HHS Public Access Author manuscript Author Manuscript
J Allergy Clin Immunol. Author manuscript; available in PMC 2016 September 01. Published in final edited form as: J Allergy Clin Immunol. 2015 September ; 136(3): 792–794.e3. doi:10.1016/j.jaci.2015.05.048.
Group 2 innate lymphocytes (ILC2s) are enriched in active eosinophilic esophagitis Taylor A. Doherty, M.D.1, Rachel Baum, B.S.1, Robert O. Newbury, M.D.6, Tom Yang, B.S. Ranjan Dohil, M.D.4,5, Melissa Aquino, B.S.2,3,5, Ashmi Doshi, M.D.1,2, Hannah H. Walford, M.D.1,2, Richard C. Kurten, Ph.D.7, David H. Broide, M.B. Ch.B.1, and Seema Aceves, M.D., Ph.D.1,2,3,5
2,3,5,
Author Manuscript
1Department
of Medicine, University of California, La Jolla, CA
2Division
of Allergy, Immunology, University of California, San Diego, Rady Children’s Hospital, San Diego 3Center
for Infection, Immunity, and Inflammation, University of California, San Diego, Rady Children’s Hospital, San Diego
4Division
of Gastroenterology and Nutrition, University of California, San Diego, Rady Children’s Hospital, San Diego 5Department
of Pediatrics, University of California, San Diego, Rady Children’s Hospital, San
Diego
Author Manuscript
6Department
of Pathology, University of California, San Diego, Rady Children’s Hospital, San
Diego 7Department
of Physiology and Biophysics, University of Arkansas for Medical Sciences and Arkansas Children’s Hospital Research Institute. Little Rock, AR
Author Manuscript
Correspondence should be addressed to: Taylor A. Doherty, M.D. UC San Diego 9500 Gilman Dr. 0635, La Jolla, CA 92093-0635. [email protected], phone: 858-822-7563 fax: 858-534-2110. Taylor A. Doherty, M.D., Department of Medicine, University of California, San Diego. La Jolla, CA Rachel Baum, B.S., Department of Medicine, University of California, San Diego. La Jolla, CA Robert O. Newbury, M.D., Department of Pathology, University of California, San Diego, Rady Children’s Hospital, San Diego. Tom Yang, B.S., Department of Pediatrics, Division of Allergy, Immunology, Center for Infection, Immunity, and Inflammation, University of California, La Jolla, CA, Rady Children’s Hospital, San Diego. Ranjan Dohil, M.D., Department of Pediatrics, Division of Gastroenterology and Nutrition, University of California, La Jolla, CA, Rady Children’s Hospital, San Diego. Melissa Aquino, B.S., Department of Pediatrics, Division of Allergy, Immunology, Center for Infection, Immunity, and Inflammation, University of California, La Jolla, CA, Rady Children’s Hospital, San Diego. Ashmi Doshi, M.D., Department of Medicine, University of California, San Diego. La Jolla, CA and Rady’s Children’s Hospital of San Diego, Division of Rheumatology, Allergy and Immunology, San Diego, CA. Hannah H. Walford, M.D., Department of Medicine, University of California, San Diego. La Jolla, CA and Rady’s Children’s Hospital of San Diego, Division of Rheumatology, Allergy and Immunology, San Diego, CA. Richard C. Kurten, Ph.D., Department of Physiology and Biophysics, University of Arkansas for Medical Sciences and Arkansas Children’s Hospital Research Institute. Little Rock, AR David H. Broide, M.B. Ch.B., Department of Medicine, University of California, San Diego. La Jolla, CA Seema Aceves, M.D., Ph.D., Department of Pediatrics and Medicine, Division of Allergy, Immunology, Center for Infection, Immunity, and Inflammation, University of California, La Jolla, CA, Rady Children’s Hospital, San Diego Publisher's Disclaimer: 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 citable 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.
Doherty et al.
