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IL-13 promotes accumulation of esophageal epithelial mitochondria with

translational implications for eosinophilic esophagitis

Jazmyne L. Jackson, B.S., Reshu Saxena, PhD, Mary Grace Murray, B.A., Abigail

J. Staub, B.A., Courtney Worrell, Jasmine Cruz, B.S., No’ad Shanas, B.S., Alena

Klochkova, MD, PhD, Travis H. Bordner, B.S., John M. Crespo, B.S., Annie D.

Fuller, B.S., B.S. William-Nazario-Lugo, Grace Davis, Hailey Golden, Andres J.

Klein-Szanto, MD, PhD, Tatiana A. Karakasheva, Adam Karami, M.S., John W.

Elrod, Gary W. Falk, MD, Zachary Wilmer Reichenbach, MD, PhD, Melanie Ruffner,

MD, PhD, Amanda B. Muir, MD, Kelly A. Whelan, PhD

PII:

S2352-345X(26)00060-3

DOI:

https://doi.org/10.1016/j.jcmgh.2026.101782

Reference:

JCMGH 101782

To appear in:

Cellular and Molecular Gastroenterology and Hepatology

Accepted Date: 31 March 2026

Please cite this article as: Jackson JL, Saxena R, Murray MG, Staub AJ, Worrell C, Cruz J, Shanas

N’a, Klochkova A, Bordner TH, Crespo JM, Fuller AD, William-Nazario-Lugo BS, Davis G, Golden H,

Klein-Szanto AJ, Karakasheva TA, Karami A, Elrod JW, Falk GW, Reichenbach ZW, Ruffner M, Muir

AB, Whelan KA, IL-13 promotes accumulation of esophageal epithelial mitochondria with translational

implications for eosinophilic esophagitis,

Cellular and Molecular Gastroenterology and Hepatology

(2026), doi:

https://doi.org/10.1016/j.jcmgh.2026.101782

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Institute.

Title: IL-13 promotes accumulation of esophageal epithelial mitochondria with

translational implications for eosinophilic esophagitis

Short Title: Mitochondria and eosinophilic esophagitis

Jazmyne L. Jackson, B.S.

, Reshu Saxena, PhD

1,2

Mary Grace Murray, B.A.

, Abigail J.

Staub, B.A.

, Courtney Worrell

, Jasmine Cruz, B.S.

, No’ad Shanas, B.S.

Alena

Klochkova, MD, PhD

, Travis H. Bordner, B.S.

, John M. Crespo, B.S.

, Annie D. Fuller,

B.S.

William-Nazario-Lugo, B.S.

, Grace Davis

, Hailey Golden

, Andres J. Klein-

Szanto, MD, PhD

, Tatiana A. Karakasheva

, Adam Karami, M.S.

, John W. Elrod

, Gary

W. Falk, MD

Zachary Wilmer Reichenbach, MD, PhD

7,8,9

, Melanie Ruffner, MD, PhD

10,

, Amanda B. Muir, MD

3,10

, Kelly A. Whelan, PhD

1,12

Fels Cancer Institute for Personalized Medicine, Lewis Katz School of Medicine, Temple

University, Philadelphia, PA 19140, USA

Amity School of Biological Sciences, Amity University Punjab, Mohali-140306, India

Division of Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital of

Philadelphia, Philadelphia, PA 19104, USA

Histopathology Facility, Fox Chase Cancer Center, Philadelphia, PA 19111, USA

Aging + Cardiovascular Discovery Center, Department of Cardiovascular Sciences,

Lewis Katz School of Medicine at Temple University, Philadelphia, PA

19140, USA

Division of Gastroenterology and Hepatology, University of Pennsylvania Perelman

School of Medicine, Philadelphia, PA 19104, USA.

Department of Medicine, Section of Gastroenterology, Temple University Hospital,

Philadelphia, Pennsylvania, USA, Philadelphia, PA 19140, USA

Center for Substance Abuse Research (CSAR), Temple University Lewis Katz School of

Medicine, Philadelphia, Pennsylvania, USA, Philadelphia, PA 19140, USA

Center for Microbiology and Immunology, Temple University Lewis Katz School of

Medicine, Philadelphia, Pennsylvania, USA.

Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA

19104, USA

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Division of Allergy and Immunology, The Children’s Hospital of Philadelphia,

Philadelphia, PA 19104, USA

Department of Cancer and Cellular Biology, Lewis Katz School of Medicine at Temple

University, Philadelphia, PA 19140, USA

Abbreviations used in this paper:

ATP, Adenosine Triphosphate;

BCH, Basal Cell

Hyperplasia; DAMP, Damage-Associated Molecular Patterns; DHTKD1, Dehydrogenase

Transketolase Domain-Containing 1; DRP1, Dynamin-Related Protein 1; ECAR,

Extracellular Acidification Rate; EGD, Esophagogastroduodenoscopy; EGID,

Eosinophilic Gastrointestinal Disease; EoE, Eosinophilic Esophagitis; Eos/HPF,

Eosinophils Per High Power Field; ETC, Electron Transport Chain; FCCP,

Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; H&E , Hematoxylin & Eosin;

HSS, Histology Scoring System; IF, Immunofluorescence; IHC, Immunohistochemistry;

IL, Interleukin; IL-4Rα, IL-4 Receptor alpha; I-SEE, Index Of Severity For Eosinophilic

Esophagitis; JAK, Janus Kinase; MFN, Mitofusin; MMP, Mitochondrial Membrane

Potential; MTCO1, Mitochondrially Encoded Cytochrome C Oxidase 1; mtDNA,

Mitochondrial DNA; OCR, Oxygen Consumption Rate; OGDHL, Oxoglutarate

Dehydrogenase-Like; OXPHOS, Oxidative Phosphorylation; OVA, Ovalbumin; STAT,

Signal Transducer And Activator Of Transcription; Th2, T Helper 2; TLR, Toll-Like

Receptor; TSLP, Thymic Stromal Lymphopoietin

Correspondence:

Kelly A. Whelan, PhD

Associate Professor and Vice Chair, Department of Cancer & Cellular Biology

Fels Cancer Institute for Personalized Medicine

Temple University Lewis Katz School of Medicine

3307 N. Broad St.

PAHB Room 206

Philadelphia, PA 19140

215-707-8467

kelly.whelan@temple.edu

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Conflict of Interest Disclosure:

Part of this study was supported by an EoE Young

Investigator Award granted to Dr. Whelan by Shire Human Genetic Therapies, Inc.

Dr.

Falk has research funding from Adare/Ellodi, Arena/Pfizer, Bristol Myers Squibb, Celldex,

Nexeos, Regeneron/Sanofi, Takeda. Dr Falk is a medical consultant for Adare/Ellodi,

AnaptysBio, Bristol Myers, Squibb, EsoCap, Uniquity, Phathom, Regeneron/Sanofi,

Takeda and Upstream Bio. Dr. Falk serves on the Speakers Bureau for

Regeneron/Sanofi. Dr. Ruffner receives research funding through her institution from

Regeneron. Dr. Muir serves as a medical consultant for Regeneron/Sanofi, EsoCap,

Apogee and Uniquity. The remaining authors disclose no conflicts.

Grant Support:

This work was supported by the National Institutes of Health:

R01DK136987 (KAW), R03AI156435 (KAW), R01AI184785 (MR), F31DK139760 (JLJ),

T32GM142606 (ADF, JMC; PI, Xavier Graña, Kelly Whelan), R01GM155497 (THB, CW;

PI, Xavier Graña), P30CA006927 (AKS; PI, Jonathan Chernoff), F31CA294914 (ADF).

Additional support was provided by: EoE Young Investigator Award from Shire Human

Genetic Therapies, Inc. (KAW; IIR-USA-002420), Washington & Jefferson College

Maxwell Internship Award (AJS) and Washington & Jefferson College Merck Internship

Award (AJS), The AGA-Dr. Harvey Young Education & Development Foundation’s Young

Guts Scholars Program (CW), and a Temple University Bridge award, an internal grant

provided by the Office of the Vice President for Research (OVPR) at Temple University

(KAW).

Preprint server:

bioRxiv; 10.1101/2025.04.02.646853

Word Count:

5171

Data Availability Statement

Data, analytic materials and study materials will be made available upon request to Dr.

Whelan.

Author Contribution

J.L.J (Data curation: Equal; Formal analysis: Lead; Funding acquisition: Supporting;

Methodology: Equal; Validation: Lead; Visualization: Lead; Writing – original draft: Lead;

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Writing – review & editing: Lead); R.S. (Data curation: Equal; Formal analysis: Equal;

Methodology: Supporting; Writing – original draft: Supporting; Writing – review & editing:

Supporting); M.G.M. (Data curation: Equal; Formal analysis: Supporting; Writing –original

draft: Supporting); A.J.S.. (Data curation: Supporting; Formal analysis: Supporting;

Visualization: Supporting; Writing – review & editing: Supporting); C.W. (Data curation:

Supporting; Funding acquisition: Supporting); J.C. (Data curation: Supporting); N.S.

(Visualization: Supporting); A.K. (Data curation: Supporting; Methodology: Supporting;

Visualization: Supporting; Writing – review & editing: Supporting); T.H.B. (Data curation:

100

Supporting; Funding acquisition: Supporting; Writing – review & editing: Supporting);

101

J.M.C. (Funding acquisition: Supporting; Writing – review & editing: Supporting); A.D.F.

102

(Funding acquisition: Supporting; Writing – review & editing: Supporting); W.N.L. (Writing

103

– review & editing: Supporting); G.D. (Data curation: Supporting); H.G. (Data curation:

104

Supporting); A.K.S (Data curation: Supporting; Formal analysis: Supporting; Validation:

105

Supporting; Writing – review & editing: Supporting); T.A.K. (Writing – review & editing:

106

Equal); A.K. (Formal analysis: Supporting; Software: Supporting); J.E. (Methodology:

107

Supporting; Writing – review & editing: Supporting); G.W.F. (Resources: Supporting);

108

Z.W.R. (Data curation: Supporting; Resources: Supporting); M.R. (Writing – review &

109

editing: Supporting); A.B.M. (Data curation: Supporting; Resources: Supporting;

110

Visualization: Supporting; Writing – review & editing: Supporting); K.A.W.

111

(Conceptualization: Lead; Funding acquisition: Lead; Investigation: Lead; Methodology:

112

Lead; Project administration: Lead; Resources: Lead; Supervision: Lead; Visualization:

113

Lead; Writing – original draft: Lead; Writing – review & editing: Lead)

114

115

Synopsis

116

Mitochondrial mass is increased in esophageal mucosa of humans and mice with EoE

117

inflammation. In esophageal keratinocytes, IL-13 acts through JAK/STAT signaling to

118

increase mitochondrial mass and extracellular mtDNA. Increased circulating mtDNA is

119

found in serum of EoE patients.

