Background: Interleukin-33 (IL-33) is involved in type 2 innate immunity by inducing type 2 cytokines, such as IL-5 and IL-13, through the activation of group 2 innate lymphoid cells (ILC2s) or T helper 2 (Th2) cells. We previously reported that mice overexpressing IL-33 (IL-33Tg) in the cornea and conjunctiva spontaneously develop atopic keratoconjunctivitis-like inflammation. Despite previous studies, it is not fully understood what types of immune cells contribute to the disease process of IL-33-induced keratoconjunctivitis. Methods: To defect Th2 cells, IL-33Tg mice were crossed with Rag2KO mice. To defect ILC2s, IL-33Tg mice received bone marrow transplantations from B6.C3(Cg)-Rorasg/J mice that lacked ILC2. Immunostaining techniques were used to determine where ILC2 is distributed in the cornea and conjunctiva. We analyzed the transcriptomes of ILC2 from the conjunctiva by using single-cell RNA-seq analysis. To investigate whether tacrolimus reduces type 2 cytokine production by ILC2, ILC2 was cultured with tacrolimus, and the percentage of cytokine-producing ILC2 was examined. To investigate whether tacrolimus can inhibit IL-33-induced keratoconjunctivitis in vivo, IL-33Tg mice were treated with tacrolimus eye drops. Results: ILC2 infiltrated the conjunctival epithelium and subepithelial tissue. Keratoconjunctivitis developed spontaneously in Rag2KO/IL-33Tg mice, but keratoconjunctivitis was abolished in IL-33Tg mice lacking ILC2. ILC2 was not a uniform cluster but a heterogeneous cluster. Tacrolimus inhibited cytokine production from ILC2s in vitro, and tacrolimus eye drops inhibited keratoconjunctivitis in IL-33Tg mice in vivo. Conclusions: ILC2 plays a pivotal role in IL-33-induced keratoconjunctivitis in mice. Keywords: Atopic dermatitis; Atopic keratoconjunctivitis; Group 2 innate lymphoid cells; Interleukin-33; Tacrolimus.

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