Page 2
SHORT SUMMARY Author Manuscript
Group 2 innate lymphoid cells (ILC2s) produce high levels of IL-5 and IL-13, both of which are important pathogenic mediators in eosinophilic esophagitis (EoE). ILC2s have not been previously described in EoE. Our study demonstrates the novel finding that ILC2s are increased in esophageal biopsies from EoE patients with active disease compared with inactive EoE and nondiseased controls, implicating these cells in EoE pathogenesis.
Keywords group 2 innate lymphoid cells; type 2 innate lymphoid cells; ILC2; ILC2s; ILC; innate lymphoid cells; eosinophilic esophagitis
Author Manuscript
To the Editor
Author Manuscript
Eosinophilic esophagitis (EoE) is a chronic, antigen mediated disease characterized by esophageal remodeling including epithelial cell hyperplasia with barrier dysfunction, subepithelial fibrosis, and smooth muscle dysmotility leading to food impactions and strictures as well as symptoms of chronic pain, feeding intolerance, and dysphagia.1 EoE pathogenesis is driven by thymic stromal lymphopoeitin (TSLP) produced by activated epithelial cells, and IL-5 and IL-13 produced by inflammatory cells. Production of IL-5 results in esophageal eosinophilia and IL-13 induces EoE-specific epithelial gene expression and remodeling changes such as angiogenesis.2 Though IL-5 and IL-13 are active in EoE, the cellular sources are not well established and may include the recently discovered group 2 innate lymphoid cells (ILC2s)3. ILC2s are lineage-negative (lack surface markers for T, B, NK, or NKT cells) but do express the chemoattractant receptor homologous molecule expressed on TH2 lymphocytes (CRTH2).4 IL-33 and TSLP induce human ILC2s produce large amounts of Th2 cytokines and ILC2s have been detected in peripheral blood, GI tract, lung, BAL, nasal polyps and in the skin of atopic dermatitis patients.3–7 Despite the potential for ILC2s to contribute to allergic diseases, there are a limited number of studies of ILC2s in human disease and no reports demonstrating the presence of ILC2s in human EoE tissue. We hypothesized that ILC2s are elevated in tissue biopsies from patients with active EoE but not in inactive EoE or control subjects. Such findings would implicate ILC2s in EoE pathogenesis.
Author Manuscript
To identify esophageal ILC2s, we obtained biopsies from patients undergoing endoscopy for routine care (UCSD IRB approved protocol 091485). Biopsy samples were transported in RPMI (Gibco) media at 7°C and cut with scissors into fine pieces. The pieces were placed on the top of a 35 μm Falcon filter tube (Corning Life Sciences, DL) and manually dispersed with a 1ml syringe plunger. Single cell suspensions were incubated with human Fc block (Miltenyi biotech) prior to staining. Cells were stained with a FITC lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56; BD, Franklin Lakes NJ), TCRγδ (BD, Franklin Lakes NJ, USA), CD4, CD11b, CD235a, FcεRI, (ebiosciences, San Diego, CA) that allows for exclusion of B, T, NK, and NKT cells as well as mast cells and basophils. Cells were also stained with CD45 PerCP (ebiosciences, San Diego, CA) and PE-conjugated CRTH2 or
J Allergy Clin Immunol. Author manuscript; available in PMC 2016 September 01.
Doherty et al.
Page 3
Author Manuscript
isotype control (Miltenyi biotech). ILC2s were identified as CD45+ FSC-low lineagenegative lymphocytes that expressed CRTH2. Biopsies from control individuals without EoE revealed a very small CD45+ hematopoietic cell population with minimal numbers of lymphocytes (Figure 1). In contrast, patients with active EoE had significant increases in CD45+ lymphocytes with approximately 25% (range: 15.1% – 50.7%) of the lineage-negative cells expressing CRTH2 (Figure 1). Thus, we were able to successfully identify ILC2s (CD45+ CRTH2+ lineage-negative lymphocytes) in biopsy specimens from patients with EoE despite the small total live cell numbers present in the biopsies (mean = 1.9 × 105 cells).