120

121

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Abstract

122

Background & Aims:

Metabolic and mitochondrial dysfunction have recently been

123

implicated in eosinophilic esophagitis (EoE) pathogenesis. However, there is a need to

124

define the influence of EoE-associated inflammatory cues upon mitochondrial biology,

125

mechanisms mediating these effects, and the clinical significance of mitochondrial

126

alterations in EoE.

127

Methods:

Mitochondria were evaluated in human biopsies, MC903/Ovalbumin-induced

128

murine EoE, and human esophageal keratinocytes stimulated with EoE-relevant

129

cytokines. Mitochondrial mediators were assessed via qRT-PCR and western blotting.

130

Metabolism, mitochondrial membrane potential, and apoptosis were measured.

131

Mitochondrial DNA (mtDNA)-encoded genes,

ND1

and

ND6

were assessed by qPCR in

132

DNA from culture media and circulating nucleic acids from human serum samples. Effects

133

of JAK inhibitor ruxolitinib or genetic inhibition of STAT3 or STAT6 on mitochondria were

134

assessed

in vitro

135

Results:

We identified evidence of increased mitochondria in esophageal mucosa of EoE

136

patients and mice with EoE-like inflammation. IL-13 consistently induced mitochondrial

137

accumulation in esophageal keratinocytes

in vitro

and this response was associated with

138

increased expression of mediators of mitochondrial biogenesis, fusion, and mitophagy.

139

IL-13 suppressed mitochondrial respiration and ATP production, without impacting

140

membrane polarization or apoptosis. Active EoE patients exhibited elevated serum

141

mtDNA levels and upregulation of mediators of mtDNA-associated inflammatory

142

signaling. Increased mitochondrial mass and accumulation of extracellular mtDNA in IL-

143

13-treated esophageal keratinocytes were dependent on JAK/STAT signaling.

144

Conclusions:

We identify IL-13 as a mediator of increased mitochondrial mass in EoE

145

through JAK/STAT signaling. We further demonstrate that IL-13 promotes accumulation

146

of extracellular mtDNA and that circulating mtDNA is elevated in EoE patients.

147

Key Words:

Eosinophilic esophagitis; mitochondria; Interleukin-13; JAK/STAT pathway

148

149

150

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Introduction

151

Eosinophilic esophagitis (EoE) is a chronic food allergen- and immune-mediated

152

disease that not only exerts significant clinical and financial burden worldwide

, but is

153

rising in prevalence

. EoE is characterized by esophageal eosinophilia and tissue

154

remodeling in esophageal epithelial, muscle, and stromal compartments

1, 3

. EoE

155

symptoms include vomiting, dysphagia and food impaction, all of which negatively impact

156

quality of life

4-8

. While dietary elimination and corticosteroids are therapeutic mainstays in

157

EoE, the monoclonal antibody dupilumab, which targets interleukin (IL)-4 receptor (IL-

158

4R)α, became the first and only FDA-approved targeted therapy for use in EoE patients

159

in 2022

9, 10

. This significant advance in EoE patient care was guided by experimental

160

evidence supporting the importance of T helper (Th)2 signaling in EoE. Presently, our

161

understanding of the molecular mechanisms through which Th2 cytokines drive EoE

162

pathobiology remains incompletely understood.

163

In response to allergens, esophageal epithelial cells secrete cytokines, including

164

thymic stromal lymphopoietin (TSLP)

11, 12

, to recruit lymphocytes that then produce Th2

165

cytokines IL-4, IL-5, and IL-13. In esophageal epithelium, IL-4 and IL-13 bind to IL-4Rα to

166

promote JAK-mediated phosphorylation of signal transducer and activator of transcription

167

(STAT) proteins

. Phosphorylated STAT proteins then dimerize and translocate to the

168

nucleus where they bind DNA and direct target gene transcription

. Targeted genetic

169

depletion of epithelial IL-4Rα subunit protects mice from experimental EoE

in vivo

170

supporting IL-4Rα-mediated signaling in esophageal epithelium as a key driver of EoE.

171

Although epithelial-associated IL-13-responsive genes, including eotaxin-3

16-18

, calpain-

172

, synaptopodin

, and follistatin

have been linked to EoE pathobiology, studies in

173

mice suggest a predominant role for IL-13 in experimental EoE

174

Genetic evidence first indicated a potential role for mitochondria in EoE

. Whole

175

exome sequencing on patients with EoE and unaffected family members revealed

176

enrichment in rare, damaging variants in two genes:

DHTKD1 (

Dehydrogenase and

177

Transketolase Domain Containing 1), which

contributes to mitochondrial lysine

178

metabolism and adenosine triphosphate (ATP) production

23, 24

, and its homolog

OGDHL

179

(Oxoglutarate Dehydrogenase-Like)

In vitro

experiments further revealed suppressed

180

mitochondrial respiration in esophageal fibroblasts from EoE patients expressing

181

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DHTKD1

OGDHL

variants

. Impaired mitochondrial respiration was also identified

182

upon DHTKD1 knockdown in non-EoE esophageal epithelial cultures that further

183

displayed increased levels of ROS

184

Two recent publications examined metabolism and mitochondria in EoE. Ryan and

185

colleagues linked hypoxia-inducing factor (HIF)-1α-mediated metabolic dysfunction to

186

differentiation defects in esophageal keratinocytes

. Our own recent collaborative work

187

linked IL-13-mediated mitochondrial structural damage and impaired respiratory capacity

188

with epithelial remodeling in EoE

. While these two studies suggest that bioenergetic

189

stress in esophageal epithelial cells contributes to EoE pathobiology, how the broader

190

EoE inflammatory milieu influences mitochondria, the direct molecular mechanisms

191

through which IL-13 influences mitochondrial mass, and the relevance of mitochondrial

192

alterations to clinical features of EoE have yet to be elucidated.

193

Beyond their metabolic functions, mitochondria also contribute to inflammation.

194

Damage-associated molecular patterns (DAMPs)

27-29

are endogenous cellular

195

components that promote an immune response upon release from cells

Mitochondrial

196

DNA (mtDNA) present in the cytosol or outside of cells can function as a DAMP, promoting

197

inflammation through toll-like receptor 9 (TLR9), cyclic GMP–AMP synthase-stimulator of

198

interferon genes (cGAS/STING) or NLR family pyrin domain containing 3 (NLRP3)

199

inflammasome pathways, the latter of which has been implicated in EoE pathogenesis

200

Circulating mtDNA has been explored as a biomarker in pathologic conditions including

201

sepsis, myocardial infarctions, and inflammatory bowel disease

32-34

; however, circulating

202

mtDNA has not been evaluated as a biomarker in EoE, a disease in which there is

203

significant interest in identifying noninvasive biomarkers as diagnosis and monitoring

204

currently rely on histological assessment of endoscopic biopsies acquired under general

205

anesthesia.

206

The current study furthers our understanding of mitochondria in EoE. We perform

207

the first systematic evaluation of protein markers of mitochondria in EoE patients and

208

explore the relationship between mitochondria and clinical features of EoE. We directly

209

assess the impact of a panel of EoE inflammatory mediators on mitochondrial mass in

210

esophageal epithelial cells, identifying IL-13 as a signal that increases expression of

211

mediators of mitochondrial dynamics to promote increased mitochondrial mass. We

212

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continue to define the impact of IL-13 on mitochondrial function, demonstrating decreased

213

OXPHOS that is not accompanied by mitochondrial depolarization or apoptosis. We

214

further demonstrate for the first time that IL-13 promotes accumulation of extracellular

215

mtDNA as well as TLR9 cleavage and increased expression of proteins in the

216

cGAS/STING pathway. In EoE patients, we then explore circulating mtDNA as a

217

biomarker. Finally, we demonstrate that IL-13-mediated increased mitochondrial mass

218

and extracellular mtDNA accumulation are dependent upon JAK/STAT signaling in

219

esophageal keratinocytes. In sum, these studies provide novel insight into the

220

mechanisms regulating mitochondrial biology in EoE as well as the potential significance

221

of these alterations in terms of EoE pathobiology and clinical care.

222

223

Results

224

Evidence of increased mitochondria in esophageal mucosa of EoE patients and

225

mice with EoE-like inflammation

226

To explore the impact of EoE inflammation on mitochondria in esophageal

227

mucosa, we evaluated expression of MTCO1 (mitochondrially encoded cytochrome c

228

oxidase I), a mitochondrially-encoded subunit of Complex IV of the electron transport

229

chain (ETC) in esophageal biopsies from human patients with EoE or control non-EoE

230

subjects with normal esophageal pathology (

Table 1

). Although EoE biopsies include

231

inflammatory cells and may also have lamina propria (LP), we assessed staining in

232

epithelial cells as staining was robust in these cells. In both non-EoE (n=5) and inactive

233

EoE (n=20) subjects, MTCO1 staining was restricted to the basal and parabasal epithelial

234

cells with no or very little stain seen in spinous and superficial cells. 22/23 (95.6%) of

235

active EoE subjects exhibited BCH, with MTCO1 positivity in the cytoplasm of all basaloid

236

cells observed across the expanded basal cell compartment and very little or no

237

immunostain in the most superficial layers (

Fig. 1A

). Average MTCO1 score was

238

significantly increased in patients with active EoE inflammation as compared to either

239

non-EoE controls or patients with inactive EoE (

Fig. 1A, B

Co-staining of MTCO1 and

240

KI67 in a representative biopsy from an active EoE patient and a non-EoE control subject

241

further suggested that increased mitochondria are not restricted to proliferative basal cells

242

(

Fig. 1C

). We then continued to assess the relationship between MTCO1 staining and

243

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EoE clinical indices, finding weak positive correlations between MTCO1 score and either

244

BCH score (R

=0.3947;

<.0001) or eosinophil count (R

=0.1792,

=.0027) (

Fig. 2A, B

)

245

across our patient cohort.

