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Yoon, Lee, Park, Yoon, Kim, Moon, and Yu: Type 2 Innate Lymphoid Cells and Skin Fibrosis in a Murine Model of Atopic Dermatitis-Like Skin Inflammation
Original Article
Immunology, Allergy & Rheumatology

Journal of Korean Medical Science 2024; 39(30): e221.

Published online: 10 July 2024

DOI: https://doi.org/10.3346/jkms.2024.39.e221

Type 2 Innate Lymphoid Cells and Skin Fibrosis in a Murine Model of Atopic Dermatitis-Like Skin Inflammation

Jisun Yoon1, 2, Jiho Lee3, Arum Park3, Jin Yoon3, Jeong Ryun Kim4, Gyeong Joon Moon4, 5, *, Jinho Yu6, *

1Department of Pediatrics, Chung-Ang University College of Medicine, Seoul, Korea.

2Clinical Trial Support Team, Chung-Ang University Gwangmyeong Hospital, Gwangmyeong, Korea.

3Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea.

4Department of Cell and Genetic Engineering, University of Ulsan College of Medicine, Seoul, Korea.

5Center for Cell Therapy, Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea.

6Department of Pediatrics, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.

Address for Correspondence: Gyeong Joon Moon, PhD. Center for Cell Therapy, Asan Institute for Life Sciences, Asan Medical Center, Department of Cell and Genetic Engineering, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea. gj.moon@amc.seoul.kr

Address for Correspondence: Jinho Yu, MD, PhD. Department of Pediatrics, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea. jinhoyu@amc.seoul.kr

Equal contributor:

*Gyeong Joon Moon and Jinho Yu contributed equally to this work.

Received 4 May 2024       Accepted 24 June 2024

© 2024 The Korean Academy of Medical Sciences.

The Korean Academy of Medical Sciences

https://creativecommons.org/licenses/by-nc/4.0/
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease. Although murine studies have demonstrated that type 2 innate lymphoid cells (ILCs) mediate type 2 skin inflammation, their role in skin fibrosis in AD remains unclear. This study investigated whether type 2 ILCs are involved in skin fibrosis using an AD-like murine model.

Methods

C57BL/6 mice were treated epicutaneously with Aspergillus fumigatus (Af) for 5 consecutive days per week for 5 weeks to induce skin fibrosis. Mature lymphocyte deficient Rag1−/− mice were also used to investigate the role of type 2 ILCs in skin fibrosis.

Results

The clinical score and transepidermal water loss (TEWL) were significantly higher in the AD group than in the control group. The AD group also showed significantly increased epidermal and dermal thicknesses and significantly higher numbers of eosinophils, neutrophils, mast cells, and lymphocytes in the lesional skin than the control group. The lesional skin of the AD group showed increased stain of collagen and significantly higher levels of collagen than the control group (10.4 ± 2.2 µg/mg vs. 1.6 ± 0.1 µg/mg, P < 0.05). The AD group showed significantly higher populations of type 2 ILCs in the lesional skin compared to the control group (0.08 ± 0.01% vs. 0.03 ± 0.01%, P < 0.05). These findings were also similar with the AD group of Rag1−/− mice compared to their control group. Depletion of type 2 ILCs with anti-CD90.2 monoclonal antibodies significantly improved clinical symptom score, TEWL, and infiltration of inflammatory cells, and significantly decreased levels of collagen were observed in the AD group of Rag1−/− mice (1.6 ± 0.0 μg/mg vs. 4.5 ± 0.3 μg/mg, P < 0.001).

Conclusion

In the Af-induced AD-like murine model, type 2 ILCs were elevated, with increased levels of collagen. Additionally, removal of type 2 ILCs resulted in decreased collagen levels and improved AD-like pathological findings. These findings suggest that type 2 ILCs play a role in the mechanism of skin fibrosis in AD.

