Role of Vasoactive Intestinal Peptide in Promoting the Pathogenesis of Eosinophilic Esophagitis (EoE)

Accepted Manuscript Role of vasoactive intestinal peptide in promoting the pathogenesis of eosinophilic esophagitis (EoE) Alok K. Verma, Murli Manohar, Sathisha Upparahalli Venkateshaiah, Uwe Blecker, Margaret H. Collins, Anil Mishra

PII: DOI: Reference:

S2352-345X(17)30140-6 10.1016/j.jcmgh.2017.09.006 JCMGH 268

To appear in: Cellular and Molecular Gastroenterology and Hepatology Accepted Date: 15 September 2017 Please cite this article as: Verma AK, Manohar M, Venkateshaiah SU, Blecker U, Collins MH, Mishra A, Role of vasoactive intestinal peptide in promoting the pathogenesis of eosinophilic esophagitis (EoE), Cellular and Molecular Gastroenterology and Hepatology (2017), doi: 10.1016/j.jcmgh.2017.09.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Role of vasoactive intestinal peptide in promoting the pathogenesis of eosinophilic esophagitis (EoE) Alok K. Verma,1 Murli Manohar,1 Sathisha Upparahalli Venkateshaiah,1 Uwe Blecker,2 Margaret H. Collins,3 and Anil Mishra1 1

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Department of Medicine, Section of Pulmonary Diseases, Tulane Eosinophilic Disorder Center 2Pediatric Gastroenterology, Tulane University School of Medicine, New Orleans, LA 70112. 3Division of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45249

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Alok K. Verma and Murli Manohar, Acquisition of data, and data statistical analysis.

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Sathisha Upparahalli Venkateshaiah, Technical support, figures and manuscript editing.

Uwe Blecker, EoE Patient material support.

Margaret Collins, Human esophageal autopsy and colon biopsy source

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Anil Mishra, Study concept and design, interpretation, supervision and funding support.

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Funding Source NIH R01 AI080581 (AM) Conflict of Interest

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There are no conflicts of interest to disclose for all authors except Dr. Mishra, who discloses past and present consultancy relationships with Axcan Pharma, Aptalis, Elite Biosciences, Calypso Biotech SA and Enumeral Biomedical.

Corresponding author: Anil Mishra, PhD., Endowed Schlieder Chair, Professor and Director of Tulane Eosinophilic Disorder Center, Department of Medicine Section of Pulmonary Medicine, Tulane University School of Medicine, New Orleans, LA 70112, Tel: 504-988-3840; Fax: 504-988-2144; E-mail: [email protected] 1|Page

ACCEPTED MANUSCRIPT Research Letter Eosinophilic esophagitis (EoE) is associated with eosinophil and mast cell accumulation that promotes dysphagia and esophageal dysmotility .1,

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Cytokines

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and chemokines implicated in eosinophil, mast cell, and basophil mediated EoE pathogenesis include IL-5, IL-13, TSLP and eotaxin-3. A correlation between IL-5 and eotaxin-3 expression and eosinophil infiltration has been observed.

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Nevertheless, it is not clear if eotaxin-3 is the only chemokine, or one of several, that

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direct eosinophil accumulation in EoE. Herein, we present evidence that a

degranulation in human EoE.

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neuroimmune pathway is involved in eosinophil and mast cell accumulation and

Morphologically, eosinophils accumulate near nerves within the muscular mucosa of the esophagus (Figure 1 A). This spatial association suggests that nerve cells may

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release chemoattractant in EoE. We hypothesize that one such chemoattractant might be vasoactive intestinal peptide (VIP), which has been implicated in eosinophil recruitment during allergic disease, e.g. asthma. VIP expression was low in control

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esophageal biopsies, i.e. without eosinophils (Figure 1 B-D). In contrast, eosinophils

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accumulated adjacent to VIP-expressing nerve cells in EoE biopsies (Figure 1 E-G). VIP performs its immunological functions via binding to 3 receptors: vasoactive intestinal peptide receptor 1 (VPAC-1), VPAC-2 and chemoattractant receptor homologous molecule expressed on Th2 lymphocytes (CRTH2).