Within our active EoE patient cohort, we compared MTCO1

246

score in patients with history of stricture or food impaction to those without and found no

247

significant difference in mean MTCO1 score (

Fig. 2C, D

248

We next assessed mitochondria in esophageal mucosa of mice with EoE-like

249

inflammation induced by MC903/OVA, a food allergen-mediated model that

250

exhibits features consistent with EoE pathophysiology as found in humans, including

251

eosinophil-rich inflammatory infiltrates in esophageal mucosa

35, 36

(

Fig 3A

). Mice treated

252

with MC903 only, which develop robust atopic dermatitis but fail to exhibit esophageal

253

inflammation, served as controls. An increase in the ratio of mtDNA relative to nuclear

254

DNA was detected in peeled esophageal epithelial sheets of mice with EoE-like

255

inflammation (

Fig. 3B

). A 1.72-fold increase in MTCO1 stain was also found in epithelium

256

of mice with EoE-like inflammation; however, this increase did not meet statistical

257

significance (

=.0648) (

Fig. 3C, D

). In our murine cohorts with and without EoE-like

258

inflammation, we observed weak positive correlations between MTCO1 score and

259

eosinophil count (R

=0.0719;

=.3154) or LP thickness (R

=0.0908;

=.2950) (

Figure

260

3E, F

261

262

IL-13 increases mitochondrial mass in esophageal keratinocytes

263

As immunostaining revealed increased MTCO1 staining in epithelium of EoE

264

patients and mice with EoE-like inflammation, we investigated the impact of EoE-

265

associated inflammatory cues on mitochondria in esophageal epithelial cells. The

266

immortalized normal esophageal cell line EPC2-hTERT was stimulated with IL-4, IL-5, IL-

267

13, IL-1



, or TNF



, for 7 days. We utilized DNA qPCR to evaluate levels of the

268

mitochondria-encoded genes

MTCO1

and

ND6

(encodes mitochondrial protein NADH-

269

ubiquinone oxidoreductase subunit 6)

as well as the nuclear-encoded gene

COXIV

270

(encodes the mitochondrial protein cytochrome c oxidase 4). Significant increases in both

271

MTCO1

and

ND6

DNA level were uniquely detected in EPC2-hTERT cells treated with

272

IL-13 (

Fig. 4A

). Although IL-4 increased levels of

MTCO1

and

ND6

DNA in EPC2-hTERT

273

cells across 6 independent experiments, this increase was not as robust as that induced

274

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by IL-13 and did not meet statistical significance (

MTCO1

=.0632;

ND6

=.4959). EoE-

275

relevant cytokines failed to significantly influence nuclear-encoded gene

COXIV,

which is

276

to be expected as EoE has not been shown to promote genomic instability

37-39

. IL-13-

277

mediated increase in mtDNA level was recapitulated in primary esophageal cell cultures

278

derived from either a non-EoE control subject or an active EoE patient (

Fig. 4B, C

279

Notably, while IL-4 failed to impact mtDNA levels in non-EoE primary cells, a significant

280

increase in

MTCO1

and

ND6

DNA level was found in EoE primary cells (

Fig. 4B, C

). In

281

EPC2-hTERT cells, co-stimulation with IL-13 and IL-4 for 7 days increased mtDNA levels

282

to a level that was similar to that observed with IL-4 stimulation (

Fig. 4D

283

As elevated mtDNA level may result from an increase in mitochondria or an

284

increase in the mtDNA copy number within each mitochondrion, we assessed

285

mitochondrial mass in IL-13-treated esophageal epithelial cells. An increase in

286

mitochondrial mass was apparent using immunostaining for mitochondrial outer

287

membrane protein TOM20 (

Fig. 5A, B

) or the mitochondrial dye MitoTracker Green (

Fig.

288

5C, D

289

290

Expression of mediators of mitochondrial dynamics in IL-13-treated esophageal

291

keratinocytes and EoE patient biopsies

292

Increase in mitochondrial mass may be reflective of enhanced biogenesis,

293

alterations in fission/fusion dynamics, or impaired mitochondrial turnover, the latter of

294

which primarily occurs via mitophagy

. To determine how IL-13 treatment may influence

295

these facets of mitochondrial dynamics, we next surveyed expression of key genes

296

involved in the regulation of each of these facets of mitochondrial biology. IL-13 induced

297

expression of

TFAM

, a critical mediator of biogenesis

, in EPC2-hTERT cells following

298

1, 3, and 7 days of treatment (

Fig. 6A

). No significant changes were detected in the

299

expression of

MFN1

MFN2

, encoding for mitofusion1 and mitofusion2, critical

300

mediators of mitochondrial fusion, or in

DRP1,

encoding for dynamin-related protein-1,

301

critical mediator of mitochondrial fission (

Fig. 6B-D

). A significant increase in expression

302

PINK1

and

PARK2

, encoding PTEN-induced kinase-1 and Parkin, mediators of

303

mitophagy, was detected in EPC2-hTERT cells following 5 days of treatment with IL-13

304

(

Fig. 6E, F

). We then assessed expression of these mediators of mitochondrial dynamics

305

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at the protein level in EPC2-hTERT cells treated with IL-13 for 7 days in which increased

306

TOM20 expression was apparent (

Fig. 6G, H

). These experiments revealed increased

307

levels of TFAM, MFN1, MFN2 and PINK1 (

Fig. 6G, H

). Neither expression of DRP1 nor

308

its phosphorylation at Serine residue 616, a site that triggers the translocation of p-DRP1

309

from the cytosol to the mitochondria where it stimulates fission

, was impacted by IL-13

310

(

Fig. 6G, H

). Notably, in EPC2-hTERT cells we were unable to detect expression of Parkin

311

protein in the presence or absence of IL-13. We further assessed expression of mediators

312

of mitochondrial dynamics in transcriptomic data from patients with active EoE and non-

313

EoE controls

TFAM, MFN1, and MFN2

were increased in active EoE patients compared

314

to normal controls while

PINK1

and

PARK2

expression

was decreased in this dataset

315

(

Fig. 6I

) in which

DRP1

was not detected.

316

317

Effects of IL-13 on mitochondrial functions in esophageal keratinocytes

318

We next aimed to determine the impact of IL-13 on mitochondrial functions in

319

esophageal epithelial cells. Seahorse respirometry revealed a decrease in oxygen

320

consumption rate (OCR) of IL-13-treated EPC2-hTERT cells, with suppression of basal,

321

ATP-linked, and maximal respiration (

Fig. 7A-D

). To gain insight into glycolytic activity,

322

extracellular acidification rate (ECAR) was assessed alongside OCR measurements.

323

Although diminished mitochondrial respiration may result in a compensatory increase in

324

glycolysis, we did not detect any change in either ECAR or lactate levels in IL-13-treated

325

EPC2-hTERT cells (

Fig. 7E, F

). Diminished overall energy level in IL-13-treated

326

esophageal keratinocytes was further supported as EPC2-hTERT cells and primary

327

esophageal epithelial cultures (both non-EoE and EoE) displayed decreased basal ATP

328

levels following 7 days of IL-13 stimulation (

Fig. 7G-I

329

To determine if decreased mitochondrial respiration is associated with

330

mitochondrial membrane depolarization, we assessed MitoTracker Red (membrane

331

potential sensitive dye) and MitoTracker Red Green (membrane potential insensitive dye)

332

staining in IL-13 treated EPC2-hTERT cells. In these experiments, we found no evidence

333

of altered mitochondrial depolarization in IL-13-treated cells (

Fig. 8A, B

)

As mitochondria

334

are key regulators of apoptosis

, we assessed cell death via Annexin-V/PI flow cytometry

335

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(

Fig. 8C, D

); however, no significant apoptosis was observed in IL-13-treated EPC2-

336

hTERT cells.

337

338

Evidence of extracellular mtDNA and activation of mtDNA-associated inflammatory

339

signaling in IL-13-treated esophageal keratinocytes

340

As IL-13 induced accumulation of mitochondria within esophageal keratinocytes,

341

we next aimed to determine if this cytokine may also influence mtDNA accumulation

342

outside of esophageal keratinocytes. To do so, we performed DNA qPCR for mtDNA-

343

encoded genes

ND1 (

encodes mitochondrial protein NADH-ubiquinone oxidoreductase

344

subunit 1) and

ND6

in culture media of IL-13-treated esophageal cells. Increased copy

345

number of

ND1

and

ND6

was detected in cell-free media from EPC2-hTERT cells as well

346

as primary esophageal keratinocytes (both non-EoE and EoE) following 7 days of IL-13

347

treatment (

Fig. 9A-D

). Notably,

COXIV,

a nuclear-encoded gene, was not detected in

348

culture media of EPC2-hTERT cells. To assess mtDNA-associated inflammatory

349

signaling, we evaluated expression of mediators of the TLR9 and cGAS/STING pathways.

350

In IL-13-treated EPC2-hTERT cells, we found marked increases in levels of cleaved

351

TLR9, indicating the active form of the protein that can recruit MyD88 and activate

352

downstream signaling

, and cGAS (

Fig. 10A, B

). Increased expression of STING as well

353

as STING phosphorylation at serine 366, indicating cGAS/STING pathway activation

354

were also increased with IL-13 treatment; however, these increases were modest and

355

failed to reach the level of statistical significance among 3 independent experiments (

Fig.

356

10A, B

). To assess TLR9- and cGAS/STING pathways in EoE patients, we mined publicly

357

available transcriptomic data

, finding that key genes within the TLR9 and cGAS-STING

358

pathways (

MYD88, TRAF6, TRAF3,

MB21D1

[cGAS],

TMEM173

[STING],

TBK1,

and

359

IRF3

)

are upregulated in active EoE patients compared to non-EoE subjects (

Fig. 10C

360

361

Evidence of increased circulating mtDNA in serum of active EoE patients

362

We continued to examine circulating mtDNA as a biomarker for EoE using DNA

363

qPCR to measure the absolute copy number of mitochondrially-encoded genes

ND1

and

364

ND6

in serum from active EoE patients or non-EoE control subjects (

Table 2

). We

365

identified increased average copy number of

ND1

and

ND6

in serum of active EoE

366

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patients compared to non-EoE controls (

Fig. 11A, B

). We further evaluated the

367

relationship between circulating mtDNA copy number and esophageal stricture or food

368

impaction. There was a trend toward decreased copy number of either

ND1

ND6

369

subjects with history of stricture; however, the data failed to reach statistical significance

370

(

Fig. 11C, D

). Serum

ND1

level was comparable in EoE patients regardless of history of

371

impaction while there was a trend toward increased serum

ND6

levels in those with history

372

of impaction (

Fig. 11E, F

373

374

Role of JAK/STAT signaling in IL-13-mediated increase in esophageal epithelial

375

mitochondria

376

IL-13-mediated activation of JAK/STAT signaling has been linked to EoE

377

pathogenesis, including by promoting STAT6-mediated eosinophilic infiltration and

378

epithelial remodeling mediated by both STAT6 and STAT3

41, 66, 179-181

. As such, we finally

379

sought to explore the functional contribution of this signaling axis to IL-13-mediated

380

increases in mtDNA levels in esophageal epithelial cells and their culture media.