Graphical Abstract

jkms-39-e221-abf001.jpg

Keywords: Type 2 Innate Lymphoid Cell, Skin Fibrosis, Atopic Dermatitis, Mouse Model

INTRODUCTION

Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease characterized by intense itch and recurrent eczematous lesion.1 The disease is highly prevalent in children and inflicts a substantial socioeconomic burden on patients.2345 The two major pathogeneses of AD are skin barrier defect and cutaneous inflammation typically defined by increase in both T helper (Th) 2-mediated and Th1-mediated immune responses.678 The chronic cutaneous inflammation induces skin fibrosis characterized by lichenification and fibrotic papules, involving various inflammatory responses including interleukin (IL)-33, thymic stromal lymphopoietin (TSLP), and IL-13, and epidermal defects such as loss of Notch1 in keratinocytes.9101112
Studies on the innate cell sources of type 2 cytokines, known to be related to allergic diseases, have identified group 2 innate lymphoid cells (ILCs) as critical sources of innate effector cells in type 2 immunity.1314 Type 2 ILCs produce type 2 cytokines such as IL-5, IL-9, and IL-13 in response to IL-25 and IL-33, leading to allergic inflammation.1415 We previously reported the involvement of type 2 ILCs in skin inflammation in a murine model of Aspergillus fumigatus (Af)-induced AD-like skin lesions.16
Recently, type 2 ILCs were reported to be involved in fibrosis in a murine model of pulmonary fibrosis, extending beyond their role in type 2 inflammation.1718 However, no studies have shown that type 2 ILCs also play a role in fibrosis induced by chronic cutaneous inflammation. Therefore, we investigated whether skin fibrosis is associated with type 2 ILCs in a murine model chronically induced by Af.

METHODS

Murine model of AD

Female 8-week-old C57BL/6 mice were purchased from Orient Bio INC (Seongnam, Korea). Af antigen was purchased from Greer Laboratories (Lenoir, NC, USA). All animal experiments were performed at a specific pathogen-free (SPF) facility. Skin fibrosis was induced by chronic AD-like cutaneous inflammation through epicutaneous application of Af extract as previously described.19 Briefly, animals were anesthetized with intraperitoneal injections of tiletamine-zolazepam (60 mg/kg, Zoletile50®; Vibac Laboratories, Carros, France) and xylazine (10 mg/kg, Rumpun®; Bayer Korea, Ansan, Korea). The back of the mice was shaved and 40 µg of Af extract was applied epicutaneously once a day to a 1 cm2 area on the dorsal skin surface for 5 consecutive days per week for 5 weeks. The control mice were treated with normal saline solution. Rag1−/− (Jackson Laboratory, Bar Harbor, ME, USA) mice which lack mature lymphocytes were used to elucidate the role of group 2 ILCs. Skin tissue, lymph nodes, and blood were collected on day 33 after sacrifice.

Antibody treatment

Isotype control (LTF-2) and anti-CD90.2 monoclonal antibodies (mAbs) (30H12) were purchased from Bio X Cell (West Lebanon, NH, USA). To remove type 2 ILCs, 30 µg/mouse anti-CD 90.2 mAb was administrated intraperitoneally (i.p.) every 2 days from 2 days before Af application until day 30 in Rag1−/− mice.

Clinical parameters

The clinical scores of the skin lesions were assessed by a single investigator on days 0, 18, 25, and 32. Dryness, scaling, erosion, excoriation, and hemorrhage were scored as 0 (absent), 1 (mild), 2 (moderate), or 3 (severe), with the sum of these items defined as the clinical score (maximum score, 15). Epidermal permeability barrier function was evaluated by measuring transepidermal water loss (TEWL) using a Vapometer® SWL-3 (DelfinTechnologies Ltd., Kuopio, Finland) on day 0, 18, 25, and 32.

Histological examination of the skin

Skin samples were cut into 1 mm × 1 mm pieces and fixed at room temperature for 18 hours in 10% formalin solution (vender). After embedding the tissue in paraffin, 5 µm thick sections were cut and stained with hematoxylin and eosin (H&E) for measuring skin thickness and counting inflammatory cells. Toluidine blue (TB) was used to identify mast cells and Masson’s trichrome (MT) was used to identify collagen fibers. The stained samples were mounted with mounting medium and analyzed using a bright-field microscopy under high power (400× magnification; Olympus, Tokyo, Japan).

