphoid cells like T cells46. Also, eosinophils could act as antigen presenting cells and induce antigen‐specific T cell proliferation47.
Overall, increased intraepithelial eosinophils could regulate both innate and adaptive immune responses in the esophagus, thus directly affect pathological development in EoE.
T Cells (T helper cells, T regulatory cells)
T cell involvement in EoE is derived from the observation of increased T cell number and Th2 cytokine secretion in patients’ esophageal epithelium48,49. It is further supported by T‐cell deficient murine model, which was protected from EoE phenotype, while B‐cell deficient mice developed EoE as wild‐type36.
More specifically, CD8‐deficient mice are not protected from the induction of experimental EoE while
CD4‐deficient mice showed less EoE phenotype36, indicating T helper cell involvement rather than cytotoxic T cell. A clinical evaluation comparing nonatopic children with pediatric EoE patients showed increased IL‐5 expressing CD4+ T cells, but not IFN‐γ CD4+ T cells, indicating a skewed immunological pathway toward Th2 immunity50. These increased Th2 cells in EoE patients then led to increased secretion of Th2 cytokines including IL‐5 and IL‐1351. IL‐5 regulates genes that are essential for
18 eosinophil growth, survival and effectors functions52, which is critical for the accumulation of intraepithelial eosinophils53 in the esophagus. IL‐13 contributes to increased expression of Eotaxin‐328, which is a chemokine that is important for eosinophils activation and recruitment, and alteration of esophageal epithelium transcriptome54.
Regulatory T cells (Treg) are a subset of T cells that negatively regulate immune responses55. Previous studies indicated a protective role of Treg in allergic disorders56. Decreased Treg numbers have been observed in adult EoE patients57. Also, in an experimental murine model of EoE, Treg number were reported to be decreased in mice following allergen challenge58, suggesting a negative regulation of anti‐ inflammatory effect in EoE. However, increased Treg numbers were reported in pediatric EoE patients59,60. It is speculated that these Tregs in EoE patients are defective33. Also, previous study indicated Th2 cell sensitivity in responding to Treg regulatory effect in allergic patients61, which might explain the reason of increased Tregs in pediatric EoE. However, further studies to compare the difference of Tregs in pediatric and adult EoE patients are needed to characterize the role of Treg in EoE better.
Basophils
Basophils, as the least abundant granulocyte in peripheral blood, have been reported deeply involved in allergic disorders such as atopic dermatitis62, acute hypersensitivity63, and asthma64. In EoE patients, basophil numbers were increased in PPI‐non‐responsive EoE patients37,65 and positively correlated with severity of eosinophilia in esophageal biopsies37. Patients who underwent steroid treatment and showed relief of symptoms showed reduced basophil counts65. Basophils are known to generate Th2 cytokines including IL‐4 and IL‐13, and basophil‐secreted IL‐4 is essential for T cell differentiation into
Th2 cells66. Studies using experimental EoE mice model further validate the importance of basophils in
Type‐2 inflammatory responses, as mice depleted of basophils showed decreased eosinophil population
19 and Th2‐cytokine skewed transcriptome compared to wild‐type mice37,67. Together, basophils may serve as a therapeutic target for EoE based on its effect on the development and progression of Th2 inflammatory responses68.
Mast cells
Although EoE is an IgE‐independent allergic disorder, mast cells are still deeply involved in the immunological pathways of EoE69. Mast cell number and degranulation are significantly increased in esophageal mucosa in both EoE patients70 and experimental EoE murine models71 and could be reversed by steroid treatment69 and food elimination therapy72. Human mast cells are shown to produce IL‐13, which is an important cytokine for EoE pathogenesis, upon high‐affinity IgE‐crosslinking73. Mast cells could also form “allergic effector unit” together with eosinophils, which resulted in augment of activity of both cells, increase expression of soluble mediators like TNF‐α and promote allergic responses74,75. By comparing mast cell‐deficient and mast cell‐reconstituted mice, a recent study indicated that mast cells contribute to the pathogenesis of allergen‐induced EoE in mice by promoting smooth muscle hyperplasia and hypertrophy71. It is also suggested that the mast cells contribute to the pathogenesis of
EoE by influencing KIT ligand expression69, secreting cytokine that leads to increasing esophageal contractility76.
ILC2s
In EoE patients, Group 2 Innate lymphoid cells (ILC2s) percentage is much higher than normal healthy control and is correlated with the level of esophageal eosinophilia77. ILC2s are linage‐negative cells that produce Type‐2 inflammatory cytokines and contribute to human allergic diseases78. Activated by epithelial secreted cytokines such as IL‐33 and TSLP79,80, ILC2 cells are considered to play a pathogenic role in EoE81 due to its ability to secrete type 2 cytokines IL‐5 and IL‐1382, which are previous characterized contribute to the progression of EoE.
20 iNKT Cells
Another cellular source for Th2 cytokines is invariant natural killer T cells (iNKTs). In pediatric EoE patients, increased iNKTs are observed in esophageal biopsies, while decreased in peripheral blood83.
CCL5 is a member of C‐C chemokine family, and previous studies showed that CCL5 important for eosinophil recruitment and activation84,85. Upon stimulation of food allergen, iNKTs isolated from EoE biopsies are activated and producing higher CCL5 compared to normal healthy control83. Consistent with other cells that contribute to EoE, patients received elimination diet therapy showed reduction of iNKTs in esophageal epithelium86. Moreover, the failure of inducing EoE phenotype in iNKT cell‐deficient mice further support the crucial effect of iNKTs in EoE pathogenesis87.
Cytokines
A variety of cytokines are elevated in EoE patients, especially Th2 cytokines including IL‐4, IL‐5, and IL‐
1349. In this section, we will focus on other cytokines, and discuss IL‐13 in the next section.
IL‐4
IL‐4 is known to activate naïve T cells and promote its proliferation and differentiation into Th2 cells88. In
EoE patients, the blood level of IL‐4 is significantly higher than healthy controls51. However, multiple studies showed no significant increase of the mRNA level of IL‐4 in biopsy samples from EoE patients51,54.
These different observations might be explained by the allergic status of EoE patients recruited. A study comparing EoE patients with or without allergic symptoms showed the increased IL‐4 level is attributed to the presence of allergy but not EoE51. Given the critical role of IL‐4 in CD4+ Th2 differentiation, it could mainly act to induce naïve T cells differentiation in EoE as in other allergic disorders89.
IL‐5
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Similar to IL‐4, IL‐5 levels are increased in EoE patients90 and significantly higher in EoE patients with allergic symptoms51. One of the main contributions of IL‐5 in the pathogenesis of EoE is its function of recruiting eosinophils into the esophageal epithelium, which is partially dependent on eotaxin expression53. Also, recent generated transgenic mice with specific epithelial IL‐5 overexpression successfully resembled an EoE phenotype that is not restricted to eosinophilia91, but also tissue remodeling. Comparison between T cell‐specific IL‐5 transgenic mice and eosinophil linage‐deficient mice suggested that IL‐5 induced eosinophilia is important for tissue remodeling including collagen deposition in lamina propria, basal layer thickening in epithelium90, while IL‐5 induces esophageal dysmotility92 is independent of eosinophilic inflammation in the esophagus. However, although clinical trial testing Anti‐IL‐5 antibody (mepolizumab) showed a significant decrease of esophageal eosinophilia, minimal symptomatic remission was observed in EoE patients93. Taken together, IL‐5 drives eosinophil recruitment and induces esophageal epithelium remodeling in EoE, but both IL‐5 and eosinophils are not the only key driver of the disease.
IL‐33
IL‐33 is a newly discovered cytokine that belongs to IL‐1 cytokine superfamily. It could be secreted by stromal cells like endothelial and epithelial cells94, as well as immune cells like macrophages95. After binding to its receptor ST2 (IL‐1RL1), IL‐33 could either act as a traditional cytokine that mediates intracellular signaling pathways like NF‐κB or serves as a nuclear factor that mediates transcription of downstream genes96. ST2 is ubiquitously expressed in all human tissue97 and was upregulated after allergen challenge98. The activation of the IL‐33/ST2 axis is known to induce Th2 cytokines secretion including IL‐4, IL‐5, and IL‐13, which are critical cytokines in EoE pathogenesis99.
Not surprisingly, the expression level of IL‐33 and ST2, are upregulated in pediatric EoE patients67,100, and
IL‐33 is primarily located in CD45‐ cells, mast cell, and epithelial cells101. Experimental murine EoE model
22 also showed elevated IL‐33 and ST2 level67, which is critical for basophil‐induced esophageal inflammation. Mice treated with rIL‐33 for a week developed early stage phenotype of EoE and showed elevated Th2 inflammatory response101. IL‐33‐deficient and ST2 deficient mice showed decreased eosinophil counts in peripheral blood compared to wild‐type mice, indicating the role of IL‐33 in the early development of eosinophils102.
Thymic stromal lymphopoietin (TSLP)
TSLP is an IL‐7 like cytokine that functions through binding to its receptor TSLPR. It is expressed by epithelial cells and could prime dendritic cells to differentiate naïve T cells into Th2 cells103. It is also showed to target NKT cells, mast cells and basophils, thus contribute to the development of various allergic disorders104,105. In EoE patients, TSLP is upregulated and primarily expressed in the suprabasal layer of esophageal epithelium106, but not elevated in serum107. The level of TSLP is dependent on NF‐kB‐ inducing kinase (NIK), as NIK knockout model which mimics EoE phenotype showed elevated TSLP level108. Interestingly, TSLP polymorphism was identified in EoE patients109 and a recent study linked this risk polymorphism with food allergen susceptibility110 and increased basophil activation in EoE37. Also, employing experimental murine model investigators demonstrate that inhibition of TSLP and basophil interaction was protective against EoE‐phenotype37, suggesting the importance of TSLP‐basophil axis in
EoE. Furthermore, TSLP is shown to induce the formation of eosinophil extracellular traps (EET)105. EET is a protective mechanism against bacteria and pathogens, a recent study indicated a possible contribution of increased EET numbers and impaired epithelial barrier in the esophagus in EoE111.
Tumor Growth Factor‐beta 1 (TGF‐β1)
TGF‐β1 secreted by eosinophils, mast cells, and epithelial cells can induce extracellular matrix secretion of fibronectin and collagen I in primary fibroblasts derived from biopsies from EoE patients112. TGF‐β1 is thought to promote subepithelial fibrosis and vascularity through phosphorylation of SMAD2/3113 and
23 p38 mitogen‐activated protein kinase (MAPK)112. TGF‐β1 is also thought to promote epithelial‐ mesenchymal transition (EMT) which contributes to fibrosis often observed in the EoE esophagus114. The lamina propria fibrosis occurring in both pediatric115 and adult116 EoE patients is thought to contribute to strictures and esophagus narrowing117.
1.1.2.5 Epithelium Involvement
Normal esophageal epithelium is composed of non‐keratinized stratified squamous epithelial cells.
Under physiological conditions, esophageal epithelial cells form a functional barrier and protect the esophagus from injury118. Under allergic conditions such as EoE, epithelium formed barrier is disrupted by protease‐active allergens or altered intrinsic proteases119, lead to antigen penetration and initiate inflammatory responses through antigen presenting cells120. This disruption of epithelium also leads to activation of the immune response of esophageal epithelial cells. Esophageal epithelial cells are capable of producing inflammatory cytokines and chemokines including CCL5, CCL26, TSLP and IL‐33 upon allergen challenges28,101,121. These molecules are important in mediating pathogenesis of EoE, which are discussed in later sections.
Transcriptome analysis comparing esophageal biopsies from EoE patients and normal healthy control using RNAseq showed 1607 dysregulated genes122 that might have related to EoE pathogenesis. Gene cluster and ontology analysis indicated that immune response and immune effector process are the two most enriched gene ontology (GO) nodes within these dysregulated genes122. Consistent with this analysis, multiple studies showed the inflammatory contribution by the esophageal epithelium123. The expression level of eotaxin‐3 is 53‐fold higher in EoE patients compared to normal health control28.
Encoded by CCL26 gene, Eotaxin‐3 is a chemokine secreted by esophageal epithelium upon the activation of IL‐1354 The local secretion of CCL26 is known to promote the recruitment of eosinophils124,125, which is a hallmark characteristic of EoE. Mice deficient of the eotaxin‐3 receptor
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(CCR3) are protected from aeroallergen induced eosinophilia in esophagus28, implicating the importance of CCR3/CCL26 axis in esophageal eosinophilia. Other immune response‐related genes, tumor necrosis factor alpha‐induced protein 6 (TNFAIP6) and arachidonate 15‐lipoxygenase (ALOX15), are also found highly expressed in the esophageal epithelium in EoE biopsies. The previous study in cancer research suggested a potential role of ALOX15 in suppressing inflammation126. Meanwhile, TNFAIP6 was also thought to play a protective role in inflammation in rheumatoid arthritis127. It is possible that they counteract with other immune responses in esophageal epithelium. However, their actual mechanism in inducing EoE phenotype are still unclear128.
Another group of genes that are significantly altered are epithelial barrier related genes. Several genes encode for junctional proteins, adhesion molecules and structural barrier proteins are downregulated in
EoE patients129. Two major components of the epithelial barrier, E‐cadherin and claudin‐1 are decreased in expression in EoE patients, while occludin is increased123. Interestingly, occludin is showed to regulate cytokine‐induced barrier permeability in epithelial cells130. Other molecules related to epithelium barrier functions such as filaggrin (FLG)128,131,132 and Desmoglein‐1 (DSG1)133 are also found decreased in expression in EoE patients. The downregulation of these barrier related genes contributes to the loss of barrier integrity and epithelium remodeling133,134. These alterations in epithelium may then lead to increased penetration of antigens in esophageal mucosa120, and lead to further immunological responses. However, either the epithelial barrier disruption in EoE is causative or consequential of immunological activation is still debatable.
1.1.3 Therapy
1.1.3.1 Steroids
Corticosteroids are the first‐line medical therapies in EoE patients135. Administration of systemic corticosteroid therapy to 20 EoE patients Induced significant remission of esophageal eosinophilia and
25 improvement of clinical symptoms in 19 out 20 patients 136. The most common steroids used to treat
EoE are fluticasone and budesonide137, all of which are topical administered steroids. A clinical study investigating the long‐term effect of steroids demonstrated that swallowed fluticasone is effective in maintaining histological, endoscopic and symptomatic improvement in pediatric EoE patients over two years, without significant adverse effect138. In the meantime, double‐blind, placebo‐controlled clinical trial suggested the effectiveness of budesonide in treating EoE in adolescent and adult EoE patients116.
The systemic steroids are now not recommended for EoE treatment, as it has no advantage on treatment efficacy compared to the topical steroid while producing systemic adverse effects such as hyperphagia and weight gain139. While topical steroids are supported by 11 randomized clinical trials for its effectiveness on histological remission in EoE140, its efficacy on resolving EoE symptoms is still debatable. A Recent clinical study showed EoE patients receiving fluticasone treatment had no significant improvement in dysphagia compared to patients receiving placebo, however also developed esophageal candidiasis141. This lack of symptomatic resolution may be related to the lack of mucosal contact time and the formulation of steroid137,142. The other caveat of this treatment is that the cessation of taking topical steroid will lead to the recurrence of disease143. However, current clinical trials showed that long‐term use of topical steroids is relatively safe and could maintain EoE in remission140.
1.1.3.2 Dietary Intervention
Food allergen or aeroallergen are used in the experimental murine model to induce EoE17, and most EoE patients showed hypersensitivity to various foods, different dietary antigen removal methods to treat
EoE are tested over last two decades.
Use dietary intervention to treat EoE was described in 1995. Ten patients with esophageal eosinophilia receiving protein‐free, amino based elemental diet and demonstrated remission of EoE symptoms144.
26
Since then numerous clinical trials have supported the effectiveness of this elemental diet and demonstrated significant improvements in both histological and symptomatic scores in EoE in all age groups145‐147. Also, elemental diet sometimes requires the insertion of nasogastric (NG) tube in pediatric and adolescent patients, which negatively impact patients’ quality of life148. Together, elemental diets are not recommended for long‐term therapy of EoE140 until other treatment options were proven ineffective.
Food elimination diet is an alternative to elemental diet for EoE therapy. Six‐food elimination diet (SFED) and food allergy testing‐directed diet are found effective to induce EoE remission149. Six foods, including dairy, wheat, egg, soy, nuts, and seafood, are known as the most common inducer for food allergy and esophageal mucosal injury10,12. A recent study indicated SFED is more cost‐efficient than steroids, combined with their similarity on efficacy, SFED might be a better option for treatment of EoE150. Food allergy testing‐directed diet is based on either skin prick tests (SPTs) or atopy patch tests (APT)10, which are based on IgE‐mediated skin response. Together with the relatively low specificity of these tests, testing‐based food elimination diet showed relatively less effectiveness compared to SFED147.
1.1.3.3 Proton Pump Inhibitor
Proton pump inhibitor (PPI) was first used as a diagnostic tool for EoE to distinguish the disease from gastroesophageal reflux disease (GERD), as earlier criteria for EoE is its unresponsiveness to PPI treatment151. In 2011, a clinical study in adults with high esophageal eosinophilia and EoE‐like symptoms reported that 50% of these patients had clinicopathologic remission152. This discovery, together with prior success cases using PPI to resolve EoE‐like symptoms153, define a new subtype of EoE, proton pump inhibitor‐ responsive eosinophilic esophagitis (PPI‐REE). PPI‐REE patients showed similar disease phenotypes immunological mechanisms and transcriptome154,155. Long‐term application of PPI in PPI‐REE showed sustained remission of EoE phenotype with no substantial side effect156. Analyses of 94 EoE
27 related genes based on EoE diagnostic panel (EDP), the patient cohort with PPI‐REE showed similar molecular overlap with patients with PPI‐unresponsive EoE155. Interestingly, comparing the transcriptional change before and after PPI‐treatment, PPI showed an effect on Th2‐inflammation‐ related gene expression157. The mechanism of how PPI influences inflammatory response is unclear. In vitro study using primary esophageal epithelial cells showed the inhibitory effect of PPI on IL‐13 induced,
STAT6‐dependent eotaxin‐3 expression158. However, this does not explain the different responses to PPI between PPI‐REE and EoE patients. Interestingly, a recent study showed that EoE patients showed remission of disease after elimination diet therapy also respond to PPI treatment159. Together with its clinically proved safety profile and easy oral administration, PPI is now used as first‐line anti‐ inflammatory therapy for EoE, although the underlying mechanism still yet to be elucidated140.
1.1.4 Summary
To summarize, EoE is a chronic, Th2‐inflammatory response‐mediated, allergic disorder in the esophagus with increasing prevalence. Multiple factors are involved in the pathogenesis of this disease, so targeting only one component always lead to the unsatisfied therapeutic outcome. Further understanding of the underlying molecular pathways that drive disease development will help to create more specific and efficient therapeutic options for EoE.
28
1.2 Interleukin‐13 (IL‐13)
1.2.1 Introduction
IL‐13 is a pleiotropic cytokine that was first described as a protein secreted abundantly by Th2 cells and influences growth and inflammatory pathways160. IL‐13 is 132 amino acids long protein that contains a four α‐helical bundle “up‐up‐down‐down”, which is characteristic of type‐I cytokine161. Structural studies have found 25% homology in amino acid sequences between IL‐4 and IL‐13 which is likely to contribute to the similar tertiary structure162. These shared homologies also explain the functional resemblance between these two cytokines. For instance, both IL‐4 and IL‐13 could signal through Type II IL4 receptor163.
1.2.2 Production of IL‐13
IL‐13 is secreted by activated Th2 cells, and later studies found additional sources of IL‐13. mRNA transcript analysis indicated that, under allergic conditions, IL‐13 could be released by mast cells, basophils, and eosinophils, and acted as an early source of Th2 cytokines164. ILC2 cells, which are recently identified as another major source for Th2 cytokine, are a prodigious source of Il‐13165. Other cell types also contribute to the production of IL‐13 during type‐2 inflammation. For example, in an oxazolone colitis model IL‐13 is produced by natural killer T (NKT) cells166 when stimulated by α‐ galactosylceramide (α‐GalCer).
1.2.3 IL‐13 signaling pathway
1.2.3.1 IL‐13 Receptors
IL‐13 achieve its biological function through binding to an IL‐13 receptor on the surface of effector cells167. IL‐13 can bind to two receptors; Type II IL4 receptor which is composed of two subunits, IL‐4 receptor α chain (IL‐4Rα)168 and IL‐13 receptor α1 chain (IL‐13Rα1)169. It can bind to both IL‐4 and IL‐13,
29 explaining the overlapping biological of IL‐4 and IL‐13161. Interestingly, the cross‐competition experiment showed that IL‐13Rα1 is specific to IL‐13 but not IL‐4, and IL‐13Rα1/IL‐4Rα receptor heterodimer showed higher affinity to IL‐13 than IL‐4170. Also, IL‐13Rα1 expression level could be regulated by microRNA‐31 and microRNA‐155, and downregulation of the receptor expression leads to an inhibitory effect on IL‐13 signaling171.
IL‐13 can also bind Type II IL‐13 receptor which is encoded by IL13RA2 gene. IL‐13Rα2 is 380 amino acid long and share some homology with IL‐5 receptor172. Different than ubiquitously expressed IL‐13Rα1, its transcript was observed in human liver, lung, thymus, placenta, brain, and heart173. Research studying murine IL‐13Rα2 (mIL‐13Rα2), which shared 59% amino acid identity with human version, showed an inhibitory effect of mIL‐13Rα2 on IL‐13 induced splenocyte activation173. The fact that injection of soluble IL‐13Rα2‐Fc fusion protein failed to expulse parasite N. brasiliensis from mice further supported the neutralizing effect of IL‐13Rα2 on IL‐13, as this parasite expulsion is dependent on IL‐13 activation174.
Human IL‐13Rα2 is proved to be an IL‐13 decoy receptor, which internalized after binding to its ligand175.
1.2.3.2 JAK/STAT pathway
When IL‐13 is secreted, it first binds to IL‐13Rα1 and leads to heterodimerization with IL‐4Rα chain, which forms Type II IL4 receptor complex. After the formation of Type II IL4 receptor, IL‐13 could signal through activating Janus kinase (JAK)/ Signal transduction and activator of transcription (STAT) pathway176.
JAKs are a group of tyrosine kinases which consists of 4 family members, JAK1‐3, and Tyk2177. IL‐13 signaling through Type II IL4 receptor activates JAK1 and Tyk2178, which results in phosphorylation in the cytoplasmic domain of IL‐4Rα chain provide docking sites for signaling molecules such as STATs179. STAT proteins are transcription factors that are presented in cytoplasmic compartments. When recruited by activated JAKs, STAT proteins are phosphorylated and form homodimers180. This activated form of STAT
30 complex will then recruited into the nucleus, binding to conserved gamma‐activated sites (GAS) motif in the promoter area of cytokine‐inducible genes and activate gene transcription181.
In 1996, IL‐13 was shown that not able to increase MHCII expression and decrease Nitro Oxide (NO) production in macrophages derived from STAT6 knockout mice, suggesting its signaling dependency on
STAT6182. When STAT6 gets recruited to IL‐4Rα chain, it will be phosphorylated and activated. The activated STAT6 monomers will then dimerized and translocate to nuclei, where it will bind to promoter areas of genes that have pro‐inflammatory functions161.
Close investigation on the cytoplasmic domain of IL‐13Rα1 chain identified tyrosine residue that had a consensus sequence for binding of STAT3 docking domain. IL‐13 treatment of myeloid cells showed an increase of STAT3 phosphorylation and activation, that was dependent on the presence of IL13Rα183,184.
IL‐13 induced STAT3 activation is thought to be dependent on p38 MAPK phosphorylation, as pharmacological inhibition of p38 MAPK leads to significant decrease of STAT3 phosphorylation and DNA binding185. The importance of IL‐13 dependent STAT3 activation remains unclear. One of its potential roles is the regulation of keratinocyte differentiation, as demonstrated by a study using JAK inhibitor that blocks STAT3 activation186.
Other stat proteins, including STA1α, 5A, 5B, are also showed increased phosphorylation in human monocytes upon IL‐13 treatment187. IL‐13‐induced STAT1 α activation is related to 15‐lipoxygenase expression, which is essential for inflammatory mediator generation in atherosclerosis185. The role of
STAT5a/b in IL‐13 signaling pathway is unclear, but the activation of STAT5 contribute to B cell proliferation and gene regulation when induced by IL‐4188.
Extracellular signal‐regulated kinase (ERK)
ERK1 and ERK2 are two protein kinases that belong to MAPK family. These two proteins share 84% sequence homology and have similar functions, thus generally referred to as ERK1/2. As most MAP
31 kinases, ERK1/2 catalyzes phosphorylation of different substrates and initiates activation of downstream pathways189. In pancreatic and ovarian cancer cells, IL‐13 promotes cancer metastasis in a STAT6‐ independent pathway, through its interactions with IL‐13Rα2 and activation of ERK1/2 pathway190,191.
Furthermore, the similar signaling pathway is observed in IL‐13 induced lung inflammation192.
1.2.3.3 Other regulators of IL‐13 signaling
IL‐13 could induce NF‐κB activation in different types of cells. In human bronchial smooth muscle cells,
IL‐13 induces NF‐κB p65 translocation in IκB kinase (IKK)‐dependent pathway193. In the meantime, in pancreatic stellate cells, IL‐13 is shown to regulate TGF‐β1 expression by suppressing NF‐κB transcription activity194.
IL‐13 can induce increased IL‐13Rα2 expression in human keratinocytes. This process is dependent on
ERK, p38 MAPK, JAK2 and STAT6 activation, which is an IL‐13Rα1‐dependent pathway195. As a result, it is fair to speculate this pathway acts as a negative feedback pathway that controls the level of IL‐13 activation. This statement is further supported by TGF‐β1’s augment effect on IL‐13, which is achieved through decreasing IL‐13Rα2 expression196.
1.2.4 IL‐13 function under physiological and pathological conditions
The primary function of IL‐13 is to promote inflammatory responses197. Under physiological conditions,
IL‐13 and other Th2 cytokines are essential for parasite expulsion198. By employing experimental murine model, a study showed IL‐13’s protective role against a variety of intestinal nematode parasite through activating both bone marrow‐derived and non‐bone marrow‐derived cells199.
Under pathological conditions, in most immunological disorders, the IL‐13 level is significantly elevated.
The increased circulating IL‐13 can promote B cell activation, induce immunoglobulin isotype switching and synthesis, and contribute to IgE‐mediate allergic diseases200 like asthma. It could also activate non‐
32 hematopoietic cells like airway epithelial cells, and lead to increased mucus secretion and airways hyperresponsiveness201.
Different than its function in promoting immune responses, IL‐13 serves as a suppressor for tumor immunosurveillance. Mouse tumor models identified IL‐13’s inhibitory role in cytotoxic T cells‐mediated tumor clearance. Also, IL‐13 is also showed to decrease E‐cadherin expression in colon cancer cell line, which decreases cell adhesion and contribute to tumor metastasis202.
