J Korean Med Sci.  2024 Aug;39(30):e221. 10.3346/jkms.2024.39.e221.

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

• Yoon J
• Lee J
• Park A
• Yoon J
• Kim JR
• Moon GJ
• Yu J

• KMID: 2558519
• DOI: http://doi.org/10.3346/jkms.2024.39.e221

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.

Keyword

Type 2 Innate Lymphoid Cell; Skin Fibrosis; Atopic Dermatitis; Mouse Model

Figure

• 

  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.

• 

  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.

• 

  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.

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