Measurement of serum total immunoglobulin (Ig) E

To collect blood samples, the mice were anesthetized, and laparotomy was performed. After laparotomy, blood was collected from the portal vein using a 1 mL syringe. Using BD Vacutainer® SST™ II Advance (BD Biosciences, San Jose, CA, USA), centrifugation was performed at 2,000 rpm, 20°C, for 10 minutes to separate serum. The remaining samples were stored at −70°C. Total IgE levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (BD OptEIA ELISA; BD Biosciences) according to the manufacturer’s protocol, and absorbance was measured at a wavelength of 450 nm using a VICTOR X3 microplate reader (PerkinElmer, Waltham, MA, USA).

Flow cytometry

Skin tissues were chopped into small pieces and incubated in RPMI-1640 media (Gibco™; Thermo Fisher Scientific, Waltham, MA, USA) containing 2.5 mg/mL collagenase IV (Worthington Biochemical, Lakewood, NJ, USA) and 0.2 mg/mL DNase 1 (Roche Molecular Systems, Somerville, NJ, USA) at 37°C for 60 minutes. ILC2 labeled antibody was used for fluorescence analysis, and the cells were analyzed using Canto II (BD Biosciences). The data were analyzed and graphically presented using FlowJo software (BD Biosciences). PI staining (APC-cy7) (Invitrogen™; Thermo Fisher Scientific) was performed to determine whether the cells were alive or dead. The markers CD45, CD3, Ly-6G, CD11b, Ter-119m, IgG2b, CD25, CD127, and IL-17A were used. IL-13 and IL-33R (BioLegend, San Diego, CA, USA) were used, along with APC, Amcyan, FITC, PE, PE-CY7, Pacific blue, and PerCP-CY5.5 for each fluorescence. ILCs were defined as lineage-negative (Lin−) CD127+ cells and type 2 ILCs were defined as CD25+IL-33R+ cells.

Collagen assay

A Sircol-soluble collagen assay kit (Biocolor, Belfast, UK) was used to measure the amount of collagen in the skin tissue. Equal weights of minced skin tissue (30 mg) were ground and incubated with pepsin and 0.5 M acetic acid (in a 1:10 ratio of pepsin to tissue wet weight) overnight at 4°C. Following this, 100 µL of Sircol dye reagent was added and shaken for 30 minutes. The mixture was then centrifuged at 12,000 rpm for 10 minutes, and the supernatant was discarded after adding 750 µL of acid-salt wash for rinsing. The resulting pellet was mixed with an alkaline reagent and gently agitated before measuring the absorbance at 555 nm using a microplate reader.

Total ribonucleic acid (RNA) preparation and quantitative real-time polymerase chain reaction (RT-PCR) analysis

After placing the skin tissue in Qiazol (Qiagen, Hilden, Germany) and homogenizing it, RNA was quantified at 1,000 ng/μL following the miRNeasy mini kit protocol (Qiagen). Subsequently, complementary deoxyribonucleic acid (cDNA) synthesis was carried out using the WizScript™ cDNA Synthesis kit (Wizbio, Sungnam, Korea), following the manufacturer’s instructions, with cDNA synthesized from 2 µg RNA. RT-PCR was performed on a Quant Studio 6 Flex (Applied Biosystems, Waltham, MA, USA) using the synthesized cDNA, and primers for IL-4 (Mm00445259_m1), interferon (IFN)-γ (Mm01168134_m1), and TSLP (Mm00498739_m1) were used, based on 18s (Mm00415898_m1), with the TaqMan® Gene Expression Assay (Thermo Fisher Scientific).

Statistical analysis

Data normality was assessed using the Shapiro–Wilk test, and subsequent testing was conducted using the Student’s unpaired t-test or Mann–Whitney U test. Statistical significance was established at P < 0.05. Statistical analyses were performed using GraphPad Prism 10 software (GraphPad Software, Inc., La Jolla, CA, USA).

Ethics statement

All animal procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Asan Medical Center and Ulsan University College of Medicine. The IACUC abides by the Institute of Laboratory Animal Resources guide (permit number: 2014-12-064).