Not only altered IL‐13 level leads to disease progression, but altered expression of IL‐13 receptor also causes diseases. In glioblastoma, IL‐13Rα2 is highly expressed and associated with increased malignancy grade and poor patient prognosis203. Recently, immunotherapeutic approaches targeting IL‐13Rα2 have been widely developed, and clinical trials showed positive outcomes in improving patients’ survival rate and quality of life204.
1.2.5 IL‐13 in EoE
The potential pathogenic role of IL‐13 in EoE is first suggested by the increased IL‐13 production T cells and eosinophils in EoE patients compared to normal healthy controls48,205. It is further supported by experimental mice experiments, as intratracheal IL‐13 treatment led to increased eosinophil infiltration and epithelial hyperplasia in a STAT6‐dependent manner206. In addition, transcriptome analysis comparing EoE‐specific dysregulated genes and IL‐13 induced genes in primary cells derived from EoE patients established IL‐13’s role in EoE pathogenesis54. This study not only demonstrated that IL‐13 directly regulates EoE‐associated genes, but it also explained the previous established eotaxin‐3 overexpression in EoE28. A study using EoE mice model showed that the deletion of decoy receptor IL‐
13Rα2 exacerbated collagen deposition and circumference in the esophageal epithelium, supporting the role of IL‐13 in epithelium remodeling in EoE207.
33
Over the last decade, more genes induced by IL‐13 have established their contribution in the pathogenesis of EoE. Increased IL‐13 in EoE patients lead to the upregulation of Leucine‐rich repeat‐ containing protein 31 (LRRC31)208 and CAPN14209. Also, Adherent proteins such as DSG‐1133, FLG131 and
SPRR330 were all decreased in EoE and downregulated by IL‐13. Together, these genes, altered by IL‐13, that contribute to barrier disruption and formation suggested the direct role of IL‐13 in influencing esophageal epithelium barrier in EoE.
1.2.6 IL‐13‐related treatment
Over last decades, different drugs have been developed to target IL‐13 in IL‐13 mediated diseases. IL‐13
IgG4 monoclonal antibody established its ability to decrease eosinophilia in both airway and esophageal epithelium210. In 2015, clinical trial using intravenous anti‐IL‐13 antibody QAX576 indeed showed improvement in esophageal eosinophilia and change in disease‐related transcriptome change211.
However, the primary endpoint for this therapy was not met. In 2017, RPC4046, a new antibody against
IL‐13 receptors, established its ability to reduce esophageal eosinophil counts in EoE patients in its phase
II clinical trial212. Also, treatment using antibody Dupilumab, that blocks IL‐4Rα subunit, also showed relief of intraepithelial eosinophilia, improved symptoms and quality of life compared to the placebo group in EoE patients213. A small number of clinical trials have been conducted and indicated its safety and potential ability to relieve symptoms like dysphagia212. More information like the clinical trial result from larger cohorts is needed, but this new antibody showed a promising result in anti‐IL‐13 therapy in
EoE.
1.2.7 Summary
IL‐13 is a Th2 cytokine that is elevated in EoE patients. It leads to transcriptome changes in EoE, which contribute to pathological phenotypes such as esophageal eosinophilia and epithelium remodeling.
Targeting IL‐13 in clinical trials showed some positive outcomes in treating EoE. As a result, further
34 understanding IL‐13 signaling pathways in EoE will be beneficial in discovering accurate treatment options.
35
1.3 Ion transporters
1.3.1 Introduction of Ion transporters
Ion channels and transporters are essential for almost all types of cells due to their functions in regulating electrolyte concentration inside and outside cells. The concentration of ions is important for generating electric potential, mediating fluid transport, adjusting cell volume and activating signaling pathways214,215. Dysregulation of ion transporter activities is pathogenic in many diseases especially in the lung, gastrointestinal tract, and kidney216‐218.
One of the critical functions of ion transporters is regulating fluid movement through changing ion concentrations219. Mucociliary clearance (MCC) plays an important role in maintaining lung functions.
Impaired MCC in juvenile mice showed increased Th2 inflammation in the airway, which contributes to the development of diseases such as asthma220. Efficient MCC requires the right volume of airway surface liquid (ASL), which is regulated by the concentration of different electrolytes221. Alteration in transportation proteins for Na+, K+, Cl‐ or Ca2+ could lead to volume changes of ASL. For instance, epithelium sodium channel (ENaC) is one of the primary mediators for sodium reabsorption in lung epithelium. In patients with cystic fibrosis, ENaC is hyper‐activated which lead to depletion of sodium and then ASL in the surface of airway epithelium, thus cause a decrease of MCC and contribute to the progression of disease222.
Ion transporters are also known to regulate intracellular signaling pathway. Calcium concentration is essential for lymphocyte functions, including mast cell degranulation, T cell proliferation and cytokine production and B cell differentiation223. One of the important calcium concentration regulators in lymphocytes is calcium release‐activated calcium (CRAC) channel. Patients with CRAC channels mutation fail to maintain sustained calcium level in lymphocytes, thus unable to induce T cell activation. This yield to immunodeficiency in patients which lead to life‐threatening infections224.
36
1.3.2 Ion transporters in the esophagus
Unlike other parts of the gastrointestinal tract, the esophagus does not undergo significant secretion and absorption which requires the deep involvement of multiple ion transportation pathways, as it mainly acts as conduct for food to pass to the lower GI tract225. However, one of the major functions of esophageal epithelium is to prevent gastric acids damage the outer esophageal epithelium. As a result, a set of ion transporters that provides buffering capacity is expressed in esophageal epithelium226,227.
Postprandial acid reflux occasionally happens in esophageal epithelium of normal individuals under physiological conditions228‐230. To prevent the acid from causing tissue injury, esophageal submucosal glands and acid‐base transporters in esophageal epithelial cells are activated, which neutralize or
‐ + 231,232 remove acid in the lumen by HCO3 delivery or H uptake . When esophageal epithelium is exposed with prolonged episodes of acid refluxate due to dysregulation of the lower esophageal sphincter (LES), the acid amount might exceed the acid clearance capacity of the esophagus and lead to diseases like gastroesophageal reflux disease (GERD) and Barrett’s esophagus (BE)233.
BE is a medical condition caused by repeated exposure to gastric acids234, it usually presents with long‐ term GERD symptoms and may progress into esophageal cancer235. The esophageal epithelial cells of BE patients changed from normal stratified squamous epithelium (ESSE) to specialized columnar epithelium
(BSCE). These two different forms of esophageal epithelium have different electrical parameters, as
BSCE has lower transmembrane electrical resistance and higher secretory capacity236. Further investigation found decrease activity of sodium‐hydrogen exchanger 1 (NHE1)237,238. The inhibition of
NHE1 leads to increased intracellular acidity, which causes DNA damage that could contribute to cancer progression239.
Esophageal adenocarcinoma (EA) is one of the most prevalent cancers worldwide with low five‐year survival rate. Under physiological condition, the apoptosis pathway in esophageal epithelial cells will be
37 activated after long‐term exposure to bile acid240. In cancerous esophageal epithelium, the resistance of bile acid‐induced apoptosis is dependent on its inhibitory effect on NHE activity241. It is suggested that
NHE‐mediated intracellular sodium increase induce the caspase 3/9 activation, which leads to cell apoptosis under physiological condition242.
1.3.3 Summary
Ion channels and transporters are expressed in the esophageal epithelium and are predominantly involved in the regulation of luminal acid. Dysregulation of ion transporters is involved in multiple diseases including esophagus related diseases. As a result, it is important and necessary to understand the involvement of ion transporters in the disease context, so they could potentially serve as therapeutic targets in esophageal diseases.
38
1.4 Sodium hydrogen exchangers (NHEs)
1.4.1 Introduction
NHEs are a group of sodium‐proton exchangers encoded by SLC9 (Solute carrier family 9) gene families243. Currently, the SLC9 family consists of 9 different isoforms, NHE1 – NHE9. These exchangers are known for their roles in regulating intracellular pH, and osmotic force mediates by sodium, as they function through exchanging extracellular Na+ with intracellular H+244.
NHE1, encoded by SLC9A1 gene, is the most widely studied isoform within the SLC9 family. NHE1 consists of 815 amino acids with 12 transmembrane domains245. In humans, it is ubiquitously expressed
246 and important for pHi regulation . It is also shown to regulate cell volume by altering osmotic force through influence sodium concentration247. In myocardial cells, NHE1 is activated by ischemia‐induced acidosis, which leads to accumulation of intracellular sodium248. This increased sodium concentration activates the Na+/Ca2+ exchanger NCX, led to cell death due to increased intracellular calcium249.
Consistent with this observation, deletion of NHE1 protects mice from ischemia/reperfusion (I/R) injury, indicating the pathogenic role of NHE1 in this process250. NHE1 is also involved in cancer progression and metastasis due to its role in regulating pH of cancer microenvironment251. In light of this, NHE1 inhibitors including Cariporide are thought to be potent anti‐cancer drugs252. However, no clinical trials have established this use of NHE1 inhibitors yet.
Other isoforms of NHEs are all restricted expressed in specific tissue types. NHE2‐5 are cytoplasmic binding proteins, while NHE6‐ NHE9 are specifically expressed on the membranes of organelles. Since most NHEs shared certain homology with NHE1, although regulated by different molecules, NHE2‐9 proteins all contribute to pH regulation in either cytoplasm or organelle compartments253.
1.4.2 NHE3
39
NHE3 is isoform 3 of SLC9 family, encoded by SLC9A3 gene. It was discovered in 1992 and shared 39% amino acid sequence to NHE1254. Different than NHE1, NHE3 is majorly expressed in the gastrointestinal tract and renal tissue255, with some expression in human ovary and testes256.
To distinguish the contribution of NHE3 from other NHEs in disease phenotypes, mice with functional ablation of NHE3 were generated257. Global NHE3 knockout mice showed slight diarrhea, acidotic blood and decreased blood pressure, suggesting the role of NHE3 in regulating fluid absorption and acid‐base homeostasis especially in kidney and gastrointestinal tract257,258.
Located in luminal side of the cellular membrane, NHE3 is responsible for Na+‐mediated fluid reabsorption in the proximal tubule of kidney259. Also, the NHE3‐dependent H+ secretion is important for bicarbonate reabsorption in the proximal tubule260. Increased NHE3 expression and activity lead to increased Na+‐reabsorption, which promotes extracellular fluid volume expansion, thus ended up causing increased blood flow261. This constant increased blood flow then leads to a supply‐demand mismatch of blood flow, causing vascular remodeling and resulted in hypertension262. As a result, the increased NHE3 expression is observed during hypertension in rat models263. Moreover, a study suggested an elevated NHE3 function in rat with heart failure, suggesting the role of NHE3 in mediating cardiac dysfuction264. In contrast, mice lacking NHE3 exhibited phenotypes such as urinary salt wasting, acidosis, reduced blood pressure and hypervolemic shock, indicating the importance of NHE3 in renal fluid volume homeostasis265.
NHE3 is also shown to be essential for the intestinal Na+ absorption and fluid volume regulation266.
Different than patients with hypertension, patients with diarrhea showed decreased NHE3 expression and activity267. This could be induced by bacteria secreted enterotoxin, which inhibited NHE3 activity in cAMP‐dependent pathway261. This decrease in NHE3 activity leads to disrupted osmolarity between intestinal epithelium, causing the fluid move to intestinal lumen268.
40
Recently, microbiota has been identified to influence the immune and metabolic homeostasis in gastrointestinal tract269. Previous studies suggested that luminal pH and altered ion transport influence the composition of gut microbiota270,271. Interestingly, recent studies indicated that altered Na+ and H+ concentration caused by inhibition of NHE3 function not only changes fluid homeostasis but also alters microbiota composition in GI tract272. This suggests the regulation of NHE3 activity could serve as a target for alteration of gut microbiota.
NHE3 expression and functions could be regulated by several factors such as hormones and growth factors273. Parathyroid hormone is shown to inhibit NHE3 activity through phosphorylation and endocytosis of the protein274, while dopamine can inhibit NHE3 expression and activity via a PKA‐ mediated pathway275,276. In contrast, Angiotensin II, glucocorticoids, and insulin are able to activate
NHE3 activity277.
1.4.3 Summary
In summary, sodium‐proton exchangers are involved in multiple physiological and pathological conditions. However, the study of their involvement in tissue aside from kidney and intestine are limited. Considering their role in regulating fluid movement and pH, NHEs could be related to pathogenesis and histological changes in other diseases, especially in the gastrointestinal tract.
41
1.5 Summary
EoE is a chronic allergic inflammatory disorder with unknown etiology. Esophageal epithelium remodeling including DIS and BZH is prominent in EoE patients. Clinical studies suggested these changes could contribute to disease pathogenesis and correlate with symptomatic remission in EoE. However, the molecular mechanisms underlie the formation of esophageal epithelium remodeling are unclear.
The Type‐2 inflammatory cytokine IL‐13 expression is elevated in EoE and is thought to drive the transcriptome changes and contributes to histopathological alterations in EoE patients. However, the molecular mechanisms of how IL‐13 regulates these histopathological changes in esophageal epithelium remains poorly understood.
The central hypothesis of this dissertation is that IL‐13 causes alteration of the transcriptome in esophageal epithelial cells and regulates esophageal epithelium remodeling in EoE. The specific aims of this dissertation are:
1. To examine the expression of SLC9A3 in EoE and the relationship between expression and onset
histopathological features of disease.
2. To determine the functional impact of IL‐13 induced SLC9A3 expression in esophageal epithelial
cells.
3. To determine the requirement of SLC9A3 in the formation of DIS in the esophageal epithelium.
4. To define the divergent roles of IL‐13‐induced STAT3 and STAT6 signaling pathways in
modulation of EoE transcriptome gene expression.
5. To determine the involvement of STAT3 and STAT6‐dependent genes in regulating IL‐13 induced
esophageal epithelium remodeling.
42
2 Chapter II: SLC9A3/NHE3 dysregulation and dilated intercellular spaces
in eosinophilic esophagitis
Chang Zeng B Sc1, Simone Vanoni PhD1, 5, David Wu PhD 1, Julie M. Caldwell PhD 1, Justin C. Wheeler MD3,
Kavisha Arora PhD2, Taeko K. Noah PhD1, Lisa Waggoner B Sc1, John A. Besse B Sc1, Amnah N. Yamani B
Sc1, Jazib Uddin B Sc1, Mark Rochman PhD1, Ting Wen PhD1, Mirna Chehade MD6, Margaret Collins MD3,
Vincent Mukkada MD4, Philip Putnam MD4, Anjaparavanda P. Naren PhD2, Marc E. Rothenberg MD PhD1 and Simon P. Hogan PhD1,7
1Division of Allergy and Immunology, 2Division of Pulmonary Medicine, 3Division of Pathology and
Laboratory Medicine, 4Division of Gastroenterology, Nutrition and Hepatology, Cincinnati Children’s
Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH, 45229; 5Institute of Pharmacology and
Toxicology, Paracelsus Medical University, Salzburg, Austria. 6Mount Sinai Center for Eosinophilic
Disorders, Jaffe Food Allergy Institute, Icahn School of Medicine at Mount Sinai, New York, NY.
7Department of Pathology, Mary H Weiser Food Allergy Center, Michigan Medicine, University of
Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109‐2200.
Correspondence: Simon P. Hogan, Ph.D., Department of Pathology, Mary H Weiser Food Allergy Center,
Michigan Medicine, University of Michigan, 109 Zina Pitcher Place
Ann Arbor, MI 48109‐2200; Email: [email protected]; Phone: 734‐647‐9923.
Disclosures: M.E.R. is a consultant for Immune Pharmaceuticals, NKT Therapeutics, Pulm One, Celgene,
Shire, GlaxoSmith Kline, Astra Zeneca and Novartis and has an equity interest in the first three companies listed and royalties from reslizumab (Teva Pharmaceuticals). M.E.R. is an inventor of several patents, owned by Cincinnati Children’s, and a set of these patents relates to molecular diagnostics. M.C. is a
43 consultant for Shire and Actelion and has received research grant support from Shire, Regeneron, and
Nutricia. The other authors have declared that they have no conflict of interest.
44
2.1 Copyright and Student Contribution
This chapter is from a publication accepted by the Journal of Allergy Immunology, which is an Elsevier
Journal. Elsevier states “Authors can include their articles in full or in part in a thesis or dissertation for non‐commercial purposes.”
Authorship is listed on the title page. I designed and performed most of the experiments and analyzed all the datasets used in this manuscript. TKN performed immunofluorescent staining of human esophageal biopsy samples, and JCW performed electron microscopy of EPC2‐ALI culture. Also, I wrote the manuscript and modified it under the guidance of other co‐authors.
2.2 Abstract
Background: Eosinophilic esophagitis (EoE) is characterized by histopathologic modifications of esophageal tissue including eosinophil‐rich inflammation, basal zone hyperplasia (BZH) and dilated intercellular spaces (DIS). The underlying molecular processes that drive the histopathologic features of
EoE remain largely unexplored.
Objective: To investigate the involvement of SLC9A3 in esophageal epithelial [pH]i and DIS formation and the histopathological features of EoE.
Methods: We examined the expression of esophageal epithelial gene networks associated with regulation of intracellular pH ([pH]i) in the EoE transcriptome of primary esophageal epithelial cells and an in vitro esophageal epithelial 3D model system (EPC2‐ALI). Molecular and cellular analyses and ion transport assays were employed to evaluate expression and function of SLC9A3.
Results: We identified altered expression of gene networks associated with regulation of intracellular pH
([pH]i) and acid protective mechanisms in esophageal biopsies from pediatric patients with EoE (normal n = 6, EoE n = 10). The most dysregulated gene central to regulating [pH]i was SLC9A3. SLC9A3
45 expression was increased within the basal layer of esophageal biopsies from patients with EoE and that expression positively correlated with disease severity (eosinophils/HPF) and DIS (normal n = 10, EoE n =
10). Analyses of esophageal epithelial cells revealed IL‐13–induced, STAT6‐dependent SLC9A3 expression and Na+‐dependent proton secretion and that SLC9A3 activity positively correlated with DIS formation.
Finally, we showed that IL‐13–mediated Na+‐dependent proton secretion was the primary intracellular acid protective mechanism within the esophageal epithelium and that blockade of SLC9A3 transport abrogated IL‐13–induced DIS formation.
Conclusions: SLC9A3 plays a functional role in DIS formation, and pharmacologic interventions targeting
SLC9A3 function may suppress the histopathologic manifestations in EoE.
46
2.3 Introduction
Eosinophilic esophagitis (EoE) is a food allergen‐induced inflammatory disease that is increasing in incidence (5 – 10 cases per 100,000) and prevalence (0.5 to 1 case per 1000)278‐281. Common symptoms of
EoE include vomiting, dysphagia, chest pain, food impaction and upper abdominal pain282 and decrease the health‐related quality of life283.
Corroborative clinical and experimental studies indicate that an underlying allergic sensitization to dietary food antigens and development of a CD4+ Th2 and ILC2 inflammatory response in the esophageal mucosa drive the eosinophilic inflammation and esophageal remodeling in EoE, which includes basal zone hyperplasia (BZH) and dilated intercellular spaces (DIS)19,77,284,285. Dietary modification (i.e., complete or targeted food antigen avoidance) and swallowed glucocorticoids alleviate much of the disease pathology116,286, suggesting a food‐induced CD4+ Type‐2 allergic inflammatory response54,136,144,287‐289.
Consistent with this, animal‐based studies have revealed important roles for CD4+ Th2 cells, pro‐allergic cytokines [interleukin 5 (IL‐5), IL‐13] and eosinophils in the histopathologic manifestations of disease17,53,206. One cytokine that seems to be central in orchestrating the EoE phenotype is IL‐1330,51,122.
IL‐13 is highly upregulated in the esophageal tissue of patients with EoE and is sufficient to alter gene expression in esophageal epithelial cells in vitro and in vivo, and the IL‐13–induced transcriptome significantly overlaps with the transcriptional changes observed in esophageal biopsies of patients with
EoE30,51,122. Importantly, treating patients with EoE with a humanized antibody against IL‐13 led to a significant decrease in esophageal eosinophil count and had a normalizing effect on the dysregulated transcriptome observed in patients with EoE211. IL‐13 has been shown to dysregulate the expression of several key epithelial barrier regulatory genes including desmosomal cadherin, desmoglein‐1 (DSG‐1),
47
Leucine‐rich repeat‐containing protein 31 (LRRC31), kallikrein (KLK) serine proteases and calpain 14
(CAPN14), which have been linked with EoE133,208,209.
Though there have been significant advances in our understanding of a link between allergic inflammation and EoE, there is a paucity of data revealing the underlying pathways that regulate epithelial BZH and DIS in EoE. DIS, also described as spongiosis, is a morphologic feature that has been identified in multiple forms of esophagitis including lymphocytic esophagitis290, gastroesophageal reflux disease (GERD)291 and
EoE19,292. Histologic comparison between GERD and EoE suggested that DIS is significantly more intense in
EoE than GERD293. Steroid therapy or elimination diet significantly decreases DIS in patients with EoE, and this decrease is associated with improvement of patients’ symptoms292, indicating an association between
DIS and the etiology of EoE. The underlying molecular pathways that drive DIS formation are currently unknown.
We recently performed RNA sequencing (RNAseq) on esophageal mucosal biopsies from normal healthy control patients (NL) and patients with active, proton pump inhibitor (PPI)–confirmed EoE. We identified a total of 1607 significantly dysregulated transcripts (1096 upregulated, 511 downregulated), with 66% of the gene signature being similar to the EoE transcript signature identified by microarray‐based expression profiling122. We have performed gene ontology enrichment network analysis of the 1607 significantly dysregulated transcripts and identified dysregulation of transmembrane transporter activity genes associated with regulation of [pH]i and acid protective mechanisms. The most dysregulated transmembrane transporter activity gene in the EoE transcriptome was the solute carrier family 9, subfamily A, member 3 (SLC9A3), which encodes sodium‐hydrogen exchanger member 3 (NHE3)294 (33‐ fold increase). We demonstrate a significant increase of SLC9A3 in the esophageal epithelium in two independent, confirmatory patient cohorts with PPI‐confirmed EoE. We show that the expression level of
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NHE3 positively correlated with the level of inflammation and the area of DIS. IL‐13 treatment of esophageal epithelial primary cells derived from patients with EoE and in a differentiated squamous esophageal epithelium model (EPC2‐ALI) increased NHE3 expression and ion transport activity.
Pharmacologic inhibition of NHE3 function substantially decreased the area of IL‐13–induced DIS. These collective data suggest that increased expression and activity of NHE3 contribute to the formation of DIS in the esophageal epithelium in EoE.
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2.4 Material and Methods
Human subjects. NL (healthy control patients) were defined as having no history of EoE diagnosis, 0 esophageal eosinophils per high‐power field (HPF) and no evidence of esophagitis within distal esophageal biopsies obtained during the same endoscopy procedure as the analyzed samples. EoE was defined as described in the recent consensus guidelines. Specifically, patients needed to have ≥15 eosinophils in at least 1 high‐power field (Eos/HPF) in a distal esophageal biopsy with other causes of esophageal eosinophilia excluded, and without a response to acid suppression. Normal control patient cohort consists of patients with a variety of non‐specific upper GI complaints including vomiting, loose stools, abdominal pain, nausea who underwent endoscopy were biopsied and demonstrated to have no histological evidence of esophageal disease. RNAseq and validation qRT‐PCR analyses and histology (Eos/hpf and DIS quantification) studies were performed on the esophageal biopsies. RNAseq and qRT‐PCR analyses and histology (Eos/hpf and DIS) were performed on human esophageal biopsy samples (normal, n = 6; EoE, n
= 10) as previously described (NCBI Gene Expression Omnibus (GEO) database under accession
GSE58640)122 (Cohort 1). The demographics of the normal control and EoE patients are described in Fig.
2‐6. The qRT‐PCR and histopathology (Eos/hpf) analyses was performed on a second independent cohort
(normal, n = 10; EoE, n = 10) (Cohort 2). The demographics of the patients and controls are described in
Fig. 2‐6.
RNA‐sequencing of human Biopsy Samples: Esophageal biopsy RNA was isolated from controls and EoE patients with active disease using the RNeasy kit (QIAGEN Incorporated, Germantown, MD) per the manufacturer's protocol. RNA libraries were prepared using standard Illumina protocols (TrueSeq RNA LS
Sample Prep V2) at the CCHMC Genetic Variation and Gene Discovery Core. RNA sequencing acquiring
100bp reads from paired‐end libraries was performed at the Genetic Variation and Gene Discovery Core
Facility at CCHMC using Illumina HiSeq 2500. The paired‐end sequencing reads were aligned against the
50
GRCh37 genome model using TopHat 2.04 with Bowtie 2.03295,296. The separate alignments were then merged using Cuffmerge297 with UCSC gene models as a reference. Raw data were assessed for statistical significance using a Welch t‐test with Benjamini‐Hochberg false discovery rate and a threshold of P < 0.05 and a 2.0‐fold cut‐off filter in GeneSpring® GX (Agilent Technologies Incorporated, Clara, CA).
RNA‐sequencing of mature EPC2‐ALI: RNA was isolated using the RNeasy kit (QIAgen Incorporated,
Germantown, MD, USA) per manufacturer instructions. Assessment of RNA quality was performed using the Agilent 2100 Expert bioanalyzer (Agilent Technologies Incorporated, Clara, CA, USA) and only those samples getting an RNA Integrity Number (RIN) above 8 were chosen for sequencing. Next‐generation sequencing analyses were performed by the CCHMC Genetic Variation and Gene Discovery Core with
Illumina HiSeq 2500. Raw data was uploaded on Biowardrobe298 and RPKM values were calculated.
Differentially expressed genes were assessed using DEseq2.
Gene Ontology Analysis: Gene list enrichment analysis and candidate gene prioritization based on molecular function using ToppGene299 with FDR B&H correction and p‐Value cutoff at 0.05. Heatmaps were generated using RStudio.
Pathology analysis.
Biopsy preparation. Formalin‐fixed, paraffin‐embedded esophageal biopsies were sectioned into 5‐mm slides. After removal of paraffin and serial hydration, sections were stained with hematoxylin and eosin
(H&E). H&E‐stained slides were then imaged using an Olympus DP‐72 microscope (Olympus Corporation,
Semrock, New York, NY, USA).
Quantification of intercellular space. Intercellular space was quantified as the percentage of the intercellular area of the total area of the biopsy sample using Image‐Pro Plus software (Media Cybernetics,
Rockville, MD, USA) automated space measurement function and calculated by the ratio of intercellular area / total tissue area.
51 qPCR analysis. RNA samples were extracted from esophageal biopsy, cultured primary cells or EPC2‐ALI cultures using the RNeasy kit (QIAGEN Sciences Incorporated, Germantown, MD, USA) according to the manufacturer’s protocol. Purified RNA (300 – 500 ng) was DNase treated and reversed transcribed to cDNA using Superscript II RNase H Reverse Transcriptase (Thermo Fisher Scientific Incorporated, Rockford,
IL, USA) per the manufacturer’s instructions. cDNA for SLC9A3 and 18S were quantified by real‐time PCR using TaqMan Universal supermix with the CFX96 Real‐Time PCR Detection System. qPCR analysis was performed using the Bio‐Rad CFX Manager Software version 3.1. Primers for SLC9A3 and 18S were purchased from TaqMan (Thermo Fisher Scientific, Waltham, MA).