381

Pharmacological inhibition of JAK signaling was achieved using ruxolitinib, which

382

effectively diminished STAT6 phosphorylation at Tyrosine residue 641, which is required

383

for STAT6 nuclear translocation

(

Fig. 12A, B

). With regard to STAT3, ruxolitinib

384

suppressed STAT3 phosphorylation at Tyrosine residue 705, which is required for nuclear

385

translocation

, but had no effect on phosphorylation at residue Serine 727 (

Fig. 12A, C,

386

), which targets STAT3 to mitochondria

. Ruxolitinib further abrogated IL-13-mediated

387

increase in mtDNA levels in EPC2-hTERT cells (

Fig. 12E

) and also in their culture media

388

(

Fig. 12F, G

389

To explore the individual contributions of STAT3 and STAT6 to IL-13-mediated

390

increase in mtDNA in esophageal epithelial cells, we generated EPC2-hTERT cells with

391

doxycycline (DOX)-inducible expression of shRNA targeting either STAT6 or STAT3. After

392

validating respective depletion of STAT3 or STAT6 (

Fig. 13A, B

), we assessed expression

393

of TOM20 fluorescence in IL-13-treated cells. Cells transfected with a non-targeting (NS)

394

shRNA exhibited increased TOM20 level and this effect was not significantly affected by

395

individual depletion of either STAT6 or STAT3 (

Fig. 13C, D

). STAT6 depletion did,

396

however, result in an increase in TOM20 level in the absence of IL-13 (

Figure 13C, D

397

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Discussion

398

Mitochondria have been implicated in various immune-mediated inflammatory

399

pathologies, including asthma and eczema

51, 52

; however, our understanding of the role

400

of this organelle in EoE is only beginning to emerge. Our initial studies revealed increased

401

expression of mitochondrial protein MTCO1 in esophageal epithelium of active EoE

402

patients. Sherrill et al. demonstrated increased expression of

DHTKD1

, which contributes

403

to mitochondrial ATP production and lysine metabolism, in active EoE patients at the level

404

of gene expression and also show a representative IHC image for DHTKD1 that suggests

405

increased expression as compared to a representative image from a non-EoE control

406

subject

. Ryan et al. showed increased expression of markers of OXPHOS at the

407

transcriptional level in EoE patients along with a representative image suggesting

408

increased level of mitochondrial complex V (ATP synthase) in EoE

. Additionally, in our

409

recent collaborative publication, an increase in mitochondrial gene expression was

410

demonstrated in esophageal epithelial basal and intermediate cell populations defined by

411

single cell RNA-sequencing in active EoE patients

. While these data collectively suggest

412

increased mitochondria in esophageal biopsies of EoE patients, the current study

413

provides the first systematic analysis of the expression of a mitochondrial protein

414

(MTCO1) in esophageal epithelium of EoE patients.

415

In a complementary analysis in mice with EoE-like inflammation, we identify

416

increased MTCO1; however, these data failed to meet statistical significance. Variability

417

in MTCO1 expression was apparent particularly in MC903/OVA-treated mice, potentially

418

as a result of the heterogeneity with regard to extent of inflammation and remodeling in

419

the epithelial and stromal compartments between animals. In both humans and mice,

420

MTCO1 correlated positively, albeit weak, with eosinophil count. MTCO1 was also

421

positively associated with BCH in humans and LP thickness in mice. These data suggest

422

that increased mitochondria is associated with increased inflammation and tissue

423

remodeling in EoE. To determine if MTCO1 score is associated with disease severity, we

424

assessed its expression in relation to history of food impaction or stricture, among the

425

most severe consequences of EoE, in EoE patients; however, these variables failed to

426

correlate. While it is possible that mitochondrial accumulation in esophageal epithelium is

427

more closely related to inflammation and reactive epithelial responses as opposed to

428

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tissue remodeling in the subepithelial space and fibrostenosis, longitudinal studies are

429

necessary to directly assess these relationships.

430

Limitations of our assessment of MTCO1 expression in esophageal mucosa of

431

mice and humans include a singular focus on esophageal epithelial cells. Indeed, our

432

finding that mtDNA level is increased in peeled esophageal epithelial sheets from mice

433

with EoE-like inflammation must be interpreted in the context of potential presence of

434

infiltrating immune cells and residual stromal cells. Although not a potential cell type

435

present in peeled murine epithelium or human biopsies, muscle cells are rich in

436

mitochondria and contribute to EoE pathobiology

, including through direct effects

437

mediated by IL-4R

, warranting investigation of the impact of EoE inflammation on

438

mitochondria in the esophageal muscle compartment. Additionally, while we provide data

439

to suggest that increased MTCO1 level in esophageal epithelium of EoE patients is not

440

merely a result of increased proliferation, these data are limited to single representative

441

non-EoE- and active EoE-derived esophageal biopsies. Our results seem to indicate that

442

mitochondrial mass may be decreased in proliferating esophageal epithelial cells;

443

however, rigorous evaluation in a larger patient cohort is necessary to formally assess

444

this relationship.

445

Although our recent collaborative work identified IL-13-mediated mitochondrial

446

function as mediator of EoE-associated BCH, the broader impact of EoE-relevant

447

inflammatory cues on mitochondrial biology as well as the mechanisms driving these

448

effects represent key knowledge gaps that we aimed to address in the current study.

449

Among a panel of EoE-associated cytokines tested, IL-13 exerted the most robust effect

450

on mitochondrial mass in both immortalized and primary esophageal keratinocytes. IL-4

451

treatment also increased mtDNA level in immortalized keratinocytes and primary EoE

452

cells but had no apparent effect on primary non-EoE cells. Additionally, IL-4-mediated

453

increase in mtDNA level reached the level of statistical significance only in primary EoE

454

cells. While it is tempting to speculate that exposure to the

in vivo

EoE inflammatory milieu

455

may prime cells for future response to IL-4, in line with the concept that EoE remission

456

patients are poised to relapse

, assessment of IL-4 mediated effects on mtDNA in

457

multiple EoE-derived cell lines is required to test this. Similarly, although the primary non-

458

EoE culture used in the current study failed to show a response to IL-4 in terms of mtDNA,

459

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it remains unclear if these findings would be recapitulated in a panel of non-EoE cultures.

460

In the context of these limitations, our findings suggest that while both IL-4 and IL-13 have

461

the capacity to increase mtDNA level in esophageal keratinocytes, IL-13 induces a more

462

robust response in both primary and immortalized esophageal keratinocytes. It will also

463

be interesting to determine how these cytokines contribute to increases in mitochondrial

464

mass in the context of the complex EoE inflammatory milieu using murine EoE models

465

with pharmacologic or genetic inhibition strategies to target IL-4 and IL-13

in vivo

466

The current study further suggests that IL-13 influences multiple aspects of

467

mitochondrial biology. We find that IL-13 promotes increased expression of mediators of

468

mitochondrial biogenesis (TFAM), fusion (MFN1, MFN2), and turnover (PINK1,

PARK2

)

469

while not influencing expression or activating phosphorylation of fission-mediator DRP1.

470

Increased expression of mitochondrial transcription factor TFAM is consistent with our

471

finding of increased mtDNA both within and outside of esophageal keratinocytes.

472

Confocal analysis further demonstrated expansion of the perinuclear mitochondrial

473

network, consistent with activation of mitochondrial fusion, which facilitates mitochondrial

474

homeostasis under conditions of energy depletion and stress

40, 56

. Thus, enhanced

475

mitochondrial fusion in response to IL-13 is consistent with our finding of diminished ATP

476

level as well as limited respiration and glycolysis in IL-13-treated esophageal

477

keratinocytes. It is important to note that our recent collaborative work demonstrates

478

diminished mitochondrial respiration and mitochondrial mass with IL-13 stimulation for 96

479

hours

. It is possible that timing contributes to the discrepancy between these two studies

480

with increased mitochondrial mass and fusion occurring as a compensatory response

481

aiming to restore energetic balance in esophageal keratinocytes. It is further possible that

482

mitochondrial metabolism is restored with long-term IL-13 treatment, promoting an

483

OXPHOS metabolic phenotype in EoE as identified by Ryan et al.

. We also observed

484

increased expression of

PARK2

and PINK1 in esophageal keratinocytes responding to

485

IL-13, which may indicate activation of mitophagy, potentially to prune unhealthy

486

mitochondria. While our own previous work showed that IL-13 promotes autophagy as a

487

cytoprotective response in esophageal keratinocytes, autophagy flux was documented at

488

72 hours of IL-13 treatment and mitophagy was not assessed

. Additionally, while

489

expression of

TFAM

MFN1

, and

MFN2

expression is increased in EoE patient biopsies,

490

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expression of both

PARK2

and

PINK 1

is decreased. Thus, further studies are required

491

to assess mitophagy in the context of both IL-13-mediated alterations in mitochondrial

492

mass and EoE pathobiology.

493

The current study provides the first evidence that IL-13 stimulation of esophageal

494

keratinocytes promotes accumulation of extracellular mtDNA. Consistent with our

495

findings, mtDNA has been shown to be released from cells independent of mitochondrial

496

damage through mechanisms that include antibacterial defense

, necroptosis

, MPTP

497

opening

, platelet activation

, cellular senescence

, and neurological inflammatory

498

responses

. While it’s well established that cells typically dispose of dysfunctional

499

mitochondria to maintain cellular homeostasis, the release of mitochondria/mitochondrial

500

components from cells is also important for communication between cells, affecting

501

cellular phenotype

and functioning as a potent DAMP to activate inflammatory

502

signaling

. In the current study, IL-13-treated epithelial cells exhibited robust increases

503

in expression of both cleaved TLR9 as well cGAS with more modest increases in STING

504

expression and activating phosphorylation that fail to meet significance. While it is

505

possible that cGAS/STING pathway is primed without sustained downstream activation,

506

time course experiments are necessary to interrogate how IL-13 influences mtDNA-

507

responsive pathways. Indeed, such studies are of high interest as transcriptomic data

508

demonstrates upregulation of key players in TLR9 and cGAS/STING signaling in EoE

509

patients. Our collaborative work has linked decreased mitochondrial mass to IL-13-

510

associated BCH

whereas the current study demonstrates that IL-13 increases

511

mitochondrial mass and accumulation of extracellular mtDNA as well as activation of

512

DNA-sensing inflammatory pathways. As such, it will be of further interest to dissect the

513

dynamic interplay between IL-13 and mitochondria in relation to EoE pathobiology to

514

determine whether there are distinct roles for decreased mitochondria in mediating BCH

515

and increased mitochondria in mediating inflammatory signaling or instead whether any

516

significant change in mitochondrial mass in esophageal keratinocytes acts as a signal to

517

drive both pathogenic features of EoE.

518

Extending our findings on extracellular mtDNA to human subjects, we demonstrate

519

for the first time that circulating mtDNA is increased in the serum of active EoE patients.