RESULTS

AD-like skin inflammation and fibrosis induced by epicutaneous application of Af

Skin fibrosis was induced by treatment with 40 μg Af for 5 days per week for 5 weeks (Fig. 1A). Erythematous and edematous skin changes with excoriation and increased dryness were observed in the saline-treated control group. Clinical symptom scores (mean ± standard deviation) were also significantly higher in the Af-treated group than those in the control group at day 18 (Af-treated group, 6.6 ± 0.4 vs. control group, 0.6 ± 0.2), day 25 (7.7 ± 0.6 vs. 0.0 ± 0.0), and day 32 (10.0 ± 1.0 vs. 0.0 ± 0.0), and degree of TEWL was significantly increased in the Af-treated group compared to the control group at day 18 (15.0 ± 0.6 g/m2h vs. 5.3 ± 0.5 g/m2h), day 25 (20.5 ± 1.1 g/m2h vs. 2.1 ± 0.2 g/m2h), and day 32 (22.0 ± 1.9 g/m2h vs. 2.8 ± 0.3 g/m2h) (all P < 0.001). Serum concentrations of total IgE were significantly higher in the Af-treated group than in the control group (P < 0.001) (Fig. 1B).
Fig. 1

Skin fibrosis induced by Af in wild type mice. (A) Schematic protocol of this study. For 5 weeks, Af was epicutaneosly applied to the dorsal skin of mice for 5 consecutive days per week. (B) Clinical symptom scores, TEWL, and serum total IgE levels in Af-induced atopic dermatitis murine model. (C) H&E (top), TB (middle), and MT (bottom) staining were performed on skin tissue sections (×400 magnification, scale bar = 200 μm). (D) mRNA expression of IFN-γ, TSLP, and IL-4 in the lesional skin of atopic dermatitis murine model. (E) Collagen levels in the lesional skin as measured by collagen assay. (F) Flow cytometry was used to measure the proportion of type 2 ILCs. ILCs were defined as lineage negative (Lin−) CD127+ cells, and type 2 ILCs were defined as CD25+IL-33R+ cells.

Af = Aspergillus fumigatus, TEWL = transepidermal water loss, Ig = immunoglobulin, H&E = hematoxylin and eosin, TB = toluidine blue, MT = Masson’s trichrome, IFN = interferon, TSLP = thymic stromal lymphopoietin, IL = interleukin, ILC = innate lymphoid cell.
*P < 0.05, ***P < 0.001.
jkms-39-e221-g001
To examine whether long-term treatment with Af leads to pathological changes, such as chronic inflammation and fibrosis, in the mouse skin, skin tissues were histopathologically examined using H&E, TB, and MT staining. Histopathological examination of the lesional skin of the Af-treated mice revealed marked hyperplasia of the epidermis (thickness of the Af-treated group, 24.5 ± 4.5 μm vs. thickness of the control group, 12.1 ± 0.8 μm, P = 0.026) and dermis (154.7 ± 12.3 μm vs. 98.0 ± 14.2 μm, P = 0.014), more infiltration into the dermis of inflammatory cells including eosinophils (Af-treated group, 5.4 ± 0.3/mm2 vs. control group, 0.6 ± 0.1/mm2), neutrophils (6.8 ± 0.5/mm2 vs. 1.2 ± 0.4/mm2), lymphocytes (4.5 ± 0.1/mm2 vs. 1.8 ± 0.3/mm2), and mast cells (5.0 ± 0.2/mm2 vs. 1.6 ± 0.2/mm2) (all P < 0.001; Fig. 1C). Moreover, IFN-γ (1.00 vs. 3.28 ± 1.51, P = 0.163), TSLP (1.00 vs. 2.34 ± 0.54, P = 0.038), and IL-4 (1.00 vs. 115.47 ± 63.29, P = 0.101) increased in the Af-treated group than in the control group (Fig. 1D). The AD group (10.4 ± 2.2 µg/mg) showed a significantly higher collagen level than the control group (1.6 ± 0.1 µg/mg) (P < 0.001; Fig. 1E).
The populations of Lin−CD127+25+IL-33R+ cells (type 2 ILCs) were significantly higher in the skin of the Af-treated group (0.08 ± 0.01%) compared to the control group (0.03 ± 0.01%), when CD45+ was measured as the denominator (P = 0.022) (Fig. 1F).