Immunofluorescence (IF) staining. For IF staining, formalin‐ or paraformaldehyde‐fixed, paraffin‐ embedded esophageal biopsies or EPC2‐ALI cultures were sectioned, mounted on slides and de‐ paraffinized using standard histological procedures. Slides were then permeabilized in Tris‐EDTA (1 mM, pH 9.0) with 0.1% Tween‐20, and antigen exposure was performed at 125°C for 30 s in a decloaking chamber using a pressure cooker. Slides were then blocked by 10% normal donkey serum for 1 h followed by overnight incubation of primary antibodies diluted in 10% normal donkey serum: NHE3 (Novus,
Littleton, CO) and CK13 (Invitrogen, Carlsbad, CA). Slides were then washed and incubated with secondary antibody at room temperature for 1 h. Slides were mounted with Fluoromount‐G (SouthernBiotech,
Birmingham, AL) mounting solution. Fluorescent imaging was performed using the Zeiss Apotome fluorescent microscope using NIKON elements software.
Primary cell preparation. Distal esophageal biopsy was obtained from NL or EoE patients who underwent routine endoscopy, suspended in 1 mL keratinocyte serum‐free media (KSFM) (Invitrogen, Carlsbad, CA) containing supplements [human epidermal growth factor (EGF) (1 ng/mL), bovine pituitary extract (50 mg/mL), and 1X penicillin/streptomycin (Invitrogen, Carlsbad, CA)] and subsequently placed in 60‐mm dish in 3 mL of filter‐sterilized (0.2 mm) Leibovitz’s L‐15 media (Invitrogen, Carlsbad, CA) containing 115
U/mL collagenase, 1.2 U/mL dispase and 1.25 mg/mL BSA. The biopsy was mechanically dispersed using
52 scissors to pieces less than 1 mm in size and then incubated at 37ºC for 1 h. The cell suspension was centrifuged at 500 g for 5 min at 4ºC and the pellet washed twice with 5 mL of supplemented KSFM media.
Cells were suspended in 1 mL of 0.05% trypsin/EDTA for 10 min at 37ºC and agitated every 2 min. Trypsin activity was inhibited with soybean trypsin inhibitor (STI) (250 mg/L in 1X DPBS; 5 mL). Cells were pelleted by centrifugation, suspended in 1 mL KSFM containing supplements, transferred to a 35‐mm dish
º containing irradiated NIH 3T3 J2 fibroblasts (162,500 cells) and cultured at 37 C and 5% CO2. Media was changed at day 5 and every other day thereafter using KSFM containing supplements. After epithelial cells became 60‐70% confluent, they were dispersed from the plate using 0.05% trypsin/EDTA for 10 min at
37ºC and agitated every 2 min. Trypsin digestion was inactivated by STI, and cells were then passaged in
KSFM containing supplements at 1‐2 x 105 cells per 3 mL in a 60‐mm dish.
pHi assay. Primary esophageal epithelial cells were cultured on Ibidi ‐Slide 4 well (ibidi GmbH, Germany) with a concentration of 25,000 cells/well. After 24 h of equilibration period, cells were stimulated with or without 100 ng/mL recombinant human IL‐13 (PeproTech, Rocky Hill, NJ) for another 48 h. pHi changes in these primary cells were measured with the pH‐sensitive fluorescent dye BCECF AM (2',7'‐bis‐(2‐ carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, acetoxymethyl ester) or SNARF‐5F AM (SNARF‐5F 5‐(and‐6)‐ carboxylic acid, acetoxymethyl ester) (Invitrogen, Carlsbad, CA). Cells were loaded with 10 M BCECF AM
‐ or SNARF‐5F AM in HCO3 ‐free Ringer’s solution [mM, 110 NaCl, 25 Na‐Gluconate, 5 KCl, 0.5 MgSO4.7H2O,
1 CaCl2.2H2O, 10 HEPES, 4 Glucose; to pH 7.4] at 37°C for 30 min prior to the experiment. To remove the
‐ extracellular dye, cells were washed two times with HCO3 ‐free Ringer’s solution at the end of the incubation period. To acidify the intracellular compartment of cells and generate the necessary H+
+ + gradient (high [H ] inside vs. low [H ] outside cell) to measure pHi recovery rate, 20 mM of NH4Cl was added to the chamber after the first 5‐min recording of the baseline pHi (Fig. 2‐2C. Stage II). To measure
+ + the Na ‐dependent pHi recovery rate, Na was first removed from the cells by replacing the buffer with
+ Na ‐free Ringer’s solution [mM, 135 NMDG‐Cl, 5 KCl, 0.5 MgSO4.7H2O, 1 CaCl2.2H2O, 10 HEPES, 4 Glucose;
53
+ to pH 7.4] for 5 min (Fig. 2‐2C. Stage III). Na ‐dependent pHi recovery rate was measured by replacing
‐ + extracellular solution to HCO3 ‐free Ringer’s solution containing 135 mM Na (Fig. 2‐2C. Stage IV) and determination of the slope of the Na+‐dependent pHi change. The pHi values are derived from the
+ calibration curves described below. The statistical significance of Na ‐dependent pHi change was determined using a Students t‐test (two‐tailed). To record the BCECF AM fluorescence change, cells were imaged by a Nikon Spectra X inverted fluorescent microscope with the excitation wavelength of 512 nm /
440 nm and emission at 535 nm. To record the SNARF‐5F AM fluorescence change, cells were imaged by
Zeiss LSM710 LIVE DUO confocal microscope with excitation wavelength at 488 nm and the emission wavelength of 640 nm / 580 nm. To inhibit NHE3 activity, 30 M of S3226 or 0.01% DMSO (vehicle) was
+ applied throughout the experiment. % S3226‐sensitive Na ‐dependent pHi recovery rate was calculated as Recovery rate of (DMSO‐treated ‐ S3226‐treated) *100/DMSO‐treated %. Quantification was performed on 10‐20 cells randomly picked in each sample, and the fluorescence intensity was measured using Nikon Elements microscope imaging software or ImageJ software. These fluorescence intensity values were then converted into pH values per calibration curves. A calibration curve was generated at the end of each experiment; BCECF AM or SNARF‐5F AM intensity was calibrated against pHi when cells were exposed to the K+/H+ ionophore nigericin (10 mM) and valinomycin (10 mM) (Invitrogen, Carlsbad,
+ + CA) in high‐K solution at four different pH values. High‐K solution [mM, 20 NaCl, 130 KCl, 1 MgCl2, 1
CaCl2.2H2O, 5 HEPES] was prepared and titrated to a pH ranging from 6.5 to 7.9. Fitting was performed with GraphPad Prism software.
EPC2‐ALI culture. The hTERT‐EPC2 cells (hTERT‐immortalized human esophageal keratinocytes) were a kind gift from Dr. Anil Rustgi (University of Pennsylvania, Philadelphia, PA, USA) as previously described300.
The air‐liquid interface (ALI) culture system was previously described and characterized together with
EPC2 cells133. EPC2 cells were grown to fully submerge on 0.4‐mM pore size, permeable transwell inserts
(Corning Incorporated, Corning, NY)) in KSFM (Life Technologies, Carlsbad, CA, USA). As depicted in Fig. 2‐
54
3A, (i) Day 0, cells were seeded on permeable membrane support and grown to single submerged layer after three days. (ii) Cells were then shifted to medium containing high [Ca2+] to induce tight junction formation ([Ca2+] = 1.8mM). (iii) On Day 7, media were removed from the top chamber in order to induce differentiation and epithelial stratification in ALI. (iv) Day 12 (5 days post‐ALI), cells were then treated with vehicle or cytokine (IL‐13) in the presence and absence of SLC9A3 inhibitor (30mM)301 as described in figure legends.
Lentiviral Transduction. EPC2 cells at 60‐70% confluence were transduced with lentiviral particles containing Mission® STAT6 shRNA TRC 0000019409 shRNA, Mission® STAT3 (TRCN0000329887) shRNA
(Sigma; St. Louis, MO, USA) or Mission® non‐target control shRNA (Sigma; St. Louis, MO, USA). All the three shRNA lentivirus were generated by the Cincinnati Children’s Hospital Medical Center Viral Core using a
4‐plasmid packaging system. Lentiviral particles were incubated with EPC2 cells for 6 hours with a
Multiplicity of Infection (MOI) from 0.5 to 10 for STAT3 or CTRL shRNA. For STAT6 shRNA, 10µL to 50µL viral particles were added to the cells. All the viral particles were added in the presence of 5µg/mL
Hexadimethrine Bromide (Polybrene®) (Sigma; St. Louis, MO, USA). During the first hour of incubation, cells were spun down at 1000*g for 1 hour at room temperature. 6 hours following transduction cells were put in fresh KSFM media, and 24 hours later media containing 1µg/mL of Puromycin (Thermo Fisher
Scientific Incorporated; Rockford, IL, USA) was used for selection. Cells were grown under selective pressure and cultured as regular EPC2 cells. Stable knockdown of STAT6 and STAT3 in EPC2‐ALI cultures were evaluated by Western blot. The results indicated an 80% reduction of STAT6 and 90% reduction of
STAT3, relatively, compared to empty control transduced cells.
Western blot. EPC2‐ALI cultures were lysed using protein lysis buffer (10% Glycerol, 20 mM Tris HCl pH 7,
137 mM NaCl, 2 mM EDTA, 1% NP40 in H2O) supplemented with Halt protease inhibitor cocktail (Thermo
Fisher Scientific Incorporated, Rockford, IL, USA). Proteins were then quantified with BCA assay, and 20
55
g of protein extracted together with protein‐reducing buffer was loaded and separated on a 4%‐12%
Bis‐Tris gel and transferred to a nitrocellulose membrane (Life Technologies, Carlsbad, CA). Antibody of
NHE3 and ‐actin were used for protein detection. The IRDye 800 CW goat anti‐rabbit IgG (H+L) (Li‐Cor,
Lincoln, NE) was used as the secondary antibody for detection. Western Blot quantification was performed using Image Studio Lite (Li‐Cor, Lincoln, NE). pH‐STAT assay. Acid secretion by confluent epithelium was quantitated using pH‐STAT (TIM856,
Radiometer Analytical, Loveland, CO) connected to an Ussing chamber system as previously described302.
EPC2‐ALI cultures were mounted into an Ussing chamber containing unbuffered Ringer’s solution [mM,
145 NaCl, 2 KCl, 1 MgCl2, 2 CaCl2, 5 Glucose] while gassed with 99.5% oxygen. Both the pH electrode and the titrating burette are placed in the apical side chamber. To measure the equilibrium extracellular pH, without any titration, the extracellular pH was measured for 10 min or until a stable pH was achieved
(<0.002 pH unit change/min). After the equilibrium period and extracellular pH measurements were obtained, the mucosal pH was adjusted by titration to a set pH (pH 7.6) to keep the extracellular pH slightly alkalized. Titration rate (amount of alkaline injected by the machine to neutralize the acid secreted by
EPC2‐ALI culture to maintain the set pH) was used to measure the acid secretion rate.
Histologic analysis for EPC2‐ALI. EPC2‐ALI cultures were treated as indicated in experiments and then fixed on transwell supports with 4% paraformaldehyde for 1 h at room temperature. Fixed membranes underwent a series of dehydration steps, cleared in Histoclear solution, embedded in paraffin and sectioned into 5‐mm slides. The slides were stained using H&E staining and imaged using an Olympus DP‐
72 microscope (Olympus Corporation, Semrock, New York, NY, USA).
Electron microscopy. EPC2‐ALI cultures were treated as indicated in experiments, fixed with 3% glutaraldehyde and submitted to CCHMC Pathology Research Core for processing, sectioning, and
56 transmission electron microscopy using a Hitachi model H‐7650 electron microscope, at 80 kV, using the
AMT‐600 image capture engine software.
BrdU assay. After EPC2‐ALI cultures were treated as indicated in the experiment, BrdU reagents dissolved in DMSO (final concentration 10 µM) were added to both the upper and lower chambers of transwells for
2 h. EPC2‐ALI cultures were then washed with PBS and fixed with 4% paraformaldehyde for another 2 h.
Statistical analysis. The statistical significance of EPC2‐ALI samples was established using an unpaired t‐ test (two‐tailed), or two‐way ANOVA if there were more than one variable. For non‐normally distributed data from patient biopsies and primary cells derived from patient biopsies, the Mann‐Whitney test was used, and the correlation analyses were assessed with a Spearman correlation test. Graphs and statistical analyses were performed using GraphPad Prism 7.02 (GraphPad Software Incorporated, La Jolla, CA, USA).
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2.5 Results
2.5.1 Transmembrane transporter SLC9A3/NHE3 specifically upregulated and correlated with
eosinophil count and DIS in EoE
To begin to determine the potential involvement of transmembrane transporter activity in the histopathologic alterations of the esophageal epithelium in EoE, we applied gene ontology (GO) enrichment analysis of the 1607 differentially expressed RNA transcripts identified by RNAseq analyses of pediatric NL and EoE biopsy samples122. GO analysis revealed 50 individual GO nodes significantly dysregulated in the EoE transcriptome based on functional annotations and protein interaction networks
(FDR‐corrected p < 0.05, Fig. 2‐7). Of these GO nodes, 5 were related to transmembrane transporter activity (Fig. 2‐1A). A combinatory comparison of all 62 genes within these 5 GO nodes revealed that the most upregulated transmembrane transporter activity gene was SLC9A3, which encodes for the sodium‐ hydrogen exchanger family member 3 (NHE3) (Fig. 2‐1B). SLC9A3 was induced 33‐fold in patients with EoE compared to NL (Fig. 2‐1C). In contrast, expression of other members of the SLC9 family, including the ubiquitously expressed sodium‐proton exchanger SLC9A1, also referred to as sodium‐hydrogen exchanger family member 1 (NHE1), were not dramatically different in expression between EoE and NL (Fig. 2‐1D).
Correlation analyses revealed a positive correlation between the level of peak distal esophageal eosinophils and SLC9A3 expression (Fig. 2‐1E, r = 0.7167, p < 0.05. Notably, this was specific to SLC9A3, as we did not observe any correlation with SLC9A1 (Fig. 2‐1E, r = 0.3201, p > 0.05), revealing a specific link between SLC9A3 expression and disease severity in EoE. To confirm these observations, we examined a second independent pediatric cohort [NL n = 10, active EoE n = 10] for which we had paired RNA and histologic biopsy samples from the same day of endoscopy. Consistent with our RNAseq analyses, qPCR analyses revealed significant SLC9A3 overexpression in the pediatric EoE cohort (Fig. 2‐1F). Furthermore, we observed a positive Pearson correlation between SLC9A3 expression level and distal peak esophageal eosinophil numbers (Fig. 2‐1G, r = 0.9172, p < 0.0001).
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We next performed immunofluorescence (IF) analyses to determine the cellular and spatial expression of
NHE3 in NL and EoE esophageal biopsy samples. Consistent with our RNAseq and PCR analyses, we observed very little expression of NHE3 in the healthy esophageal epithelium (Fig. 2‐1H, upper panel). The positive staining observed was restricted to the CK13‐ single‐cell basal esophageal epithelial layer (Fig. 2‐
1H, upper panel). In EoE, NHE3 protein expression was remarkably increased, localized to the esophageal basal cell layer and also expanded into the CK13+ suprabasal layer (Fig. 2‐1H, lower panel). The localization of NHE3 to the suprabasal zone, an area associated with DIS formation, lead us to examine the relationship between SLC9A3 expression and DIS in EoE esophageal biopsies. Notably, SLC9A3, but not SLC9A1, expression positively correlated with the percentage of DIS area in EoE individuals (r = 0.8095, p < 0.05)
(Fig. 2‐1I). These cumulative data indicate a specific upregulation of SLC9A3/NHE3 in the suprabasal layer of esophageal epithelium in patients with EoE and that SLC9A3 levels correlate with esophageal eosinophilic inflammation and DIS.
2.5.2 Increased NHE3 function in IL‐13–stimulated primary esophageal epithelial cells.
The epithelial sodium‐proton exchanger, NHE3, is predominantly expressed in the apical membrane of epithelial and is the principal mechanism for the electroneutral exchange of (Apical Baso) Na+ and (Baso
+ Apical) H and plays an important role in the maintenance of intracellular pH (pHi) and regulation of cell volume303. Given IL‐13’s known role in upregulating the EoE transcriptome54 and that humanized anti–
IL‐13 monoclonal antibody (QAX576) has been shown to modulate expression of an anion transport activity node211, we examined the impact of IL‐13 exposure on SLC9A3 expression in primary esophageal epithelial cells. We demonstrate upregulation of SLC9A3 expression in primary esophageal epithelial cells following IL‐13 stimulation (Fig. 2‐2A). To determine whether increased SLC9A3 expression was associated with altered NHE3 activity, we examined the effect of IL‐13 exposure on intracellular pH ([pH]i) in primary esophageal epithelial cells. Notably, increased SLC9A3 expression coincided with a significant increase in
59
+ baseline pHi (Fig. 2‐2B) and Na ‐dependent pHi recovery rate (Fig. 2‐2C‐D), supporting an overall increase of Na+/H+ exchange activity. Notably, the recovery rate positively correlated with SLC9A3 expression in primary esophageal epithelial cells (r = 0.9503 p < 0.01; Fig. 2‐2E), suggesting that the increase of pHi recovery rate in IL‐13–treated primary esophageal epithelial cells is predominantly due to increased NHE3 expression. Addition of the NHE3‐specific inhibitor S3226 (30 mM) confirmed that the IL‐13–induced
+ increase in Na ‐dependent pHi recovery rate was predominantly mediated by NHE3 (Fig. 2‐2F).
2.5.3 IL‐13 induces an EoE‐like transcriptome including increased transmembrane transporter
activity and SLC9A3 overexpression in an in vitro, matured esophageal epithelium model
system.
In order to define the involvement of SLC9A3/NHE3 in the regulation of [pH]i and DIS formation in a mature esophageal epithelial model system, we adapted an in vitro model developed from keratinocyte esophageal epithelial cells (EPC2) grown in air‐liquid interface (ALI)133. EPC2 cells were grown under submerged conditions in low‐calcium media (0.09 mM; days 0‐3), changed to high‐calcium media (1.8 mM, days 3‐7) and then exposed to the ALI for 5 days in the presence of high calcium (1.8 mM, days 7‐12) to induce differentiation and formation of a mature, stratified epithelium (Fig. 2‐3A). Following maturation, EPC2‐ALI cells were stimulated with vehicle or IL‐13 and RNAseq analyses performed (Fig. 2‐
3A, days 12‐14). We show that IL‐13 significantly dysregulated a total of 572 genes (p < 0.05, fold change
> 2.0); notably, many of the most highly dysregulated genes included inflammatory genes associated with
EoE, including CCL26 (7‐fold), TNFAIP6 (9‐fold), CDH26 (3‐fold) and CAPN14 (5‐fold), and also gene families located in the epidermal differentiation cluster (EDC) on chromosome 1q21 (e.g., IVL, LOR, S100A4,
S100A6) (Fig. 2‐3B and Fig. 2‐6). Consistent with these findings, comparative analyses of the IL‐13–induced transcriptome changes in EPC2‐ALI cells with that of the EoE‐specific transcriptome revealed significant overlap with the EoE‐specific transcriptome (Fig. 2‐3B, p < 0.0001). GO analysis based on the biological process on IL‐13–dysregulated genes revealed the most significant 15 individual GO biological process
60 nodes were associated with keratinization, epidermis development, skin development, keratinocyte differentiation, epidermal cell differentiation and inflammatory response and GO Pathways associated with formation of the cornified envelope, keratinization and interleukin‐4 and 13 signaling (Table 2‐2, FDR‐ corrected p < 0.05). To examine the effect of IL‐13 on transmembrane transporter activity, we performed
GO analysis based on the molecular function of the 572 genes and identified 6 nodes significantly dysregulated related to transmembrane transport activity (Fig. 2‐3C, FDR‐corrected p < 0.05). Of the 28 differentially expressed transmembrane transport activity genes in these 6 GO nodes, SLC9A3 was one of the most highly upregulated genes (Fig. 2‐3D).
IL‐13 is known to signal through the JAK/STAT pathway, in particular through a STAT3‐ and STAT6‐ dependent signaling pathway304. To determine the involvement of STAT‐3 and STAT‐6 in the IL‐13 induction of SLC9A3, we examined SLC9A3 mRNA expression in STAT3 and STAT6 shRNA–transduced
EPC2‐ALI cells following IL‐13 stimulation. Notably, STAT‐3 knockdown in EPC2‐ALI cells caused no significant reduction in IL‐13–induced SLC9A3 expression compared to control shRNA–transduced EPC2‐
ALI cells (Fig. 2‐3E). In contrast, STAT6 knockdown in EPC2‐ALI cells significantly ablated IL‐13–induced
SLC9A3 expression (50% reduction), suggesting that IL‐13–induced STAT6 signaling is important for
SLC9A3 expression in EPC2‐ALI cells (Fig. 2‐3E). These studies demonstrate that IL‐13 induces SLC9A3 expression in EPC2‐ALI cells in part by the STAT6‐dependent mechanism.
2.5.4 IL‐13–induced NHE3 expression and function in differentiated esophageal epithelial cells.
Employing this mature EPC2‐ALI model system, we show that IL‐13 stimulation of EPC2‐ALI cells induced an increase in SLC9A3 mRNA (Fig. 2‐4A) and NHE3 protein (Fig. 2‐4B‐C) expression. Immunofluorescence analyses revealed that NHE3 was barely expressed in vehicle‐treated EPC2‐ALI cells (Fig. 2‐4D, top panel).
In contrast, we observed a significant increase in NHE3 expression in EPC2‐ALI cells following IL‐13
61 exposure; comparable to what we observed in EoE biopsy samples, NHE3 was predominantly localized to the basal and suprabasal layer of epithelium in IL‐13–treated EPC2‐ALI cultures (Fig. 2‐4D, lower panel).
To examine NHE3 function in the mature EPC2‐ALI cultures, we measured proton secretion rates in an
Ussing chamber system fitted with pH‐STAT (Fig. 2‐4E). We show that IL‐13 stimulation reduced the extracellular pH (pHe) compared with vehicle‐treated control (pHe 6.82 vs. 6.97, respectively, p < 0.05), indicating altered acid‐base transport. Notably, the reduction in pHe was abrogated with exposure to the specific NHE3 inhibitor S3226 (Fig. 2‐4F), indicating NHE3‐dependent proton extrusion. To measure the rate of acid extrusion by the mature EPC2‐ALI, the apical side buffer was adjusted to an alkaline pH [pH
7.6; Ba(OH)2] to generate an ion concentration gradient and the amount of alkali [Ba(OH)2] required to maintain this condition (pH 7.6) was continuously monitored using a pH‐STAT (Fig. 2‐4G). We show that
+ IL‐13 stimulation of the mature EPC2‐ALI cells led to increased Ba(OH)2 injection to counterbalance H secretion from the tissue and maintain pH 7.6 (Fig. 2‐4G). To determine the involvement of NHE3 in apical acid secretion function in the mature EPC2‐ALI, S3226 was added to the apical side of the buffer to identify the NHE3‐mediated fraction of acid secretion. Notably, the acid‐secretion rate in IL‐13–stimulated mature
EPC2‐ALI cells was significantly abrogated with S3226, indicating NHE3‐dependent secretion (Fig. 2‐4H).
These observations indicate an IL‐13–induced increase in NHE3‐dependent acid secretion in EPC2‐ALI cells.
2.5.5 Increased SLC9A3 expression and activity is linked with DIS formation.
Given the observed association between NHE3 and acid secretion in EPC2‐ALI cells and the correlation between SLC9A3 expression and DIS formation in esophageal biopsy samples from patients with EoE (Fig.
2‐4H and 2‐1I, respectively), we examined the relationship between NHE3 function and DIS formation in the mature EPC2‐ALI cells. IL‐13 stimulation of EPC2‐ALI cells induced DIS formation within the basal and suprabasal layer of EPC2‐ALI cells as evidenced by spaces between cells (Fig. 2‐5A). Electron microscopy analyses revealed alteration to the intercellular junctional structures of esophageal cells, with the
62 appearance of expanded or dilated intercellular areas (Fig. 2‐5B). Notably, the DIS is sealed by lateral membranes that are of close apposition and tethered by intercellular junctional proteins such as desmosomes (Fig. 2‐5B). To determine the requirement of NHE3 activity in DIS formation, we stimulated
EPC‐ALI cells with IL‐13 in the presence of the NHE3 inhibitor S3226 (Fig. 2‐5C‐D). Notably, we show that the IL‐13–induced DIS within the suprabasal layer in EPC2‐ALI cells were diminished in the presence of
S3226 (Fig. 2‐5C‐D). Collectively, we concluded that NHE3 has an important role in IL‐13–induced DIS formation in esophageal cells.
63
2.6 Discussion
EoE is characterized by histopathologic manifestations including BZH and DIS. The underlying molecular processes that drive these pathologic manifestations remain largely unexplored. Herein, we demonstrate
1) dysregulation of transmembrane transporter activity gene networks in esophageal biopsies from patients with EoE; 2) increased expression of the Na+‐H+ exchanger SLC9A3/NHE3 in EoE esophageal biopsies and positive correlation of this increased expression with DIS area and eosinophil infiltration; 3) increased SLC9A3 expression and NHE3 activity in primary esophageal epithelial cells and in response to
IL‐13 stimulation in a mature EPC2‐ALI model system; and 4) reduction of IL‐13–induced DIS formation in
EPC2‐ALI cells by pharmacological antagonism of NHE3 activity. Collectively, we have identified a role for
SLC9A3/NHE3 in IL‐13–induced DIS formation in the esophageal epithelium and provide evidence for the involvement of this pathway in EoE.
The cytokine IL‐13 has an important role in driving the underlying allergic inflammatory cascade and the histopathologic features of EoE30,51,122. This notion is supported by the observations that stimulating esophageal cells with IL‐13 leads to a transcript signature that partially overlaps the esophageal EoE transcriptome54. Furthermore, while the primary outcome of greater than 75% decrease in peak eosinophil counts at week 12 was not met, treating patients with EoE with anti–IL‐13 (QAX576) reduced intraepithelial esophageal eosinophil counts and led to an improvement in the EoE transcriptome and clinical symptom such as dysphagia in adults with EoE211. Our observation of elevated SLC9A3 and NHE3 expression in EoE tissue samples and in both primary esophageal epithelial and EPC2‐ALI cultures following IL‐13 stimulation was surprising given that SLC9A3/NHE3 expression and function is predominantly associated with induction of Th1 proinflammatory cytokines, including IFN‐ and TNF305.