520

It must be acknowledged, however, that there was a significant degree of overlap in terms

521

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of copy numbers of both genes in EoE subjects and controls, limiting the potential value

522

of circulating mtDNA as a diagnostic biomarker. However, it is possible that circulating

523

mtDNA may have utility as a biomarker for a specific subset of EoE patients (e.g., a

524

phenotypic biomarker) or may serve as a biomarker for therapeutic response, perhaps in

525

the context of dupilumab. We further assessed the relationship between mtDNA and

526

history of stricture or food impaction in EoE patients, observing trends toward a negative

527

association with stricture. As a progressive shift from inflammatory to fibrostenotic EoE

528

has been proposed

, it is possible that declining mtDNA is indicative of diminished

529

inflammatory signaling. As such, longitudinal studies to assess mtDNA levels in the

530

context of EoE progression to fibrostenosis are of high interest. There are several

531

limitations to this analysis, including its cross-sectional design with a limited number of

532

patients with history of stricture and also failure to account for atopy status in control

533

patients, the latter of which is important as circulating mtDNA has been demonstrated in

534

patients with atopic dermatitis

. Future studies should also evaluate mtDNA in relation to

535

established clinical indices, including Endoscopic Reference Score (EREFS)

65, 66

536

Histology Scoring System (HSS)

Index of Severity for Eosinophilic Esophagitis (I-

537

SEE)

and Pediatric Eosinophilic Esophagitis Symptom Scores (PEES)

. Given the

538

costs and risks associated with general anesthesia, prospective, longitudinal studies with

539

well-characterized comparison groups are needed to determine the utility of circulating

540

mtDNA as a noninvasive biomarker.

541

Finally, our findings demonstrate that JAK/STAT signaling is necessary for mtDNA

542

accumulation within and outside of esophageal keratinocytes. As STAT3 localizes to

543

mitochondria following Serine727 phosphorylation

, we speculated mitochondrial STAT3

544

may contribute to IL-13-mediated increases in mtDNA. However, while JAK inhibition

545

abrogated IL-13-mediated increases in both intracellular and extracellular mtDNA, it had

546

no appreciable impact on STAT3 Serine727 phosphorylation, suggesting that

547

mitochondrial STAT3 may not contribute to IL-13-induced mtDNA accumulation. STAT6

548

has also been shown to translocate to mitochondria where it regulates mitochondrial

549

function and fusion in non-epithelial cells

. As such, future studies will assess the effect

550

of IL-13 on subcellular localization of STAT6. We further found that genetic depletion of

551

either STAT3 or STAT6 alone was insufficient to suppress IL-13-mediated increase in

552

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mitochondria, suggesting that they may act in a redundant fashion to induce this effect in

553

esophageal keratinocytes. As STAT1 localizes to mitochondria and has been implicated

554

in EoE

71-73

, it is further possible that STAT1 or other members of the STAT family may

555

contribute to mitochondrial alterations in EoE.

556

As we failed to observe any significant effect of IL-13 on mtDNA levels prior to 7

557

days of stimulation, it is unlikely that JAK/STAT signaling mediates its effects on

558

mitochondria through an immediate transcriptional response. Among the panel of

559

mediators of mitochondrial dynamics that we assessed at the level of RNA only

TFAM

560

showed a response at early time points. To the best of our knowledge, STAT-mediated

561

transcriptional regulation of TFAM has not been reported. STAT3 has been implicated in

562

mitochondrial gene regulation and interactions with TFAM

74, 75

. STAT3-mediated

563

transcriptional induction of

Mfn2

downstream of protein kinase Cε has been demonstrated

564

to protect against DOX-induced cytotoxicity in rats

. Indirect regulation of

MFN2

565

STAT3 has also been demonstrated following STAT3-mediated transcriptional

566

upregulation of

BCL6

(encoding B-cell lymphoma 6)

. In this case BCL6 acted as a

567

negative regulator of

MFN2

transcription to promote senescence in endothelial cells

568

Mitochondrial STAT6 has also been shown to inhibit mitochondrial fusion by blocking

569

MFN2 dimerization

. In addition to its effects on mediators of mitochondrial dynamics,

570

mitochondrial STAT3 has been shown to inhibit mitochondria respiration

, limiting ROS

571

production and oxidative damage during cardiac ischemia

. We also found an increase

572

in mtDNA with STAT6 depletion in EPC2-hTERT cells in the absence of IL-13, suggesting

573

STAT6 as a negative regulator in this context. These findings underscore the complexity

574

of STAT-mediated regulation of mitochondrial biology and the need for further mechanistic

575

studies to define the precise molecular mechanisms through which IL-13-mediated STAT

576

signaling promotes mitochondrial accumulation in esophageal keratinocytes.

577

Mitochondria have been linked to EoE-associated esophageal epithelial defects in

578

terms of impaired epithelial differentiation

and BCH

. The current study adds to our

579

understanding of mitochondria in EoE, linking IL-13 to increased mitochondria both inside

580

and outside of esophageal epithelial cells, the latter of which unveils a novel IL-13-

581

mitochondria-immune axis with relevance to pathobiology and biomarker discovery in

582

EoE. Although the body of literature related to mitochondria in EoE is presently limited

583

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and there are areas of discrepancy that must be reconciled, this emerging area of

584

investigation highlights the potential translational and clinical relevance of mitochondria

585

in EoE.

586

587

Methods

588

Human Subjects

589

Biological specimens were obtained from patients via Children’s Hospital of Philadelphia

590

(CHOP), Hospital at the University of Pennsylvania (HUP), or at Temple University

591

Hospital (TUH). Human subjects underwent endoscopic biopsy collection via

592

esophagogastroduodenoscopy (EGD) during routine clinical care under protocols

593

approved by the Institutional Review Board at HUP or TUH (HUP Protocol Number:

594

813841; TUH Protocol Number: 29721). At the time of diagnostic EGD, pinch biopsies

595

were obtained for clinical evaluation. For circulating mtDNA studies, blood was drawn

596

from each subject at the time of diagnostic EGD under protocols approved by the

597

Institutional Review Board at CHOP and HUP (CHOP Protocol Number: 10-007737; HUP

598

Protocol Number: 813841). Serum was isolated from whole blood using standard

599

techniques. Written informed consent was obtained from each subject or their legal

600

guardian (where appropriate). Patients who met clinical criteria for EoE

, with ≥15

601

mucosal eosinophils per high power field (eos/HPF), were classified as active EoE.

602

Inactive EoE patients had a history of active EoE, and clinical examination showed

603

resolution of esophageal eosinophilia (<15 eos/HPF) at the time of follow-up EGD. Non-

604

EoE subjects included those reporting symptoms warranting EGD, but those who

605

demonstrated no endoscopic or histological abnormalities in the upper gastrointestinal

606

tract. Subjects with a history of inflammatory bowel disease, celiac disease, GI bleeding,

607

or any other acute or chronic intestinal disorders were excluded from recruitment.

608

Demographic and clinical information on human subjects whose esophageal biopsies or

609

serum were evaluated in the current study is provided in

Table 1

and

Table 2,

610

611

Murine Studies

612

All animals were purchased, bred, and treated under Temple University Institutional

613

Animal Care and Use Committee-approved protocols (Protocol Number: 5018). EoE was

614

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induced in wild type C57/B6 mice using the MC903/Ovalbumin (OVA) protocol

35, 36

with

615

the following modifications: sensitization period during which MC903 (2 nmol; Tocris

616

Bioscience, Cat# 2700) and OVA (100 mg; Sigma-Aldrich, Cat# A5503-50G) are applied

617

to ears was shortened to from 14 days to 12 days; no treatments were administered on

618

days 13 and 14; OVA in drinking water was increased from 1.5 g/mL to 15 g/mL; challenge

619

period was extended from 4 days to 18 days. Mice treated with MC903 only were used

620

as controls. We further used a cohort of mice including from our published study

for

621

immunostaining. This cohort was treated as described above and included groups treated

622

with MC903 alone or in combination with OVA. Following experimental protocols,

623

esophagi were harvested and processed as described below.

624

625

Immunohistochemistry (IHC) & Histological Evaluation

626

Formalin-fixed paraffin-embedded tissue sections on glass slides were stained with the

627

hematoxylin & eosin (H&E) or the following primary antibodies as previously described

628

A pathologist blinded to clinical parameters (AKS) scored MTCO1 cytoplasmic staining

629

on a scale from 0 (negative) to 3, taking into account stain intensity and distribution. A list

630

of antibodies with used dilutions is provided in

Table 3

631

632

Immunofluorescence

633

Paraffin-embedded tissue sections were deparaffinized. Antigen retrieval was achieved

634

by heating sections in 10mM Citric Acid Buffer (pH 6.0). Sections were washed with PBS,

635

and blocked with StartingBlock 20 blocking buffer (37539, Thermo Scientific) for 10

636

minutes. Sections were then incubated overnight at 4°C with primary antibody and for 1

637

hour with secondary at room temperature. A list of antibodies with used dilutions is

638

provided in

Table 3

. Sections were mounted with Antifade Mounting Medium with DAPI

639

(VectaShield, Cat # H-1200-10). Images were captured with a LEICA SP8 Lacer Scanning

640

Microscope and analyzed using LAS X Life Science Microscope Software.

641

642

Cell Culture

643

The immortalized normal esophageal keratinocyte cell line EPC2-hTERT was provided

644

as a generous gift from Drs. Anil Rustgi and Hiroshi Nakagawa (Columbia University).

645

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Primary cell cultures generated from esophageal biopsies from a non-EoE- human

646

subject (EPC203) and an active EoE patient (EPC128) were obtained through CHOP

647

Gastrointestinal Epithelium Remodeling Program. All cells were cultured in keratinocyte

648

serum free media (KSFM; Gibco, Cat# 17005042) supplemented with recombinant

649

epidermal growth factor (1ng/mL), bovine pituitary extract, (50 mg/mL), and

650

penicillin/streptomycin (1% v/v, Gibco, Cat# 15140-122), as previously described

. For

651

experiments utilizing EPC2-hTERT cells with TNFα (R&D Systems, Cat# 210-TA-005/CF;

652

40 ng/mL), IL-4 (R&D Systems, Cat# 204-IL-010; 10 ng/mL), IL-5 (R&D Systems, Cat#

653

205-IL-005; 10 ng/mL), IL-13 (R&D Systems, Cat# 213-ILB-005/CF; 10 ng/mL), or IL-



654

(R&D Systems, Cat# 201-lB-005; 10 ng/mL), 30,000 cells (25,000 for primary cells) were

655

plated in 6-well plates, or 100,000 cells were plated on 10-cm plates, 24 hours prior to

656

stimulation with cytokines. Media was changed every 48 hours for a total of 7 days. 10

657

ng/ml stocks of cytokines (20 ng/ml of TNFα) were dissolved in 0.1% Bovine Serum

658

Albumin (Fisher, Cat# BP9703-100) in 1X D-PBS (Gibco, Cat# 14190-144).

