Temporal dynamics of cytokine production balance between innate and adaptive lymphocytes in atopic dermatitis model mice

Abstract

Atopic dermatitis (AD) involves a complex interplay between innate and adaptive immune responses. In the initial phase of disease onset, innate immunity predominates. Activated group 2 innate lymphoid cells (ILC2s) release type 2 cytokines, including IL-13, IL-5, and IL-4, which further disrupt the skin barrier, facilitating the entry of various antigens into the epidermis and thereby promoting the induction of antigen-specific effector T helper (Th) 2 cells. This process represents a transition from innate to adaptive immunity. Previous studies have examined the changes in mRNA expression of various cytokines during the acute and chronic phases of human AD and investigated the presence of ILCs, cytokines, and cytokine receptors in human AD (Supplementary Information 1). Although previous murine studies have compared the numerical balance of cytokine-producing ILCs and Th cells 1 , no reports have tracked their temporal dynamics during the progression of dermatitis.Here, using three distinct spontaneous AD model mouse strains, we longitudinally profiled cytokine production by ILCs and Th cells in skin-infiltrating lymphocytes, skin-draining cervical lymph nodes, and spleens at disease onset, the acute phase, and the chronic phase. As AD models, keratin 14 promoter-driven IL-33-overexpressing transgenic (IL-33Tg): IL-33-induced acute-phase dermatitis 2 , keratin 14 promoterdriven IL-18-overexpressing transgenic (IL-18Tg): models of chronic-phase AD 3 , keratin 14 promoter-driven caspase-1-overexpression transgenic (KCASP1Tg) mice, which are regarded as combined features of both AD and psoriasis histologically and immune profiles 4 , were used. 2-, 4-, and 6-month-old female transgenic for IL-33Tg and KCASP1Tg mice were used. For IL-18Tg, 4-, 6-, and 8-month-old mice were used, as dermatitis typically develops late in life. The Mie University Board Committee approved the experimental protocol for Animal Care and Use (#22-39-7).Purified mononuclear cells were cultured in the presence of Phorbol Myristate Acetate (25 ng/mL), ionomycin (1µg/mL), and brefeldin A (1 g/mL) for 4 hours, and then LIVE/DEAD TM Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, antibodies. The expression patterns of inflammatory cytokines were measured using a BD Lyric flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (v10.10.0, Tree Star Inc., Ashland, OR, USA). As a limitation, the number of cells obtained was small, and the data is aggregated from five animals. Therefore, statistical analysis could not be performed, and this is a descriptive analysis.Analysis of skin-infiltrating lymphocytes revealed, consistent with previous data 5 , that ILCs produced a high proportion of type 2 cytokines (IL-13 and IL-5), and this profile remained consistent across onset, acute, and chronic phases in all three AD models. IL-17A was produced comparatively, and its proportion relative to other cytokines did not change over time. The production of IL-17F was substantially very low. IFN-γ production in the acute phase was detectable, whereas it showed an increasing tendency during disease evolution (Figure 1A-C).By contrast, in skin-infiltrating Th cells, type 2, type 1, and type 3 cytokines were produced at roughly similar frequencies, and no time-dependent changes were observed across all strains. IFN-γ-producing Th cells increased in the chronic phase in IL-33Tg.Overall, the ratio of cytokine production by Th cells was lower than that of ILCs. These findings indicate that ILCs and Th cells in the skin may be subject to distinct regulatory influences, with ILCs specialized for robust cytokine secretion (Figure 1A-C).In skin-draining lymph nodes, ILCs produced type 2 cytokines alongside substantial IFN-γ and detectable IL-17A, whereas IL-17F remained at low levels.Lymph node Th cells exhibited IFN-γ production but generally displayed a poor cytokine-producing capacity, with no specific shifts in cytokine balance over time (Figure 1D-F).Finally, in the spleen, both ILCs and Th cells included a high fraction of IFN-γproducing cells. Splenic ILCs from KCASP1Tg mice produced IL-5.ILCs and Th cells showed uniformly low cytokine production (Figure 1G-I). Considering the absolute number of cytokine-producing cells, Th cells are more numerous compared to those of ILCs in all mice (Supplemental Figure 1). Due to the low number of infiltrating cells in normal skin, control analyses were not feasible. Moreover, murine immune mechanisms may not fully recapitulate those in humans, warranting the use of confirmatory studies with human samples.In summary, lymphocytes infiltrating into the skin in AD models tend to have a higher overall rate of cytokine production, while those in the lymph nodes and spleen tend to have a lower rate. Skin-infiltrating ILCs consistently exhibit a dominant type 2 cytokine production profile. In all investigated samples, both ILCs and Th cells show a trend toward increased IFN-γ production over time. The late rise of IFN-γ likely represents a layer superimposed on a persistent type 2 milieu rather than a polarity switch. This can reinforce chronicity by licensing antigen presentation and CXCR3ligand trafficking, while preserving type 2 dominance of ILCs. The pattern aligns with the clinical spectrum of extrinsic and intrinsic AD, suggesting consideration of IFN-γresponsive pathways (e.g., JAK-STAT). The overall balance among type 1, type 2, and type 3 cytokines remains remarkably unchanged during disease onset, acute, and chronic phases. The cytokines were abbreviated as follows: IL-5; 5, IL-13; 13, IL-17A; 17A, IL-17F; 17F, IFN-γ; IFN.