Monitoring Cellular Movement with Photoconvertible Fluorescent Protein and Single-Cell RNA Sequencing Reveals Cutaneous Group 2 Innate Lymphoid Cell Subtypes, Circulating ILC2 and Skin-Resident ILC2

Abstract

We previously generated a transgenic mouse line expressing skin-specific IL-33 (IL33tg mice) and showed that IL-33 elicits group 2 innate lymphoid cell (ILC2)-dependent atopic dermatitis-like skin inflammation. ILC2s are believed to be tissue-resident cells under steady-state conditions, but the dynamics of ILC2 migration are not fully understood. We sorted ILC2s from the skin and draining lymph nodes of IL33tg mice and analyzed their transcriptomes using the single-cell RNA sequencing technique, which revealed that the skin ILC2s had split into two clusters: circulating ILC2 and skin-resident ILC2. The circulating ILC2s expressed H2-related major histocompatibility complex class II genes. Conversely, the skin-resident ILC2s demonstrated increased mRNA expression of the ICOS, IL-5, and IL-13. Next, we tracked ILC2 migration using IL33tg-Kikume Green-Red mice. Exposing the IL33tg-Kikume Green-Red mice's inflamed skin to violet light allowed us to label the circulating ILC2s in their skin and track the ILC2 migration from the skin to the draining lymph nodes. Cutaneous local innate responses could transition to systemic type 2 responses by migrating the activated ILC2s from the skin into the draining lymph node. Conversely, the skin-resident ILC2s produced a large number of cytokines. Thus, the skin ILC2s turned out to be a heterogeneous cell population.

Keywords: AD, atopic dermatitis; DC, dendritic cell; ILC2, group 2 innate lymphoid cell; KikGR, Kikume Green-Red; MHC, major histocompatibility complex; RNA-seq, RNA sequencing; dLN, draining lymph node.

Figure 1

Single-cell RNA-seq analysis of sorted ILC2s from the skin and the dLNs of transgenic mice expressing IL-33 (IL33tg). (a) Single-cell RNA-seq identifies two clusters. t-SNE plot of the single-cell RNA-seq data of 1,052 ILC2s combined from three mice. Hierarchical clustering based on gene expression profiles was performed, and the ILC2s split into two clusters: cluster 1 (circulating ILC2s) and cluster 2 (skin-resident ILC2s). (b) Heatmaps of the representative ILC2-related genes and chemokine receptor‒related genes from each cluster. (c) Single-gene expression t-SNE plots of the single-cell RNA-seq data. Note that Icos, Il1rl1, Klrg1, and Il7r are markers of ILC2s. Representative data are from two independent experiments. dLN, draining lymph node; Il1rl1, IL-33 receptor alpha chain; ILC2, group 2 innate lymphoid cells; RNA-seq, RNA sequencing; t-SNE, t-distributed stochastic neighbor embedding.

Figure 2

Features of transgenic mice expressing IL-33 (IL33tg)– KikGR photoconvertible fluorescent protein. (a) Cutaneous manifestations of the IL33tg–KikGR mice. (b) The concept of photoconversion. The KikGR photoconvertible fluorescent protein changes irreversibly to red (KikGR-Red) on exposure to violet light. (c) Conceptual figure of the experimental system. KikGR-Red cells were observed in the dLN after photoconversion, and these KikGR-Red cells can be distinguished from the KikGR-Green cells. (d) The IL33tg–KikGR mice were killed immediately after skin photoconversion, and the cells from their skin and dLNs were isolated for analysis by flow cytometry. We used nonphotoconverted mice as the control. At this point, the cells in violet light‒exposed skin were KikGR-Red, and the cells in the dLNs remained KikGR-Green. dLN, draining lymph node; KikGR, Kikume Green-Red.