Author Manuscript
To confirm that human esophageal ILC2s expand in response to cytokines IL-33 and TSLP, similar to ILC2s in other tissues3, we performed culture studies with lymphoid aggregate cells from human donor esophagi (Fig E1 and supplemental methods). Lymphoid aggregates were digested with Collagenase D and DNAase into single cell suspensions and cultured with IL-2 (10ng/ml) with or without IL-33 (50ng/ml) and TSLP (50 ng/ml). After one week, the cells were collected and ILC2s enumerated. We found that ILC2s were increased in total number and percent after a week of stimulation with IL-33 and TSLP compared with IL-2 only treatment (Fig E2). Thus, ILC2s are present in the human esophagus and respond to cytokines known to induce ILC2 expansion.
Author Manuscript
Using the gating strategy in Figure 1, we next investigated potential differences in esophageal ILC2 numbers in biopsies from patients with active EoE (n= 15, age= 6.8 +/− 3.7 years, biopsy eosinophils= 76.3 +/− 40.8 per hpf) compared to individuals with inactive EoE (n= 12, age= 9.9 +/− 4.8 years, biopsy eosinophils= 1.6 +/− 2.8 per hpf), proton pump inhibitor-responsive esophageal eosinophilia (PPIREE, n= 3, age= 5.0 +/− 3.5 years, biopsy eosinophils= 2.0 +/− 3.5 per hpf), and non-EoE control samples (n= 3, age= 6.33 +/− 6.7 years, biopsy eosinophils= 0.0) (Table 1). Strikingly, patients with active EoE had significantly increased ILC2s (% of live cells) from biopsies compared with all other groups (p = 0.005, one-way ANOVA) (Figure 2A). We further assessed whether a correlation was present between levels of esophageal ILC2s and the degree of epithelial eosinophilia from all subjects. ILC2 numbers positively correlated with mean eosinophils per high power field (r=0.7, p
J Allergy Clin Immunol. Author manuscript; available in PMC 2016 September 01. Published in final edited form as: J Allergy Clin Immunol. 2015 September ; 136(3): 792–794.e3. doi:10.1016/j.jaci.2015.05.048.
Group 2 innate lymphocytes (ILC2s) are enriched in active eosinophilic esophagitis Taylor A. Doherty, M.D.1, Rachel Baum, B.S.1, Robert O. Newbury, M.D.6, Tom Yang, B.S. Ranjan Dohil, M.D.4,5, Melissa Aquino, B.S.2,3,5, Ashmi Doshi, M.D.1,2, Hannah H. Walford, M.D.1,2, Richard C. Kurten, Ph.D.7, David H. Broide, M.B. Ch.B.1, and Seema Aceves, M.D., Ph.D.1,2,3,5
2,3,5,
Author Manuscript
1Department
of Medicine, University of California, La Jolla, CA
2Division
of Allergy, Immunology, University of California, San Diego, Rady Children’s Hospital, San Diego 3Center
for Infection, Immunity, and Inflammation, University of California, San Diego, Rady Children’s Hospital, San Diego
4Division
of Gastroenterology and Nutrition, University of California, San Diego, Rady Children’s Hospital, San Diego 5Department
of Pediatrics, University of California, San Diego, Rady Children’s Hospital, San
Diego
Author Manuscript
6Department
of Pathology, University of California, San Diego, Rady Children’s Hospital, San
Diego 7Department
of Physiology and Biophysics, University of Arkansas for Medical Sciences and Arkansas Children’s Hospital Research Institute. Little Rock, AR
Author Manuscript
Correspondence should be addressed to: Taylor A. Doherty, M.D. UC San Diego 9500 Gilman Dr. 0635, La Jolla, CA 92093-0635. [email protected], phone: 858-822-7563 fax: 858-534-2110. Taylor A. Doherty, M.D., Department of Medicine, University of California, San Diego. La Jolla, CA Rachel Baum, B.S., Department of Medicine, University of California, San Diego. La Jolla, CA Robert O. Newbury, M.D., Department of Pathology, University of California, San Diego, Rady Children’s Hospital, San Diego. Tom Yang, B.S., Department of Pediatrics, Division of Allergy, Immunology, Center for Infection, Immunity, and Inflammation, University