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. 2018 Jun 19;115(25):E5736-E5745.
doi: 10.1073/pnas.1800303115. Epub 2018 Jun 5.

Cell-intrinsic Regulation of Murine Epidermal Langerhans Cells by Protein S

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Free PMC article

Cell-intrinsic Regulation of Murine Epidermal Langerhans Cells by Protein S

Yaara Tabib et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

AXL, a member of the TYRO3, AXL, and MERTK (TAM) receptor tyrosine kinase family, has been shown to play a role in the differentiation and activation of epidermal Langerhans cells (LCs). Here, we demonstrate that growth arrest-specific 6 (GAS6) protein, the predominant ligand of AXL, has no impact on LC differentiation and homeostasis. We thus examined the role of protein S (PROS1), the other TAM ligand acting primarily via TYRO3 and MERTK, in LC function. Genetic ablation of PROS1 in keratinocytes resulted in a typical postnatal differentiation of LCs; however, a significant reduction in LC frequencies was observed in adult mice due to increased apoptosis. This was attributed to altered expression of cytokines involved in LC development and tissue homeostasis within keratinocytes. PROS1 was then excised in LysM+ cells to target LCs at early embryonic developmental stages, as well as in adult monocytes that also give rise to LCs. Differentiation and homeostasis of LCs derived from embryonic precursors was not affected following Pros1 ablation. However, differentiation of LCs from bone marrow (BM) precursors in vitro was accelerated, as was their capability to reconstitute epidermal LCs in vivo. These reveal an inhibitory role for PROS1 on BM-derived LCs. Collectively, this study highlights a cell-specific regulation of LC differentiation and homeostasis by TAM signaling.