IFN‐ and TNF are thought to modulate SLC9A3 expression by PKA‐mediated phosphorylation of Sp1 and
Sp3 transcription factors305. TNF has also been shown to alter NHE3 activity by stimulating PKCa‐
64 dependent internalization of NHE3306. Stimulation of EPC2‐ALI cultures with other pro‐Type 2 cytokines such as IL‐25 and IL‐33 did not lead to induction of SLC9A3/NHE3 mRNA expression. Notably, a recent study in kidney and intestinal epithelial cells (Caco‐2) reported a STAT3‐dependent increase in SLC9A3 expression through the recruitment of transcriptional factor Sp1 and Sp3307. IL‐13 is known to signal through the JAK/STAT pathway, in particular through a STAT3‐ and STAT6‐dependent signaling pathway304. We reveal that STAT6 signaling plays a significant role in IL‐13–induced SLC9A3 expression in
EPC2‐ALI cells. Examining the SLC9A3 promoter did not reveal the presence of STAT6 gamma‐interferon‐ activation site (GAS) elements, suggesting that STAT6 may indirectly modulate SLC9A3 expression.
Notably, there are recent reports of IL‐13–induced, STAT6‐dependent activation of EGR1 signaling pathways in EoE308,309, and previous studies in intestinal epithelial cells have revealed that overexpression of EGR1 promotes SLC9A3/NHE3 expression and activity310. We are currently further pursuing the molecular basis of IL‐13 transcriptional regulation of SLC9A3 expression.
SLC9A3, as a member of the Na+/H+ exchanger family, drives Na+‐dependent extrusion of H+ and is
256,257,311,312 primarily involved in the regulation of [pH]i and acid protective mechanisms . A consequence of
Na+‐dependent acid extrusion in a multilayered stratified epithelium such as the esophageal epithelium is acidification of the intercellular spaces313. In well‐perfused tissues where there are short diffusion distances and good cell‐to‐capillary diffusive coupling, the acid is rapidly buffered by phosphates, proteins,
‐314 and HCO3 . However, in the stratified epithelium that is undergoing rapid and sustained cellular proliferation, the diffusion distances are increased leading to often‐inadequate capillary perfusion, which limits the capacity of the intercellular acid‐protective mechanisms and neutralization of the acidified intercellular spaces. The accumulation of acid [H+] in the intercellular spaces permits the formation of an electrochemical gradient and Cl‐ diffusion, creating an osmotic force for water flux and dilation of the intercellular spaces315,316. In EoE, there is significant esophageal epithelial basal zone (BZ) expansion, and
65 the basal cell layer can exceed 15% of the total epithelial thickness317. We speculate that the esophageal proliferative response and the thickening of the suprabasal layer of the esophageal epithelium in EoE increase the diffusion distances, thus causing a loss in the intercellular acid‐protective mechanisms and leading to DIS. Consistent with the concept of esophageal epithelial intercellular acid as a primary driver for DIS in EoE, luminal acid has been shown to drive acidification of the intercellular spaces and DIS in non‐ erosive reflux disease314‐316,318. Further, support for this concept, a recent study reports a strong positive correlation between BZH and DIS (r2 ≥ 0.67) in both proximal and distal biopsy samples from pediatric patients with EoE319. Interestingly, the increased esophageal intercellular acid in non‐erosive reflux disease is thought to activate afferent neurons (nociceptors) within the esophageal epithelium leading to the development of heartburn318,320. Intriguingly, though not common, EoE is also associated with the development of heartburn4.
SLC9A3’s role in the regulation of [pH]i may not simply in response to dysregulation of [pH]i but also in part fulfilling a larger role in the regulation of esophageal epithelial proliferation. Moreover, intracellular pH plays an important role in many cellular functions including proliferation321 and apoptosis322. In tumor cells, the [pH]i is often elevated as compared to normal cells, and it is thought that the alkaline [pH]i provides an optimal environment for DNA synthesis relative to enzyme function323. Consistent with this, growth factors such as EGF and PDGF stimulate a rapid rise in [pH]i that is a critical requirement for entry of mitogen‐stimulated quiescent cells into the S phase of the cell cycle324,325. Experimental studies have identified an important role for NHE family members in the growth factor‐induced increase in [pH]i and cellular proliferation326,327. The pharmacologic abrogation of mitogen‐stimulated, Na+‐dependent
+ extrusion of H and increase in [pH]i‐inhibited growth factor‐induced mouse bone marrow‐derived macrophage DNA synthesis and prevents progression into the S phase326. Furthermore, the rapid and transient mitogen‐induced increase in NHE1 activity and [pH]i during G2/M entry and transition is ablated
66
327 in NHE1 mutant fibroblasts . Notably, increasing the [pH]i in the absence of NHE1 activity was sufficient to restore CDC2 activity and cyclin B1 expression and to promote G2/M entry and transition and cellular proliferation, indicating that the NHE1‐driven increase in [pH]i at the completion of S phase is an important
327 checkpoint for progression to G2 and mitosis . We speculate that IL‐13 induction of SLC9A3/NHE3 may be a critical requirement for esophageal epithelial cell proliferation via regulating [pH]i. Notably, we have previously demonstrated that overexpression of IL‐13 in mice leads to esophageal cell proliferation207.
Herein, we show co‐localization of NHE3 expression within the esophageal basal proliferative zone in EoE esophageal biopsy samples and IL‐13–treated EPC2‐ALI cultures. Furthermore, we show that stimulating
EPC2‐ALI cells with IL‐13 induces SLC9A3, but not SLC9A1, expression and that treating mature EPC2‐ALI cells with the pan NHE inhibitor ethylisopropyl amiloride (EIPA) attenuated IL‐13–induced proliferation
(Fig. 2‐8). These findings support the necessity of an NHE in IL‐13–induced proliferation in an esophageal epithelial model system in vitro and given the overexpression, localization, and function of NHE3, we conjecture that [pH]i balance and regulation is a critical component of the observed proliferative effect that is induced by IL‐13 and mediated by an NHE in esophageal epithelial cells.
+ + Multiple compensatory mechanisms regulate [pH]i in mammalian cells and involve Na /H exchangers,
‐ + + 328,329 HCO3 transporters, lactate‐H transporters and vacuolar H ‐ATPase . Our gene ontology analysis supports dysregulation of these mechanisms in EoE, identifying that 5 out of the 50 individual GO nodes generated from genes significantly dysregulated in EoE are tightly correlated with the transmembrane ion transport activity. Notably, several of the most dysregulated genes were part of the [pH]i regulatory
‐ ‐ circuit, including Cl /HCO3 exchangers (SLC26A4, SLC4A2, SLC4A8) and carbonic anhydrases (CA2). These functional analyses support the concept of [pH]i pathways being active in the primary esophageal epithelial cells in EoE.
67
The significant decrease in DIS with steroid therapy or elimination diet in patients with EoE being associated with symptom improvement292 indicates an association between DIS and the etiology of EoE.
We demonstrate a role for SLC9A3/NHE3 and Na+/H+ exchange in DIS formation, one of the histopathologic manifestations of EoE. Given our observations, one would predict that utilizing NHE3 antagonists (systemically or topically) may be a therapeutic approach for reducing DIS and thus normalize associated esophageal caliber dimensions in EoE. Notably, an NHE3‐specific inhibitor, tenapanor, is in phase 3 clinical trials for the treatment of cardiorenal and gastrointestinal disease330. Tenapanor has been
330 shown to reduce sodium uptake, resulting in a reduction in [pH]i . Given the contribution of DIS to esophageal barrier dysfunction and facilitating food allergen exposure, considering the potential usage of
NHE3 inhibitors for EoE is warranted.
In summary, we identified a relationship between SLC9A3/NHE3 expression and activity with DIS in EoE.
Mechanistically, we show that IL‐13 stimulates SLC9A3 expression and NHE3 activity (via [pH]i) and that these were associated with esophageal epithelial DIS. Inhibiting NHE activity attenuated esophageal epithelial DIS formation, providing a rationale for the therapeutic utilization of NHE3 antagonists for reducing DIS and DIS‐associated esophageal pathophysiologic manifestations in EoE.
68
2.7 Figures
Figure 2‐1. SLC9A3 is the most upregulated transmembrane transporter activity gene in the EoE transcriptome, and levels correlate with EoE severity and DIS
69
70
Figure 2‐1. SLC9A3 is the most upregulated transmembrane transporter activity gene in the EoE transcriptome, and levels correlate with EoE severity and DIS. A) GO nodes associated with transmembrane transporter activity‐related genes identified by Gene Ontology (GO) enrichment analysis of 1610 dysregulated genes from RNA sequencing. B) Heatmap depicting the expression level of
62 individual genes within the transmembrane transporter activity GO nodes. C) Individual FPKM values of SLC9A3 and D) Heatmap depicting expression level of SLC9A1‐9, E) Correlation analysis of SLC9A3 or
SLC9A1 expression and matched peak distal eosinophil counts/HPF in esophageal biopsies (NL = 6; EoE =
10). F) qPCR analysis of SLC9A3 expression and G) Spearman correlation relating SLC9A3 expression and eosinophil count/HPF in an independent validation cohort (NL = 10; EoE = 10). H) Immunofluorescence staining of esophageal biopsy sections from NL (top panel) and patients with EoE (lower panel). NHE3
(red), CK13 (green) and nuclei (DAPI, blue) are shown. Images are representative of 7 patients per group.
Magnification x40. I) Spearman correlation between SLC9A3 or SLC9A1 expression and percentage of dilated intercellular spaces (DIS) in esophageal biopsies from patients with active EoE (n = 8). (C, F) Data are represented as the average ± S.E.M. (E, G, I) Data are presented as relative expression over 18S. (C,
E‐G, I) Individual symbols represent an individual patient **p < 0.01, ***p < 0.001.
71
Figure 2‐2. Increased SLC9A3/NHE3 expression and activity in primary esophageal epithelial cells derived from EoE biopsy in response to IL‐13.
72
Figure 2‐2. Increased SLC9A3/NHE3 expression and activity in primary esophageal epithelial cells derived from EoE biopsy in response to IL‐13. A) qPCR analysis of SLC9A3, B) Baseline intercellular pH
+ (pHi), C) Representative curves of pHi change over time and D) Na ‐dependent pHi recovery rate of cells derived from biopsies of patients with active EoE (n = 6) treated with vehicle (Veh) or IL‐13. E) Spearman
+ correlation analysis comparing the SLC9A3 expression level and Na ‐dependent pHi recovery rate (n = 5 individual patients stimulated with vehicle and IL‐13). F) Percentage of S3226‐sensitive Na+‐dependent intracellular recovery rate in cells treated with vehicle or IL‐13. See Material and Methods for a detailed protocol. B) Data represent the average pHi ± S.E.M of cells from patients with active EoE (n = 8). C)
Each data point represents the average value of 40 individual cells from 2 individual experiments. Data points for D) represent the average value of 120 individual cells from 6 different patients with active EoE and F) 50 individual cells from 5 different patients with active EoE. (A‐B, D, F) Data are represented as the average ± S.E.M. *p < 0.05, **p < 0.01, ****p < 0.0001.
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Figure 2‐3. Mature stratified squamous esophageal epithelium model using EPC‐ALI culture system.
74
Figure 2‐3. Mature stratified squamous esophageal epithelium model using EPC‐ALI culture system. A)
Schematic diagram of the esophageal epithelium (EPC2) air‐liquid interface (ALI) (EPC2‐ALI) differentiation protocol (See Material and Methods). B) Gene expression change of IL‐13–stimulated
EPC2‐ALI versus non‐treated EPC2‐ALI compared to that of patients with active EoE versus normal control (NL) patients. Fold changes were calculated from RNA sequencing of EPC2‐ALI and patient samples. Spearman correlation analysis was applied to analyze these 23660 genes. C) Gene ontology analysis of 572 genes that were significantly dysregulated by IL‐13 treatment of EPC2‐ALI cells (Fold change > 2, p < 0.05) identified 6 GO nodes related to transmembrane transporter activity. D) Heatmap depicting expression level of 46 individual genes within the transmembrane transporter activity GO nodes that are significantly dysregulated in EPC2‐ALI cells following IL‐13 stimulation. E) RPKM value is indicating SLC9A3 expression level in empty control (CTRL), STAT3 lentiviral knockdown (STAT3KD) and
STAT6 lentiviral knockdown (STAT6KD) EPC2‐ALI cultures treated with vehicle (Veh) or IL‐13; n = 3 per treatment. **padj < 0.01, ***padj < 0.001.
75
Figure 2‐4. Increased SLC9A3/NHE3 expression and activity in IL‐13–stimulated EPC2‐ALI.
76
Figure 2‐4. Increased SLC9A3/NHE3 expression and activity in IL‐13–stimulated EPC2‐ALI. A) qPCR, B)
Western blot analysis and C) Western blot quantification of SLC9A3/NHE3 expression in EPC2‐ALI following a 72 h–treatment with vehicle (Veh) or IL‐13 (100 ng/mL). D) Immunofluorescence staining of the vehicle (top panel) or 100 ng/mL of IL‐13 (lower panel) stimulated EPC2‐ALI cultures. NHE3 (red) and nuclei (blue) are shown. Images are presentative of 3 samples per group. Magnification X400 E)
Schematic view of the pH‐STAT assay. See Material and Methods for a detailed protocol. F) Baseline extracellular pH (pHe) of EPC2‐ALI cells treated with vehicle or IL‐13 for 72 h. DMSO (0.1%) or S3226 (30 mM) was added to both sides of the solution during the experiment (n = 6‐11 samples per group from 5 individual experiments). G) Amount of Ba(OH)2 injection over time and H) Ba(OH)2 injection rate measured for EPC2‐ALI cells treated with vehicle or IL‐13 (100 ng/mL) for 72 h. DMSO (0.1%) or S3226
(30 mM) is added to both sides of the solution during the experiment (n = 6‐11 samples per group from
5 individual experiments). (A, C, F, H) Data are represented as the average ± S.E.M. *p < 0.05; n.s. not significant
77
Figure 2‐5. Blockade of NHE3 protected EPC2‐ALI from IL‐13–induced dilated intercellular spaces (DIS).
78
Figure 2‐5. Blockade of NHE3 protected EPC2‐ALI from IL‐13–induced dilated intercellular spaces (DIS).
A) H&E staining and B) electron microscopy of EPC2‐ALI cultures stimulated with vehicle or IL‐13 (100 ng/mL) for 72 h and showing DIS (black arrows) in only IL‐13–treated cells. C) H&E staining of EPC2‐ALI cultures stimulated with vehicle or IL‐13 (100 ng/mL) in the presence and absence of S3226 (30 mM) for
72 h. D) DIS formation (% of total area) was quantitated by morphometric analyses and expressed as the mean ± S.E.M; n = 3 independent experiments; ****p < 0.0001. Magnification A) X200, B) X5000 and C)
X300.
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Figure 2‐6. Demographics of patient cohorts examined in RNAseq analysis or qPCR.
Biopsy set 1 (Cohort 1): RNAseq cohort (NL=6, EoE=10)
Age Male Female Max Eosinophils per high powered field (hpf)
NL 12.7 ± 6.0 50% 50% 0 ± 0 EoE 15.1 ± 12.4 60% 40% 163.6 ± 92.4
Biopsy set 2 (Cohort 2): qPCR cohort (NL=10, EoE=10)
Age Male Female Max Eosinophils per high powered field (hpf)
NL 9.7 ± 3.1 50% 50% 0 ± 0 EoE 10.7 ± 5.3 80% 20% 124. 4 ± 73.5
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Figure 2‐6. Patient characteristics of EoE and control patients in cohort 1 and cohort 2. Description of the age of biopsy collected, gender percentage and maximum eosinophils per high power field of the patient in cohort 1 (Biopsy set 1) and 2 (Biopsy set 2).
81
Figure 2‐7. GO analysis of dysregulated genes in EoE patients
82
Figure 2‐7. The 1610 genes that were identified to be specifically dysregulated by RNA sequencing of esophageal biopsies from patients with eosinophilic esophagitis (EoE) (n = 10) and normal controls (NL)
(n = 6) underwent gene ontology (GO) enrichment analysis and were clustered into 50 individual GO nodes per their molecular function (with FDR‐corrected p < 0.05). GO enrichment based on molecular function is shown, 50 individual GO nodes are identified with FDR B&H p < 0.05. Blue indicates the total number of genes as part of the GO annotation; red indicates number of genes common between EoE transcriptome and GO node. Red line indicates FDR B and H p‐value cutoff; orange dotted line indicates
FDR B and Y p‐value; light green dotted line indicates FDR B and H p‐value. Figure is generated by
Toppgene.
83
Figure 2‐8. IL‐13–induced proliferation in EPC2‐ALI is attenuated by NHE3 inhibitor EIPA.
84
Figure 2‐8. IL‐13–induced proliferation in EPC2‐ALI is attenuated by NHE3 inhibitor EIPA. A) EPC2‐ALI cultures treated with the four combinations of vehicle (Veh) or IL‐13 (100 ng/mL) with DMSO or EIPA (20
M) for 72 h and stained for BrdU incorporation and DAPI (nuclei). B) Quantification of BrdU‐positive cells in EPC2‐ALI cultures. Data are presented as mean ± SEM; n = 6 per treatment. ****p < 0.0001.
Magnification X300.
85
Table 2‐1. Expression level of 572 genes dysregulated in mature EPC2‐ALI by IL‐13.
gene_id 48hr Veh 48hr IL‐13 LOGFC pvalue NTRK1 0 12.8962 12.0048 7.72E‐25 ATP5J2‐PTCD1 0 2.89614 10.92178 4.59E‐15 TREML2 0 1.59001 9.500339 1.66E‐07 FGF19 0 2.40941 9.285919 9.07E‐07 DYX1C1‐CCPG1 0 0.996634 9.175073 2.04E‐06 PPT2‐EGFL8 0 1.44493 8.900974 1.26E‐05 TNFAIP6 0.417235 183.29 8.662212 2.90E‐45 ARPC4‐TTLL3 0 0.708783 8.407443 0.000188 ZNF20 0 0.923428 8.407443 0.000188 CSF3 0 1.32101 7.949604 0.001337 SHC4 0 0.446474 7.949604 0.001337 C17orf61‐ PLSCR3 0 0.896016 7.757792 0.002667 FAM26D 0 1.11879 7.65139 0.003799 C8orf44‐SGK3 0 0.348911 7.595093 0.004545 NELL2 0 0.479493 7.475448 0.006542 SERPINB4 1.15211 214.884 7.426299 6.27E‐40 SLC5A5 0 0.337742 7.201519 0.013904 MAOB 0 0.391933 6.955428 0.025126 CCDC103 0 0.597608 6.863093 0.030786 LRRC31 0 0.386815 6.863093 0.030786 CADM2 0 0.10635 6.764443 0.037847 CCL26 0.37199 21.8657 6.751451 7.30E‐11 ALOX15 0 0.307886 6.658548 0.046698 TMEM191A 0 0.422046 6.658548 0.046698 FAM26E 0.111923 8.87311 6.192024 1.08E‐19 ANO1 0.233214 10.1932 5.662446 8.53E‐23 CAPN14 11.5813 546.537 5.443614 5.67E‐15 LBH 0.337045 13.5442 5.211747 1.96E‐19 DPP4 0.964848 23.7153 4.502532 9.19E‐22 BCL2L15 0.0199123 0.48573 4.491584 0.002442 FCGBP 0.0120834 0.259852 4.309746 0.000157 OSR1 0.104566 2.08085 4.197853 0.000294 C1QTNF1 0.457086 7.90129 4.14251 9.17E‐12 SOCS1 0.488605 8.46859 3.998544 3.81E‐07 EML5 0.0828536 1.35625 3.916082 8.80E‐07 SLCO2A1 0.0937948 1.35472 3.735509 8.01E‐05 GLDC 0.0519947 0.734296 3.703088 0.003112 NFE2 0.17579 1.58641 3.601208 0.004677 LMO2 0.1237 0.836398 3.565584 0.04179
86
TMEM176A 0.0953987 1.16355 3.491584 0.049478 PSMC3IP 0.257843 1.37932 3.197853 0.018798 CDH26 2.91177 25.124 3.134618 4.55E‐13 CISH 4.78419 44.7131 3.125573 2.49E‐13 EDNRA 0.400461 3.44915 3.098317 1.49E‐06 TMPRSS2 0.184351 1.71595 3.084959 0.000545 S100A7A 0.0694255 0.638786 3.084959 0.008248 ATP13A5 0.108312 0.973403 3.051011 0.003533 EDAR 0.140993 1.22186 2.998544 0.000899 ROR1 0.0496481 0.490605 2.944096 0.038502 KRT27 0.184288 1.49847 2.906621 0.015563 KAL1 0.0940993 0.745001 2.868147 0.00182 C16orf59 0.182476 1.36661 2.787977 0.02287 PDE6A 0.0526536 0.371801 2.703088 0.02961 TNC 16.9492 119.52 2.701126 3.33E‐07 LYPD6B 2.51809 17.6597 2.693218 7.44E‐08 CYP1B1 3.28606 22.9077 2.684562 7.77E‐11 FAM115C 0.222967 1.49912 2.663195 0.000713 SUSD2 0.404433 2.73598 2.641249 0.000245 NBL1 2.58312 17.0047 2.63068 2.78E‐08 FETUB 4.44299 29.8103 2.629371 3.67E‐09 UGT2B17 0.143098 0.918593 2.565584 0.043728 SECTM1 1.82253 11.5601 2.548306 3.63E‐07 IL32 0.438112 2.78624 2.529059 0.025765 LURAP1L 5.66902 34.0519 2.469724 2.17E‐09 RASGRP1 1.29847 7.50046 2.45311 9.42E‐08 PRICKLE4 1.92513 11.1222 2.413581 1.09E‐05 MB 0.874594 4.3936 2.395659 0.004687 MKX 0.27223 1.55531 2.38116 0.003579 GLI1 0.125642 0.631713 2.372939 0.041457 UNC93A 0.281245 1.32395 2.347993 0.027743 ST6GAL1 2.75556 14.4354 2.287356 4.93E‐08 ANKRD33B 0.604775 3.16028 2.268747 1.91E‐06 RNF122 0.424085 2.2119 2.266024 0.01191 TGM3 5.88151 30.6317 2.263929 3.70E‐08 IL2RG 0.322764 1.65754 2.243656 0.038637 SERPINB3 177.206 904.027 2.234098 0.000169 ABLIM2 0.583637 2.64277 2.209441 0.001872 GPC6 0.292805 1.44999 2.191189 0.000726 NPNT 0.438648 2.14552 2.188515 0.000906 SLC7A4 0.256871 1.26419 2.182256 0.032189 SLC26A2 2.39129 11.1783 2.108001 2.06E‐07 LOC645638 23.5999 110.244 2.107006 3.56E‐07