659

EPC2-hTERT cells with DOX-inducible expression of short hairpin RNAs

660

(shRNAs) targeting STAT6 (Horizon Discovery, Cat.# V3IHSPGG_5029170) or STAT3

661

(Horizon Discovery, Cat.# V3IHSPGR_5162692) were generated by lentiviral

662

transduction. Cells expressing a non-targeting (NS) shRNA (Horizon Discovery, Cat#

663

VSC11653) served as controls. 500,000 cells were plated in 100 mm dishes and were

664

treated with DOX for 5 days to initiate knockdown before being treated with IL-13 (10

665

ng/mL) for 7 days. DOX treatment was continuous throughout the period of IL-13

666

treatment to maintain knockdown. To suppress the JAK/STAT signaling EPC2-hTERT

667

cells were treated with JAK inhibitor, ruxolitinib (100 ng/mL; Invitrogen, Cat# tlrl-rux-3) .

668

Ruxolitinib was refreshed every 48 hours throughout the 7-day period of IL-13 (10 ng/mL)

669

stimulation.

670

671

Quantitative Polymerase Chain Reaction (qPCR)

672

For mitochondrial DNA (mtDNA) assays

, DNA isolation was performed on esophageal

673

keratinocytes or peeled murine esophageal epithelium using DNeasy Blood and Tissue

674

Kit (Qiagen, Cat# 69506) according to the manufacturer’s instructions. DNA concentration

675

was determined using Qubit™ dsDNA HS Assay Kit according to the manufacturer’s

676

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instructions (Invitrogen, Cat# Q32851). qPCR was performed using PowerUp™ SYBR™

677

green master mix (ThermoFisher Scientific, Cat# A25743).

678

To assess mtDNA levels, primers for mtDNA D-Loop (murine),

MTCO1

(human)

679

and

ND6

(human) were used. To assess nuclear DNA, primers for

Ikbβ

(murine),

COXIV

680

(human), and

GAPDH

(human) were used. The relative fold change between the noted

681

mtDNA-encoded genes and nuclear DNA-encoded genes (

Ikbβ

GAPDH

) was

682

calculated using the delta-delta CT method. In human specimens, the relative levels of

683

nuclear DNA-encoded gene

COXIV

was used as an additional control.

684

mtDNA copy numbers were evaluated in conditioned media and patient serum

685

samples. DNA was extracted from 1.5 mL serum samples using QiAamp Circulating

686

Nucleic Acid Kit (Qiagen Cat# 55114) according to the manufacturer’s protocol. qPCR

687

was performed using Green Real-Time PCR Master Mix Kit (Life Technologies). DNA was

688

extracted from 1.5 mL of cell-free media collected from EPC2-hTERT cells treated with

689

IL-13 for 7 days using DNeasy Mini Prep Kit (Qiagen Cat# 69506). qPCR was performed

690

using PowerUp™ SYBR™ green master mix (ThermoFisher Scientific, Cat# A25743).

691

Primers for

ND6

and

ND1

were used. Copy number of each gene was calculated in

692

individual subjects using a standard curve generated for each gene

693

For reverse transcriptase PCR (RT-PCR), RNA extraction was performed with

694

RNeasy Mini Kit (Qiagen, Cat# 74106) according to manufacturer’s instructions. RNA

695

concentration was measured using Qubit™ RNA HS Assay Kit (Invitrogen, Cat# Q32852).

696

Reverse transcription was performed using the High-Capacity cDNA Reverse

697

Transcription Kit for RT-qPCR (Thermo Fisher Scientific, Cat# 4368814). RT-qPCR was

698

performed using PowerUp™ SYBR™ green master mix (Thermo Fisher Scientific, Cat#

699

A25743). The relative expression of each gene normalized to

β-

Actin was calculated

700

using delta delta CT method. All primer sequences are listed in

Table 4.

701

702

Immunocytochemistry

703

Esophageal keratinocytes were grown in 100 mm tissue culture plates in the presence or

704

absence of IL-13 (10 ng/mL) for 7 days. On the 6

day, cells were plated in 6-well plates

705

with 25 mm collagen-coated cover slips overnight. In brief, cells were fixed with 4%

706

Paraformaldehyde, washed with 1X PBS, then permeabilized with PBSTx (0.2% Triton-X

707

Journal Pre-proof

in 1X PBS) for 30 minutes. Cells were then blocked with 3% BSA for 1 hour and incubated

708

in primary antibodies overnight at 4°C. Cells were washed in PBSTx and incubated in

709

secondary antibodies for 40 minutes in the dark at room temperature. After washing with

710

PBSTx, cells were quickly counterstained with 2 µM Hoechst (Invitrogen, Cat# 33342),

711

mounted with Prolong Gold Antifade Mounting media and stored at 4°C until visualization.

712

Images were captured with an AMG Evos Fl Color Microscope. Mean integrated

713

intensities of images after background subtraction were interpreted as a quantitative

714

measure of mitochondria. Images were quantified using ImageJ software. A list of

715

antibodies with used dilutions is provided in

Table 3

716

717

718

Live Cell Imaging

719

Mitochondria were measured using MitoTracker Green dye (Invitrogen, Cat#

720

M7514).

Esophageal keratinocytes were grown in 100 mm tissue culture plates in the

721

presence or absence of IL-13 (10 ng/mL) for 7 days. On the 6

day, cells were plated on

722

30 mm glass bottom collagen coated plates overnight, then stained with indicated dyes

723

according to the manufacturer’s protocol. In brief, cells were stained with KSFM

724

containing 200 nM MitoTracker Green and counterstained with 2 µM Hoechst dye

725

(Invitrogen, Cat# 33342), for 30 min at 37



C, 5% CO

. After staining, cells were washed

726

3X with HBSS and live cell images were captured with a LEICA SP8 Lacer Scanning

727

Microscope using a 63X oil objective and analyzed using LAS X Life Science Microscope

728

Software. Cells were maintained at 37



C, 5% CO

during imaging. Mean integrated

729

intensities of images after background subtraction were interpreted as a quantitative

730

measure of mitochondria. Images were quantified using ImageJ software.

731

732

Immunoblotting

733

Whole cell lysates from esophageal keratinocyte cultures or tissue lysates from peeled

734

murine esophageal epithelium were subjected to immunoblotting as previously

735

described

35, 85

. A list of antibodies with used dilutions is provided in

Table 3

. Targeted

736

proteins were visualized using chemiluminescence detection reagents (ProSignal Femto

737

ECL Reagent, Cat# 20-302) and imaged on an iBright imaging system (Invitrogen).

738

Journal Pre-proof

739

Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) Assay

740

Cellular oxidative phosphorylation (OXPHOS) and glycolysis was monitored using a

741

Seahorse Bioscience Extracellular Flux Analyzer (XF96e, Seahorse Bioscience) by real

742

time OCR and ECAR measurements, respectively, following the protocol of XF

743

Cell Mito Stress Kit (Agilent Technologies, Cat# 103015-100). In brief, EPC2-hTERT

744

cells were grown in 100 mm tissue culture plates with 10 ng/mL IL-13 treatment for 7

745

days. On the 6

day, 100,000 cells/well were seeded onto XF96 96-well plates in

746

200 μL of growth medium and incubated overnight at 37°C, 5% CO

One hour prior to

747

assay, culture media was replaced by XF assay media (180 μL; Agilent Technologies,

748

Cat# 102353-100) and cells were incubated at 37°C in an incubator, without

749

regulation, for 1 hour to allow pre-equilibration with the XF assay medium, which was

750

further supplemented with 25 mM glucose (Sigma, Cat# D8270) and 1 mM sodium

751

pyruvate (pH 7.4; Sigma, Cat# S8636). Cells were sequentially exposed to oligomycin

752

(1 μM), carbonyl cyanide p-triflouromethoxyphenylhydrazone (FCCP; 3 μM), and

753

Rotenone plus Antimycin A (1 μM). Basal OCR and ECAR levels were recorded, followed

754

by OCR and ECAR levels after the injection of individual compounds that inhibit

755

respiratory mitochondrial ETC complexes. Results were normalized according to cell

756

protein concentration. Sequential addition of oligomycin, FCCP, as well as rotenone &

757

antimycin A, allowed an estimation of the contribution of individual parameters for basal

758

respiration, maximal respiration, ATP-linked respiration and glycolysis/lactate production

759

(ECAR).

760

ATP Assay

761

ATP production was assessed by using the CellTiter-Glo 2.0 Kit (Promega, Cat# G9241)

762

according to the manufacturer’s protocol. In brief, EPC2-hTERT cells were grown in 100

763

mm tissue culture plates in the presence or absence of IL-13 (10 ng/mL) for 7 days. On

764

the 6

day, 100,000 cells/well were seeded onto 96-well solid white plates in 100 μL of

765

growth medium per well and incubated overnight at 37 °C, 5% CO

. Then, 100µL

766

of CellTiter-Glo® 2.0 Reagent was added to 100 µl of medium containing cells, followed

767

by mixing for 2 minutes and incubation at room temperature for 10 minutes to stabilize

768

the luminescent signal. Luminescence was measured by using Promega GloMax-Multi+

769

Journal Pre-proof

detection system. Results are expressed as fold change of percent change from

770

untreated controls.

771

772

Measurement of Lactate Levels

773

Lactate level was measured using glycolysis assay kit (Cayman Chemical, Cat# 600450)

774

according to the manufacturer’s instructions. EPC2-hTERT cells were grown in 100 mm

775

tissue culture plates in the presence or absence of IL-13 (10 ng/mL) for 7 days. On the

776

day, cells were plated in a 96-well plate overnight. In brief, the 96-well plate was

777

centrifuged at 1000 RPM for 5 min and supernatant was collected. 90 µl of assay buffer

778

was added to a new plate and 10 µl of supernatant was transferred. 100 µl of reaction

779

solution was added to each well and the plate was incubated at room temperature on a

780

shaker for 30 min. The absorbance (490 nm) was recorded using Modulus microplate

781

reader (Promega).

