Interleukin-2-induced skin inflammation

Abstract

Recombinant human IL-2 has been used to treat inflammatory diseases and cancer; however, side effects like skin rashes limit the use of this therapeutic. To identify key molecules and cells inducing this side effect, we characterized IL-2-induced cutaneous immune reactions and investigated the relevance of CD25 (IL-2 receptor α) in the process. We injected IL-2 intradermally into WT mice and observed increases in immune cell subsets in the skin with preferential increases in frequencies of IL-4- and IL-13-producing group 2 innate lymphoid cells and IL-17-producing dermal γδ T cells. This overall led to a shift toward type 2/type 17 immune responses. In addition, using a novel topical genetic deletion approach, we reduced CD25 on skin, specifically on all cutaneous cells, and found that IL-2-dependent effects were reduced, hinting that CD25 - at least partly - induces this skin inflammation. Reduction of CD25 specifically on skin Tregs further augmented IL-2-induced immune cell infiltration, hinting that CD25 on skin Tregs is crucial to restrain IL-2-induced inflammation. Overall, our data support that innate lymphoid immune cells are key cells inducing side effects during IL-2 therapy and underline the significance of CD25 in this process.

Keywords: Drug‐induced skin inflammation; IL‐2 therapy; Immunotherapy; Innate lymphoid cells; γδ T cells.

Figure 1:. Intradermal IL-2 injections induce skin inflammation characterized by considerable immune cell infiltrates.

A Wild type mice were intradermally injected with IL-2 (aldesleukin) daily for 7 consecutive days before analysis of skin. Created with BioRender.com. Representative histological images of injections sides from control (B) and IL-2-treated skin (C). Overview pictures (upper images; scale bars 200 μm) and close-ups (middle and lower images; scale bares 40 μm) are shown.

Figure 2:. IL-2-induced skin inflammation is characterized by increases in frequencies of innate immune cells.

A Wild type mice were intradermally injected with IL-2 (aldesleukin) daily for 7 consecutive days before analysis of skin and skin-draining lymph nodes (SDLN). Created with BioRender.com. B Absolute CD45+ immune cell numbers in skin and SDLN. C Absolute numbers of T cell subsets (Teffs: CD4+ T effector cells; dGDs: dermal γδ T cells; DETCs: dendritic epidermal T cells), innate lymphoid cells (ILCs), and CD3-NK1.1+ group 1 ILCs (ILC1s). D Absolute numbers of neutrophils and eosinophils in skin. E Frequencies of innate immune cells upon IL-2 injection. F CD25 expression as shown by CD25+ frequencies of the respective cells. cDC: conventional dendritic cell. n=5 mice from two independent experiments; * p<0.05, ** p<0.01, *** p<0.001 as determined by paired t-tests. All other conditions n.s.

Figure 3:. Reduction of CD25 expression on skin cells reduces IL-2-induced increases of skin immune cell numbers.

A Cre recombinase of Rosa26^CreERT2xCD25^fl/fl mice was induced by topical 4-Hydroxytamoxifen (4-OHT) application prior to IL-2 injections on both sides to decrease CD25 on all skin cells. Acetone on the contralateral side of the back of the same mouse served as vehicle control. Created with BioRender.com. B CD25 expression as shown by CD25+ cell frequencies of the respective cell subsets. Teffs: CD4+ T effector cells; dGDs: dermal γδ T cells; DETCs: dendritic epidermal T cells; ILCs: innate lymphoid cells; ILC1s: CD3-NK1.1+ group 1 ILCs; cDC1: type 1 conventional dendritic cell. C Mean fluorescence intensity (MFI) of CD25 on respective cells. D Immune cell numbers on control and 4-OHT-treated sides in skin and skin-draining lymph nodes (SDLN). E Absolute cell numbers of lymphocyte immune cell subsets in skin tissue. F Absolute numbers of neutrophils and eosinophils in skin. G Expression of T-bet at GATA-3 transcription factors in Teffs and ILCs. n=4–8 mice from three independent experiments; * p<0.05, ** p<0.01, **** p<0.0001 as determined by paired t-tests. All other conditions n.s.

Figure 4:. Reduction of CD25 on Tregs leads to significant increases in IL-2-induced skin inflammation.

A Cre recombinase of FoxP3^CreERT2xCD25^fl/fl mice was induced by topical 4-Hydroxytamoxifen (4-OHT) application to delete CD25 on skin Tregs prior to IL-2 injections on both sides. Acetone on the contralateral side of the back of the same mouse served as vehicle control. Created with BioRender.com. B CD25 expression as shown by CD25+ frequencies of the respective cells. Teffs: CD4+ T effector cells; dGDs: dermal γδ T cells; DETCs: dendritic epidermal T cells; ILCs: innate lymphoid cells; ILC1s: CD3-NK1.1+ group 1 ILCs; cDC1: type 1 conventional DC. C CD25 mean fluorescence intensity (MFI) of CD25+ Tregs (left) and representative CD25 gating on Tregs (right). D Immune cell numbers on control and 4-OHT-treated sides in skin and skin-draining lymph nodes (SDLN). E Absolute cell numbers of immune cell subsets in skin tissue. F Absolute numbers of neutrophils and eosinophils in skin. G MFI of FoxP3 in Tregs in skin. H Frequencies of skin Teffs and Tregs of CD4+ cells. I Expression of T-bet at GATA-3 transcription factors in Teffs and ILCs. n=7 mice; * p<0.05, ** p<0.01, *** p<0.001 as determined by paired t-tests. All other conditions n.s.

Figure 5:. IL-2 injections induce a mixed type2/type 17 immune reaction in skin, associated with dermal γδ T cells and ILCs.

A Experimental set-up for injection of wild type mice with IL-2. Created with BioRender.com. B Expression of cytokines in CD45+ cells upon IL-2 injection. C Relative cytokine expression in immune cells in IL-2-treated vs. Ctrl skin. Frequencies of cytokine expression in CD45+ cells (type 1: IFN-γ + TNF-α; type 2: IL-4 + IL-13; type 17: IL-17) upon IL-2 injection was normalized to control conditions to yield fold changes. D Cytokine expression in different lymphocyte populations. Teffs: CD4+ T effector cells; dGDs: dermal γδ T cells; ILCs: innate lymphoid cells; ILC1s: CD3-NK1.1+ group 1 ILCs. n=4–5 mice representative for two independent experiments; * p<0.05, ** p<0.01 as determined by paired t-tests. All other conditions n.s.