87
PPYR1 0.405006 1.8849 2.101637 0.022824 CTSC 86.6005 389.937 2.090948 5.58E‐05 DNER 0.423696 1.92332 2.065658 0.006451 HECW2 0.157272 0.67917 1.993678 0.01682 LINC00476 0.562423 2.07078 1.959863 0.048518 MAP3K14 2.10046 8.56304 1.920289 1.37E‐05 AKAP2 3.79869 15.4173 1.90406 1.94E‐06 CHI3L2 1.64993 6.31169 1.900004 0.003662 LOX 0.400767 1.55909 1.899008 0.005648 GCNT3 19.0739 76.0969 1.8794 2.98E‐06 PTCHD4 1.25931 4.84785 1.85371 0.000866 HS3ST1 3.88032 14.8807 1.82236 7.76E‐05 DPYD 1.37435 4.88437 1.815443 0.00029 PRR9 3.30348 12.4651 1.798994 0.001009 SEMA3A 0.22748 0.853696 1.791144 0.026732 NMI 3.21376 12.0343 1.787977 0.000618 ALDH9A1 18.1412 67.8892 1.787077 8.53E‐06 LAMP3 1.24484 4.60435 1.770202 0.00116 CFB 0.678504 2.49234 1.760232 0.015186 PDCD1LG2 0.73715 2.70776 1.760232 0.015186 TGM2 1.99162 7.11691 1.751657 0.000158 DIO2 2.24815 8.1191 1.724783 3.73E‐05 SFRP1 7.29323 25.9703 1.715393 1.57E‐05 SLC16A14 0.382766 1.34418 1.695352 0.023025 RPTN 130.765 456.312 1.686209 0.005013 ZNF860 0.432419 1.48748 1.66512 0.037828 C1S 0.619963 1.97069 1.653047 0.03097 SLC9A3 3.1023 10.3236 1.617697 0.000391 PKD2 4.13331 13.1789 1.556026 0.000116 CLGN 0.831392 2.55356 1.552409 0.025808 SCLY 1.12276 3.49355 1.544826 0.016068 ADAM8 3.94848 12.3019 1.529059 0.000355 PPP1R3C 2.694 8.4245 1.528001 0.001579 SLC45A4 3.43181 10.5442 1.502575 0.00055 ID3 22.8578 69.6206 1.48999 0.000173 PHF17 1.05748 3.16967 1.48615 0.004017 PAPSS2 0.927162 2.72897 1.471925 0.014292 CDC45 1.45115 4.32246 1.466049 0.025636 EHD3 5.62808 16.7135 1.453461 0.000308 CDC6 0.746004 2.20703 1.448015 0.037464 MUTED‐TXNDC5 2.10105 6.20834 1.447056 0.002885 PPP1R3D 12.3208 36.1941 1.438288 0.000267 SLC16A9 0.620917 1.78567 1.407155 0.038559
88
NUDT3 1.61044 4.5133 1.396608 0.01802 BTBD3 6.46411 18.8548 1.395777 0.000416 SDSL 1.84296 5.18726 1.376107 0.041176 PGBD5 1.62383 4.56755 1.375186 0.008955 MME 9.26011 25.6182 1.355959 0.000607 ST8SIA1 0.417951 1.15826 1.35371 0.019406 TNFRSF19 0.992628 2.7798 1.345536 0.020287 TRIM16L 3.04766 8.33872 1.335287 0.009187 PDZK1IP1 30.0173 81.8403 1.330179 0.000825 NCF1 4.03745 11.0037 1.32963 0.011062 MAOA 11.7049 31.5023 1.311863 0.000988 PPARGC1B 1.25664 3.36902 1.309424 0.002308 CA2 345.751 919.198 1.293803 0.03045 IRF1 2.47978 6.4926 1.271749 0.006422 BARD1 1.48879 3.89634 1.271137 0.032774 PP7080 8.46504 21.9548 1.258108 0.002991 CALCRL 0.753184 1.9467 1.253117 0.037427 NNMT 4.89472 12.5683 1.243656 0.010111 CAT 22.1727 56.8231 1.240852 0.001614 EEF1E1‐MUTED 2.22758 5.6761 1.238263 0.01409 LTA4H 75.9864 193.943 1.235562 0.00523 ITPRIPL2 5.03939 12.7608 1.217592 0.001929 CANT1 23.4506 59.2563 1.214236 0.002587 SAA1 15.5576 39.0059 1.212913 0.011045 FAM217B 7.83605 19.6528 1.21259 0.001995 SGK1 24.1875 60.2575 1.20568 0.002264 DDX58 1.31171 3.27457 1.203014 0.019629 PIK3R3 1.39546 3.36954 1.196038 0.014162 MSRB1 7.53364 18.6003 1.187073 0.008886 ORAI1 7.44987 18.2405 1.175021 0.008484 CDCA5 1.61452 3.94824 1.173267 0.046253 NINJ1 12.9094 31.4112 1.166014 0.005354 SPSB1 5.59134 13.6025 1.165768 0.005048 ENTPD2 5.09431 12.1684 1.161602 0.010615 NPDC1 3.36992 8.11224 1.150547 0.036634 GPRIN2 4.34553 10.391 1.140897 0.018744 BIRC3 0.931928 2.36181 1.122155 0.02956 PARP12 3.29122 7.76348 1.121245 0.010631 CD14 5.92275 13.752 1.119566 0.01736 DUOX2 1.93491 4.53761 1.112827 0.011514 GJA9‐MYCBP 2.64351 6.47864 1.111019 0.025506 MFHAS1 2.92078 6.82238 1.107083 0.009092 RUNX2 1.15696 2.67917 1.106153 0.031511
89
ASB1 6.79675 15.787 1.098983 0.004983 NANOS1 2.05453 4.57482 1.038072 0.026567 LRP12 6.96458 15.3995 1.035181 0.009178 TMTC3 6.61259 14.676 1.033336 0.008207 RAB27A 3.6526 8.01938 1.027928 0.01993 VWF 2.3094 5.08792 1.022722 0.012611 ALOX15B 6.9515 14.6907 1.021935 0.014344 CST3 193.576 423.969 1.014224 0.01734 TXN 124.236 269.499 1.001292 0.014368 SLC22A23 33.8029 18.2844 ‐1.0035 0.013808 ID1 69.9144 37.4873 ‐1.00924 0.010038 WNT7A 15.2522 8.19617 ‐1.01284 0.020001 TNS1 1.35874 0.72994 ‐1.01325 0.044735 ANXA9 50.8149 27.2877 ‐1.01384 0.009358 ITGB6 15.0787 8.08837 ‐1.01543 0.014282 NLRX1 22.6021 12.1086 ‐1.01668 0.009196 WWC1 12.1696 6.52788 ‐1.01798 0.009139 C6orf15 56.5726 30.2725 ‐1.01893 0.009637 LDLR 93.8748 49.8872 ‐1.01944 0.026684 PLEKHA6 1.74968 0.934552 ‐1.02158 0.047762 CLIC3 132.641 70.8381 ‐1.02176 0.008955 GBA 35.577 18.5995 ‐1.022 0.008824 MANSC1 18.9684 10.1286 ‐1.022 0.011745 POF1B 67.1948 35.7603 ‐1.02683 0.014242 S100A10 407.64 216.368 ‐1.03065 0.022404 PPP1R12B 3.05278 1.72252 ‐1.03402 0.015542 PIM1 77.64 41.1106 ‐1.03412 0.011265 KLK13 18.4194 9.75228 ‐1.03425 0.020384 AKR1C2 9.38037 5.25946 ‐1.0344 0.018703 RNF222 6.36259 3.35871 ‐1.03855 0.024965 CTGF 6.55088 3.44557 ‐1.04378 0.034145 LCE3E 493.985 259.781 ‐1.04401 0.014283 LPIN1 19.0839 9.85132 ‐1.0449 0.007464 PPARD 43.0156 22.4969 ‐1.04655 0.008629 IRS1 10.6324 5.581 ‐1.0467 0.007332 KIAA1239 1.58926 0.833159 ‐1.04852 0.048315 TP53INP2 36.9779 19.3598 ‐1.05044 0.008195 ARHGEF4 72.9352 38.0041 ‐1.05271 0.012202 WNT10A 8.63071 4.49651 ‐1.05751 0.021095 TUFT1 24.4823 12.7784 ‐1.05765 0.006907 NFASC 3.93257 2.17929 ‐1.0605 0.045469 LRP1 10.7551 5.58044 ‐1.0634 0.007641 GRB7 26.5239 13.6355 ‐1.06389 0.007208
90
AIM1L 28.2062 14.6282 ‐1.0641 0.007288 TIMP3 6.37175 3.30232 ‐1.06505 0.010695 TCP11L2 16.251 8.39722 ‐1.06938 0.010085 SPIN4 5.27389 2.72075 ‐1.0717 0.018055 C9orf169 56.4704 29.1204 ‐1.0723 0.00863 TPRG1 8.4483 4.33235 ‐1.07711 0.011312 SYT8 58.2452 29.7508 ‐1.08605 0.005476 WFDC5 102.818 52.3894 ‐1.08958 0.005332 UGCG 29.0211 14.7322 ‐1.09497 0.006647 ARID5B 8.17082 3.89986 ‐1.10212 0.00538 SRPX2 6.14943 3.10577 ‐1.10234 0.031874 DUSP1 28.67 14.4784 ‐1.10248 0.005485 IGFBP3 4.24878 2.15771 ‐1.10427 0.042708 NCCRP1 96.0627 48.3596 ‐1.10701 0.006143 KAT2B 20.1373 10.0805 ‐1.11514 0.004334 CPA4 21.3155 10.6498 ‐1.11755 0.00481 PLEKHM1P 5.35447 2.6758 ‐1.11761 0.023359 KRT2 5.53779 2.76491 ‐1.11891 0.029953 SLC39A2 15.204 7.5445 ‐1.11891 0.01414 SERPINB1 67.5999 33.7513 ‐1.11891 0.004216 PLK3 28.2309 14.0973 ‐1.12076 0.004459 MYOM1 1.74526 0.868805 ‐1.12744 0.048521 GLRX 33.8623 17.0859 ‐1.12831 0.006211 AADACL2 7.19573 3.55973 ‐1.13221 0.03983 CCDC88B 3.32092 1.64085 ‐1.13398 0.02007 RORA 3.55452 1.75489 ‐1.1341 0.006191 KLK5 118.993 58.9909 ‐1.13451 0.00456 CAMK2N1 31.8426 15.7092 ‐1.13619 0.003789 HS3ST6 15.2918 7.54181 ‐1.13662 0.020009 HMOX1 53.996 26.626 ‐1.13685 0.003664 KIFC3 25.733 12.6092 ‐1.14554 0.003418 KIAA1199 3.66444 1.79567 ‐1.14591 0.009087 MYLK 2.73077 1.06278 ‐1.14919 0.017331 OTUB2 3.42608 1.6741 ‐1.15001 0.026372 ASPRV1 27.1928 13.2455 ‐1.15456 0.003652 SLC37A2 57.6277 28.055 ‐1.15551 0.005243 OSBP2 6.79934 3.29551 ‐1.16173 0.006903 SH3D21 4.31603 2.08801 ‐1.16198 0.039352 PADI3 3.88268 1.8743 ‐1.16754 0.027452 FBXO32 4.16856 2.02648 ‐1.16969 0.006959 IFITM1 20.4401 9.84087 ‐1.17138 0.020119 TRIB2 5.63118 2.47007 ‐1.17138 0.00959 LRRC20 26.212 12.6197 ‐1.17196 0.002779
91
LGALSL 60.4183 28.9347 ‐1.17902 0.004201 MAP3K9 7.74508 3.68914 ‐1.18683 0.003677 INHBA 11.2455 5.34837 ‐1.18901 0.007588 GDPD3 26.4244 12.5628 ‐1.18955 0.005881 GLTP 345.911 163.935 ‐1.19411 0.021022 IMPA2 60.4967 28.6608 ‐1.19461 0.002277 TNFAIP8L3 10.3497 4.89841 ‐1.19604 0.007817 PADI1 26.8216 12.6902 ‐1.19652 0.002251 CALML5 116.432 54.9315 ‐1.20062 0.002173 DUSP16 27.5642 12.9913 ‐1.20209 0.002739 SLC6A6 2.30587 1.27922 ‐1.2038 0.016779 SCNN1B 26.2716 12.2874 ‐1.21316 0.002117 PTPRH 2.52051 1.18113 ‐1.21578 0.033522 SRPX 4.87309 2.2492 ‐1.21578 0.040778 SASH1 17.2383 8.03138 ‐1.21873 0.002016 FOSL1 13.5356 6.28807 ‐1.22291 0.007033 DUSP6 46.8348 21.7271 ‐1.22541 0.0019 GJB4 4.39331 2.03682 ‐1.22583 0.020898 PCSK5 3.59228 1.37804 ‐1.22697 0.009194 SPARC 35.0307 16.1307 ‐1.23565 0.001705 NFATC2 2.56734 1.15494 ‐1.23715 0.009305 KRT79 5.7037 2.61528 ‐1.24177 0.021659 TMPRSS13 19.267 9.35643 ‐1.24444 0.001936 SH3TC2 0.648238 0.296549 ‐1.24509 0.010234 LIPN 18.1171 8.28438 ‐1.24573 0.006581 TINAGL1 50.7916 23.1182 ‐1.25105 0.001422 UCA1 5.30638 2.4174 ‐1.25111 0.019222 KRT78 223.834 101.964 ‐1.25121 0.004783 NDRG1 447.095 203.287 ‐1.25432 0.03493 LIPG 8.05869 3.65343 ‐1.25813 0.003009 FLG2 90.5457 41.0166 ‐1.25928 0.01509 C10orf116 70.1305 31.7259 ‐1.26011 0.001932 MALL 21.6099 9.78108 ‐1.26046 0.001456 TNFSF9 8.20738 3.70328 ‐1.26496 0.014626 LYNX1 1381.19 620.621 ‐1.26535 0.014294 MBOAT2 38.2663 17.2118 ‐1.26952 0.001376 SPRR2G 1148.07 515.868 ‐1.27097 0.011861 IL6R 8.33919 3.67472 ‐1.27134 0.001737 MATN2 2.96072 1.33777 ‐1.28289 0.017476 PNPLA1 3.27661 1.49227 ‐1.28884 0.035963 SLURP1 306.661 135.656 ‐1.29352 0.001219 KIF13B 16.4814 7.27326 ‐1.297 0.001078 IL18 43.2525 19.0087 ‐1.30428 0.001285
92
S100A12 164.048 72.024 ‐1.30441 0.000931 ADSSL1 6.82477 2.959 ‐1.30568 0.016037 HSPA2 8.0106 3.51235 ‐1.30631 0.00434 H19 153.395 67.2493 ‐1.30681 0.002661 GLUL 195.441 86.4826 ‐1.3122 0.011754 EEPD1 1.58455 0.691679 ‐1.31274 0.03769 GABRE 14.8599 6.4333 ‐1.32463 0.001158 CRCT1 846.618 364.754 ‐1.33163 0.005276 RASAL1 2.18686 0.96451 ‐1.33254 0.030061 PEG10 5.88086 2.51982 ‐1.34131 0.001307 SPON2 6.26296 2.67543 ‐1.34131 0.014396 IL22RA1 13.2388 5.62271 ‐1.35228 0.001173 ARHGAP29 9.01501 3.82781 ‐1.35265 0.000591 FGFBP1 243.257 102.98 ‐1.35696 0.001617 TRPV3 3.71045 1.56017 ‐1.36041 0.002965 GLA 26.3971 11.1174 ‐1.36439 0.001144 GNAO1 1.86537 0.805934 ‐1.3664 0.013094 FAM49A 2.44451 1.02811 ‐1.3664 0.013094 B4GALNT3 27.6506 11.5929 ‐1.37091 0.000487 DNAJB5 8.80537 3.71198 ‐1.37432 0.0029 SPNS2 70.9613 29.6095 ‐1.37812 0.001225 PNLIPRP3 75.8125 31.6181 ‐1.37852 0.000605 DSG1 75.8771 31.5732 ‐1.3818 0.001931 SMAD3 19.8634 8.22074 ‐1.38942 0.000429 CDSN 85.951 35.5609 ‐1.39006 0.000703 PRSS22 16.7898 6.92537 ‐1.39446 0.002221 GRAMD1C 6.75636 2.82685 ‐1.39575 0.002047 CLCA4 28.7374 11.7299 ‐1.4042 0.000359 RUSC2 7.6773 3.09819 ‐1.4065 0.000671 IL37 8.52204 3.3792 ‐1.4117 0.036442 ARRB1 0.765803 0.313581 ‐1.41931 0.041956 BHLHE41 1.41463 0.571764 ‐1.42377 0.045085 SLC15A1 7.34683 2.96794 ‐1.4245 0.001871 THEM5 13.0476 5.26949 ‐1.42489 0.005312 MUC20 3.88438 1.56977 ‐1.42728 0.001667 SLC16A5 6.22436 2.50965 ‐1.42728 0.00898 CRLF1 2.79946 1.12757 ‐1.42877 0.048549 CDA 24.7818 9.87636 ‐1.44407 0.001515 OVOL2 5.79497 2.28922 ‐1.45678 0.015159 CSGALNACT1 3.14939 1.32582 ‐1.46089 0.006164 NOS3 1.46849 0.581862 ‐1.47094 0.029232 SPTSSB 16.5755 6.47796 ‐1.47228 0.000464 FOS 6.79126 2.65107 ‐1.47394 0.004294
93
FHDC1 7.86995 3.07043 ‐1.47475 0.00029 C1QTNF6 5.80248 2.26082 ‐1.48217 0.002758 MYCT1 3.02636 1.16981 ‐1.48815 0.012706 KLK9 16.5958 6.40971 ‐1.48932 0.001097 FAM43A 21.1272 8.12521 ‐1.49546 0.000179 ARNT2 1.08718 0.416799 ‐1.5 0.021107 MUC21 27.0311 10.3626 ‐1.50007 0.000142 SERPINA12 10.0417 3.82449 ‐1.50951 0.001381 CCBP2 1.63925 0.622782 ‐1.51307 0.040646 RAPGEF3 1.14625 0.439459 ‐1.51377 0.020679 CEACAM6 94.2063 35.6128 ‐1.52027 0.000251 SEMA7A 4.99729 1.96415 ‐1.52188 0.002253 SHF 9.78356 3.68687 ‐1.5248 0.000803 CEACAM7 13.8434 5.19308 ‐1.53138 0.000416 LGALS1 180.302 67.5538 ‐1.53314 0.000102 CTSL2 66.0985 24.758 ‐1.53416 0.000302 KRT80 157.066 58.3824 ‐1.54012 0.0013 CLEC2B 3.0125 1.12287 ‐1.54061 0.024653 ECM1 256.631 94.7383 ‐1.54175 0.00093 MLPH 1.80645 0.674938 ‐1.55281 0.020929 KLK14 4.71958 1.73925 ‐1.55703 0.030191 MIR210HG 4.42877 1.6285 ‐1.5602 0.007271 ESYT3 2.80571 1.02919 ‐1.5637 0.004155 3‐Mar 2.94486 1.07584 ‐1.56958 0.004315 RNF39 19.5417 7.22099 ‐1.57091 0.000187 GPR111 2.62724 0.954864 ‐1.57702 0.003123 SPTBN5 0.44769 0.162672 ‐1.57737 0.029351 ZNF555 2.06264 0.75378 ‐1.58822 0.00132 SLC6A14 20.5034 7.38176 ‐1.59067 5.75E‐05 SERPINB9 4.84382 1.73085 ‐1.6015 0.000816 SLC26A9 4.24966 1.55532 ‐1.6015 0.000816 SPRR2F 27.4731 9.78694 ‐1.60593 0.001105 EMP3 13.6173 4.83076 ‐1.61195 0.004343 SLC5A1 19.0366 6.73827 ‐1.61628 4.39E‐05 SDR9C7 21.8245 7.70723 ‐1.6185 0.000132 SLC13A4 1.64241 0.57109 ‐1.64087 0.029154 PSAPL1 8.71749 3.02892 ‐1.64194 9.47E‐05 NPR2 2.20855 0.769098 ‐1.64672 0.010917 HCG22 3.35319 1.15474 ‐1.65481 0.000682 CST6 46.1786 15.8656 ‐1.65816 0.000202 LINC00319 1.91319 0.656418 ‐1.66013 0.019003 ADRB2 6.93458 2.36585 ‐1.66829 0.001631 KRT15 325.81 110.793 ‐1.67301 0.000452
94
PGLYRP4 79.4136 26.8069 ‐1.68362 2.48E‐05 VSIG10L 46.2237 15.4332 ‐1.69944 2.46E‐05 FAM25A 67.8396 22.4766 ‐1.71054 0.000201 C21orf7 1.63799 0.541671 ‐1.71327 0.046707 HSPB8 9.24948 3.04815 ‐1.71828 0.00049 LCE2A 59.0463 19.4351 ‐1.72002 6.77E‐05 AGAP11 2.52904 0.828227 ‐1.72072 0.007186 HYAL1 5.38962 1.68809 ‐1.73081 0.00257 CNFN 721.862 235.606 ‐1.73218 8.08E‐05 LGALS9B 3.34594 1.07393 ‐1.75634 0.027117 C7orf57 3.04559 1.0273 ‐1.75634 0.011893 IFI6 8.68132 2.84889 ‐1.75634 0.008246 BDKRB1 6.01298 1.90553 ‐1.77472 0.005993 TCN1 4.48672 1.4198 ‐1.77681 0.007916 FCHSD1 21.9994 6.9484 ‐1.77909 7.66E‐06 SMPD3 24.6573 7.74526 ‐1.78747 7.27E‐06 FGD2 1.39837 0.438391 ‐1.79029 0.023567 CRISP3 5.88265 1.83092 ‐1.80074 0.001026 XKRX 7.95386 2.47489 ‐1.80113 0.000133 ATG9B 23.2719 7.24138 ‐1.80132 5.86E‐06 CSPG4 0.716699 0.222368 ‐1.80525 0.010854 PTCD1 3.72798 1.15905 ‐1.80597 0.000186 IL36RN 34.2847 10.6961 ‐1.81317 5.30E‐06 SPRR2C 35.6369 10.9695 ‐1.81672 9.38E‐05 MMP2 5.50197 1.69338 ‐1.82452 0.000205 RNASE7 124.077 37.8939 ‐1.82804 6.79E‐06 KLK6 11.6747 3.60692 ‐1.83077 0.000273 C18orf26 1.85639 0.56448 ‐1.83435 0.02679 ZNF365 4.17548 1.24415 ‐1.84488 0.000319 TGM5 9.61207 2.80098 ‐1.84871 5.95E‐05 SERPINE1 38.9626 11.7549 ‐1.85007 3.46E‐06 LIPM 29.2906 8.81263 ‐1.85165 1.14E‐05 RNF223 8.20786 2.43616 ‐1.86924 0.002448 GDA 5.94488 2.17955 ‐1.87109 2.20E‐05 IL23A 2.47085 0.732054 ‐1.87182 0.049354 FN1 27.64 8.17514 ‐1.88108 5.43E‐06 NKPD1 7.52134 2.21066 ‐1.88335 1.51E‐05 DLX2 2.53577 0.744922 ‐1.8841 0.008628 CXCR7 8.3463 2.45186 ‐1.8841 0.0002 IL1R2 3.08575 0.920779 ‐1.89385 0.016743 TMEM132B 0.378951 0.109048 ‐1.91388 0.037156 LCE2B 156.558 44.944 ‐1.91734 1.58E‐06 RHBG 1.49563 0.466034 ‐1.92627 0.041236
95
GPSM1 12.1397 3.21886 ‐1.92714 6.38E‐06 FLG 47.8845 13.589 ‐1.93395 5.26E‐05 SCNN1D 1.17016 0.343151 ‐1.93692 0.025845 LCE1A 198.523 56.1421 ‐1.93899 1.23E‐06 LCE1C 117.831 33.0181 ‐1.95223 1.18E‐06 LCE1B 68.3343 19.1426 ‐1.95266 1.24E‐06 S100A6 567.321 158.126 ‐1.95993 8.86E‐06 GSDMA 67.9073 18.5941 ‐1.98556 7.94E‐07 LOXL2 2.57306 0.700711 ‐2.0135 0.001041 EGFL8 3.67043 1.06475 ‐2.01938 0.009366 ST6GALNAC1 3.46428 0.924515 ‐2.02262 0.001429 KCTD4 5.18719 1.37993 ‐2.0272 0.000595 COX6B2 1.72681 0.458689 ‐2.02936 0.028748 ABCG4 2.162 0.598133 ‐2.04392 0.001671 ELMOD1 4.00286 1.0624 ‐2.04852 0.000383 FGF5 0.513723 0.144004 ‐2.0518 0.031587 CASP14 16.315 4.24587 ‐2.05891 8.41E‐07 ALDH3A1 16.1135 4.18195 ‐2.06589 5.90E‐06 LCE2D 93.3328 24.0071 ‐2.07576 5.34E‐07 IGLON5 0.79858 0.205054 ‐2.07827 0.034776 SLC47A2 6.67977 1.75362 ‐2.08299 0.000104 KIAA1644 0.998906 0.254606 ‐2.08892 0.002732 SULT1B1 3.63113 0.922664 ‐2.09338 0.007449 CHRNA9 2.65398 0.670299 ‐2.10212 0.003647 USP2 5.23575 1.45218 ‐2.10568 3.45E‐05 DHRS9 27.3434 6.86805 ‐2.11069 7.34E‐07 ACSBG1 1.23198 0.320613 ‐2.11891 0.014395 CXCL14 105.699 26.3868 ‐2.11891 2.73E‐07 DSC1 9.08248 2.29473 ‐2.12157 1.09E‐06 IGFBP6 14.5563 3.61922 ‐2.12473 0.000115 TMEM86A 21.1973 5.26239 ‐2.12809 1.60E‐07 MSH5‐SAPCD1 0.972757 0.235653 ‐2.13486 0.011337 PSG2 1.29001 0.315467 ‐2.14866 0.042388 LCE6A 84.9878 20.3968 ‐2.17575 1.91E‐07 ENDOU 18.9507 4.59805 ‐2.17999 3.53E‐07 MT2A 203.72 48.699 ‐2.18146 7.05E‐08 CYP4F22 12.3353 2.94809 ‐2.18178 1.23E‐06 DNASE1L2 2.01632 0.479387 ‐2.1893 0.023534 CHI3L1 5.98348 1.41874 ‐2.19321 0.000249 HYAL4 2.0167 0.475561 ‐2.20113 0.005011 NDUFA4L2 5.91809 1.39119 ‐2.20565 0.001491 NPR3 0.607935 0.144264 ‐2.21578 0.006529 CCL24 6.05146 1.41259 ‐2.21578 0.034239
96
CFTR 0.403784 0.0933127 ‐2.23027 0.025522 LCE2C 101.124 23.2283 ‐2.23901 6.38E‐08 KCNE1 0.877545 0.209704 ‐2.24177 0.019355 LCE1F 146.714 32.8463 ‐2.27604 2.05E‐08 EGR3 8.70421 1.94984 ‐2.28181 2.28E‐07 DKK1 1.42638 0.316953 ‐2.28686 0.020813 ENO2 1.30779 0.288582 ‐2.29691 0.012338 ADAMTS1 10.2196 2.25511 ‐2.29691 7.51E‐08 IGFL1 138.427 30.1714 ‐2.31471 1.05E‐08 TREX2 7.57473 1.57017 ‐2.38711 0.000185 LCE1D 65.8624 13.6003 ‐2.39265 3.07E‐07 PLA2G4B 40.2165 8.30458 ‐2.39291 3.65E‐09 FRY 0.323396 0.0652453 ‐2.4262 0.007011 PLA2G4D 2.98398 0.585296 ‐2.46684 6.65E‐05 MXRA5 6.40136 1.2529 ‐2.46995 3.56E‐09 KCNMA1 1.7114 0.337059 ‐2.47059 7.61E‐05 TNFRSF6B 2.01445 0.402936 ‐2.47255 0.019084 BPIFC 11.3027 2.1831 ‐2.48906 5.53E‐07 HAL 4.9103 0.960323 ‐2.49082 1.56E‐06 KIF26A 0.2931 0.0564452 ‐2.49331 0.025404 ZNF662 4.5797 0.861328 ‐2.51964 1.76E‐07 RTN1 1.04577 0.223707 ‐2.52188 0.034496 IGFL3 11.3683 2.11249 ‐2.54484 0.000275 MMP1 5.088 0.967246 ‐2.5637 6.87E‐05 S100A4 4.25436 0.868585 ‐2.5637 0.020033 MMP9 1.78039 0.326541 ‐2.5637 0.00271 PCDHGA5 0.307647 0.0608375 ‐2.5637 0.048024 LCE5A 3.38543 0.62092 ‐2.5637 0.009517 MPP2 0.493773 0.0864461 ‐2.63081 0.015806 NGEF 0.563221 0.11282 ‐2.66323 0.036488 SLC16A6 0.516864 0.0989214 ‐2.68234 0.020781 FCHO1 0.908266 0.166263 ‐2.70388 0.007798 TIAF1 1.36314 0.220365 ‐2.75634 0.006241 PRB2 1.10951 0.178058 ‐2.75634 0.02776 RGCC 2.14492 0.344224 ‐2.75634 0.009855 AQP9 0.524456 0.0841664 ‐2.75634 0.02776 PTGER3 1.98846 0.36334 ‐2.77804 2.74E‐06 LCE1E 33.5119 5.17591 ‐2.81163 4.98E‐10 MEF2B 1.19178 0.208542 ‐2.84381 0.021148 KLHL6 0.203688 0.0301741 ‐2.87182 0.037763 LOR 80.7339 11.741 ‐2.89846 3.00E‐12 CCL22 1.66284 0.239628 ‐2.91162 0.000509 CYP26B1 0.787292 0.112309 ‐2.92627 0.001752
97
IGSF22 0.433619 0.0617884 ‐2.92627 0.016133 MMP10 2.81478 0.397518 ‐2.94077 0.000423 IVL 71.659 9.91461 ‐2.97036 9.68E‐13 NR4A1 0.586822 0.100288 ‐2.97874 0.02802 KCNN4 0.839935 0.113512 ‐3.00427 0.012324 PSG7 5.98116 0.836074 ‐3.00788 6.18E‐06 KRT37 3.91945 0.518005 ‐3.03645 6.30E‐05 KPRP 124.81 16.2759 ‐3.05576 3.66E‐12 PSG5 1.00735 0.161457 ‐3.07827 0.02084 MMP3 2.7627 0.347739 ‐3.10684 0.000224 PAPL 20.6342 2.55469 ‐3.13065 6.05E‐13 EPHA8 2.98593 0.322206 ‐3.15489 9.70E‐05 CYP4F2 1.59445 0.188545 ‐3.19692 0.000693 FRMPD1 2.41718 0.284472 ‐3.2038 4.98E‐07 KLHL30 0.295594 0.0345002 ‐3.21578 0.037528 HLA‐F‐AS1 0.675554 0.0667367 ‐3.34131 0.026904 LOC100507564 1.01634 0.116087 ‐3.34131 0.026904 STEAP1B 1.06805 0.0979456 ‐3.45678 0.019374 APOD 2.80422 0.225015 ‐3.75634 0.00042 SCNN1G 0.735816 0.0545013 ‐3.87182 0.00092 C1orf68 11.451 0.840088 ‐3.88563 4.39E‐08 GPBAR1 0.603274 0.0915109 ‐3.92627 0.034093 MYO1H 0.209304 0.0149288 ‐3.92627 0.034093 POU5F1 0.622654 0.0730543 ‐3.92627 0.034093 ARC 0.302825 0.0215993 ‐3.92627 0.034093 FAM65C 0.855848 0.0593942 ‐3.9658 0.000121 GP1BB 2.08565 0.136895 ‐4.07827 0.002148 FOSB 0.296567 0.0340534 ‐4.07827 0.023276 MYPN 0.204422 0.0221812 ‐4.07827 0.023276 LOC149086 0.847807 0.0544235 ‐4.07827 0.023276 HNRNPH2 8.45215 0.542771 ‐4.10684 1.90E‐12 GYS2 0.347785 0.0202959 ‐4.21578 0.016015 ARG1 5.60549 0.257025 ‐4.5637 2.61E‐08 SYNPO2L 0.623297 0.0387408 ‐4.66323 0.000116 FSBP 0.355492 0.0171707 ‐5.00427 0.000995 AQP5 3.15397 0.104723 ‐5.02936 3.05E‐07 CAPN8 0.989767 0.0302554 ‐5.14866 0.000518 C4orf26 0.374112 0 ‐6.30345 0.038212 LY75 0.0989903 0 ‐6.30345 0.038212 RELN 0.0513609 0 ‐6.30345 0.038212 IFNK 0.521178 0 ‐6.30345 0.038212 SYT14L 0.263222 0 ‐6.30345 0.038212 MYCBPAP 0.217294 0 ‐6.52323 0.023764
98
CACNA1D 0.116176 0 ‐6.71391 0.015043 CCL20 0.942526 0 ‐6.71391 0.015043 NPPA‐AS1 0.497653 0 ‐6.71391 0.015043 C1orf177 0.557644 0 ‐6.88231 0.009667 NWD1 0.129528 0 ‐7.03309 0.006294 KRTAP5‐4 0.838475 0 ‐7.03309 0.006294 XCR1 1.02833 0 ‐7.29428 0.002758 CA9 0.764663 0 ‐7.29428 0.002758 SENP3‐EIF4A1 0.293999 0 ‐7.29428 0.002758 C9orf131 0.489218 0 ‐7.51536 0.001255 ZNF321P 0.678821 0 ‐7.61437 0.000856 PTPN5 0.54883 0 ‐7.70702 0.000589 SLC6A2 0.649817 0 ‐7.95386 0.000198 UBE2F‐SCLY 1.60147 0 ‐9.28737 2.64E‐08 MUTED 2.36062 0 ‐9.63266 8.66E‐10 PALM2‐AKAP2 0.984738 0 ‐9.91105 3.59E‐11 TMX2‐CTNND1 1.51256 0 ‐10.2711 2.87E‐13
99
Table 2‐1. Expression level of 572 genes dysregulated in mature EPC2‐ALI by IL‐13. RPKM value of gene expression level in mature EPC2‐ALI treated with vehicle (Veh) or IL‐13 (100 ng/mL) for 48 h. Log2 fold change (LOGFC) indicates the gene expression fold change induced by IL‐13. n = 3 per treatment; 572 genes were selected with |LOGFC| > 1 and p < 0.05.