Type 2 ILCs in skin fibrosis of Af-treated Rag1−/− mice

To elucidate the role of type 2 ILCs in skin fibrosis induced by Af, Rag1−/− mice lacking in mature lymphocytes were used. It was similarly treated with 40 μg Af for 5 days, resting for 2 days, for 5 weeks (Fig. 2A). The lesional skin of Af-treated Rag1−/− mice showed gross erythematous and edematous changes with excoriation and greater dryness than that of saline-treated Rag1−/− mice. Similar to the AD group of wild type mice, both clinical symptom score and TEWL were significantly increased in the Af-treated Rag1−/− mice at day 18 (6.2 ± 0.4 g/m2h and 16.2 ± 1.4 g/m2h vs. 0.4 ± 0.2 g/m2h and 4.2 ± 0.6 g/m2h, respectively), day 25 (8.2 ± 0.7 g/m2h and 19.6 ± 1.4 g/m2h vs. 0.0 ± 0.0 g/m2h, 4.1 ± 0.4 g/m2h, respectively), and day 32 (8.6 ± 1.0 g/m2h and 16.6 ± 3.1 g/m2h vs. 0.0 ± 0.0 g/m2h and 2.7 ± 0.2 g/m2h, respectively) compared to the control group (all P < 0.001; Fig. 2B).
Fig. 2

Skin fibrosis induced by Af in Rag1−/− mice. (A) Schematic protocol of this study. For 5 weeks, Af was epicutaneosly applied to the dorsal skin of mice for 5 consecutive days per week. (B) Clinical symptom scores and TEWL in Af-induced atopic dermatitis murine model. (C) H&E (top), TB (middle), and MT (bottom) staining were performed on skin tissue sections (×400 magnification, scale bar = 200 μm). (D) Collagen levels in the lesional skin as measured by collagen assay. (E) Flow cytometry was used to measure the proportion of type 2 ILCs. ILCs were defined as lineage negative (Lin−) CD127+ cells, and type 2 ILCs were defined as CD25+IL-33R+ cells.

Af = Aspergillus fumigatus, TEWL = transepidermal water loss, Ig = immunoglobulin, H&E = hematoxylin and eosin, TB = toluidine blue, MT = Masson’s trichrome, ILC = innate lymphoid cell, IL = interleukin.
***P < 0.001.
jkms-39-e221-g002
Histopathological examination of the lesional skin of the Af-treated Rag1−/− mice showed increased epidermal (Af-treated group, 28.5 ± 2.1 μm vs. control group, 16.7 ± 1.3 μm, P = 0.001) and dermal thickness (189.4 ± 11.3 μm vs. 100.3 ± 5.8 μm, P = 0.048), and increased mast cell (7.7 ± 1.2/mm2 vs. 2.6 ± 0.6/mm2, P = 0.004), eosinophil (6.8 ± 1.0/mm2 vs. 1.9 ± 0.3/mm2, P = 0.002), neutrophil (9.6 ± 0.9/mm2 vs. 2.9 ± 0.6/mm2, P < 0.001), and lymphocyte (5.8 ± 0.6/mm2 vs. 2.4 ± 0.3/mm2, P < 0.001) infiltration, and increased collagen stain in H&E, TB and MT staining compared to the control group (Fig. 2C). In addition, the collagen was measured to determine the degree of fibrosis in the lesional skin of the Af-treated Rag1−/− mice model (Fig. 2D). The AD group (3.9 ± 1.2 µg/mg) showed a higher collagen level tendency than the control group (1.6 ± 0.0 µg/mg) (P = 0.174).
The populations of Lin−CD127+CD25+IL-33R+ cells were higher in the skin of the Af-treated Rag1−/− mice (0.1 ± 0.04%) compared to the control Rag1−/− mice (0.03 ± 0.01%), when CD45+ was measured as the denominator; however, the difference was not significant (P = 0.240; Fig. 2E).