8, 9

Our in vitro

analyses indicated that eosinophils mainly express CRTH2 receptor, not VAPC1, VAPC2 and VIP (Supplementary Figure 1 A-D). Further, eosinophil motility in response to VIP is comparable to that induced by eotaxin (Supplementary Figure 1 E), and that anti-CRTH2 pre-treatment restricted human eosinophil motility

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ACCEPTED MANUSCRIPT (Supplementary Figure 1 F). In EoE biopsies, we show that CRTH2 expressing eosinophils accumulated in the epithelial mucosa (Supplementary Figure 2 A-F). Further, mast cells contribution to the pathogenesis of EoE is also reported; but the mechanism

of

mast

cell

recruitment

to

the

esophagus

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Immunofluorescence analyses confirmed CRTH2 receptor on tryptase-positive mast cells in the esophageal mucosa of EoE patients (Supplementary Figure 2 G-L). These findings suggest that similar to eosinophils, mast cells accumulate via

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interaction of the CRTH2 receptor with neurally-derived VIP. The details of control

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and EoE patients clinical characteristics are provided in supplementary table 1. Since previous clinical trial demonstrated reduction of peak eosinophil levels in adult EoE patients treated with a CRTH2 antagonist.10 But, the effect of the CRTH2 antagonist on mast cells that may be critical to the esophageal functional abnormalities observed in EoE, was not examined. Therefore, to better assess the

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potential therapeutic utility of in vivo CRTH2 blockade, we initially assessed eosinophil and mast cell distributions in mice after induction of experimental EoE

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(Supplementary Figure 3 A, B).11 As in the human study, eosinophil infiltration in each segment of the esophagus was reduced in CRTH2 antagonist-treated mice

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(Figure 2 A-D). This was paralleled by reduced mast cell numbers (Figure 2 E-H). Morphometric quantification analysis indicated that CRTH2 antagonist treatment significantly reduced the number of both eosinophils and mast cells (Supplementary Figure 4 B, C).

Taken together, the current studies identify a novel and important chemoattractant role for VIP in the accumulation of eosinophils and mast cells in the pathogenesis of EoE. Moreover, we suggest that inhibiting the VIP-CRTH2 axis may ameliorate the dysphagia, stricture and motility dysfunction of chronic EoE. 3|Page

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Acknowledgements This work was supported in part by the grants NIH R01 AI080581 (AM) and by the

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Tulane Edward G. Schlieder Educational Foundation.

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Figure legends

Figure 1. Eosinophils accumulate adjacent to nerve cells in the muscular mucosa of human gastrointestinal disorder. Eosinophil accumulation in the

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esophageal muscularis propria of undiagnosed human autopsy sample (A). AntiMBP-positive eosinophils (red) were not detected in control esophageal biopsies, and VIP expression (green) in nerves was low (B-D). Increased eosinophils numbers and VIP expression were observed in EoE (E-G). Arrows indicate anti-MBP and antiVIP immunostained eosinophils and nerve cells, respectively.

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Figure 2: CRTH2 antagonist pretreatment reduces the accumulation of both eosinophils and mast cells in Aspergillus-induced experimental EoE. CRTH2 antagonist pre-treatment significantly reduced the number of eosinophils (D) and

H).

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mast cells (H) relative to Aspergillus-challenged mice that were not pre-treated (C, No eosinophils (A, B) and few baseline mast cells (E, F) were detected in

sections from mice treated with saline or saline+CRTH2 antagonist. Data is

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expressed as mean ± SD, n=8-10 mice/group. EP, epithelial mucosa; LP, lamina propria; LU, Lumen; MP, Muscularis propria.

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ACCEPTED MANUSCRIPT References

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5. 6. 7. 8. 9. 10. 11.

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3. 4.

Attwood SE, Smyrk TC, Demeester TR, et al. Dig Dis Sci 1993; 38:109-16. Martin Martin L, Santander C, Lopez Martin MC, et al. J Gastroenterol Hepatol 2011; 26:1447-50. Attwood SE, Furuta GT. Gastroenterol Clin North Am 2014; 43:185-99. Nonevski I, Fiocchi C, Falk GW. Gastroenterology 2006; 131:2018-20; discussion 2020. Mishra A, Hogan SP, Brandt EB, et al. J Immunol 2002; 168:2464-9. Mishra A, Hogan SP, Brandt EB, et al. J Biol Chem 2002; 277:4406-12. Blanchard C, Wang N, Stringer KF, et al. J Clin Invest 2006; 116:536-47. Dunzendorfer S, Meierhofer C, Wiedermann CJ. J Leukoc Biol 1998; 64:828-34. El-Shazly AE, Begon DY, Kustermans G, et al. J Biol Chem 2013; 288:1374-84. Straumann A, Hoesli S, Bussmann C, et al. Allergy 2013; 68:375-85. Niranjan R, Mavi P, Rayapudi M, et al. Am J Physiol Gastrointest Liver Physiol 2013; 304:G1087-94.