100
Table 2‐2. GO analysis of IL‐13–induced dysregulated genes in mature EPC2‐ALI.
ID Name pValue Genes from Genes in Input Annotation
1 GO:0005509 calcium ion binding 5.388E-7 45 708
2 GO:0038024 cargo receptor activity 7.485E-7 12 71
3 GO:0008236 serine-type peptidase activity 1.986E-6 22 242
4 GO:0017171 serine hydrolase activity 2.435E-6 22 245
5 GO:0004252 serine-type endopeptidase activity 4.074E-6 20 215
6 GO:0005044 scavenger receptor activity 6.312E-6 9 47
7 GO:0004175 endopeptidase activity 2.395E-5 30 457
8 GO:0015291 secondary active transmembrane transporter activity 5.357E-5 19 236
9 GO:0042802 identical protein binding 5.841E-5 64 1359
10 GO:0005125 cytokine activity 7.640E-5 18 222
11 GO:0002020 protease binding 1.151E-4 12 115
12 GO:0016638 oxidoreductase activity, acting on the CH-NH2 group 1.584E-4 5 19 of donors
13 GO:0019955 cytokine binding 1.790E-4 11 103
14 GO:0005518 collagen binding 2.090E-4 9 72
15 GO:0015075 ion transmembrane transporter activity 2.108E-4 44 873 GO: Molecular Function
GO: Biological Process
ID Name pValue Genes from Input Genes in Annotation
1 GO:0018149 peptide cross-linking 3.322E-21 23 57
2 GO:0031424 keratinization 1.154E-20 22 53
3 GO:0008544 epidermis development 2.465E-20 48 340
4 GO:0043588 skin development 7.283E-20 43 278
5 GO:0030216 keratinocyte differentiation 1.824E-18 30 138
6 GO:0009913 epidermal cell differentiation 1.631E-15 32 200
7 GO:0016477 cell migration 4.759E-12 82 1300
8 GO:0040011 locomotion 7.712E-12 99 1735
9 GO:0060429 epithelium development 1.047E-11 81 1296
10 GO:1901700 response to oxygen-containing compound 2.252E-11 93 1614
11 GO:0006954 inflammatory response 3.784E-10 52 711
12 GO:0051674 localization of cell 5.066E-10 82 1428
13 GO:0048870 cell motility 5.066E-10 82 1428
14 GO:0030334 regulation of cell migration 5.906E-10 53 742
15 GO:1902533 positive regulation of intracellular signal transduction 9.700E-10 62 958
GO: Pathway
ID Name P Value Genes Genes in from Input Annotation
1 1457791 Formation of the cornified envelope 1.343E-22 27 71
2 M5889 Ensemble of genes encoding extracellular matrix and 9.224E-18 87 1028 extracellular matrix-associated proteins
3 1457790 Keratinization 1.006E-15 35 214
4 M5885 Ensemble of genes encoding ECM-associated proteins 2.839E-15 68 753 including ECM-affiliated proteins, ECM regulators and secreted factors
5 1470923 Interleukin-4 and 13 signaling 1.916E-8 18 114
101
6 M3468 Genes encoding enzymes and their regulators involved in the 4.142E-8 26 238 remodeling of the extracellular matrix
7 1270302 Developmental Biology 1.709E-6 63 1081
8 M5883 Genes encoding secreted soluble factors 1.786E-6 29 344
9 1269310 Cytokine Signaling in Immune system 4.550E-6 48 763
10 1269318 Signaling by Interleukins 6.491E-6 37 531
11 169349 Validated transcriptional targets of AP1 family members Fra1 8.732E-6 8 34 and Fra2
12 M3008 Genes encoding structural ECM glycoproteins 1.623E-5 19 196
13 PW:0000051 histidine metabolic 4.113E-4 4 12
14 83051 Cytokine-cytokine receptor interaction 4.145E-4 20 270
15 1268755 Regulation of Insulin-like Growth Factor (IGF) transport and 4.287E-4 5 21 uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs)
102
Table 2‐2. GO analysis of IL‐13–induced dysregulated genes in mature EPC2‐ALI. GO enrichment based on molecular function, biological pathway and pathway are shown. The p‐value was calculated on the basis of probability density function
103
3 Chapter III: IL-13 activated STAT3 and STAT6 pathways in Eosinophilic
Esophagitis
Chang Zeng B Sc1, Simone Vanoni PhD1, 2, Simon P. Hogan PhD1,3
1Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave,
Cincinnati, OH, 45229; 2Institute of Pharmacology and Toxicology, Paracelsus Medical University,
Salzburg, Austria. 3Department of Pathology, Mary H Weiser Food Allergy Center, Michigan Medicine,
University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109‐2200.
Correspondence: Simon P. Hogan, Ph.D., Department of Pathology, Mary H Weiser Food Allergy Center,
Michigan Medicine, University of Michigan, 109 Zina Pitcher Place
Ann Arbor, MI 48109‐2200; Email: [email protected]; Phone: 734‐647‐9923.
Disclosures: The authors have declared that they have no conflict of interest
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3.1 Copyright and Student Contribution
Authorship is listed on the title page. I performed all the bioinformatic analysis used in this manuscript and experiments related to EPC2‐ALIΔSTAT6 cultures. SV generated knockout cell lines, performed western blot analysis and BrdU assay in EPC2‐ALIΔSTAT3 cultures. Also, I generated figures, wrote the manuscript and modified it under the guidance of SPH.
3.2 Abstract
Eosinophilic Esophagitis (EoE) is a food allergen‐related disorder driven by Type‐2 inflammatory responses characterized by esophageal eosinophilia and epithelial remodeling. Previous studies have demonstrated that increased Interleukin‐13 (IL‐13) level in EoE patients contributes to transcriptome changes and led to histological changes. While anti‐IL‐13 therapy has demonstrated significant improvement in treating EoE, the molecular mechanism of IL‐13‐driven dysregulation in EoE remains unclear.
Janus kinases (JAK) /Signaling transducer and activator of transcription (STAT) pathways are the most well‐known interleukin signaling pathways, and IL‐13 mainly signals through binding to IL‐4Rα/IL‐13Rα1 complex and activate JAK/STAT3 or STAT6 pathways. Herein, we identified STAT protein binding motif is one of the most enriched transcription factors binding site (TFBS) in the dysregulated genes in both EoE biopsies and IL‐13 treated in vitro esophageal epithelium (EPC2‐ALI). In particular, we identified the
STAT3 binding site as the most enriched TFBS in IL‐13 induced upregulated genes in EPC2‐ALI.
To study the involvement of STAT signaling in IL‐13 induced esophageal epithelial remodeling, we employed shRNA technique to knockdown STAT3 and STAT6. Using RNA sequencing and gene ontology
(GO) analysis, we identified the involvement of STAT3‐dependent genes in regulating epithelial development and proliferation. Experimental analysis showed that STAT3 knockdown in EPC2‐ALI led to increased filaggrin expression and epithelial barrier resistance, suggesting the important role of STAT3 in
105 epithelial integrity. Further analysis showed that IL‐13 induced increased proliferation is abrogated in
STAT3KD cells, we thus conclude that STAT3 is essential for IL‐13 induced basal cell hyperplasia in EoE. In contrast, we found that IL‐13‐induced STAT6‐dependent genes are related to cytokine and chemokine production in esophageal epithelium.
Taken together, these data suggested an essential role of STAT proteins in EoE pathogenesis and defines the distinct role of STAT3 and STAT6 in IL‐13 induced epithelium dysregulation in EoE.
106
3.3 Introduction
Eosinophilic esophagitis (EoE) is a chronic inflammatory disease of the esophagus, characterized by upper gastrointestinal symptoms such as food impaction and dysphagia in both adults and children140,331.
Analysis of both patient biopsies and experimental murine model indicated EoE is driven by Th2‐ mediated inflammatory responses48,50,332,333.
One of the Th2 cytokines that is elevated in EoE patients is IL‐1348,51,54. Interestingly, intratracheal delivery of IL‐13 led to eosinophils accumulation and esophageal epithelial hyperplasia in a murine model of EoE206. Moreover, inducible, lung‐specific IL‐13 transgenic mice model showed EoE phenotypes including esophageal eosinophilia and epithelium remodeling207.Conversely, IL‐13‐deficient mice fail to develop histological EoE phenotypes upon repetitive epicutaneous antigen challenges to induce experimental EoE18. Together, these experimental models have demonstrated a pathogenic role of IL‐13 in EoE. Furthermore, IL‐13 is shown to induce EoE‐like transcriptome changes in vitro54, leading to dysregulation of inflammatory and epithelial barrier regulatory genes including Chemokine (C‐C motif) ligand 26 (CCL26)54, cadherin‐26 (CDH26)334 desmoglein‐1 (DSG‐1)133, Leucine‐rich repeat‐containing protein 31 (LRRC31)208, calpain‐14 (CAPN14)209 and synaptopodin (SYNPO)335.
Given the importance of IL‐13 in EoE pathogenesis, several clinical trials were performed to examine the efficacy of anti‐IL‐13 therapy in treating EoE. In clinical trial studying anti‐IL‐13 mAb QAX576, inhibition of IL‐13 in EoE patients showed decreased esophageal eosinophilia, together with the improvement of
EoE‐relevant transcriptome211. Aside from direct inhibition of IL‐13, antibodies against IL‐13 receptor subunits also showed promising outcome in treating EoE. RPC4046, an antibody against both IL13Rα1 and IL13Rα2, recently established its ability to reduce esophageal eosinophil counts in EoE patients in its phase II clinical trial212. In addition, in a recent clinical trial, treatment using antibody Dupilumab, that
107 blocks IL‐4Rα subunit, also showed relief of intraepithelial eosinophilia, improved symptoms and quality of life compared to the placebo group in EoE patients213.
Considering the critical role of IL‐13 in the exacerbation of esophageal inflammation and remodeling, we began to define the involvement of signaling pathways in IL‐13 effects on esophageal cells. IL‐13 mainly signals upon binding to IL4Rα/IL13Rα1 complex. It is known to activate Janus kinase (JAK)/Signaling transducer and activator of transcription 6 (STAT6) pathway and regulate transcription of STAT6‐ dependent genes176. In the meantime, recent studies also identified that IL‐13 could also signal through
JAK2/STAT3 pathway336,337.
Although all being STAT6 family members, STAT3 and STAT6 seem to have a distinct influence on cell homeostasis. STAT6‐dependent signaling is critical for various allergic inflammatory disorders on both hematopoietic and non‐hematopoietic compartments338. Mice with STAT6 knockout in eosinophils are protected from allergic airway inflammation339. Also, epithelial STAT6 activation is essential for airway hyperreactivity and mucus production in asthma340. Meanwhile, STAT3 signaling is proven important for regulating barrier permeability341, epithelium differentiation342, and proliferation343. Together, both signaling pathways could either regulate inflammatory responses or epithelial changes, which are important pathogenic mechanisms and phenotypes in EoE. Given this, we examined the involvement of
STAT6 and STAT3 pathways in IL‐13‐mediated responses in esophageal epithelial cells.
We show that established that STAT binding site is one of the most enriched transcription factors binding sites (TFBS) in EoE transcriptome and that STAT3 binding motif is the most significantly enriched
TFBS in IL‐13‐mediated upregulated genes. Bioinformatic and network analyses identified an enrichment of genes that contain the STAT‐3 binding site that is involved in epithelium barrier regulation (e.g., LOR and EMP1) and proliferation (e.g., WNT7A and IRS1). Using EPC2‐ALIΔSTAT3, we further proved the influence of STAT3 on barrier function and IL‐13 induced proliferation in esophageal epithelium. In
108 contrast, we show that IL‐13‐induced STAT6‐dependent genes (e.g., POSTN and CCL26) are related pro‐ inflammatory esophageal function promoting cytokine production.
109
3.4 Materials and methods
RNAseq analysis of Human subjects ‐ NL (healthy control patients) were defined as having no history of
EoE diagnosis, 0 esophageal eosinophils per high‐power field (HPF) and no evidence of esophagitis within distal esophageal biopsies obtained during the same endoscopy procedure as the analyzed samples. EoE was defined as described in the recent consensus guidelines. Specifically, patients needed to have ≥15 eosinophils in at least 1 high‐power field (Eos/hpf) in a distal esophageal biopsy with other causes of esophageal eosinophilia excluded, and without a response to acid suppression. RNAseq analyses were performed on human esophageal biopsy samples (normal, n = 6; EoE, n = 10) as previously described (NCBI Gene Expression Omnibus (GEO) database under accession GSE58640)122.
Transcription factor binding site (TFBS) analysis – TFBS analyses were performed using the University of
California Santa Cruz transcription factor binding sites (UCSC_TFBS) database which belongs to Database of Annotation, Visualization, and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/)344,345.
EPC2‐ALI culture ‐ The cells used were immortalized human esophageal epithelial cell line (EPC2‐hTERT), a kind gift from Dr. Anil Rustgi (University of Pennsylvania, Philadelphia, PA, USA), and were cultured as previously described346. Air‐liquid interface (ALI) culture system was previously described and characterized together with EPC2 cells133. EPC2‐hTERT cells were seeded onto permeable (0.4 µm) transwell support (Corning Incorporated, Corning, NY, USA) and grown to confluence while fully submerged in low‐calcium ([Ca2+] = 0.09 mM) keratinocyte serum‐free media (K‐SFM) (Life Technologies;
Carlsbad, CA). Epithelial differentiation was induced by culturing submerged cell monolayers in high‐ calcium K‐SFM ([Ca2+] = 1.8 mM) for 4 days (day 3 to 7). Cells were then exposed for 5 days (day 7 to 12) at ALI by removing cell media from the top chamber, in order to induce stratification. Stimulation with
IL‐13 (100ng/mL) in the lower chamber occurred when the epithelial differentiation was complete. Cells
110 were collected for RNA or fixed for Chromatin Immunoprecipitation (ChIP) at different IL‐13 exposure times as indicated.
H3K4me3 Histone Mark Chromatin Immunoprecipitation‐sequencing (ChIP‐seq) ‐ EPC2 cells were stimulated at day 5 of ALI culture with IL‐13 (100 ng/mL) for 4 hours. Following exposure to IL‐13, cells were cross‐linked with 0.8% formaldehyde for 10 minutes at RT. After lysis, cells were sonicated for 10 minutes (Peak power 175W, Duty factor 10%, Cycles/burst 200 count) at 4 °C using Covaris S series
Sonicator (Covaris; Woburn, MA, USA), yielding chromatin fragments of 200‐300 base pairs in size. To avoid the presence of unspecific binding, pre‐clearing with 10µL Dynabeads® Protein G magnetic beads
(Life Technologies; Carlsbad, CA) was performed on each chromatin sample for 45 minutes at 4 °C on rotating wheel. 20µL of Dynabeads® Protein G magnetic beads (Life Technologies; Carlsbad, CA, USA) were combined for 1 hour at RT on a rotating wheel with 1 µL of anti‐H3K4me3 (cat#17‐614, Millipore corporation; Temecula, CA, USA) per sample. Beads coated with antibody were then incubated for 14‐16 hours at 4 °C on a rotating wheel with 5µg of chromatin complexes to perform immunoprecipitation.
Chromatin‐antibody‐beads complexes were washed with 500µL of each of the following buffers in succession: low salt wash buffer (10 mM Tris‐HCl pH7.6, 1 mM EDTA, 0.1% SDS, 0.1% sodium
Deoxycholate, 1% Triton X‐100, 150 mM NaCl), high salt wash buffer (10 mM Tris‐HCl pH7.6, 1 mM
EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X‐100, 400 mM NaCl), LiCl wash buffer (10 mM
Tris‐HCl pH7.6, 1 mM EDTA, 250 mM LiCl, 0.5% sodium deoxycholate, 0.5% Nonidet P‐40) and Triton X‐
100 buffer (10 mM Tris‐HCl pH7.6, 1 mM EDTA, 0.2% Triton X‐100). Elution of the immunoprecipitated chromatin was obtained following 1‐hour incubation at 37 °C with shaking using 100 uL elution buffer
(10 mM Tris‐HCl pH7.6, 1 mM EDTA, 250 mM NaCl, 0.3% SDS). 5 µL of Proteinase K solution (Life
Technologies; Carlsbad, CA, USA) was added to the eluates and incubated at 37°C for 1 hour for protein degradation. Incubation at 65 °C for 6 hours was then performed for reversing the cross‐link. DNA was purified with QIAquick® PCR purification kit (Qiagen; Valencia, CA, USA) and eluted in 30 µL molecular
111 biology grade water (Sigma; St. Louis, MO, USA) and processed for qPCR prior to sequencing. Libraries were created as described previously347 and sequenced by CCHMC Gene Discovery and Genetic
Variation Core. Raw data was uploaded on Biowardrobe298, and peak values were calculated.
Differentially enrichment of binding sequences was assessed using MAnorm method348. Alteration of
H3K4me3 enrichment is identified by unique peaks in a certain group, with the criteria that log10 [p value] > 1.3.
Lentiviral Transduction ‐ EPC2 cells were transduced at 60‐70% confluency with lentiviral particles containing Mission® STAT6 shRNA (TRC0000019409, Sigma; St. Louis, MO, USA), Mission® STAT3 shRNA
(TRCN0000329887, Sigma; St. Louis, MO, USA) or Mission® non‐target control shRNA (Sigma; St. Louis,
MO, USA). All the shRNA lentiviruses were generated by the Cincinnati Children’s Hospital Medical
Center Viral Core using a 4‐plasmid packaging system. Lentiviral particles were incubated with EPC2 cells for 6 hours. All the viral particles were added in the presence of 5 µg/mL Hexadimethrine Bromide
(Polybrene®) (Sigma; St. Louis, MO, USA). During the first hour of incubation, cells were spun down at
1000*g for 1 hour at room temperature. 6 hours following transduction cells were put in fresh KSFM media, and 24 hours later media containing 1 µg/mL of Puromycin (Thermo Fisher Scientific
Incorporated; Rockford, IL, USA) was used for selection. Cells were grown under selective pressure and cultured as regular EPC2 cells. Stable knockdown for STAT6 and STAT3 was demonstrated by western blot and RNAseq analyses.
Western Blot ‐ EPC2 ALI cells were lysed following stimulation, using a protein extraction reagent (10%
Glycerol, 20 mM Tris HCl pH7, 137 mM NaCl, 2 mM EDTA, 1% NP‐40 in H2O) with Halt™ protease inhibitor cocktail (Thermo Fisher Scientific Incorporated; Rockford, IL, USA). Approximately 40 μg of protein extract were separated on a 4%‐12% Bis‐Tris gel and transferred to a nitrocellulose membrane
(Life Technologies; Carlsbad, CA). The following antibodies were used: anti‐STAT3 (1:1000, #9132, Cell
Signaling Technology; Danvers, MA), anti‐STAT6 (1:1000, sc‐621, Santa Cruz Biotechnology; Santa Cruz,
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CA, USA), anti‐α‐actin (1:2000, A2066, Sigma; St. Louis, MO, USA) or anti‐GAPDH (1:2000, clone 2D9,
TA802519, Origene; Rockville, MD). The IRDye® 800 CW goat anti‐rabbit or goat anti‐mouse IgG (H+L)
(Li‐Cor; Lincoln, NE) were used as secondary antibody for detection.
RNAseq analyses and Gene ontology (GO) analysis ‐ RNAseq analysis was performed as previously described. Briefly, RNA was isolated using the RNeasy kit (QIAgen Incorporated, Germantown, MD, USA) according to manufacturer instructions. Assessment of RNA quality was performed using the Agilent
2100 Expert bioanalyzer (Agilent Technologies Incorporated, Clara, CA, USA) and only those samples getting an RNA Integrity Number (RIN) above 8 were chosen for sequencing. Next‐generation sequencing analyses were performed by the CCHMC Genetic Variation and Gene Discovery Core. Raw data was uploaded on Biowardrobe298, and RPKM values were calculated. Differentially expressed genes were assessed using DEseq2 under p<0.05 cut‐off conditions. Criteria of DEseq2 analysis for identification of IL‐13‐induced STAT3 dependent genes: (1) RPKM>2 in any of the 4 groups (vehicle and
IL‐13 treated EPC2‐ALIctrl, vehicle and IL‐13 treated EPC2‐ALIΔSTAT3); (2) compare EPC2‐ALIctrl treated with or without IL‐13, |Fold Change (FC)|>2, p<0.05; (3) compare EPC2‐ALIΔSTAT3 treated with or without IL‐13,
|Fold Change (FC)|<2, p>0.05. Similar criteria were applied for IL‐13‐induced STAT6 dependent genes.
Gene list enrichment analysis and candidate gene prioritization based on molecular function using
ToppGene299 with FDR B&H correction and p‐Value cutoff at 0.05.
5‐Bromo‐2’‐deoxyuridine (BrdU) assay and Immunofluorescent (IF) staining ‐ After EPC2‐ALI cultures were treated as indicated in the experiment, BrdU reagents dissolved in DMSO (final concentration 10
µM) were added to both the upper and lower chambers of transwells for 2 hours. EPC2‐ALI cultures were then washed with PBS and fixed with 4% paraformaldehyde for another 2 hours. To examine BrdU incorporation, IF staining was performed as previously described349. In brief, formalin or paraformaldehyde fixed, paraffin‐embedded EPC2‐ALI cultures were sectioned, mounted on slides and de‐paraffinized using xylene followed by serial hydration using graded ethanol and water. Slides were
113 then permeabilized in Tris‐EDTA (1 mM, pH 9.0) with 0.1% Tween‐20, and antigen exposure performed at 125°C for 30 seconds in a decloaking chamber using pressure cooker. Slides were then blocked by 4%
Normal Donkey serum for 1 hour followed by overnight incubation of primary antibody (mouse anti‐
BrdU (2.5 µg/mL)) diluted in 10% normal donkey serum. Slides were then washed and incubated with secondary antibody (1:200, Donkey anti‐Mouse IgG (H+L) Secondary antibody, Alexa Fluor 488, Thermo
Fischer Scientific Inc., Waltham, MA, USA) at RT for 1hr. Slides were mounted with Fluoromount‐G
(SouthernBiotech, Birmingham, AL, USA) mounting solution. Fluorescent imaging was performed using the Zeiss Apotome fluorescent microscope with consistent exposure time under the same excitation wavelength.
Trans‐epithelial electrical resistance (TEER) measurement – TEER measurements were performed on
EPC2‐ALI cultures using epithelial volt/ohm meter (EVOM, World Precision Instrument, Sarasota, FL,
USA) together with chopstick electrode set (#STX2, World Precision Instrument, Sarasota, FL, USA).
Before the measurement, media was removed from the lower chamber of the transwell insert and the inserts were washed with 1x PBS on both top and lower chamber. Fresh 1x PBS was added to both top and lower chamber for measurement. TEER readings were recorded immediately, and three measurements were performed for each culture insert. The TEER values were reported as ohms per square centimeter (Ω/cm2).
Statistical Analysis ‐ Statistical significance of EPC2‐ALI samples were established using unpaired t‐test
(two‐tailed), or two‐way ANOVA when there was more than one variable. For non‐normally distributed data from patient biopsies, Mann‐Whitney test was used. Graphs and statistical analyses were performed using GraphPad Prism 7.02 (GraphPad Software Incorporated, La Jolla, CA, USA).
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3.5 Results
3.5.1 STAT binding site is one of the most enriched TFBS in EoE dysregulated genes
To begin to define the Il‐13‐induced signaling pathways involved in esophageal inflammation and remodeling in EoE, we analyzed the enrichment of transcription factor binding motifs (TFBM) in the
1607 dysregulated genes identified by RNAseq analyses comparing biopsy samples from normal healthy controls and pediatric EoE patients122. We identified the significant enrichment of genes that possess nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB), nuclear factor of activated T‐cells
(NFAT), peroxisome proliferator‐activated receptor gamma (PPARG), CCAAT‐enhancer‐binding proteins
(CEBP), STAT and interferon regulatory factor 1 (IRF‐1) binding motifs (Fig. 3‐1A). Notably, STAT family binding site was one of the most enriched binding motifs (Fig. 3‐1A) within all the dysregulated genes in
EoE patients (448 genes, 27.9% of the total, p = 0.038). As the STAT family consists of seven family members, we examined the expression of STAT proteins (STAT‐1‐STAT‐6) between NL and EoE patients, and demonstrate significant differential expression in STAT1, STAT3 and STAT6 expression in EoE patients (Fig. 3‐1B). Notably, we observed ~ 2‐fold increase in interferon‐induced STAT1 and IL‐4 and IL‐
13‐induced STAT3 and STAT6 mRNA in esophageal biopsy samples in patients with active EoE (Fig. 3‐1C,
D, E respectively). These studies show that IL‐13‐induced transcription factor STAT3 and STAT6 are significantly elevated in EoE and that genes that possess STAT‐binding motifs are enriched in the EoE transcriptome.