Elimination of type 2 ILCs and skin fibrosis in Af-treated Rag1−/− mice

For further investigating the role of type 2 ILCs in skin fibrosis, an anti-CD90.2 mAb was used to deplete type 2 ILC2 in Rag1−/− mice.20 Anti-CD90.2 and isotype of rat IgG2b were intraperitoneally injected in the control group from 2 days before Af application to day 30 at an interval of 2 days (Fig. 3A). Both clinical symptom score and TEWL at day 18 (2.6 ± 0.5 g/m2h and 8.4 ± 1.1 g/m2h, respectively), day 25 (2.5 ± 0.3 g/m2h and 6.6 ± 0.8 g/m2h, respectively), and day 32 (2.4 ± 0.5 g/m2h and 7.7 ± 0.6 g/m2h, respectively) were significantly decreased in anti-CD90.2-treated Rag1−/− mice compared to those at day 18 (6.4 ± 0.5 g/m2h and 18.1 ± 2.0 g/m2h, respectively), day 25 (5.3 ± 0.9 g/m2h and 16.0 ± 2.0 g/m2h, respectively), and day 32 (6.5 ± 0.3 g/m2h and 18.9 ± 2.1 g/m2h, respectively) in isotype-treated control mice (all P < 0.001; Fig. 3B). Both epidermal (22.0 ± 4.7 μm vs. 29.1 ± 1.8 μm) and dermal (130.9 ± 10.1 μm vs. 202.0 ± 30.4 μm) thickness in anti-CD90.2-treated Rag1−/− mice were not significantly different from those in isotype-treated control mice (P = 0.188 and P = 0.057, respectively; Fig. 3C). The depletion of type 2 ILCs in the anti-CD90.2-treated Rag1−/− mice was accompanied by significantly reduced immune cells including mast cell (control group, 9.4 ± 1.0/mm2 vs. anti-CD90.2-treated group, 3.5 ± 0.6/mm2, P < 0.001), eosinophil (8.3 ± 0.8/mm2 vs. 2.1 ± 0.6/mm2, P < 0.001), neutrophil (12.6 ± 1.3/mm2, 5.0 ± 0.9/mm2, P = 0.001), and lymphocyte (7.8 ± 0.9 mm2 vs. 2.4 ± 0.6/mm2, P < 0.001). Collagen levels were significantly decreased in the anti-CD90.2-treated Rag1−/− mice (1.6 ± 0.0 μg/mg) than in the isotype-treated group (4.5 ± 0.3 μg/mg) (P < 0.001; Fig. 3D).
Fig. 3

Depletion of type 2 ILCsimproved skin fibrosis in Rag1−/− mice. (A) Schematic protocol of this study. For 5 weeks, Af was epicutaneosly applied to the dorsal skin of mice for 5 consecutive days per week. (B) Clinical symptom scores and TEWL in Af-induced atopic dermatitis murine model. (C) H&E (top), TB (middle), and MT (bottom) staining were performed on skin tissue sections (×400 magnification, scale bar = 200 μm). (D) Collagen levels in the lesional skin as measured by collagen assay. (E) Flow cytometry was used to measure the proportion of ILCs. ILCs were defined as lineage negative (Lin−) CD127+ cells, and type 2 ILCs were defined as CD25+IL-33R+ cells.

ILC = innate lymphoid cell, Af = Aspergillus fumigatus, TEWL = transepidermal water loss, Ig = immunoglobulin, H&E = hematoxylin and eosin, TB = toluidine blue, MT = Masson’s trichrome, IL = interleukin.
***P < 0.001.
jkms-39-e221-g003
The populations of Lin−CD127+CD25+IL-33R+ cells were lower in the skin of anti-CD90.2-treated Rag1−/− mice (0.008 ± 0.01%) compared to isotype-treated Rag1−/− mice (0.03 ± 0.01%), when CD45+ was measured as the denominator; however, the difference was not significant (P = 0.29; Fig. 3E).