782

783

Flow Cytometry

784

Mitochondrial membrane potential (depolarized mitochondria) was determined by

785

staining EPC2-hTERT cells with MitoTracker Green (ThermoFisher Scientific, Cat#

786

M7514) at a final concentration of 25 nM and MitoTracker Deep Red (ThermoFisher

787

Scientific, Cat# M22426) at a final concentration of 100 nM. Cells were stained with each

788

dye for 30 minutes at 37°C. Cells were washed with and resuspended in 1X HBSS (Gibco,

789

Cat# 14175095). Apoptosis was examined with FITC Annexin-V and propidium iodide (PI)

790

assay kit (BD Biosciences, Cat #556547). Cells were initially washed with cold phosphate-

791

buffered saline (PBS; Gibco, Cat #14190-144), stained for 15 minutes in the dark at room

792

temperature, and resuspended in 1X Annexin-V binding buffer. Flow cytometry was

793

performed using Symphony A5 flow cytometer (BD Biosciences), and data was analyzed

794

with FlowJo version 10.0.

795

796

EGIDExpress Database

797

To assess the expression of mediators of mitochondrial biology and mtDNA-associated

798

inflammatory pathways in esophageal biopsies, we accessed the “EoE Transcriptome by

799

RNA

Sequencing

”

section

the

EGIDExpress

database

800

Journal Pre-proof

(https://egidexpress.research.cchmc.org/data/), a site containing datasets relating to

801

Eosinophilic Gastrointestinal Diseases (EGIDs).

802

803

Statistics

804

Descriptive statistics are presented as mean ± standard error of the mean (SEM).

805

Student’s t-test was used for comparison of two groups. Pairwise Wilcoxon signed rank

806

test or Mann-Whitman t-test was used to assess differences in average mtDNA copy

807

number between non-EoE and active EoE subjects or disease severity (strictures,

808

impactions) within EoE subjects, respectively. For groups of three or more, one-way

809

ANOVA followed by Tukey’s or Dunn’s post-hoc test was performed. Pearson’s correlation

810

tests were used to evaluate the linear relationship between two variables. Data were

811

analyzed using GraphPad Prism version 9.0.2 for macOS (GraphPad Software, San

812

Diego, California USA).

<.05 was considered statistically significant. For all data, the

813

following indicators of significance are used: *

<.05; **

<.01; ***

<.001; ****

<.0001.

814

815

Acknowledgments:

We thank the following former members of the Whelan lab for their

816

support: Anbin Mu, Timothy Hall, Anne-Laure Monéger, Julie Gang, and M. Faujul Kabir.

817

We acknowledge the staff of the following core facilities for technical assistance: Temple

818

University Lewis Katz School of Medicine Flow Cytometry Core, (Director, Amir

819

Yarmahoodi); Fox Chase Cancer Center Histopathology Core; Children’s Hospital of

820

Philadelphia Gastrointestinal Epithelium Modeling Program Core (RRID: SCR_026402)

821

and the University of Pennsylvania Center for Molecular Studies in Digestive and Liver

822

Diseases (NIHP30DK050306). The graphical abstract was created using

823

https://BioRender.com

824

825

826

827

828

829

830

831

Journal Pre-proof

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Figure Legends

1169

Figure 1. Evidence of increased mitochondria in EoE patients.

(

A, B

)

1170

Immunohistochemistry (IHC) staining for mitochondrially-encoded cytochrome oxidase 1

1171

(MTCO1) was performed in esophageal biopsies from human subjects classified as non-

1172

EoE (n=5), active EoE (n=23) or inactive EoE (n=20). (

) Representative images of

1173

immunohistochemistry (IHC) staining for mitochondrially-encoded cytochrome oxidase 1

1174

(MTCO1) in esophageal biopsies from human subjects classified as non-EoE (n=5),

1175

active EoE (n=23) or inactive EoE (n=20). Images taken at 200X and 400X. Scale bars,

1176

50 µm. (

) Average cytoplasmic MTCO1 score in esophageal mucosa of indicated

1177

groups. *

<.05; **

<.01 by one-way ANOVA with Tukey’s post-hoc test. Data in graph

1178

presented as mean per group ± SEM with each dot representing an individual subject.

1179

(

) Immunofluorescence (IF) for Ki67 (green), MTCO1 (red), and nuclei (DAPI, blue) in

1180

esophageal epithelial biopsies from a non-EoE subject or active EoE subject. Images

1181

taken with 63X objective. Scale bars, 10 µm.

1182

1183

Figure 2. Relationship between MTCO1 expression and clinical features of EoE.

(

1184

)

Correlation graphs depicting relationship between cytoplasmic MTCO1 score and

1185

basal cell hyperplasia (BCH) score (

) or eosinophils per high power field (eos/HPF) (

)

1186

for non-EoE controls (n=5) and patients classified as active EoE (n=23) or inactive EoE

1187

(n=20). Linear regression analysis determined correlation coefficient, R

, and Pearson’s

1188

correlation test determined

values. (

C, D

) Average MTCO1 score is shown for active

1189

EoE patients (n=23) with and without history of stricture (

) or endoscopically removed

1190

food impaction (

). Data in dot plots (

C, D

) represent mean per group ± SEM with each

1191

dot representing an individual subject. Mann-Whitman U test determined p values.

1192

1193

Figure 3. Evidence of increased mitochondria in esophageal mucosa of mice with

1194

EoE-like inflammation.

(

A-F

) Experimental EoE was induced in wild type C57BL/6 mice

1195

using MC903/Ovalbumin (OVA). Mice treated with MC903 only served as controls. (

)

1196

Representative H&E images of esophageal mucosa. White arrows indicate eosinophils.

1197

Inset shows an individual eosinophil. Scale bars, 50 µm. (

) Peeled esophageal epithelial

1198

sheets were subjected to qPCR to assess mtDNA levels (

mito D-loop

) with

Ikbβ

as an

1199

Journal Pre-proof

internal nuclear control. *

<.05 by two-tailed student t-test. (

) Representative images

1200

of MTCO1 IHC staining for MTCO1 esophageal epithelium. Images taken at 400X. Scale

1201

bars, 50 µm. (

) Average cytoplasmic MTCO1 score. Two-tailed student t-test test

1202

determined noted p value. (

E, F

) Correlation graphs depicting relationship between

1203

cytoplasmic MTCO1 score and eosinophils per high power field (eos/HPF) (

) or lamina

1204

propria (LP) thickness (

). Linear regression analysis determined correlation coefficient,

1205

and Pearson’s correlation test determined

values. Data in bar diagrams (

B, D

)

1206

represent mean per group ± SEM. Each dot represents an individual mouse.

1207

1208

Figure 4. Effects of EoE-relevant cytokines on mitochondrial DNA levels in human

1209

esophageal keratinocytes in vitro.

(

A-D

) qPCR evaluated levels of mitochondrially-

1210

encoded genes,

MTCO1

and

ND6

, and nuclear-encoded gene,

COXIV

in esophageal

1211

keratinocytes following cytokine stimulation for 7 days. All genes shown are normalized

1212

to nuclear-encoded gene,

GAPDH

. All interleukin (IL) treatments were at a final

1213

concentration of 10 ng/mL. Tumor necrosis factor (TNF)α treatment was at a final

1214

concentration of 40 ng/mL. (

) EPC2-hTERT cells were treated with indicated cytokines.

1215

One-way ANOVA with Tukey's post-hoc determined p values. (

B, C

) mtDNA levels were

1216

evaluated in primary esophageal cultures derived from a non-EoE subject (

) or an active

1217

EoE patient (

). Two-tailed student t-test determined p values. (

) mtDNA levels were

1218

evaluated in EPC2-hTERT treated with IL-13 and IL-4 alone or in combination. One-way

1219

ANOVA with Tukey’s post-hoc test determined p values. Data in all bar diagrams

1220

represent mean per group ± SEM. Each dot represents an individual experiment. *

<.05;

1221

<.01; ***

<.001; ****

<.0001.

1222

1223

Figure 5. IL-13 promotes increased mitochondrial mass in human esophageal

1224

keratinocytes in vitro.

(

A-D

) EPC2-hTERT cells were treated with IL-13 (10 ng/mL) for

1225

7 days. (

A, B

) Immunocytochemistry for TOM20 (red) and nuclei (Hoechst 33342, blue)

1226

was performed with representative images (

) and relative quantitative fluorescence

1227

intensity of TOM20 (

) shown. Scale bars, 50 µm. ****

<.0001 by two-tailed student t-

1228

test. (

C, D

) Live cell imaging for MitoTracker Green (green) and nuclei (Hoechst 33342,

1229

blue) was performed with representative images (

) and relative quantitative

1230

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fluorescence intensity of MitoTracker Green (

) shown. Scale bars, 25 µm (left), 8 µm

1231

(right). **

<.01 by two-tailed student t-test. Data in bar diagrams (

B, D

) represent mean

1232

per group ± SEM. Each dot represents an individual experiment.

1233

1234

Figure 6. Time-dependent effects of IL-13 on mediators of mitochondrial dynamics

1235

in esophageal keratinocytes.

(

A-G

) EPC2-hTERT cells were treated with IL-13 (10

1236

ng/mL) for the indicated number of days with day 0 serving as a control. (

A-F

) qRT-PCR

1237

determined relative expression of

TFAM

(

MFN1

(

MFN2

(

DRP1

(

PINK1

(

1238

and

PARK2

(

) normalized to

ACTB

. (

G, H

) Immunoblotting assessed levels of indicated

1239

total and phosphorylated (p) proteins with Actin as a loading control. Representative blot

1240

(

) and bar diagram of densitometric analysis values (

) are shown. (

) RNA level of

1241

TFAM, MFN1, MFN2

and

PINK1

in esophageal epithelium of active EoE patients (n=10)

1242

and non-EoE subjects (n=6) were obtained from EGIDExpress database

. Data in bar

1243

diagrams (

A-F, H

) and dot plot (

) represent mean per group ± SEM. Each dot represents

1244

an individual experiment in bar diagrams (

A-F, H

) and individual subject in dot plot (

1245

Two-tailed student t-test determined p values. *

<.05; **

<.01; ***

<.001; ****

<.0001.

1246

1247

Figure 7. Effects of IL-13 on mitochondrial respiration, glycolysis and ATP in

1248

esophageal keratinocytes.

(

A-I

) Esophageal epithelial cells were treated with IL-13 (10

1249

ng/mL) for 7 days. (

A-D

) In EPC2-hTERT cells, Seahorse respirometry assessed oxygen

1250

consumption rate (OCR) (

), basal respiration (

), ATP-linked respiration (

), maximal

1251

respiration (

) and extracellular acidification rate (ECAR) (in

). (

) Lactate production

1252

was assessed via a colorimetric assay. (

G-I

) Relative ATP was measured in EPC2-hTERT

1253

cells (

), primary non-EoE esophageal epithelial cells (

), or primary active EoE

1254

esophageal epithelial cells (

). Line graphs show OCR (

) and ECAR (

) data from 1

1255

representative experiment; n=3. Data in bar diagrams (

B-D, F-I

) represent mean per

1256

group ± SEM. Each dot represents an individual experiment in bar diagrams. Two-tailed

1257

student t-test determined p values. *

<.05; **

<.01; ***

<.001; ****

<.0001.