3.5.2 STAT binding site is the most enriched in both dysregulated genes and transcriptionally active
genes following 4 hours incubation with IL‐13 in EPC2‐ALI
In order to better understand the involvement of STAT proteins in regulating esophageal epithelium remodeling, we employed the previously established in vitro stratified squamous epithelium model
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(EPC2‐ALI). We have previously demonstrated that stimulation of EPC2‐ALI with IL‐13 induces an EoE like transcriptome similar to what is observed in primary cells derived from EoE patients’ biopsies54,350.
RNAseq analysis of EPC2‐ALI showed that 4 hours treatment of IL‐13 led to significant dysregulation of
712 genes, with 39.04% upregulated and 60.96% downregulated genes (Fig. 3‐2A). TFBS analysis indicated that STAT binding motif is the most significantly enriched binding site after short‐term IL‐13 exposure (Fig. 3‐2B, 220 genes, 30.1% of the total, p‐value 5.6e‐4). In order to better understand the cell signaling network involved in the IL‐13 mediated transcriptome dysregulation, we also analyzed trimethylation of lysine 4 on histone H3 (H3K4me3) modification on the chromatin of EPC2 ALI cultures.
The H3K4me3 modification is highly enriched in open chromatin regions, normally correlated with transcriptionally active genes351. Using ChIP‐seq analysis, we identified 858 genes showing active transcription following 4 hours IL‐13 treatment. TFBS analysis of these genes showing unique H3K4me3 modification revealed significant enrichment of genes that possess binding motifs of only STAT, ETS domain‐containing protein (ELK1) and AP1FJ. Indeed, STAT binding motifs are the most significantly enriched binding motif within the genes with at least one transcriptionally active region (Fig. 3‐2B, 310 genes, 36.1% of the total, p‐value 1.5e‐13).
3.5.3 STAT3 is the most enriched TFBS in IL‐13‐induced upregulated genes.
As STAT family binding motif is the most significantly enriched binding sites in both dysregulated and transcriptionally activated genes in EPC2‐ALI cells after 4hr IL‐13 treatment, we’re interested in identifying the specific STAT protein next involved in IL‐13‐induced dysregulation in EoE. We first compared the expression level of STAT family members upon 4hr IL‐13 treatment. RNAseq analysis indicated that STAT3 is the most upregulated STAT protein and also the only significantly upregulated
STAT within the family (Fig. 3‐3A, p=0.049 for STAT3). Interestingly, when analyzing subsets of IL‐13
116 dysregulated genes, we also found that STAT3 binding motif is the most significantly enriched binding site in IL‐13 mediated upregulated genes (Fig. 3‐3B, 52% of the total, p‐value 5.9e‐9). Based on the studies, we concluded that IL‐13 ‐induced STAT3 in esophageal epithelial cells and that IL‐13 induced the enrichment of genes that possess STAT3 binding motifs.
3.5.4 STAT3 and STAT3‐related genes regulate barrier integrity of the esophageal epithelium
In order to better understand the role of STAT3 in esophageal epithelium, we employed the lentiviral shRNA gene silencing system and generated stable knockdown (KD) EPC2 cell line with diminished STAT3 expression level. Western blot (Fig. 3‐4A) and RNAseq (Fig. 3‐4B) analyses indicated an 80% reduction in both protein and mRNA level of STAT3 in STAT3 shRNA lentiviral transduced EPC2‐ALI cells (EPC2‐
ALIΔSTAT3) compared to empty control transduced group (EPC2‐ALICTRL).
We’re first examined the impact of STAT3 on EPC2‐ALI steady state function. RNAseq analysis comparing transcriptome differences between EPC2‐ALICTRL and EPC2‐ALIΔSTAT3 cultures identified 493 genes that are altered following STAT3 knockdown. GO analysis based on biological process annotation on these dysregulated genes showed that within the top 5 GO nodes that were significantly dysregulated, 2 of them are related to epidermal or skin development (Fig. 3‐4C), indicating a potential role of STAT3‐ dependent genes in the regulation of squamous epithelial differentiation maturation and barrier integrity. Several junctional proteins, including epithelial membrane protein 1 (EMP1) and loricrin (LOR), are significantly upregulated in EPC2‐ALIΔSTAT3 cells (Fig. 3‐4D).
To validate the altered expression of barrier integrity genes in EPC2‐ALIΔSTAT3 cells, we examined the alteration of filaggrin (FLG) expression in EPC2‐ALI cultures. The FLG expression is significantly decreased in EoE according to RNAseq analysis in our patient cohort (Fig. 3‐4E) and also been established in other cohorts131. We showed that FLG expression in both mRNA and protein level are increased in EPC2‐
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ALIΔSTAT3 cells (Fig. 3‐4F and 4G, mRNA and protein respectively), suggesting the role of STAT3 in regulating the expression of barrier integrity genes such as filaggrin under steady state.
Given the role of STAT3 in regulating junctional proteins, we speculated that STAT3 could influence the barrier function in esophageal epithelium. To verify this hypothesis, we compared the TEER between
EPC2‐ALICTRL and EPC2‐ALIΔSTAT3 cultures, as TEER is one of the important parameters that are used to indicate epithelial barrier integrity. Consistent with our hypothesis, EPC2‐ALIΔSTAT3 cells showed significantly higher TEER than the EPC2‐ALICTRL group (Fig. 3‐4H).
3.5.5 IL‐13 induced STAT3‐dependent genes are involved in IL‐13 induced cell proliferation
We next examined the contribution of STAT3 in IL‐13 induced esophageal epithelial dysregulation. By performing DeSeq2 analysis on RNAseq results, we identified 129 genes that are regulated in an IL‐13‐ induced and STAT3‐dependent manner.
To further understand these genes, we performed GO analysis to determine gene clustering based on biological processes. Within the top 10 individual GO nodes, there’s three GO nodes that are directly related to proliferation, together with one node related to DNA replication, which is an essential requirement of cellular proliferation (Fig. 3‐5A). Of the 27 IL‐13‐induced STAT3‐dependent, cell proliferation regulation related genes included WNT7A and IRS1(Fig. 3‐5B).
To define the requirement of STAT3 in IL‐13 induced proliferation in EPC to ALI cells we performed a
BrdU assay. Consistent with previous studies IL‐13 stimulation of EPC2‐ALICTRL cells increased proliferation rate (Fig. 3‐5C, top panel). We did not observe the IL‐13 ‐induced increase in proliferation rate in EPC2‐ALIΔSTAT3 cultures (Fig. 3‐5C, lower panel). In the meantime, the level of EPC2‐ALI cell proliferation at steady state was comparable between EPC2‐ALICTRL and EPC2‐ALIΔSTAT3 cultures,
118 suggesting STAT3 alteration doesn’t influence baseline proliferation rate (Fig. 3‐5D). Collectively, we conclude that STAT3 activated gene transcription is essential for the IL‐13 induced esophageal epithelium proliferation.
3.5.6 IL‐13 induced STAT6‐dependent genes are involved in cytokine production but not IL‐13
induced cell proliferation
We and others have previously reported a role for IL‐13‐induced STAT6 activation in allergic inflammatory disorders206,352,353. To examine the IL‐13‐induced STAT6‐driven transcriptome in the esophageal epithelium, we generated EPC2‐ALIΔSTAT6 cultures using lentiviral particles containing STAT6 shRNA. The efficiency of knockdown is verified by Western blot and RNAseq, showing a 70% knockdown rate compared to EPC2‐ALICTRL (Fig. 3‐6A & 6B). RNAseq analyses were also performed to compare EPC2‐
ALIΔSTAT6 with EPC2‐ALICTRL, resulting in 166 genes that are IL‐13‐induced and STAT6‐dependent. In contrast to the STAT3 bioinformatics analysis, GO analysis identified the top node for this group of genes is positive regulation of cytokine production (Fig. 3‐6C), including periostin (POSTN) (Fig. 3‐6D). As
POSTN is previously showed to induced in IL‐13 JAK/STAT6 pathway in airway epithelium354, this supported our criteria for the IL‐13‐induced STAT6 pathway. In addition, we checked several inflammatory and cytokine‐related genes listed on EDP panels129, including CCL26 and TNFAIP6, and found them also being induced by IL‐13 and showed repressed expression when STAT6 is knocked down in EPC2‐ALI (Fig. 3‐6E).
Similar as STAT3, we were also interested in examining the contribution of STAT6 to the proliferation. To understand if STAT6 contributes to IL‐13‐dependent proliferative response, we also performed proliferation assay in EPC2‐ALICTRL and EPC2‐ALIΔSTAT6. Different from the STAT3 knockdown, knocking down STAT6 did not prevent IL‐13 from inducing increase proliferation (Fig. 3‐6F & 6G). However,
119 interestingly, STAT6KD EPC2‐ALI showed an overall decrease of proliferation rate in absence or presence of IL‐13. Based on these studies, we concluded that IL‐13 induced STAT6‐dependent transcriptome changes are important for cytokine and chemokine production in esophageal epithelium.
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3.6 Discussion
In this study, by analyzing the conserved binding motifs in both dysregulated genes in EoE transcriptome and altered genes induced by IL‐13 in vitro model of esophageal epithelium, we identified the involvement of STAT proteins in the regulation of EoE transcriptome and esophageal epithelial function.
Specifically, we demonstrated the role of STAT3 in modifying genes related to barrier integrity and cell proliferation. Using genetic modification, we reveal that STAT3 negatively regulates barrier integrity gene expression such as LOR and FLG, and suppression of STAT3 expression showed altered epithelial barrier function. In addition, knocking down STAT3 protects against IL‐13 induced basal zone hyperplasia
(BZH) in esophageal epithelial cells. In contrast, we showed that STAT6 regulates IL‐13 dependent cytokine and chemokine producing genes. Collectively, we showed the divergent function of IL‐13‐ induced STAT3 and STAT6 signaling pathways in influencing esophageal epithelium functions, suggesting new potential therapeutic targets for EoE.
STAT3, a 92kDa transcription factor and a member of STAT family, is found important for regulating intercellular permeability. STAT3 activated retinal endothelial cells have been associated with increased endothelial permeability and decreased expression of junctional protein like Zonulin‐1 (ZO‐1) and
Occludin (OCLN)341. In lung carcinoma cells, downregulation of STAT3 led to decreased junctional permeability, causing suppressed ability for intercellular communication355. Consistent with previous observations, we demonstrated that, under steady state, knocking down STAT3 in esophageal epithelial cells led to elevated expression of junctional proteins such as LOR, EMP1, and FLG. Previous studies suggested that expressional alteration of these proteins was related to dysregulation of barrier function356‐358. Specifically, FLG expression was decreased in the esophageal biopsies from patients with active EoE131. We showed that downregulation of STAT3 in esophageal epithelial cells leads to increased expression of FLG on both mRNA and protein level, as well as increased epithelial barrier function, suggesting an important role of STAT3 in regulating barrier integrity in esophageal epithelium.
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Interestingly, a recent study in atopic dermatitis showed that IL‐33 could also activate STAT3 and lead to decrease expression of FLG359. IL‐33 is a cytokine that is found elevated in EoE patients101, mediating EoE pathogenesis in experimental mice model67,101. Together with our observations, we speculated that IL‐33 could signal through STAT3 and mediate impaired barrier function in EoE. However, further experiments are needed for further validation.
Aside from its involvement in regulating barrier function, STAT3 is widely studied in anti‐cancer therapies360,361. Increased expression and activation of STAT3 is observed in various cancers including colitis‐associated cancer362, neuroblastoma363and gastric carcinoma364. Upon IL‐6 activation, STAT3 is known to promote cancer inflammation and inflammation‐induced carcinogenesis in coordination with
NF‐κB365. Clinical and experimental studies suggested that STAT3 targets a variety of genes, including
Cyclin D1 (CCND1), Vascular endothelial growth factor (VEGF) and B‐cell lymphoma‐extra large (Bcl‐xL), which contribute to tumor proliferation, angiogenesis, and metastasis366. Specifically, in esophageal carcinoma cell line, down‐regulation of STAT3 induced increased Caspase‐3 and promote apoptosis367.
Together, these studies suggested that activation of STAT3 pathways is tightly related to cell proliferation. In esophageal epithelial cells, we showed that IL‐13‐mediated decrease expression of genes like WNT7A and IRS1, which are previously showed to increase cell proliferation rate368,369.
Knockdown STAT3 in esophageal epithelial cells prevents these genes from decreasing, which contribute to the abrogation of IL‐13 induced proliferation we observed in EPC2‐ALIΔSTAT3 cultures.
One other characteristic of esophageal remodeling in EoE is epithelial‐mesenchymal transition (EMT).
EMT occurs more frequently in patients with EoE compared to NL and is correlated with subepithelial fibrosis in esophagus114. In esophageal epithelial cells, EMT could be induced by exposure to transforming growth factor – beta 1 (TGF‐β1), a profibrotic factor that is increased in EoE patients113.
Interestingly, in lung cancer cells, the TGF‐β1 induced EMT is dependent on JAK/STAT3 pathway370.
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Overall, these observations further support the role of STAT3 in regulating epithelium remodeling induced by various cytokines involved in EoE.
Our work primarily demonstrated that STAT3’s involvement in esophageal epithelium remodeling and other previous studies had suggested its activating role in modulating allergic inflammatory pathways in hematopoietic cells. Autosomal‐dominant hyper‐IgE syndrome (AD‐HIES) is a disease caused by dominant negative STAT3 mutation. Patients with AD‐HIES are characterized by skin and lung infections and always showed increased IgE level371. A recent study comparing patients with AD‐HIES and patients with atopic dermatitis showed that AD‐HIES patients have a significant lower rate of food allergies and anaphylaxis372. This is proved to be related to decreased mast cell degranulation and basophil activation caused by STAT3 deficiency372. In malignant T cells that secreted a high level of IL‐5, knockdown of STAT3 using siRNA led to decrease of IL‐5 production373, which could further decrease eosinophil infiltration in situ. In eosinophils, STAT3 could be activated by IL‐5 and induced increased Pim‐1 and Cyclin D3 expression, which are important for eosinophil viability374. Interestingly, mast cells70, basophils65, and eosinophils are all important effector cells in EoE with an increased level of activation and infiltration. In light of this, further investigation of the involvement of STAT3 in hematopoietic cells would be beneficial in understanding the pathogenesis of EoE.
Considering the role of STAT3 in modulating tissue remodeling and immunological pathways in EoE, it is a potential target for EoE therapy. Pharmacological inhibition of STAT3 has been widely studied given its involvement in cancer pathogenesis. A variety of natural and synthetic blockers has been established and tested in targeting STAT3 in cancer375. A potent STAT3 specific inhibitor, C188‐9376, is currently under clinical trial. Toxicity tests in murine models indicated that C188‐9 was well‐tolerated with good oral bioavailability377. Importantly, a recent study using C188‐9 in mice showed significant reduction of cell proliferation378. These extensive selections of STAT3 inhibitors, together with a proven effect on cell proliferation, will easier allow the exploration of targeting STAT3 in esophageal epithelium.
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In addition, we also identified the role of STAT6 in IL‐13 mediated transcriptome changes in esophageal epithelium. JAK/STAT6 pathway is characterized as the canonical signaling pathway of IL‐13176. Previous studies in animal models showed that STAT6 is important for inflammatory responses, as OVA‐induced airway eosinophilia was abrogated in STAT6 KO mice, together with decreased peri‐bronchial inflammation379‐381. Mice experiment transferring STAT6+/+ antigen‐specific Th2 cells to STAT6‐/‐ mice failed to develop experimental allergic asthma, suggesting the importance of STAT6 in inducing inflammatory responses in resident cells382. Consistent with it, by analyzing IL‐13‐induced STAT6‐ dependent genes in EPC2‐ALI, we showed that, in the esophageal epithelium, STAT6 is regulating the expression of genes that are related to cytokine and chemokine production, such as CCL26, POSTN and
TNFAIP6. Collectively, our observations indicated that STAT6 is the key regulator for the production of cytokines and chemokines in esophageal epithelium.
In conclusion, we described the transcriptome and phenotypical change of mature EPC2 ALI cells following IL‐13 stimulation, showing correlation with some of the signatures of EoE. In particular, we identified that barrier disruption and epithelial remodeling are driven by divergent pathways starting with IL‐13 signaling and leading to the activation of STAT3 and STAT6, which control proliferation rate and inflammatory chemokines production, respectively (Fig.7).
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3.7 Figures
Figure 3‐1. STAT binding site is one of the most enriched TFBS in EoE dysregulated genes
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126
Figure 3‐1. STAT binding site is one of the most enriched TFBS in EoE dysregulated genes. A)
Transcription factor binding site (TFBS) analysis of 1607 dysregulated genes from RNA sequencing.
Number of genes enriched with specific TFBS (grey bar) and p‐value (blue dot) is presented. B) Heatmap depicting expression level of all family members of STAT proteins. C) Individual RPKM value of STAT1, D)
STAT3 and E) STAT6 in esophageal biopsies (NL = 6; EoE = 10). (C‐E) Data are represented as the average
± S.E.M. Individual symbols represent an individual patient ***p < 0.001
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Figure 3‐2. STAT binding site is the most enriched in both dysregulated genes and transcriptionally active genes following 4hr incubation with IL‐13 in EPC2‐ALI
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Figure 3‐2. STAT binding site is the most enriched in both dysregulated genes and transcriptionally active genes following 4hr incubation with IL‐13 in EPC2‐ALI. A) 712 genes are dysregulated in 4hr IL‐13
(100ng/mL) treated in EPC2‐ALI culture. TFBS analysis of B) these 712 significantly altered genes by RNA sequencing and C) 858 genes highly enriched with H3K4me3 chromatin modification specific in 4hr IL‐13
(100ng/mL) treated in EPC2‐ALI culture identified by Chromatin Immunoprecipitation sequencing.
Number of genes enriched with specific TFBS (grey bar) and p‐value (blue dot) are presented in each graph.
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Figure 3‐3. STAT3 is the most upregulated STAT protein in 4hr IL‐13 treated EPC2‐ALI. And STAT3 binding site is the most enriched TFBS in IL‐13‐induced upregulated genes.
130
Figure 3‐3. STAT3 is the most upregulated STAT protein in 4hr IL‐13 treated EPC2‐ALI. And the STAT3 binding site is the most enriched TFBS in IL‐13‐induced upregulated genes. A) Heatmap depicting expression level of all family members of STAT proteins in EPC2‐ALI cultures treated with vehicle (Veh) and IL‐13 (100ng/mL) for 4 h. B) TFBS analysis of 278 upregulated genes in EPC2‐ALI treated with IL‐13
(100ng/mL) for 4 h from RNA sequencing. Number of genes enriched with specific TFBS (grey bar) and p‐ value (blue dot) are presented.
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Figure 3‐4. STAT3 and STAT3‐related genes regulate barrier integrity of esophageal epithelium
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Figure 3‐4. STAT3 and STAT3‐related genes regulate barrier integrity of esophageal epithelium. A)
Western Blot analysis and B) RNA sequencing analysis of EPC2‐ALI cultures transduced with empty control vector (EPC2‐ALICTRL) and vector with STAT3 shRNA (EPC2‐ALIΔSTAT3). C) Gene ontology (GO) enrichment analysis of 493 STAT3‐dependent genes from RNA sequencing. D) Heatmap depicting STAT3‐ dependent genes that are related to epidermal development. E) Filaggrin (FLG) expression level identified by RNA sequencing in esophageal biopsies (NL = 6; EoE = 10) and F) EPC2‐ALI. G) Expression level of filaggrin and pro‐filaggrin identified by western blot analysis in EPC2‐ALI. H) Trans‐epithelial electrical resistance (TEER) in EPC2‐ALI. (E, H) Data are represented as the average ± S.E.M. ***p <
0.001, ****p < 0.0001.
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Figure 3‐5. IL‐13 induced STAT3‐dependent genes are involved in IL‐13 induced cell proliferation
135
136
Figure 3‐5. IL‐13 induced STAT3‐dependent genes are involved in IL‐13 induced cell proliferation. A)
Gene ontology (GO) enrichment analysis of 129 IL‐13 induced STAT3 dependent genes identified by RNA sequencing analysis. B) Heatmap depicting 27 IL‐13‐induced STAT3‐dependent, cell proliferation regulation related genes in EPC2‐ALICTRL and EPC2‐ALIΔSTAT3 cultures treated with or without IL‐13 (100 ng/mL) for 48 hours. C) EPC2‐ALICTRL and EPC2‐ALIΔSTAT3 cultures treated with vehicle (Veh) or IL‐13 (100 ng/mL) for 48 hours and stained for BrdU incorporation and DAPI (nuclei). D) Quantification of BrdU‐ positive cells in EPC2‐ALI cultures. Data are presented as mean ± SEM; n = 3 per treatment. **p < 0.01, n.s. not significant. Magnification X300.
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Figure 3‐6. IL‐13 induced STAT6‐dependent genes are involved in cytokine production but not IL‐13 induced cell proliferation
138
139
140
Figure 3‐6. IL‐13 induced STAT6‐dependent genes are involved in cytokine production but not IL‐13 induced cell proliferation. A) Western Blot analysis and B) RNA sequencing analysis of EPC2‐ALI cultures transduced with empty control vector (EPC2‐ALICTRL) and vector with STAT6 shRNA (EPC2‐ALIΔSTAT6). C)
Gene ontology (GO) enrichment analysis of 166 genes that are IL‐13‐induced STAT6‐dependent identified by RNA sequencing analysis. D) Heatmap depicting 27 IL‐13‐induced STAT6‐dependent, cytokine production related genes and E) 5 representative inflammatory and cytokine‐related genes in
EPC2‐ALICTRL and EPC2‐ALIΔSTAT6 cultures treated with or without IL‐13 (100 ng/mL) for 48 hours. F) EPC2‐
ALICTRL and EPC2‐ALIΔSTAT6 cultures treated with vehicle (Veh) or IL‐13 (100 ng/mL) for 48 hours and stained for BrdU incorporation and DAPI (nuclei). G) Quantification of BrdU‐positive cells in EPC2‐ALI cultures. Data are presented as mean ± SEM; n = 3 per treatment. **p < 0.01. Magnification X300
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Figure 3‐7. Proposed mechanism of IL‐13 driven STAT3 and STAT6 dependent transcriptome alteration in esophageal epithelium
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Figure 3‐7. Proposed mechanism of IL‐13 driven STAT3 and STAT6 dependent transcriptome alteration in esophageal epithelium
IL‐13 signals through binding to IL4Rα/IL13Rα1 complex, which lead to the activation of Janus kinases.
Phosphorylation of JAKs lead to the phosphorylation of STAT3 and STAT6, which form homodimers, translocated to the nuclear compartments, bind to protein‐specific DNA elements and activate downstream gene transcription. Activation of STAT3 leads to alteration of genes related to proliferation including WTN7A and IRS1, while STAT6 activation leads to dysregulation of genes regulating inflammatory chemokines production.
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4 Chapter IV: General Discussion and Summary
The aim of this dissertation was 1) to define the molecular mechanism of IL‐13‐induced epithelium remodeling in esophageal epithelial cells; 2) to identify the underlying molecular processes that control these cellular functions; 3) to determine the significance of these molecular processes in the histological manifestations of EoE; and 3) to illustrate if specific targeting of these molecular pathways and functions could serve as therapeutic target for EoE treatment. Herein, our studies demonstrated that:
1. A group of genes which are part of the transmembrane transporter function group protein
expression and function is dysregulated in EoE.
2. SLC9A3 (NHE3) is the most upregulated transmembrane transporter in esophageal biopsies from
EoE patients and is primarily expressed in the basal and suprabasal layer of esophageal
epithelium.
3. NHE3 expression level positively correlated with severity of EoE including esophageal eosinophil
numbers and histopathological features such as esophageal DIS.
4. In both primary cells derived from EoE patients and in vitro stratified squamous esophageal
epithelium model, EPC2‐ALI, IL‐13 stimulation induced an increase in NHE3 mRNA and protein
expression and function.
5. Increased expression and activity of NHE3 led to changes in intracellular pH and elevated acid‐
secretion capacity in esophageal epithelial cells.
6. IL‐13 induced DIS was abrogated by pharmacological inhibition of NHE3 function.
7. STAT binding motif is one of the most enriched TFBS within the dysregulated genes in the EoE
biopsies, and IL‐13 stimulated mature stratified squamous esophageal epithelium.
8. The STAT3 binding motif is the most enriched STAT TFBS in IL‐13‐induced upregulated genes in
the esophageal epithelium.
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9. STAT3 plays an important role in IL‐13‐mediated dysregulation of esophageal barrier function
and esophageal epithelial proliferation.
10. IL‐13 induced STAT6 dependent genes contribute to cytokine and chemokine production by
esophageal epithelial cells.
The significances of these collective findings are further discussed below.
4.1 Alteration of transmembrane transporters expression in EoE
Performing gene ontology analysis on genes that were identified to be dysregulated in EoE patients, we identified 5 individual GO clusters of genes that were functional related to transmembrane transporter activities (Chapter 2). Active transmembrane transporter activities are commonly described in gastric and intestinal epithelial cells, as they are critical for the physiological functions for the lower GI tract383,384. However, the importance of transmembrane transporters in the esophagus is not fully understood.
Unlike other segments of the GI tract, the esophagus is not actively involved in fluid absorption and secretion, as the main function of the esophagus is to act as conduct for food to pass from the external environment to the stomach385. The esophageal epithelium possesses defensive mechanisms including luminal acid clearance that protects against toxic factors from causing esophageal tissue injury118.
Under physiological conditions, the esophageal epithelium of normal individuals is occasionally exposed to acidic contents from the stomach especially following meals228,229. The luminal acid is neutralized by
‐ + HCO3 secretion or H uptake by esophageal submucosal glands and esophageal epithelial cells, which protects against acid injury231,232. When the esophageal epithelium is exposed to prolonged episodes of acid refluxate due to dysregulation of the lower esophageal sphincter (LES), the acid may exceed the acid clearance capacity of esophagus leading to esophageal acid injury and development of esophageal diseases including gastroesophageal reflux disease (GERD)386. In response to this acid overload, a group
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+ + + ‐ of acid‐base transporters, including Na /H exchanger isotype 1 (NHE1), Na /HCO3 cotransporter (NBC) and sodium‐dependent chloride‐bicarbonate exchanger (e.g. SLC4A8), are induced to maintain a neutral pH in both intercellular spaces and cytosolic compartments227,237,268,387,388 (Fig. 4‐1).