Figure 3

ILC2s migrate from the skin to dLNs. (a) Flow cytometry plots displaying KikGR-Green⁺ and KikGR-Red⁺ ILC2s in the dLNs 72 hours after skin photoconversion from the transgenic mice expressing IL-33 (IL33tg) or the IL33tg–KikGR mice. Cervical dLN cells were gated on ILC2s (see Figure 7a for the gating strategy). The top right number (8.6%) indicates the proportion of ILC2s that migrated from the skin, which were KikGR-Red⁺. The data are representative of three mice and three independent experiments. (b) Cells from the dLNs were stained to analyze the skin-derived ILC2s by flow cytometry 0–72 h (right panel) or 72 h (left panel) after skin photoconversion. The data represent the proportion of skin-derived ILC2s in the dLNs (labeled KikGR-Red) from the IL33tg–KikGR mice (n = 3–7). (c) Flow cytometry plots displaying the KikGR-Green⁺ and KikGR-Red⁺ skin-derived DCs in the dLNs 72 h after skin photoconversion from IL33tg–KikGR mice. Cervical dLN cells were gated on skin-derived DCs (see Figure 7b for the gating strategy). (d) Cells from the dLNs were stained to analyze the skin-derived DCs by flow cytometry 72 h after skin photoconversion. The data represent the proportion of skin-derived DCs in the dLNs labeled KikGR-Red from the IL33tg–KikGR mice (n = 3). Unpaired t-test was used to assess the statistical significance. ∗∗∗P < 0.001, ∗P < 0.05. Each dot represents a value for each mouse. The bold lines represent the estimated mean values, and the thin lines indicate the SEM values. DC, dendritic cell; dLN, draining lymph node; h, hour; ILC2, group 2 innate lymphoid cell; KikGR, Kikume Green-Red.

Figure 4

Differential expression of genes of sorted lymph node-derived ILC2s KikGR-Green⁺ and skin-derived ILC2s KikGR-Red⁺ in lymph nodes 72 hours after skin photoconversion from transgenic mice expressing IL-33 (IL33tg)–KikGR. (a) t-SNE plot of the single-cell RNA sequencing data of 1,068 KikGR-Green⁺ and KikGR-Red⁺ ILC2s. (b) Correlation plot for the log10 mean gene expression levels in the KikGR-Green⁺ ILC2s versus that in KikGR-Red⁺ ILC2s. R is the correlation coefficient obtained by Pearson’s product-moment correlation test (R² = 0.980). Note that the KikGR-Green⁺ ILC2s and KikGR-Red⁺ cells shared the same gene signature. (c) Heat map displaying the level of expression of each chemokine receptor‒related gene in each cell. Cells are ordered by hierarchical clustering. Representative data are from two independent experiments. ID, identification; ILC2, group 2 innate lymphoid cell; KikGR, Kikume Green-Red; t-SNE, t-distributed stochastic neighbor embedding.

Figure 5

Features of circulating ILC2s and skin-resident ILC2s. (a) Flow cytometry of ILC2s from the lymph node (thin line) and skin (bold line) of IL33tg mice. Cells were gated on lineage marker (Lin)⁻ Sca-1⁺ IL-33R (ST2)⁺ cells for ILC2s. (b) Flow cytometry of cells from the skin of IL33tg mice. The cells were gated on an ILC2 fraction. Note that the ICOS⁺ KLRG1⁺ IA/IE⁺ cells are circulating ILC2s, and the ICOS⁺ KLRG1^‒ IA/IE^‒ cells are skin-resident ILC2s. (c) Immunofluorescence of IA/IE and ICOS in Rag2 knockout IL33tg mouse skin. Note that the ICOS⁺ IA/IE⁺ cells (red/green double‒positive cells, arrowheads) are circulating ILC2s, and the ICOS⁺ IA/IE^‒ cells (green single‒positive cells) are skin-resident ILC2s. Representative data are from three independent experiments. Bar = 100 μm (inset, 10 μm). ILC2, group 2 innate lymphoid cell; Max, maximum.

Figure 6

Skin-resident ILC2s (IA/IE^‒) produce large numbers of cytokines. Type 2 cytokine production by the sorted ILC2s (IA/IE⁺ or IA/IE^‒ population; 2 × 10⁴ cells per well) from the skin of transgenic mice expressing IL-33 (IL33tg mice) were cultured 24 hours after stimulation with phorbol myristate acetate/ionomycin. The type 2 cytokines (IL-4, IL-5, IL-13) in each supernatant were measured using ELISA (n = 4). Paired t-test was used to assess the statistical significance. ∗∗P < 0.01; ∗P < 0.05. ILC2, group 2 innate lymphoid cell.

Figure 7

Gating strategy for flow cytometry. (a) Gating strategy for the analysis of FSC^lowSSC^lowCD45⁺ lineage (Lin)⁻Thy1.2⁺Sca-1⁺ IL-33R (ST2)⁺ ILC2s. The numbers indicate the percentage of cells in each gate. (b) Gating strategy for the analysis of CD45⁺ B220⁻ DCs within the draining LNs 72 hours after the photoconversion of the skin. The LN-resident DCs are CD11c^high IA/IE^med, and the skin-derived DCs are CD11c^med IA/IE^high. Note that only skin-derived DCs contain KikGR-Red⁺ cells. The numbers indicate the percentage of cells in each quadrant. Similar results were obtained from two independent experiments. DC, dendritic cell; ILC2, group 2 innate lymphoid cell; KikGR, Kikume Green-Red; LN, lymph node.