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1. 2.

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ACCEPTED MANUSCRIPT Supplementary Material

Methods

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Clinical Characteristics of EoE and non-EoE patients Formalin-fixed, paraffin-embedded biopsy samples were obtained from the esophagus of control patients or EoE patients as per an Institutional Review Board (IRB)-approved protocol. The comparison groups include control patients (non-EoE),

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EoE and dysphagia patients were selected without regard to age, atopic status or

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gender. The diagnosis was established based on the maximum eosinophil count per high power field (HPF) (×400). Control patients (non-EoE) were defined as having 02 esophageal eosinophils/HPF and no basal layer expansion. The normal biopsies were obtained from patients who came to the clinic with symptoms typically noticed in EoE but were found to have completely normal esophageal endoscopic and

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microscopic analyses. Patients with EoE were defined as having ≥ 15-esophageal eosinophils/HPF. The EoE with dysphagia patients have more esophageal

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eosinophilia as compared to patients with only EoE along with basal cell hyperplasia. Blood was drawn in citrate-coated tubes from each control patient, EoE and

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dysphagia patient at the time of scheduled endoscopy (including before and after treatment of EoE patients) at Tulane University School of Medicine (TUSM). All samples were used according to the patients’ consent and IRB approved protocol of TUSM, New Orleans, LA (Year 2013-18). Detailed control, EoE and dysphagia patients’ characteristics including treatment are listed in Supplementary Table 1.

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ACCEPTED MANUSCRIPT Eosinophils detection in the muscular mucosa of autopsy and biopsy tissue sections Eosinophils were detected in the muscular mucosa of 10% buffered formalin fixed esophageal autopsy (provided by Dr. Margaret Collins, Cincinnati Children’s) and

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colon biopsy tissue sections stained with haematoxylin and eosin (H&E). The tissue sections were stained with haematoxylin followed by differentiation with 0.3% acid alcohol. The stained slides were further washed with running water, dehydrated with

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70% alcohol and counterstained with eosin made in 70% alcohol. Slides were further dehydrated with 100% alcohol and cleared with xylene and mounted for images

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captured using Olympus BX51 microscope.

Immunofluorescence tissue staining for eosinophils, mast cells, nerve cells, VIP and CRTH2 receptor on esophageal biopsies of human EoE patients

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Immunofluorescence staining of formalin-fixed, paraffin-embedded human control patient and EoE esophageal biopsy sections was performed to analyze CRTH2 receptor expression on eosinophils, and mast cells. Further, eosinophils accumulation

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nearby nerve cells derived VIP following the immunofluorescence tissue staining 1, 2

Briefly, in formalin-fixed, paraffin-

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methods was performed as described earlier.

embedded sections from esophageal biopsies, endogenous peroxide was quenched using 0.3% hydrogen peroxide in methanol, followed by antigen retrieval with pepsin and blocking with 3% goat serum to reduce the non-specific binding. Esophageal eosinophils were detected using rat anti-MBP (kindly provided by Dr. Jamie Lee, Mayo Clinic Scottsdale, AZ) antibody followed by TRITC labeled anti-rat IgG that is used as secondary antibody. Further, anti-CRTH2 antibody (ProSci) followed by FITC labeled anti-rabbit IgG (Biolegend) were used as secondary antibody to detect CRTH2

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ACCEPTED MANUSCRIPT receptor expression on eosinophils on esophageal biopsies. VIP expressing nerve fibres were detected using goat anti-VIP antibody followed by FITC-anti goat IgG antibody that was used as secondary antibodies to detect VIP producing nerve cells. Additionally, CRTH2 receptor expression on anti-human tryptase (Bio-Rad) positive

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mast cells was detected using anti-tryptase antibody followed by PE labeled antimouse IgG that is used as secondary antibody for mast cell and anti-CRTH2 antibody (ProSci) followed by FITC labeled anti-rabbit IgG (Biolegend) as secondary antibody

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for CRTH2 expression on mast cells in esophageal biopsies. The double immunostained each tissue sections slides were mounted with DAPI mounting

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material (Thermo Fisher Scientific) and the Images were captured using an Olympus BX51 microscope.