Unlike GERD, an altered esophageal luminal pH is rarely observed in EoE patients389, as most EoE patients have normal LES tone390. Also, EoE patients have much less acid reflux events compared to
GERD patients391. Somewhat paradoxically, our RNAseq analysis comparing EoE and NL transcriptome identified that a large number of the transmembrane transporter genes that are dysregulated in EoE, that are tightly related to acid‐base transport circuit. For example, the SLC4 family which consists of a
‐ ‐ 392 group of HCO3 transporters are involved in HCO3 secretion to neutralize extracellular acid .
Furthermore, SLC26A4 is anion transporter that is known to facilitate chloride and bicarbonate transport and alter the acid‐base balance in kidney393,394. Given the lack of evidence to support altered esophageal lumen acidity in EoE, we speculated that dysregulation of the acid‐base transporters is associated with an esophageal epithelial response independent of luminal acidity.
Esophageal epithelial dilated intercellular spaces, basal zone hyperplasia, subepithelial fibrosis, and angiogenesis, together with smooth muscle hypertrophy are some of the most representative histopathological features related to tissue remodeling in EoE patients113,395. Previous studies suggest that altered activities of ion transporters are tightly connected with tissue remodeling396. For example, cytoplasmic Cl‐ concentration regulated by calcium‐activated chloride channel TMEM16A is required for tissue architecture and junctional remodeling396, while alteration of sodium hydrogen exchanger influences tissue proliferation and apoptosis397.
Previous study has reported that IL‐13 is able to induce EoE‐like transcriptome changes in primary esophageal epithelial cells54, and we also identified a similar group of ion transporters that are important for maintaining acid‐base homeostasis. Using this ex vivo model, we demonstrated that
146 intracellular pH is significantly increased in IL‐13‐treated primary esophageal cells derived from EoE patients. The alteration of intracellular pH has been shown to initiate a variety of signaling pathways and crucial for cell survival, function, and proliferation398. Since basal zone hyperplasia is an important histopathological feature in EoE399, one speculation is that the altered ion transporter gene network is related to the induction of intracellular pH change and increased esophageal epithelium proliferation .
Previous research suggested that elevated intracellular pH mediated by sodium‐proton exchanger contributes to the initiation of DNA synthesis321, which is directly related to increased cell proliferation.
+ + + ‐ Furthermore, Na /H exchanger and Na /HCO3 exchanger have been shown to be important in affecting alkalized pHi which is essential for cancer cell proliferation400. Alternatively, the alteration of acid‐base regulating ion transporters could be a consequence of the increased proliferation of esophageal epithelium. Cellular proliferation is energetically expensive, and in some fast proliferating cells, anaerobic glycolytic metabolic pathways are utilized to maintain cellular proliferation which often leads to the accumulation of lactate and H+ inside the cells401. While the accumulation of acid can cause cellular apoptosis402,403, intracellular pH regulation is essential for continuous proliferation. As a result,
‐ + + increased expression of proton pumps, HCO3 transporter family, Na /H exchanger family and monocarboxylate transporter (MCT) family are all observed in response to altered pH in cancer cells404,405. Given our observations that most of these transporters are also altered in EoE transcriptome, it is difficult to determine if these alterations are causative or consequential of basal zone hyperplasia.
To address this question, genetic overexpression or deletion of specific ion transporters and examination of the cell proliferation rate could be examined. In addition, assessment of the cell cycle to determine whether cells become trapped prior to proliferation or apoptosis could be performed.
Together, our findings suggested pHi mediated by these altered acid‐base transporters could play a role in modulating esophageal epithelium remodeling in EoE.
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Interestingly, within all the dysregulated transmembrane transporters in EoE patients, we identified several genes in the SLC16 family that encodes proteins responsible for transporting monocarboxylic acid. MCTs transport lactate and pyruvates across cellular membrane thus are important for energy metabolism406. At steady state, they are highly expressed in the epithelial cells both in the small and large intestine407, but not previously reported in the esophagus. Interestingly, SLC16A1 and SLC16A3 are observed increased in patients with Barrett’s esophagus and esophageal adenocarcinoma (EAC) and are thought to contribute to tumor metabolism408. Studies in other cancer cells demonstrated that the suppression of SLC16A1 and SLC16A3 led to decreased cell proliferation due to the accumulation of lactate inside cells which induces cell apoptosis409,410. Considering with some of EoE features especially esophageal epithelial hyperplasia, we speculated that increased proliferation in esophageal epithelium leads to elevated expression of these lactate transporters, which efflux lactate as a consequence of
411 glycolysis to maintain pHi and cellular proliferation . However, future studies are still needed to understand the contribution of MCTs in EoE pathogenesis.
Given the importance of pH in regulating cell physiology, one of the limitations of our research is we did not measure the intercellular pH alteration in the esophageal epithelium in EoE. In GERD studies, the diffusion of acid from the lumen to intercellular spaces is considered as one of the mechanisms of the disruption of epithelial barrier and formation of dilated intercellular spaces412. Also, a previous study indicated that increased paracellular permeability, leading to decreased pH promotes to increased tight junction permeability413. Intraluminal impedance measurement in EoE patients showed a much lower impedance compared to both NL and GERD patients, suggesting an increased permeability in the esophageal epithlium412. We demonstrated that IL‐13 stimulation of esophageal epithelium resulted in elevated intracellular pH and led to increased acid secretion. This supports the hypothesis that IL‐13 altered acid‐base transporters in esophagus could counteract the intracellular acid accumulation and increased pHi, as a consequence led to extracellular pH changes. However, further studies are needed to
148 confirm the intercellular pH changes between esophageal epithelial cells before we could speculate the contribution of altered pH in changing barrier permeability.
4.2 SLC9A3/NHE3 dysregulation in esophageal epithelium
Within all the dysregulated transmembrane transporters, we identified sodium hydrogen exchanger 3
(NHE3), encoded by SLC9A3 gene, as the most upregulated transporter in the esophageal biopsies derived from EoE patients. NHE3 is widely studied in the intestinal epithelium and proximal renal tubule due to its function in regulating ion homeostasis414. When coupled with Cl‐/HCO3‐ exchangers, NHE3 can effectively regulate NaCl absorption which drives the water movement across the epithelium415.
However, NHE3 expression and function have never been established in esophageal epithelium. Our study, for the first time, identified the expression pattern of NHE3 in the esophagus.
Using immunofluorescent staining, we identified that NHE3 is located primarily in the basal layer of esophageal epithelium in normal individuals, however expression expanded to the suprabasal layers in
EoE patients. This expressional expansion of NHE3 is mirroring the thickened basal layer in esophageal epithelium of EoE patients19. Also, we observed that the other histopathological change, dilated intercellular spaces (DIS), is also primarily localized in the suprabasal layer of esophageal epithelium. In light of these observations, we speculated that NHE3 is related to DIS formation and basal zone hyperplasia in EoE.
Correlation analysis first supported that NHE3 expression positively correlated with the severity of DIS spaces in EoE. To understand if elevated NHE3 expression directly contributes to the DIS formation, primary esophageal epithelial cells derived from EoE patients and EPC2‐ALI were treated with IL‐13.
Surprisingly, we observed an increase of NHE3 expression in both models upon IL‐13 treatment.
Previous studies showed that NHE3 expression are usually altered by Th1 cytokines including interferon‐ gamma (IFN‐γ) and tumor necrosis factor‐alpha (TNF‐α)305. This new finding of the ability of IL‐13
149 inducing NHE3 expression might explain the differential expression pattern of NHE3 between ulcerative colitis (UC) and Crohn’s disease (CD), two subtypes of inflammatory bowel diseases (IBD). Previous studies have reported that NHE3 activity decreased in UC and CD. However, the NHE3 mRNA level was only decreased in CD patients but not UC patients267,416,417. Interestingly, previous cytokine profiling indicated that IL‐13 is only increased in biopsy samples from UC patients but not CD patients418, potentially due to the Th2 predominance in the pathogenesis of UC419. In combination with our findings, the elevated IL‐13 level, which counterbalances the IFN‐γ effect in decreasing NHE3 expression, might explain the consistent NHE3 level in UC patients. Together, our observation showed an alternative pathway for NHE3 induction, which might be important to understand NHE3‐related disordered associated with Type‐2 inflammation.
As indicated in IBD patients, the alteration of NHE3 expression doesn’t seem necessarily correlate with dysregulation of NHE3 activity267. The NHE3 function can be post‐translationally modified through translocation between cytoplasmic compartments and plasma membrane as recycling endosome compartment, thus regulating activity in the absence of protein or mRNA regulation420. NHE3 only achieves its function of Na+ uptake and H+ secretion when binds to the plasma membrane. Previous study showed that Ezrin, a cytoskeletal linker protein, is essential for NHE3 translocation and activation421. Inhibition of Ezrin phosphorylation directly decreases NHE3 activity422. Also, NHE3 activity could be directly regulated by direct kinase phosphorylation423. As a result, simple observation of increased NHE3 expression could not conclude the functional involvement of NHE3 in EoE.
However, we also observed that NHE3 activity is increased upon IL‐13 treatment in esophageal epithelial cells. As NHE3 is responsible for Na+ absorption, dysregulation of NHE3 activity led to the disruption of
Na+ homeostasis417,424. Previous studies examining ion transport of the esophagus have revealed that
Na+ transport accounts for the majority of potential differences in esophageal epithelium predominantly
150 through epithelial sodium channel (ENaC)425. Dysregulation of Na+ concentration leads to changes in osmotic forces and altered fluid movement 426. Moreover, an increase of intracellular Na+ concentration, which facilitates Cl‐ influx will cause increased water movement from outside of the cell, which lead to fluid absorption 427. A consequence of fluid absorption and this hypertonicity causes increased cell volume. Fluid movement and increased cell volume, possible together with the disruption of junctional protein, will lead to the change of epithelium structure, for example, the formation of DIS. Currently, the mechanism of DIS formation is unclear. The previous study exposing esophageal epithelium in hypertonic NaCl solution on rabbit esophagus led to increased DIS area, suggesting that altered Na+ concentration in esophagus contributes to the DIS formation428,429. Aside from its role in regulating water r movement, sodium concentration is also related to uptake of other ions and substance430,431.
Together, we speculated that increase NHE3 activity led to increased intracellular Na+ concentration, which directly contributes to the increased presence of DIS in EoE patients. Indeed, we showed that IL‐
13 induction of DIS formation was NHE3‐dependent in EPC2‐ALI cells. To further validate this theory, intracellular Na+ concentration should be measured in esophageal biopsies in NL and EoE patients. Also, we showed that increased NHE3 activity is related to alteration of pH changes and acid‐secretion in esophageal epithelium. In previous studies in GERD, esophageal acid perfusion is considered as one of the drivers for DIS432. However, instead of being acidic, the pH in the esophageal lumen of EoE patients tends to be slightly alkalined433. We speculated, together with increase NHE3 activity and its localization in the basal and suprabasal layer of esophageal epithelium in EoE, consistent with the observation of increased intracellular pH, there’s acidification in the intercellular spaces in the esophageal epithelium in
EoE. The decrease of intercellular pH can then contribute to the disruption of tight junction protein and also initiate water movement caused by the change of osmotic gradient388. Together, the mechanism of
NHE3 in inducing DIS formation could be a combination of increased extracellular water accumulation and acidification in intercellular spaces (Fig. 4‐3).
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DIS is observed in the esophageal epithelium in both pediatric and adult EoE patients292, is associated intraepithelial antigen exposure434. Successful treatment of EoE using corticosteroids showed a reduction of DIS292, indicating DIS as a therapeutic marker. Our study showed that pharmacological inhibition of NHE3 protected esophageal epithelium from IL‐13 induced DIS, suggesting NHE3 could serve as a therapeutic target in EoE.
Basal cell hyperplasia was observed in EoE patients along the esophagus, characterized by more than
25% thickness of epithelial compared to NL19,435. Previous studies employing murine model of EoE revealed that IL‐13 induces esophageal epithelium hyperplasia206,207. We showed that inhibition of NHE3 activity prevents the IL‐13 induced proliferation in EPC2‐ALI. We speculated that IL‐13‐induced increased
NHE3 activity could either act as a protective mechanism to neutralize proliferation generated intracellular acid, or an initiator to generate an alkalinized intracellular environment to promote proliferation. Previous studies on esophageal NHE1 suggest that NHE1 can contribute to the hyperproliferation of esophageal tissue in both Barrett’s disorders and esophageal adenocarcinoma through the regulation of intracellular pH239,436. Also, intracellular pH is essential for cell proliferation and apoptosis437,438. As NHE3 can regulate intracellular pH, and we did show an elevated intracellular pH in IL‐13 treated esophageal epithelial cells with overexpressed NHE3, one hypothesis could be NHE3 is required for IL‐13 mediated proliferation in esophageal epithelium. Alternatively, the increased expression of NHE3 could be merely a consequence of increased proliferation. Fast proliferating cells like cancer cells have a preference towards lactic acid‐generating energy consuming pathways401. The conversion of lactic acid will lead to accumulation of proton inside cells, which decreased the glycolytic rate439 and induce cell apoptosis403. As a result, proton regulators that exporting intracellular H+ to prevent intracellular acidosis play a protective role in cells, and inhibition of these proton regulators could prevent increased cell proliferation440. Together, we conclude that IL‐13 upregulation of NHE3 is part of the cellular proliferative response, which is related to esophageal epithelium hyperplasia.
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However, further investigations are needed to elucidate whether this observation is causative or consequential of increased proliferation rate.
Interestingly, previous works indicated that NHE3‐deficient mice showed alteration of inflammatory‐ related pathways. Increased IFN‐γ was observed in small intestine441, while elevated expression of TNF‐ alpha and IL‐18 were shown in distal colon442. Also, inflammatory cells including NK cells and CD8+ T cells were elevated in NHE3KO mice443. It is still unclear how did the alteration of NHE3 expression contribute to changes of the inflammatory response. Previous study suggested that disruption of barrier integrity could result in the onset of inflammatory in gut444. It is possible that NHE3 alteration led to barrier disruption, which causes inflammatory responses. However, it would be still interesting to see if the increase of NHE3 would also cause any inflammatory changes and if it would have related to EoE pathogenesis.
4.3 STAT involvement in EoE transcriptome
Several cytokines have been shown to be involved in EoE pathogenesis, as increased level of cytokines including IL‐5, IL‐13, IL‐33, TNF‐α, IFN‐γ, and TSLP were observed in EoE patients48,332,445. After binding to their receptors on effector cells, these cytokines contribute to the progression of EoE through influencing downstream gene transcription including CCL26, CAPN14 and SLC9A3 (Chapter 2), which contribute to different aspects of EoE pathophysiology28,209. However, the molecular pathways involved in the regulation of the proinflammatory cytokines signaling remains unclear.
Janus kinase (JAK)‐ signal transducer and activator of transcription (STAT) pathway is a well‐ characterized pathway that plays a crucial role in a wide range of cytokines signaling, especially in interferons and interleukins446. After phosphorylated by JAKs, STAT monomers dimerize, translocate to nucleus and bind to conserved binding motif to activate gene transcriptions447. EoE related cytokines,
153 especially IL‐5, IL‐13 and IFN‐γ, all signaling through JAK/STAT pathway. (Fig. 4‐2) As a result, we were interested in the contribution of JAK/STAT pathway to the dysregulation of in EoE pathogenesis.
Since conserved sequences of STAT binding motifs has been identified448, we employed transcription factor binding site analysis among the dysregulated genes in EoE patients. Similar to what we expected, we identified STAT binding motif as one of the most significantly enriched binding motifs (Chapter 3).
STAT pathways are involved in multiple inflammatory responses, contributing to immune cell proliferation and differentiation, also related to the development of autoimmune and allergic disorders449. Mice models and cell lines with genetic modification on STAT expression suggest that alteration of STAT proteins leads to dysregulated cytokine functions and immune responses. For example, STAT1KO mice showed severe bacterial infection in multiple organs while WT mice remain healthy450. On the other hand, constitutively active STAT6 promoted T cell hyperproliferation451. With the increased enrichment of STAT regulating genes in EoE patients, we speculated an increased expression of STAT at least in mRNA level.
Further examination of RNAseq data supported our hypothesis, that increased level of STAT1 STAT3 and
STAT6 were observed in EoE patients compared to NL. Interestingly, all these three STAT proteins have previously been showed to relate to allergic disorders452‐454. STAT1KO mice showed decreased nasal eosinophilia compared to WT mice when sensitized with aeroallergen, and they were protected against experimental induction of allergic rhinitis452. Also, both STAT3KO and STAT6KO mice showed decreased airway eosinophilia and suppressed Th2 responses compared to WT mice upon antigen challenges453,455.
As IL‐13 has been previously identified as one the major driver for EoE phenotypes and transcriptome changes54, we’re more interested in the contribution of STAT3 and STAT6 in EoE pathogenesis due to their involvement in IL‐13 signaling pathways185,456. However, since IFN‐γ is also elevated in EoE332, future studies on IFN/STAT1 pathway should be conducted to understand the etiology of EoE.
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Aside from STAT proteins, the binding motifs of other transcription factors are found significantly enriched in our analysis in dysregulated genes in EoE patients. Some of these transcription factors were previously characterized and closely related to Th2 allergic disorders. For example, NF‐κB binding motif is the second most significantly enriched TFBS, and its activation has been widely established in various inflammatory disorders457. A recent study showed that the deletion of NF‐κB ‐inducing kinase (NIK), an essential molecular for the non‐canonical NF‐κB pathway, drives spontaneous EoE in the experimental murine model, suggesting an important role of NF‐κB in the pathogenesis of EoE108. Interestingly, NF‐κB has previously shown to coordinate with STAT proteins to regulate cancer immunology458. In this sense, aside from STAT proteins, it would be beneficial to explore the molecular mechanism of other enriched transcription factors we observed in the pathogenesis of EoE.
4.4 STAT3 regulates tissue remodeling in esophageal epithelium
To better understand how STAT proteins regulated EoE‐related gene dysfunction and histopathological changes, we employed IL‐13 induced EPC2‐ALI model, which is previously established to mimic EoE dysregulation in transcriptome level. By applying TFBS analysis of genes that are transcriptionally active or altered by IL‐13, we identified STAT as an important regulator in IL‐13‐induced genes. Previous GO analysis identified these genes were important for epithelium development and inflammatory responses
(Chapter 2). As a result, we speculated that STAT signaling pathways are involved in these processes.
Closer examination of IL‐13 regulated subsets of genes, we identified the STAT3 binding site as the most enriched TFBS in IL‐13 induced upregulated genes. Studies using tissue‐specific STAT3 knockout mice suggested that STAT3 is important in mediating various biological process including cell survival, apoptosis, and motility459. Specifically, previous studies indicated that STAT3 is involved in various allergic pathways. For example, patients with the autosomal‐dominant hyper‐IgE syndrome (AD‐HIES) with the dominant negative STAT3 mutation have a significant lower rate of food allergies and
155 anaphylaxis371,372. This is consistent with the essential role of STAT3 in mediating mast cell degranulation460,461. STAT3 has also been shown to activate Th2 cell development and is required for Th2 cytokines expression462. Thus mice with STAT3 deficiency in T cells fail to develop allergic inflammation in OVA‐induced mice model462. Moreover, Conditional knockout of STAT3 in mice airway epithelium led to decrease of house dust mite‐induced airway eosinophilia, suggesting the role of STAT3 in mediating eosinophils recruitment453. However, the role of STAT3 has never been identified in EoE. Comparing the transcriptome differences between STAT3 knockdown and non‐transduced EPC2‐ALI cultures, we identified a group of genes that are functionally related to the epidermis and skin development being
STAT3‐dependent. Previous studies have revealed that activation of STAT3 in endothelial cells leads to downregulation of tight junction proteins such as zonulin‐1 (ZO‐1) and occludin together with elevated paracellular leakage341. Also, STAT3 mediates epidermal growth factor (EGF)‐induced downregulation of claudin‐2 and claudin‐4, which emphasized the importance of STAT3 in modulating junctional proteins463.
One of the clinical features of EoE is the impaired barrier function of esophageal epithelium indicated by decreased impedance412. Earlier studies have established the role of IL‐13 in decreasing epithelium barrier functions through regulating junctional proteins like DSG‐1 and FLG133,464. The DSG‐1 expression is previously established regulated in a STAT6‐dependent manner465, while FLG could be induced by IL‐
33 in a STAT3‐dependent manner359. As a result, we speculated that STAT3 might influence esophageal barrier function by affecting filaggrin expression. Consistent with our hypothesis, the FLG expression is increased upon STAT3 knocking down. In addition, STAT3KD EPC2‐ALI cultures also showed higher trans‐ epithelial electrical resistance compared to WT group. Overall, we established that STAT3 inhibition could increase filaggrin expression contribute to epithelial barrier integrity. However, given our study is limited due to the presence of residual STAT3 caused by the incomplete knockdown, we could not conclude the necessity of STAT3 in epithelial function. Also, STAT3 must alter the epithelial resistance by
156 changing proteins other than filaggrin. Thus future studies should check on other STAT3‐dependent junctional proteins.
In addition, by examine IL‐13 induced STAT3‐dependent genes in EPC2‐ALI model, we speculated that
STAT3 might contribute to IL‐13‐mediated basal cell proliferation in esophageal epithelium. A previous study in limbal epithelial cells indicated that STAT3 is crucial for keratinocyte proliferation466. Studies on human and mouse epithelial cells further supported the role of IL‐13 in epithelial differentiation and proliferation342,467. In EPC2‐ALI, we showed that IL‐13/STAT3 pathway regulates oncogenes like IRS1 and
WNT7A, which are widely known to promote DNA replication and cell proliferation368,369. Our results in
IL‐13 treated STAT3KD EPC2‐ALI models further supported the role of STAT3 in regulating cell proliferation. We showed that deletion of STAT3 in EPC2‐ALI prevented IL‐13‐induced proliferation, suggesting a potential role of STAT3 in basal cell hyperplasia in EoE.
Furthermore, a recent investigation identified the presence epithelial‐mesenchymal transition (EMT) of in pediatric EoE patients114, and they also suggested the contribution of EMT to tissue remodeling in
EoE. Further work indicated the development of EMT in EoE is mediated by the CXCR4/SDF‐1 axis in experimental murine model468. Previous studies have demonstrated the role of STAT3 in promoting EMT in different cancer cells469. Interestingly, the CXCR4/SDF‐1 pathway is tightly related to STAT3 activation470‐472. We speculated that IL‐13 induced STAT3 is contributing to EMT in EoE. However, future experiments and analysis are required to prove this hypothesis.
4.5 STAT6 regulates cytokine production in esophageal epithelium
JAK/STAT6 pathway has long been characterized as the canonical IL‐13 signaling pathway176. Studies showed that knocking down STAT6 resulted in significant impairment of IL‐13 effect on cells including macrophage182 and epithelial cells473. Previous animal studies showed the alteration of inflammatory responses in mice with STAT6 deficiency. OVA‐induced airway eosinophilia was abrogated in STAT6 KO
157 mice, together with decreased peri‐bronchial inflammation379‐381. In addition, STAT6 is also essential in
Th2 cell differentiation474. Deficiency of STAT6 that fails to induce Th2 cell differentiation led to the development of severe experimental autoimmune encephalomyelitis (EAE) in mice475. Together, these suggested a critical role of STAT6 in modulating inflammatory responses. In both EoE patients and IL‐13 induced EPC2‐ALI, we observed increased expression of STAT6 (Chapter 3). We speculated that this increase of STAT6 contributes to changes in inflammatory genes.
Previous studies have suggested that esophageal epithelium secretion of CCL26, a chemokine induced by IL‐13 that is important for eosinophil activation and recruitment in EoE28, is dependent on STAT6476.
By analyzing IL‐13‐induced STAT6‐dependent genes in EPC2‐ALI, we showed that, in the esophageal epithelium, STAT6 is regulating CCL26 expression, as well as genes including POSTN and TNFAIP6 that contribute to the regulation of cytokine production. Mice experiment transferring STAT6+/+ antigen‐ specific Th2 cells to STAT6‐/‐ mice failed to develop experimental allergic asthma, suggesting the importance of STAT6 in inducing inflammatory responses in resident cells382. Consistent with it, we also showed that the impairment of STAT6 leads to decreased expression of several IL‐13‐induced cytokine and chemokine in esophageal epithelium.
Moreover, previous works demonstrated the primary effector cells of STAT6 functions on inflammatory responses are hematopoietic cells such as B cell, Th2 cells, and mast cells477. Limited by our current model, we’re not able to fully demonstrate the importance of IL‐13/STAT6 pathway in inflammatory responses in EoE. Future works are required in a model containing hematopoietic cells to prove our hypothesis.
4.6 Summary
In this dissertation, we identified the importance of both transmembrane transporter SLC9A3 and transcription factor STAT3 in the pathogenesis of EoE. Comprehensive analysis of SLC9A3 in three
158 different models relevant to EoE suggests that increased SLC9A3 expression and activity induced by IL‐
13 is important for the formation of dilated intercellular spaces in EoE patients. While the SLC9A3 expression in the esophageal epithelium is dependent on STAT6, IL‐13 induced STAT3 activation is also important for tissue remodeling in EoE, as STAT3 deletion esophageal epithelium showed increased barrier function and is protected against IL‐13 induced esophageal hyperplasia. Together, the studies in this dissertation provide rationales to targeting SLC9A3 and IL‐13/STAT3 pathways for the treatment of
EoE, especially in the effort to address esophageal epithelium remodeling.
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4.7 Figures
Figure 4‐1. Acid protection mechanism in esophagus
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Figure 4‐1. Acid protection mechanism in esophagus.
In esophageal epithelium, when exposed to excessive acidic contents, ion transporters related to H+ and
+ + + ‐ HCO3‐ transportation are activated. Some of the major transporters are Na /H exchangers, Na /HCO3
‐ ‐ cotransporters, Cl / HCO3 exchangers and sodium‐dependent chloride‐bicarbonate exchangers.
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Figure 4‐2. JAK/STAT signaling pathway
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Figure 4‐2. JAK/STAT signaling pathway.
After binding to their receptors on the transmembrane domain, cytokines such as IL‐13, IL‐5, and IFN‐γ led to the phosphorylation of Janus kinases (JAKs). Phosphorylated JAKs then phosphorylate STAT proteins, which then dimerized and translocated to the promoter area of their downstream genes. The binding of STAT dimers will then activate or suppress gene transcription.
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Figure 4‐3. Proposed Mechanism of IL‐13 induced NHE3‐dependent DIS formation in EoE
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Figure 4‐3. Proposed Mechanism of IL‐13 induced NHE3‐dependent DIS formation in EoE
(Left) In normal stratified squamous epithelium cells, transmembrane transporters are not dysregulated intracellular pH remains normal. (Right) Elevated IL‐13 secretion in EoE patients drives increased NHE3 transcription. This increased expression and activity of NHE3 on the plasma membrane, together with other acid‐base regulating transporters, contribute to the altered concentration of H+ and Cl‐ both intracellularly and intercellularly, which led to the changed equilibrium of osmotic balance. Alteration in osmotic force drove water influx into intercellular spaces and formed DIS in the esophageal epithelium in EoE.
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