1258

1259

Figure 8. IL-13 does not impact mitochondrial integrity or induce apoptosis in

1260

esophageal keratinocytes.

(

A-D

)

EPC2-hTERT cells were treated with IL-13 (10 ng/mL)

1261

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for 7 days. (

A, B

) MitoTracker Red/Green staining evaluated cells with mitochondrial

1262

depolarization with representative flow dot plot showing gate for depolarization-positive

1263

fraction (

) and bar diagram quantifying percent of cells with depolarized mitochondria

1264

(

) shown. (

C, D

) Annexin-V/Propidium iodide (PI) flow cytometry evaluated apoptotic

1265

cells with representative flow dot plot (

) and bar diagram quantifying distribution of live

1266

(events in Q1 gate shown in

) and apoptotic cells (sum of events in Q2 and Q3 gates

1267

shown in

). Data in bar diagrams (

B, D

) represents mean per group ± SEM. Each dot

1268

represents an individual experiment. Two-tailed student t-test determined p values.

1269

1270

Figure 9. IL-13 promotes the release of mtDNA from esophageal keratinocytes in

1271

vitro.

(

A-D

) Esophageal keratinocytes were treated with IL-13 (10 ng/mL) for 7 days.

1272

qPCR determined average copy number of mitochondrially encoded genes

ND1

and

ND6

1273

in DNA extracted from cell-free culture medium of EPC2-hTERT cells (

A, B

), primary non-

1274

EoE cells (

) and primary active EoE cells (

). Data in all bar diagrams represent mean

1275

per group ± SEM. Each dot represents an individual experiment. Two-tailed student t-test

1276

determined p values. *

<.05; **

<.01; ***

<.001.

1277

1278

Figure 10. mtDNA-associated inflammatory signaling components are upregulated

1279

in esophageal keratinocytes responding to IL-13 and in EoE patient biopsies.

(

A-B

)

1280

EPC2-hTERT cells were treated with IL-13 (10 ng/mL) for the indicated number of days

1281

with day 0 serving as a control. Immunoblotting assessed cleavage of TLR9, levels of

1282

cGAS, and STING, and phosphorylation (p) of STING. Actin serves as a loading control.

1283

Representative blot (

) and bar diagram of densitometric analysis values (

) are shown.

1284

(

) RNA expression of genes relevant to TLR9 signaling (

MYD88, IRAK4, IRF5, IRAK1,

1285

IRAK2, TRAF6, TRAF3)

and cGAS/STING signaling

(MB21D1, TMEM173, TRAF6,

1286

TBK1, IRF3)

in esophageal epithelium of active EoE patients (n=10) and non-EoE

1287

controls (n=6) was obtained from the EGIDExpress database

. Data in bar diagram (

)

1288

and dot plot (

) represent mean per group ± SEM. Each dot represents an individual

1289

experiment in bar diagram (

) and individual subject in dot plot (

). Two-tailed student t-

1290

test determined p values. *

<.05; **

<.01; ***

<.001; ****

<.0001.

1291

1292

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Figure 11. Circulating mtDNA is increased in serum of active EoE patients.

(

A-F

)

1293

DNA extracted from serum was evaluated for absolute copy number of mtDNA-encoded

1294

genes via qPCR with data from non-EoE subjects (n=49) and active EoE patients (n=65)

1295

for

ND1

(

) and

ND6

(

) shown. (

C-F

) Average copy number of

ND1

and

ND6

is shown

1296

for EoE patients with and without history of stricture (

C, D

) or endoscopically removed

1297

food impaction (

E, F

). Data in dot plots (

A-F

) represent mean per group ± SEM with each

1298

dot representing an individual subject. Mann-Whitman U test determined p values. *

1299

<.05; **

<.01.

1300

1301

Figure 12. Ruxolitinib limits IL-13-mediated increases in intracellular and

1302

extracellular mtDNA in vitro.

(

A-G

) EPC2-hTERT cells were treated with IL-13 (10

1303

ng/mL) for 7 days in the presence or absence of JAK inhibitor, ruxolitinib (100 ng/mL). (

A-

1304

) Immunoblotting was used to assess expression of indicated total and phosphorylated

1305

(p) proteins. Actin serves as a loading control. Representative blot (

) and bar diagram

1306

of densitometric analysis values (

B-D

) are shown. (

) qPCR evaluated relative levels of

1307

mtDNA-encoded genes,

MTCO1

and

ND6

, and nuclear-encoded gene,

COXIV

. (

)

1308

DNA extracted from cell-free media was assessed by qPCR to determine average copy

1309

number of mitochondrially encoded genes

ND1

(

) and

ND6

(

). Data in bar graphs (

B-

1310

) represent mean per group ± SEM with each dot representing an individual experiment.

1311

One-way ANOVA with Tukey’s post-hoc test determined p values. *

<.05; **

<.01; ***

1312

<.001; ****

<.0001.

1313

1314

Figure 13. Effects of genetic STAT3 or STAT6 depletion on mitochondrial mass in

1315

IL-13-treated esophageal keratinocytes.

(

A-D

) EPC2-hTERT cells were lentivirally

1316

engineered to express doxycycline (DOX)-inducible shRNA targeting STAT3 or STAT6 or

1317

non-targeting (NS) control shRNA. (

A, B

) Immunoblotting confirmed DOX-mediated

1318

depletion of STAT3 (

) or STAT6 (

). Actin serves as a loading control. (

C, D

) Cells with

1319

DOX-mediated STAT3 or STAT6 depletion were cultured in the presence of DOX (0.5

1320

ng/µL) and/or IL-13 (10 ng/mL) for 7 days. Immunocytochemistry for TOM20 (red) and

1321

nuclei (Hoechst 33342, blue) was performed with representative images (

) and relative

1322

quantitative fluorescence intensity of TOM20 (

) shown. Scale bars, 200 µm. Data in bar

1323

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Status

Age (years),

median (IQR)

Eos/HPF,

median (IQR)

Basal Cell

Hyperplasia

(BCH) Score,

median (IQR)

Male, n (%)

Non-EoE (n=5)

36 (33-51)

0 (0-0)

0 (0-1)

1 (20%)

Active EoE

(n=23)

29 (23.5-44)

50 (34.5-65.5)

2 (0-3)

12 (52%)

Inactive EoE

(n=20)

37 (29.25-45)

0 (0-10)

0.5 (0-1)

12 (60%)

Table 1. Characteristics of Human Subjects (MTCO1 Staining).

IQR: interquartile range; Eos: eosinophils; HPF: high powered field

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Status

Age, mean ± SD

(IQR)

Eos/HPF, mean

(IQR)

Male, n (%)

Non-EoE (n=49)

19 ± 7.1 (15.2-55.6)

0 (0-0)

15 (34.7)

Active EoE (n=65)

29 ± 5.6 (2.3-34.3)

52 (15-100)

41 (64.4)

Table 2. Characteristics of Human Subjects (mtDNA Assessment).

IQR: interquartile range; Eos: eosinophils; HPF: high powered field

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Antibody

Source

Catalog number

Application

(dilution)

MTCO1

Abcam

ab14705

IHC (1:250)
IF (1:500)

Ki67

Cell Signaling

9129S

IF (1:400)

TOM20

Abcam

AB289670

IF (1:100)

TFAM

Sigma

7495S

IHC (1:250)
WB (1:1000)

p-STAT6

Y641

Cell Signaling

56554S

WB (1:1000)

STAT6

Cell Signaling

5397S

WB (1:1000)

p-STAT3

Y705

Cell Signaling

9145S

WB (1:1000)

p-STAT3

S727

Cell Signaling

9134S

WB (1:1000)

STAT3

Cell Signaling

9139S

WB (1:1000)

MFN1

Cell Signaling

14739S

WB (1:1000)

MFN2

Cell Signaling

9482S

WB (1:1000)

PINK1

Cell Signaling

6946S

WB (1:1000)

Parkin

Cell Signaling

4211S

WB (1:1000)

p-DRP1

S616

Cell Signaling

3455S

WB (1:1000)

DRP1

Cell Signaling

8570S

WB (1:1000)

Toll-Like Receptor 9

Cell Signaling

2254S

WB (1:1000)

cGAS

Cell Signaling

79978S

WB (1:1000)

p-STING

S366

Cell Signaling

19781S

WB (1:1000)

STING

Cell Signaling

13647S

WB (1:1000)

Actin

Invitrogen

MA1-744

WB (1:10000)

Table 3 Antibodies for Immunohistochemistry (IHC), Immunofluorescence (IF), and
Western Blotting (WB)

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Forward (5’

→

3’)

Reverse (5’

→

3’)

MTCO1

(human)

CCC ACC GGC GTC AAA
GTA T

TGC AGC AGA TCA TTT
CAT ATT GC

ND1

(human)

CTC GCT ACC ACT CTC
GAT TCC A

TGG AAT CGA GAG TGG
TAG CGA

ND6

(human)

CCC CCA TGC CTC AGG
AT

GGA ATG ATG GTT GTC
TTT GGA TAT ACT

COXIV

(human)

GGG CGG TGC CAT GTT
CT

CAT AGT GCT TCT GCC
ACA TGA

GAPDH

(human)

CCA GGT GGT CTC CTC
TGA CTT C

TCA TAC CAG GAA ATG
AGC TTG ACA

mtDNA

D-Loop

(murine)

ACT ATC CCC TTC CCC
ATT TG

TGT TGG TCA TGG GCT
GAT TA

Ikbβ

(murine)

GCT GGT GTC TGG GGT
ACA GT

ATC CTT GGG GAG GCA
TCT AC

TFAM

(human)

GAG GGA ACT TCC TGA
TTC AAA GA

AGC TTT CCT TTT TAA
ATG TTT GTC C

MFN1

(human)

TGT TTT GGT CGC AAA
CTC TG

CTG TCT GCG TAC GTC
TTC CA

MFN2

(human)

AGC TGG ACA GCT GGA
TTG AC

GCT TTT CCG TCT GCA
TCA GG

DRP1

(human)

TGC TTC CCA GAG GTA
CTG GA

TCT GCT TCC ACC CCA
TTT TCT

PINK1

(human)

TAC CAG TGC ACC AGG
AGA AG

GCT TGG GAC CTC TCT
TGG AT

PARKIN

(human)

AAA TCA GGT GGC TCC
CTT CTG TCA

CAT GCA GAT TGG GAA
GGC GCA ATA

ACTB

(human)

TTG CCG ACA GGA TGC
AGA A

GCC GAT CCA CAC GGA
GTA CT

Table 4. Primer Sequences for qPCR and qRT-PCR.

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