CRTH2 antagonist treatment in Experimental EoE

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Specific pathogen-free BALB/c mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All of the experiments were performed on age- and gender-matched mice 6–8 weeks of age. The Tulane Institutional Animal Care and Use Committee

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approved the animal protocols. Experimental EoE in mice was induced using methods

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described previously3-5 that were used in accordance with National Institute of Health guidelines. In brief, mice were lightly anesthetized with isoflurane (Iso-Flo; Abbott Laboratories, North Chicago, IL), and 100 µg of Aspergillus fumigatus (Greer Laboratories, Lenoir, NC) in 50 µl normal saline or 50 µl of normal saline alone was given intranasal using a micropipette with the mouse held in the supine position for 3 times/week for three weeks. In addition, 100 µg/mice CRTH2 antagonist (OC000459) (Cayman Chemical, Ann Arbor, MI) was given through intravenous role on an alternate day up to last Aspergillus challenge. The mice were euthanized after 24 hours of last

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ACCEPTED MANUSCRIPT intranasal allergen or saline challenge. Esophageal tissues sections were analyzed for eosinophils by anti-MBP immunostaining and for mast cells by chloroacetate esterase staining as per the earlier described protocol.2, 6, 7

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Flow cytometer analysis for VIP and VIP receptors expression on blood eosinophils

VIP, VPAC-1, VPAC-2, and CRTH2 receptors expression on blood eosinophils of EoE

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patients were performed by flow cytometric analysis as describe earlier.8, 9 The blood cells were stained with different florescence tagged anti-hCCR3 (Biolegend, San

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Diego, CA), anti-hSiglec-8 (Biolegend, San Diego, CA), anti-hVIP, anti-hVPAC-1, antihVPAC-2, and anti-hCRTH2 antibodies (Santa Cruz Biotechnology, Dallas, TX and Biolegend, San Diego, CA). Depending on availability, we used both fluorescence tagged antibodies or the combination of both primary and secondary antibodies tagged

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with different florescent tagged IgG (Santa Cruz Biotechnology, Biolegend or eBioscience). Respective labelled IgG antibodies were used as isotype control. FACS analysis was performed using a FACS Calibur (BD Biosciences, San Diego, CA) and

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analyzed by FlowJo software (BD Biosciences).

Eosinophil migration assay The chemoattractant behaviour of VIP for eosinophils was analyzed in vitro using Transwell units (24 wells) with 5-µm porosity polycarbonate filters (Corning Inc, NY) following the previously described protocol.

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The human blood eosinophils were

incubated with the anti-human CCR3 and anti-human Siglec-8 antibodies for 45 min, washed and eosinophils were separated by FACS. The purified human eosinophils (105 cells/well) in HBSS, pH 7.2 (Life Technologies, Carlsbad, CA) were placed in 4|Page

ACCEPTED MANUSCRIPT the upper chamber and different concentrations of recombinant VIP (1, 10, 100, and 500 ng/ml) were added to the lower chamber. Eotaxin-2 (200 ng/ml), a known chemoattractant for eosinophils, was used as a positive control. The Transwell unit was kept at 37°C for 4 hours in a humidified 95% ai r-5% CO2 atmosphere. After 4

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hours, media from the lower chamber was centrifuged at 250 g, and cells were resuspended in PBS. The number of migrated cells in the lower chamber was counted with a hemocytometer. Each assay was set up in triplicate and repeated three times.

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The data is expressed as eosinophils migration index, which is defined as the ratio of migration of eosinophils in the presence of the chemoattractant VIP and the

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migration of eosinophils to the medium control.

Statistical analysis

Parametric data were compared using t-tests between two groups. Values are

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reported as mean ± SD. P-values < 0.05 were considered statistically significant.

