Group 2 innate lymphocytes (ILC2s) are enriched in active eosinophilic esophagitis

SHORT SUMMARY

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 non-diseased controls, implicating these cells in EoE pathogenesis.

To the Editor

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.

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 isotype control (Miltenyi biotech). ILC2s were identified as CD45+ FSC-low lineage-negative 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).

Figure 1. Identification of esophageal ILC2s.

Single cell suspensions from esophageal biopsies from a patient with active EoE (top) and a non-EoE control individual (bottom) were stained for CD45, lineage (CD3, CD14, CD16, CD19, CD20, CD56, TCRγδ, CD4, CD11b, CD235a, and FCeRI) and CRTH2 or isotype. ILC2s are identified as CD45+ low FSC lymphocytes that are lineage-negative and express CRTH2.

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.

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<0.0001, CI=0.45–0.84, Pearson r correlation) enumerated by independent pathologic analysis of H&E sections (Figure 2B). Thus, esophageal ILC2s correlated strongly with eosinophilia and were increased in samples from patients with active EoE compared with inactive EoE, PPI-REE, and control non-EoE tissue.

Table 1.

Patient characteristics. PPI=proton pump inhibitor, OVB=oral viscous budesonide, AR=allergic rhinitis, FA=food allergy.

Category	Age	Gender	Peak Eos/hpf	Meds/Treatment	AR	Asthma	Eczema	FA
EoE Active	6	Male	20	PPI	N	Y	N	N
EoE Active	4	Male	108	OVB	Y	N	N	N
EoE Active	1	Male	100	PPI	N	N	N	Y
EoE Active	2	Male	100	OVB	N	N	N	N
EoE Active	7	Male	175	OVB + PPI	N	N	N	N
EoE Active	12	Male	30	OVB + PPI	Y	N	N	Y
EoE Active	7	Female	37	OVB + PPI	Y	Y	N	Y
EoE Active	15	Male	44	OVB + PPI	Y	Y	N	N
EoE Active	5	Male	100	No Tx	Y	N	Y	Y
EoE Active	7	Female	60	OVB	Y	N	N	N
EoE Active	5	Male	90	No Tx	Y	Y	Y	Y
EoE Active	8	Male	32	OVB + PPI	N	N	N	Y
EoE Active	11	Male	92	OVB + PPI	Y	Y	N	Y
EoE Active	8	Female	66	PPI	Y	Y	N	N
EoE Active	5	Female	90	Noncompliant with OVB	N	N	N	N
EoE Inactive	17	Male	0	Fluticasone	Y	N	N	N
EoE Inactive	6	Male	9	Milk Elimination Diet	N	N	N	N
EoE Inactive	7	Female	0	No Current Tx	N	N	Y	N
EoE Inactive	11	Male	0	OVB	N	N	N	N
EoE Inactive	6	Male	0	OVB + PPI	N	Y	N	N
EoE Inactive	15	Male	5	OVB	N	N	N	N
EoE Inactive	16	Male	0	OVB + PPI	Y	Y	N	Y
EoE Inactive	8	Male	3	OVB + PPI	N	N	N	Y
EoE Inactive	2	Female	2	OVB + PPI	N	N	Y	Y
EoE Inactive	9	Female	0	Food Elimination Diet	Y	N	N	Y
EoE Inactive	15	Male	0	Fluticasone + PPI	Y	N	N	N
EoE Inactive	7	Male	0	OVB + PPI	N	N	Y	Y
Non-EoE	14	Female	0	No Tx	N	N	N	N
Non-EoE	3	Female	0	No Tx	N	N	Y	N
Non-EoE	2	Male	0	No Tx	N	N	Y	N
PPIREE Inactive	7	Male	6	PPI	N	N	N	N
PPIREE Inactive	1	Male	0	PPI	Y	N	N	N
PPIREE Inactive	7	Male	0	PPI	Y	Y	N	N

Figure 2. Increased ILC2s correlate with active EoE and tissue eosinophilia.

(A) ILC2s were quantifed (% of live cells) in control non-EoE biopsy tissue as well as in patients with active EoE, inactive EoE, and PPI-REE. P=0.005, one-way ANOVA. (B) %ILC2s of live cells correlated with mean (bottom, Pearson r=0.7, p<0.0001, 95% confidence interval 0.45–0.84) eosinophils.

Our studies demonstrate several novel findings. First, the presence of ILC2s in human esophagus has not been previously reported. Second, we have shown that ILC2s can be successfully quantified in biopsy specimens taken during routine care of patients undergoing endoscopy for EoE. Finally, we found that biopsy ILC2s were significantly increased in tissue from patients with active EoE versus inactive EoE and also higher than ILC2s in PPIREE and controls. Importantly, levels of biopsy ILC2s correlated with numbers of eosinophils detected by independent analysis. These findings support a role for ILC2s in active EoE pathogenesis given their capacity for robust production of IL-5 and IL-13 as demonstrated in other tissues including BAL, lung and nasal polyps.^{3, 4, 6} Additionally, mast cells and IL-9 have been implicated in EoE and ILC2s are a source of IL-9 and co-localize with mast cells in human lung suggesting that meaningful crosstalk occurs.^8–10 Whether ILC2s and mast cells interact through IL-9 to promote EoE will require future investigation.

One limitation of our work is the lack of functional ILC2 studies from patient biopsy specimens. On average, we obtained 1.9 × 10⁵ total cells from the esophageal biopsies and ILC2s are approximately 0.0–0.4% of the live cells. Thus, adequate ILC2 functional analysis for cytokine production or proliferation is not possible given the very limited biopsy cell numbers. We did, however, demonstrate that IL-33+TSLP can activate ILC2s from esophageal lymphoid aggregates, providing proof of concept that ILC2s could be induced in EoE. Indeed, TSLP is highly expressed in EoE.^{1, 5} Since ILC2s are not antigen specific, activation of ILC2s via EoE-relevant cytokines may represent an antigen-independent mechanism for propagation of chronic esophageal eosinophilia and structural changes in EoE.