TIM-4 is differentially expressed in the distinct subsets of dendritic cells in skin and skin-draining lymph nodes and controls skin Langerhans cell homeostasis

Abstract

T cell immunoglobulin and mucin-4 (TIM-4), mainly expressed on dendritic cells (DC) and macrophages, plays an essential role in regulating immune responses. Langerhans cells (LC), which are the sole DC subpopulation residing at the epidermis, are potent mediators of immune surveillance and tolerance. However, the significance of TIM-4 on epidermal LCs, along with other cutaneous DCs, remains totally unexplored. For the first time, we discovered that epidermal LCs expressed TIM-4 and displayed an increased level of TIM-4 expression upon migration. We also found that dermal CD207+ DCs and lymph node (LN) resident CD207-CD4+ DCs highly expressed TIM-4, while dermal CD207- DCs and LN CD207-CD4- DCs had limited TIM-4 expressions. Using TIM-4-deficient mice, we further demonstrated that loss of TIM-4 significantly upregulated the frequencies of epidermal LCs and LN resident CD207-CD4+ DCs. In spite of this, the epidermal LCs of TIM-4-deficient mice displayed normal phagocytic and migratory abilities, comparable maturation status upon the stimulation as well as normal repopulation under the inflamed state. Moreover, lack of TIM-4 did not affect dinitrofluorobenzene-induced contact hypersensitivity response. In conclusion, our results indicated that TIM-4 was differentially expressed in the distinct subsets of DCs in skin and skin-draining LNs, and specifically regulated epidermal LC and LN CD207-CD4+ DC homeostasis.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; Langerhans cells; TIM-4; dendritic cells; skin; skin-draining lymph node.

Figure 1. TIM-4 is differentially expressed by the distinct subsets of cutaneous DCs

Epidermal and dermal cells were freshly-isolated from the skin TIM-4 WT and KO mice. Representative FACS dot plots for epidermal LC (MHC-II⁺CD45.2⁺), dermal LCs in transit (MHC-II⁺CD207⁺CD103⁻), dermal CD207⁺ DCs (MHC-II⁺CD207⁺CD103⁺), dermal CD207⁻CD11b⁻ DCs (MHC-II⁺CD207⁻ CD11b⁻) and dermal CD207⁻CD11b⁺ DCs (MHC-II⁺CD207⁻CD11b⁺) (upper panel). The MFI of TIM-4 expression in each cutaneous DC subset (lower panel). All data represent one of at least four independent experiments, with 2-5 mice per experiment.

Figure 2. Disparate expressions of TIM-4 in the different subsets of skin-draining LN DCs

LN cells were harvested from the skin-draining LNs of TIM-4 WT and KO mice. a. Representative FACS dot plots for LN mLCs (MHC-II⁺CD207⁺CD8⁻CD103⁻), CD207⁺ mDDCs (MHC-II⁺CD207⁺CD8⁻CD103⁺) and CD207⁺CD8⁺ DCs (MHC-II⁺CD207⁺CD8⁺) (upper panel). The MFI of each LN DC subset (lower panel). All data represent one of three independent experiments, with 3-5 mice per experiment. b. Representative FACS dot plots for the LN CD4⁻CD8⁺ DCs, CD4⁻CD8⁻ DCs, CD4⁺CD8⁻ DCs and CD4⁺CD8⁺ DCs gated on CD11c⁺CD207⁻ LN cells (upper panel). Representative FACS analysis of TIM-4 expression (middle panel) and MFI (lower panel) of each DC subset. All data represent one of two independent experiments, with 3-4 mice per experiment.

Figure 3. TIM-4 deficiency increases the ratio of epidermal LCs

Epidermal and dermal cells were freshly-isolated from the skin of TIM-4 WT and KO mice. a.-c. Representative FACS analysis of epidermal LCs (a), dermal LCs in transit and dermal CD207⁺ DCs (b), dermal CD207⁻CD11b⁻ DCs and dermal CD207⁻CD11b⁺ DCs (c) from the trunk skin of TIM-4 WT and KO mice (left panel). The ratios of different skin DC populations in TIM-4 WT and KO mice (right panel). Data represent the results of at least four independent experiments, with 2-5 mice per experiment.

Figure 4. Lack of TIM-4 upregulates the frequencies of LN CD207-CD4⁺ DCs

LN cells were harvested from the skin-draining LNs of TIM-4 WT and KO mice. a. Representative FACS analysis of LN mLCs, CD207⁺CD8⁺ DCs, CD207⁺ mDDCs from skin-draining LNs of TIM-4 WT and KO mice (upper panel). The ratios of different LN DC populations (lower panel). Data represent the results of three independent experiments, with 3-5 mice per experiment. b. Representative FACS analysis of LN CD207⁻CD4⁺CD8⁻ DCs, CD207⁻CD4⁺CD8⁺ DCs, CD207⁻CD4⁻CD8⁺ DCs and CD207⁻CD4⁻CD8⁻ DCs from skin-draining LNs of TIM-4 WT and KO mice (upper panel). The ratios of different LN DC populations (lower panel). Data represent the results of two independent experiments, with 3-4 mice per experiment.

Figure 5. TIM-4 is not required for LC maturation, phagocytosis and migration

Epidermal cells were freshly-isolated from the trunk skin of TIM-4 WT and KO mice in (a-c). a. Epidermal suspensions were stained with anti-MHC-II, CD45.2, CD80, and CD86 Abs and analyzed by flow cytometry. Data represent two independent experiments, with 4-5 mice per experiment. b. Epidermal suspensions from the trunk skin of TIM-4 KO and WT littermates were cultured in complete culture medium for 60 h, and then stained as described in (a). Data represent three independent experiments, with 2-3 mice per experiment. c. Epidermal cells were incubated with 0.025% Dextran-FITC for 45 minutes at 37°C or 4°C, which were then stained with anti-CD45.2 and anti-MHC-II. Data represent three independent experiments, with 3-4 mice per experiment (Control: grey filled graph). d. TIM-4 WT and KO mice were painted with 200 μl of 5mg per ml FITC in acetone/dibutylphthalate (1:1), and 24 h later the draining LN cells were then stained with anti-MHC-II, anti-CD8, anti-CD207 and anti-CD103. Seven mice were analyzed.

Figure 6. TIM-4 deficiency does not affect contact hypersensitivity response

Mice were sensitized with DNFB (0.5%) in the abdominal area and then challenged with DNFB (0.2%) 5 days later to induce delayed type hypersensitivity in the ears. Ear thicknesses were measured at different time points after challenge (24h, 48h, 72h) by comparing challenged and unchallenged ears using a thickness gauge in a blinded manner. Ten mice were analyzed.

Figure 7. TIM-4 deficiency does not alter LC repopulation

a. Schematic representation of the experimental plan. b.-c. The mice were given 15 minutes of UV exposure: b. Analysis of the proportions of short-term LCs (MHC-II⁺ langerin^low) and long-term LCs (MHC-II⁺ langerin⁺) on day 7 and 21 after UV treatment. c. Charts represent the frequencies of short-term LCs (MHC-II⁺ langerin^low) and long-term (MHC-II⁺ langerin⁺) cells from each genotype at different time points. The data represent the results of two independent experiments, with 5-7 mice per experiment.