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ACCEPTED MANUSCRIPT Supplementary Table 1: Patients Clinical and pathological characteristics

Age

gender

Esophageal disease

Allergic diseases

Other diseases

Eos/HPF

Current Treatment

1

9

M

NL

None

None

0

-

2

11

F

NL

None

None

0

-

3

12

F

NL

None

None

0

-

4

9

F

NL

None

None

0

-

5

4

M

NL

None

None

0

-

6

13

M

NL

None

None

7

11

M

NL

None

None

8

2

M

EoE

None

9

9

M

EoE

10

8

M

11

10

M

12

12

M

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Patients

Steroids

Elimination diet

Nasocort, Flovent

0

Food Trial

-

None

35 eos/HPF

Elimination

-

None

None

40 eos/HPF

Elimination

-

EoE

None

None

31 eos/HPF

Ad Lib

-

EoE

None

None

41 eos/HPF

Ad Lib

Flovent

None

None

63 eos/HPF

Elimination

Nasocort

EoE

None

30 eos/HPF

-

Flovent

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None

14

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EoE

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0

3

M

EoE

None

None

30 Eos/ HPF

Ad Lib

Pulmicort

15

5

M

EoE with dysphagia

None

None

63 eos/HPF

-

Flovent

16

10

F

EoE with dysphagia

None

None

80 eos/HPF

Ad Lib

-

17

6

M

EoE with

None

None

64 eos/HPF

-

Rhinocort,

13

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ACCEPTED MANUSCRIPT dysphagia

Flovent

7

M

EoE with dysphagia

None

non-specific colitis with focal cryptitis

73 eos/HPF

Elimination

Flonase

19

15

M

EoE with dysphagia

None

None

70 eos/HPF

Elimination

-

20

8

M

EoE with dysphagia

None

None

50 eos/HPF

Ad Lib

-

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18

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Abbreviations: NL=normal, M=male, F= female, eos= eosinophils, HPF=high power field, EoE= eosinophilic esophagitis,

Reference Cited

9. 10. 11. 12. 13. 14.

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Mavi P, Niranjan R, Dutt P, et al. Am J Physiol Gastrointest Liver Physiol 2014;307:G499-507. Rayapudi M, Rajavelu P, Zhu X, et al. Clin Transl Immunology 2014;3:e9. Dutt P, Shukla JS, Ventateshaiah SU, et al. Immunol Cell Biol 2015;93:849-57. Rayapudi M, Mavi P, Zhu X, et al. J Leukoc Biol 2010;88:337-46. Rajavelu P, Rayapudi M, Moffitt M, et al. Am J Physiol Gastrointest Liver Physiol 2012;302:G645-54. Niranjan R, Rayapudi M, Mishra A, et al. Immunol Cell Biol 2013;91:408-15. Venkateshaiah SU, Zhu X, Rajavelu P, et al. J Allergy Clin Immunol 2017. Zhu X, Wang M, Crump CH, et al. Am J Physiol Gastrointest Liver Physiol 2009;297:G550-8. Zhu X, Wang M, Mavi P, et al. Gastroenterology 2010;139:182-93 e7. Metwali A, Blum AM, Ferraris L, et al. J Neuroimmunol 1994;52:69-78. Lacy P, Willetts L, Kim JD, et al. Int Arch Allergy Immunol 2011;156:137-47. Hornung D, Dohrn K, Sotlar K, et al. J Clin Endocrinol Metab 2000;85:2604-8. Niranjan R, Mavi P, Rayapudi M, et al. Am J Physiol Gastrointest Liver Physiol 2013;304:G1087-94. Mavi P, Rajavelu P, Rayapudi M, et al. Am J Physiol Gastrointest Liver Physiol 2012;302:G1347-55.

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Supplementary Figures

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Supplementary Figure 1: Eosinophils highly express VIP receptor CRTH2 compared to the VPAC-1 or VPAC-2. An earlier report indicated that intestinal eosinophils produce VIP;10 therefore, we examined whether blood eosinophils of EoE patients also produce VIP and express VIP specific receptors. Accordingly, human eosinophils were examined for the expression of VIP, and VIP associated receptors using anti-VIP anti-VAPC1, anti-VACP2 and anti-CRTH2 antibodies. The flow cytometer analysis indicated none to very low expression of VIP and other VIP receptors VPAC-1 and VPAC-2 on eosinophils (A-C). But our analysis detected highly expressed VIP receptor CRTH2 on eosinophils (D). Further, we tested the hypothesis whether CRTH2 receptor has a critical role in VIP induced eosinophil motility. Notably, eotaxins and the interaction of its receptor CCR3 are reported to be major motility factor for eosinophils.11, 12 Accordingly, we kinetically examined human eosinophil motility in response to different concentrations of VIP recombinant protein (0, 1, 10, 100, 500 ng/ml) using transwell chemotactic chambers for 4 hours. Established eosinophil chemokine eotaxin-2 (200 ng/ml) recombinant protein was used as positive control. The analysis indicated that VIP indeed has eosinophil chemo-attraction activity similar to the eotaxin-2 (E). The critical role of CRTH2 in eosinophil motility was established by examining anti-CRTH2 pretreated eosinophils motility in response to VIP. The eosinophils were treated anti-CRTH2 for 1 hour, and both treated and non-treated eosinophils motility was again examined using transwell chambers in response to VIP protein (500 ng/ml) and eotaxin-2 protein (200 ng/ml). Analysis indicated that anti-CRTH2 antibody blocks in vitro migration of eosinophils in response to VIP (F); whereas, anti-CRTH2 treatment did not restrict eosinophils motility in response to eotaxin-2. The analysis establishes the critical role of CRTH2 and VIP interaction in eosinophil motility. Data is expressed as mean ± SD, n=3 experiments.