of California, La Jolla, CA, Rady Children’s Hospital, San Diego. Ranjan Dohil, M.D., Department of Pediatrics, Division of Gastroenterology and Nutrition, University of California, La Jolla, CA, Rady Children’s Hospital, San Diego. Melissa Aquino, B.S., Department of Pediatrics, Division of Allergy, Immunology, Center for Infection, Immunity, and Inflammation, University of California, La Jolla, CA, Rady Children’s Hospital, San Diego. Ashmi Doshi, M.D., Department of Medicine, University of California, San Diego. La Jolla, CA and Rady’s Children’s Hospital of San Diego, Division of Rheumatology, Allergy and Immunology, San Diego, CA. Hannah H. Walford, M.D., Department of Medicine, University of California, San Diego. La Jolla, CA and Rady’s Children’s Hospital of San Diego, Division of Rheumatology, Allergy and Immunology, San Diego, CA. Richard C. Kurten, Ph.D., Department of Physiology and Biophysics, University of Arkansas for Medical Sciences and Arkansas Children’s Hospital Research Institute. Little Rock, AR David H. Broide, M.B. Ch.B., Department of Medicine, University of California, San Diego. La Jolla, CA Seema Aceves, M.D., Ph.D., Department of Pediatrics and Medicine, Division of Allergy, Immunology, Center for Infection, Immunity, and Inflammation, University of California, La Jolla, CA, Rady Children’s Hospital, San Diego Publisher's Disclaimer: 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 citable 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.
Doherty et al.
Page 2
SHORT SUMMARY Author Manuscript
Group 2 innate lymphoid cells (ILC2s) produce high levels of IL-5 and IL-13, both of which are important pathogenic mediators in eosinophilic esophagitis (EoE). ILC2s have not been previously described in EoE. Our study demonstrates the novel finding that ILC2s are increased in esophageal biopsies from EoE patients with active disease compared with inactive EoE and nondiseased controls, implicating these cells in EoE pathogenesis.
Keywords group 2 innate lymphoid cells; type 2 innate lymphoid cells; ILC2; ILC2s; ILC; innate lymphoid cells; eosinophilic esophagitis
Author Manuscript
To the Editor
Author Manuscript
Eosinophilic esophagitis (EoE) is a chronic, antigen mediated disease characterized by esophageal remodeling including epithelial cell hyperplasia with barrier dysfunction, subepithelial fibrosis, and smooth muscle dysmotility leading to food impactions and strictures as well as symptoms of chronic pain, feeding intolerance, and dysphagia.1 EoE pathogenesis is driven by thymic stromal lymphopoeitin (TSLP) produced by activated epithelial cells, and IL-5 and IL-13 produced by inflammatory cells. Production of IL-5 results in esophageal eosinophilia and IL-13 induces EoE-specific epithelial gene expression and remodeling changes such as angiogenesis.2 Though IL-5 and IL-13 are active in EoE, the cellular sources are not well established and may include the recently discovered group 2 innate lymphoid cells (ILC2s)3. ILC2s are lineage-negative (lack surface markers for T, B, NK, or NKT cells) but do express the chemoattractant receptor homologous molecule expressed on TH2 lymphocytes (CRTH2).4 IL-33 and TSLP induce human ILC2s produce large amounts of Th2 cytokines and ILC2s have been detected in peripheral blood, GI tract, lung, BAL, nasal polyps and in the skin of atopic dermatitis patients.3–7 Despite the potential for ILC2s to contribute to allergic diseases, there are a limited number of studies of ILC2s in human disease and no reports demonstrating the presence of ILC2s in human EoE tissue. We hypothesized that ILC2s are elevated in tissue biopsies from patients with active EoE but not in inactive EoE or control subjects. Such findings would implicate ILC2s in EoE pathogenesis.