DISCUSSION

This study investigated the role of type 2 ILCs in skin fibrosis using a murine model of AD-like skin inflammation. Epicutaneous exposure to Af extract for 5 weeks induced skin fibrosis, as demonstrated by increased collagen levels and AD-like skin inflammation. We found a trend of increased type 2 ILCs in the lesional skin of Af-treated mice, although the difference was not statistically significant. Similar to the results in wild-type mice, collagen levels significantly increased with an increase in type 2 ILCs in lymphocyte-depleted Rag1−/− mice treated with Af compared to control Rag1−/− mice. Depletion of type 2 ILCs using anti-CD90.2 significantly decreased both staining and collagen levels.
According to previous mouse model studies, type 2 ILCs play a role in the development of AD even in the absence of adaptive immunity.212223 As expected from conventional immunobiology of type 2 inflammation, type 2 ILC-derived type 2 cytokines, including IL-4, IL-5, and IL-13, are responsible for skin inflammation and barrier dysfunction in mice and human studies.242526 TSLP, IL-33, and IL-25 have the capacity to activate type 2 ILCs, and these cytokines can be amplified when exposed to allergens such as house dust mite extracts,2627 viruses such as respiratory syncytial virus, rhinovirus, and influenza,282930 and fungus.31
However, the mechanisms underlying AD fibrosis are not fully understood. Similar to other diseases, it is known to be associated with Th2 and Th17 immune responses.3233 It was demonstrated that IL-13 was associated with the development of skin fibrosis in AD.34 Under the influence of Th2 immune response, the secretion of transforming growth factor (TGF)-β leads to fibroblasts differentiating into myofibroblasts, and secreting a significant amount of collagen.3536 Using Rag1−/− mice with depleted lymphocytes, such as Th2 and Th17 cells, this study elucidated that type 2 ILCs in fibrosis is independent of adaptive immunity, highlighting a potential role of type 2 ILCs in the development of skin fibrosis. Studies investigating the association between type 2 ILCs and skin fibrosis in AD are scarce. Type 2 ILCs are implicated in the induction of fibrosis in other organs. Type 2 ILCs are involved in fibrosis in a murine model of pulmonary fibrosis and have been found in bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis.1837 Other studies have explained the role of type 2 ILCs in organ fibrosis in mucosal immune response.38 The elucidated mechanism involves the production of Th2 cytokines by type 2 ILCs in response to IL-25, IL-33, and TSLP. Given that Th2 cytokines such as IL-13 and IL-5 also mediate tissue fibrosis, type 2 ILCs can play a role in modulating remodeling and fibrosis, particularly in mucosal tissues. Additionally, Th2 cytokines recruit and stimulate eosinophils to release the profibrotic cytokines such as TGF-β, platelet-derived growth factor, and IL-13. This promotes the differentiation of fibroblasts to myofibroblasts,39 which could potentially induce intestinal40 and hepatic fibrosis.41 However, whether fibrosis is a consequence of chronic inflammation caused by type 2 ILCs or whether type 2 ILCs are directly involved in the development of fibrosis in our study remains unclear. Therefore, more in-depth mechanistic research is required to determine the interrelationship between skin fibrosis caused by type 2 ILCs.
We used Af-induced AD model with histological findings and immune responses similar to those in humans. Epicutaneous application of Af for 5 weeks induced skin fibrosis concomitant with chronic skin inflammation, including both neutrophil and eosinophil infiltration, involving both type 2 and type 1 immune responses. Therefore, this model may be well suited for investigating fibrosis in human AD, which manifests as a combination of both immune response types.
In conclusion, our study implicates the role of type 2 ILCs in the development of skin fibrosis in a murine model of AD-like skin inflammation. Further investigations are warranted to elucidate the underlying mechanisms and to evaluate the therapeutic potential of targeting type 2 ILCs in the management of AD-associated fibrosis.

Notes

Funding: This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A01060084) and research grant from Chung-Ang University Research Grants in 2021.

Disclosure: The authors have no potential conflicts of interest to disclose.

Author Contributions:

  • Conceptualization: Yoon J1, Moon GJ, Yu J.

  • Data curation: Lee J.

  • Formal analysis: Lee J, Yoon J.2

  • Investigation: Yoon J,1 Lee J, Park A, Yoon J.2

  • Methodology: Lee J, Park A, Yoon J.2

  • Software: Lee J.

  • Supervision: Yoon J,2 Yu J.

  • Validation: Park A, Kim JR, Moon GJ.

  • Visualization: Kim JR, Moon GJ.

  • Writing - original draft: Yoon J,1 Lee J.

  • Writing - review & editing: Yoon J,1 Moon GJ, Yu J.

Yoon J,1 Jisun Yoon; Yoon J,2 Jin Yoon.

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