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Supplementary Figure 2: CRTH2 receptor is expressed on the eosinophils and tissue accumulated mast cells in the esophageal biopsy of human EoE. The in vitro analysis indicated that VIP receptor CRTH2 is critical for eosinophil motility. Therefore, we examined whether tissue eosinophils in human EoE similarly expresses CRTH2 receptors. Accordingly, immunofluorescence analysis was performed using anti-CRTH2 receptor on anti-MBP positive tissue eosinophils in human EoE biopsies. The anti-MBP (A, B) and anti-CRTH2 (C, D) expressed eosinophils are detected in the biopsies of human EoE (A-D, original magnification 400x and 1000x). The merged photomicrograph of anti-MBP and anti-CRTH2 stained and DAPI mounted tissue sections shows that tissue eosinophils expressed VIP receptor CRTH2 in the esophageal biopsies of human EoE patients (E-F, original magnification 400x and 1000x). Further, induced mast cells numbers and their role in the pathogenesis of EoE is well established.4, 13 However, it is not clear which chemokine receptors are responsible for mast cells recruitment in the human EoE. Therefore, we examined VIP receptor CRTH2 expression on the tissue accumulated mast cells in the esophageal biopsies of human EoE by performing immunofluorescence analyses using anti-tryptase and anti-CRTH2 antibodies. We demonstrated the presence of anti-tryptase positive mast cells in the esophageal biopsies (G, H) and CRTH2 stained receptors (I, J). DAPI mounted merged tissue section detected the co-localized tryptase expressed mast cell expressing CRTH2 receptor in esophageal biopsy of EoE patients (K, L). The arrows indicate tissue accumulated mast cells and CRTH2 receptors on mast cells in respective photomicrographs. The photomicrograph presented are 400x (A, C, E) and 1000x (B, D, F) of the original magnification photomicrographs are presented. Data is expressed mean ± SEM, n= 6-7.

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Supplementary Figure 3: Accumulation of eosinophils and mast cells in muscular mucosa following the induction of experimental EoE. Induced eosinophils and mast cell accumulation is implicated in the induction of esophageal functional abnormalities, including stricture and motility dysfunction in human and experimental EOE.14 However, the accumulation and mechanism of eosinophils and mast cells beyond the epithelial mucosa has not been examined. Since it is difficult to obtain deep mucosal biopsies in human EoE, we examined the accumulation of eosinophils and mast cells in each segment of the mouse esophagus in experimental EoE. The mouse esophageal tissue sections were examined for eosinophils and mast cells following anti-MBP and chloroacetate esterase staining, respectively. Both eosinophils and mast cells were detected in each segment of mouse esophagus following the induction of experimental EoE. A number of eosinophil accumulations in low and high magnification are shown in the epithelial mucosa, lamina propria and muscular mucosa (A). Similarly, mast cell accumulation in low and high magnification is shown mostly in the lamina propria and muscular mucosa (B). Photomicrographs presented are in 100x and 400x of the original magnification, respectively. EP, epithelial mucosa; LP, lamina propria; LU, Lumen; MP, Muscularis propria.

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Supplementary Figure 4:

BALB/c mice challenged with Aspergillus were treated with a CRTH2 antagonist (OC000459) as per the protocol was shown (A). Morphometric quantification indicated that CRTH2 antagonist treatment reduced the number of eosinophils (B) and mast cells (C) that accumulate in the esophagus of Aspergillus-challenged mice relative to the untreated Aspergillus-challenged control mice. The levels of eosinophils and mast cells in the esophageal sections are expressed as eosinophils/mm2 and mast cells/mm2, respectively. Data is expressed as mean ± SD, n=8-10 mice/group.