The Interplay of Type 1, Type 2, and Type 3 Lymphocytes and Cytokines in Atopic Dermatitis

Abstract

Atopic dermatitis (AD) is classified as a type 2 disease owing to the majority of type 2 lymphocytes that constitute the skin-infiltrating leukocytes. However, all of the type 1-3 lymphocytes intermingle in inflamed skin lesions. Here, using an AD mouse model where caspase-1 was specifically amplified under keratin-14 induction, we analyzed the sequential changes in type 1-3 inflammatory cytokines in lymphocytes purified from the cervical lymph nodes. Cells were cultured and stained for CD4, CD8, and γδTCR, followed by intracellular cytokines. Cytokine production in innate lymphocyte cells (ILCs) and the protein expression of type 2 cytokine IL-17E (IL-25) were investigated. We observed that, as inflammation progresses, the cytokine-producing T cells increased and abundant IL-13 but low levels of IL-4 are produced in CD4-positive T cells and ILCs. TNF-α and IFN-γ levels increased continuously. The total number of T cells and ILCs peaked at 4 months and decreased in the chronic phase. In addition, IL-25 may be simultaneously produced by IL-17F-producing cells. IL-25-producing cells increased in a time-dependent manner during the chronic phase and may work specifically for the prolongation of type 2 inflammation. Altogether, these findings suggest that inhibition of IL-25 may be a potential target in the treatment of inflammation.

Keywords: atopic dermatitis; cytokine; inflammatory skin mouse model; interleukin-17E; interleukin-25; type 2 inflammation.

Figure 1

Cytokine production by T helper cells. (A) The plots represent the production of specified cytokines by the three types of T cells namely, CD4 T, CD8 T, and γδ-T cells, in 4-month-old KCASP1Tg mice. CD4T cells produce IL-13 and a small amount of IL-4; TNFα-producing cells are predominant for type 1 cytokines, especially from CD8T cells and CD4T cells; CD4T cells are divided into IL-17A alone, IL-17F alone, and IL-17A/F-producing cells. On the other hand, γδ T cells do not produce IL-17F alone. (B,C) The number of indicated cytokine-producing T cells was measured at 2, 4, and 6 months of age (M). All cytokine-producing cells were high in number at the peak of the dermatitis at 4 months of age, with a decreasing trend in cell number at 6 months of age when the skin rash became chronic. (C) Pie chart of the data shown in (B). The size of the graph in (C) reflects the total number of T helper cells. Asterisks indicate a significance difference based on a one-way ANOVA and Tukey’s multiple comparison test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 2

Cytokine production by ILCs. (A) The plots represent type 1, 2, and 3 cytokine production by ILCs in 4-month-old KCASP1Tg mice as observed by flow cytometry. (B,C) The number of indicated cytokine-producing ILCs was measured at 2, 4, and 6 months of age (M). (C) Pie chart of the data shown in (B). The total number of ILCs peaked at 4 months and decreased at 6 months. The size of the graph in (C) is representative of the number of ILCs. Asterisks indicate significance difference based on a one-way ANOVA and Tukey’s multiple comparison test (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001).

Figure 3

Expression of nuclear transcription factors. The graphs represent the relative expression of the indicated transcription factors with respect to the housing-keeping gene-GAPDH of mononuclear cells before and 4 h post-culture. In KCASP1Tg, with prolonged inflammation, T-bet, GATA3, RoRa, and RoRc transcription factors were enhanced in the unstimulated condition, probably owing to the constitutive activation, which was suppressed upon simulation.

Figure 4

IL-25 production by mononuclear cells. (A) Focusing on the lymphocyte fraction of cultured cells, we detected CD4T cells, CD8T cells, γδ-T cells, and cells producing IL-25 from ILCs. The plots represent the production of IL-25 by indicated cell types as observed by flow cytometry. (B) The number of indicated cytokine-producing cells was measured at 2, 4, and 6 months of age (M). IL-25-producing cells increased in a time-dependent manner even at 6 months of age. Asterisks indicate significance difference based on a one-way ANOVA and Tukey’s multiple comparison test (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 5

IL-25 production by type 2 and type 3 cells. The plots represent the co-production of indicated type 2 and type 3 cytokines as observed by flow cytometry. Intracellular staining results for type 2 and type 3 show some overlap between IL-4- and IL-13-producing cells. However, IL-25-producing cells also proved to be distinct from IL-4- and IL-13-producing cells. As for type 3 cytokines, IL-25 might be simultaneously produced by IL-17F-producing cells and not by IL-17A-producing cells.

Figure 6

The immunological balance between four factors. Based on our analyses, this cartoon represents the balance between type 1, type 2, and type 3 immunity with respect to the regulatory cells in AD. The size of the rounds represents the abundance in the indicated cell types.