Author Manuscript
To identify esophageal ILC2s, we obtained biopsies from patients undergoing endoscopy for routine care (UCSD IRB approved protocol 091485). Biopsy samples were transported in RPMI (Gibco) media at 7°C and cut with scissors into fine pieces. The pieces were placed on the top of a 35 μm Falcon filter tube (Corning Life Sciences, DL) and manually dispersed with a 1ml syringe plunger. Single cell suspensions were incubated with human Fc block (Miltenyi biotech) prior to staining. Cells were stained with a FITC lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56; BD, Franklin Lakes NJ), TCRγδ (BD, Franklin Lakes NJ, USA), CD4, CD11b, CD235a, FcεRI, (ebiosciences, San Diego, CA) that allows for exclusion of B, T, NK, and NKT cells as well as mast cells and basophils. Cells were also stained with CD45 PerCP (ebiosciences, San Diego, CA) and PE-conjugated CRTH2 or
J Allergy Clin Immunol. Author manuscript; available in PMC 2016 September 01.
Doherty et al.
Page 3
Author Manuscript
isotype control (Miltenyi biotech). ILC2s were identified as CD45+ FSC-low lineagenegative lymphocytes that expressed CRTH2. Biopsies from control individuals without EoE revealed a very small CD45+ hematopoietic cell population with minimal numbers of lymphocytes (Figure 1). In contrast, patients with active EoE had significant increases in CD45+ lymphocytes with approximately 25% (range: 15.1% – 50.7%) of the lineage-negative cells expressing CRTH2 (Figure 1). Thus, we were able to successfully identify ILC2s (CD45+ CRTH2+ lineage-negative lymphocytes) in biopsy specimens from patients with EoE despite the small total live cell numbers present in the biopsies (mean = 1.9 × 105 cells).
Author Manuscript
To confirm that human esophageal ILC2s expand in response to cytokines IL-33 and TSLP, similar to ILC2s in other tissues3, we performed culture studies with lymphoid aggregate cells from human donor esophagi (Fig E1 and supplemental methods). Lymphoid aggregates were digested with Collagenase D and DNAase into single cell suspensions and cultured with IL-2 (10ng/ml) with or without IL-33 (50ng/ml) and TSLP (50 ng/ml). After one week, the cells were collected and ILC2s enumerated. We found that ILC2s were increased in total number and percent after a week of stimulation with IL-33 and TSLP compared with IL-2 only treatment (Fig E2). Thus, ILC2s are present in the human esophagus and respond to cytokines known to induce ILC2 expansion.
Author Manuscript
Using the gating strategy in Figure 1, we next investigated potential differences in esophageal ILC2 numbers in biopsies from patients with active EoE (n= 15, age= 6.8 +/− 3.7 years, biopsy eosinophils= 76.3 +/− 40.8 per hpf) compared to individuals with inactive EoE (n= 12, age= 9.9 +/− 4.8 years, biopsy eosinophils= 1.6 +/− 2.8 per hpf), proton pump inhibitor-responsive esophageal eosinophilia (PPIREE, n= 3, age= 5.0 +/− 3.5 years, biopsy eosinophils= 2.0 +/− 3.5 per hpf), and non-EoE control samples (n= 3, age= 6.33 +/− 6.7 years, biopsy eosinophils= 0.0) (Table 1). Strikingly, patients with active EoE had significantly increased ILC2s (% of live cells) from biopsies compared with all other groups (p = 0.005, one-way ANOVA) (Figure 2A). We further assessed whether a correlation was present between levels of esophageal ILC2s and the degree of epithelial eosinophilia from all subjects. ILC2 numbers positively correlated with mean eosinophils per high power field (r=0.7, p