Keywords: Langerhans cells; Pros1; TAM signaling; epidermis; protein S.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Expression of TAM receptors and ligands in the murine skin epidermis. Epidermal cells were prepared from 8-wk-old mice, and representative flow cytometry plots present the expression of AXL, MERTK, and TYRO3 receptors on (A) LCs and (B) keratinocytes. Data of one of three independent experiments are provided, and each experiment included at least five separately analyzed mice. (C) Western blot analysis showing the presence of MERTK, TYRO3, PROS1, GAS6, and GAPDH as a control in lysates of total epidermal cells from adult WT Gas6+/+ and Gas6−/− mice or sorted keratinocytes, LCs, and dendritic epidermal T cells (DETC) from 8-wk-old WT mice. +, positive control. Representative results of one of three independent experiments are shown. (DF) Ear cross-sections (D, Upper image presents the actual image while the Lower image provides a magnification of a selected area) or whole epidermal sheets (E) were stained for PROS1 (green), langerin (red), and DAPI (blue) for nuclear visualization. The white dotted line demarcates the basal membrane and the arrowhead indicates the epidermis. (F) Whole epidermal sheets were stained with GAS6 (red) and langerin (green). Immunofluorescence microscopy images are shown representing one of three independent experiments. (G) Relative expression levels of Pros1 and Gas6 in whole epidermal tissues and sorted cells isolated from 8-wk-old mice were analyzed by RT-qPCR. Bar graphs present the fold change in mRNA levels among the various groups, which was normalized to Gas6+/+ or Cre mice, respectively, ±SD (n = 5). D, dermis; E, epidermis. *P < 0.05. Results of one of two independent experiments are presented.
Fig. 2.
Fig. 2.
LC differentiation is not impaired due to the lack of GAS6. (A) Epidermal cells were purified from adult Gas6−/− mice and littermate controls (WT). Representative flow cytometry plots present the frequencies of LCs (langerin+) and γδ T cells (γδ TCR+) among total CD45+ leukocytes. Data are representative of three independent experiments, and each experiment included at least four separately analyzed mice. (B) Whole-mount immunofluorescence staining on epidermal layers prepared from adult Gas6−/− mice and littermate controls, stained for MHCII (red) and langerin (green). Representative confocal microscopy images are presented, representing one of four independent experiments (n = 3 mice in each experiment). (C) Representative flow cytometry plots demonstrating the expression levels of MHCII, CD86, and langerin on epidermal LC cells of adult Gas6−/− mice and littermate controls. Data of one of three independent experiments are provided (n = 5 in each experiment). (D and E) Relative expression of various cytokines or TAM receptors and ligands in epidermal layers of adult Gas6−/− mice and littermate controls were analyzed by RT-qPCR (n = 5 mice). RNA expression data of one of two independent experiments are presented as the mean values ± SD. *P < 0.05.
Fig. 3.
Fig. 3.
Ablation of PROS1 in keratinocytes reduces LC frequencies. (A) Whole-mount immunofluorescence staining on epidermal layers prepared from adult K5/Pros1fl/fl and littermate Cre mice, stained for PROS1 (green) and DAPI (blue). Representative fluorescent images are shown, representing one of two independent experiments (n = 3 mice in each experiment). Negative control, primary antibody was omitted. (B) Quantification of mRNA levels of PROS1 in epidermal layers of adult K5/Pros1fl/fl and Cre mice using RT-qPCR (n = 5 mice). Data of one of two independent experiments are presented as the mean values ± SD. (C) Frequencies and absolute numbers (per a half ear) of LCs (CD45+MHCII+CD11c+ cells) in epidermal cells prepared from 2-, 5-, and 10-mo-old K5/Pros1fl/fl and Cre mice. Representative flow cytometry plots, as well as bar graphs, present the fold change in LC frequencies (normalized to Cre mice) or absolute LC numbers in the epidermis of these mice. Data are representative of four independent experiments, and each experiment included at least five separately analyzed mice. (D and E) Whole-mount immunofluorescence staining on epidermal layers prepared from adult K5/Pros1fl/fl and littermate Cre mice, stained for langerin (green) and MHCII (red). Representative fluorescent images are presented representing one of three independent experiments (n = 3 mice in each experiment). **P < 0.01. (Scale bars: A and E, 50 μm; D, 500 μm.)
Fig. 4.
Fig. 4.
LCs are not activated in K5/Pros1fl/fl. (A) Epidermal cells were prepared from adult K5/Pros1fl/fl and littermate Cre mice. Representative flow cytometry plots present the expression of AXL and TYRO3 on LCs and keratinocytes. Data of one of three independent experiments are provided, and each experiment included at least five separately analyzed mice. (B and C) Relative expression of various genes in epidermal layers of adult K5/Pros1fl/fl and littermate Cre mice were analyzed by RT-qPCR (n = 5 mice). RNA expression data of one of two independent experiments are presented as the mean values ± SD. Representative FACS plot demonstrates MHCII expression levels on keratinocytes of adult K5/Pros1fl/fl and littermate Cre mice. (D) Expression levels of MHCII, langerin, and CD86 on epidermal LCs of adult K5/Pros1fl/fl and littermate Cre mice (n = 5). Representative FACS plots of one of three independent experiments are presented. (E) Relative expression of genes involved in LC maturation and migration to the LNs as measured by RT-qPCR. Data are representative of two independent experiments, and each experiment included five separately analyzed mice. (F) K5/Pros1fl/fl and littermate Cre mice were sensitized with 1% TNCB on the shaved abdomen, and, 5 d later, the mice were challenged on one ear with 0.5% TNCB while the unchallenged ear was used as control. The graph presents the change in ear swelling at the indicated time points and represents the mean values ± SD (n = 5). Data of one of two independent experiments are provided. Ctrl, control. *P < 0.05.
Fig. 5.
Fig. 5.
Intact postnatal differentiation of LCs in K5/Pros1fl/fl. Epidermal cells were prepared from 9-d-old (P9) K5/Pros1fl/fl mice and littermate Cre controls. (A) Representative flow cytometry plots and graph present the frequencies of CD45+MHCII+ cells in the epidermis. (B) Frequencies and absolute numbers (per a half ear) of LCs (CD45+MHCII+langerin+) in the epidermis. Representative flow cytometry histogram demonstrates langerin expression in P9 K5/Pros1fl/fl and Cre mice, as well as adult Cre mice. Data are representative of two independent experiments, and each experiment included at least three separately analyzed mice. (CE) Relative expression of Pros1 and various genes in epidermal layers of (C and D) P9 K5/Pros1fl/fl and Cre mice, or (E) adult vs. P9 Cre mice were analyzed by RT-qPCR (n = 5 mice). RNA expression data of one of two independent experiments are presented as the mean values ± SD. Ctrl, control. *P < 0.05.
Fig. 6.
Fig. 6.
Increased apoptosis rate of LCs in K5/Pros1fl/fl mice. (A) Three-week-old K5/Pros1fl/fl and Cre mice were treated with BrdU in the drinking water for 3 or 8 wk. Representative flow cytometry plots demonstrate the frequencies of CD45+MHCII+ (LCs) in the epidermis, as well as the frequencies of BrdU-labeled LCs at the end of the treatments. Bar graphs present the frequencies of BrdU-labeled LCs and the mean florescence intensity (MFI) of BrdU staining in LCs (n = 5). Data of one of two independent experiments are provided and present the mean values ± SD. (B) Epidermal sheets of 6- to 8-wk-old mice were stained against langerin and anti-caspase 3 to detect LCs undergoing apoptosis. Representative images of whole-mount immunofluorescence staining and graph demonstrating the numbers of active-caspase 3+ langerin+ cells are presented and present the mean values ± SD. Data are representative of three independent experiments. Each experiment included at least three separately analyzed mice, and 20 fields per mouse were analyzed. White circles exemplify langerin+ cells stained positively to active-caspase 3. (C) Representative flow cytometry plots and graph present the frequencies of dead PI+ LCs in the epidermis of K5/Pros1fl/fl and Cre mice (n = 5). Results of one of two independent experiments are provided and present the mean values ± SD. ctrl, control. *P < 0.05.
Fig. 7.
Fig. 7.
Normal development and homeostasis of LCs in LysM/Pros1fl/flEyfp mice. (A) Epidermal cells were prepared from adult LysM/Pros1fl/flEyfp and littermate Cre mice. Representative flow cytometry plots present the percentages of YFP+ LCs among total epidermal LCs. Representative whole-mount immunofluorescence staining on epidermal sheet stained against langerin (red) and YFP (green). Data are representative of four independent experiments, and each experiment included at least three separately analyzed mice. (B) Blood and back skin epidermis of E18 embryos were sampled from LysM/Pros1fl/flEyfp and Cre mice. Representative flow cytometry plots present the percentages of YFP+ cells among LC precursors in the blood (CD45+MHCII+) and epidermis (CD45+MHCII+F4/80+). Results of one of two independent experiments are presented. (C) YFP+ LCs were analyzed in the epidermis of LysM/Pros1fl/flEyfp mice at the indicated time points. Representative flow cytometry plots and graph present the frequencies of YFP+ LCs among total LCs (n = 3–5). Data of one of three independent experiments are provided and present the mean values ± SD. (D) Adult LysM/Pros1fl/flEyfp mice were lethally irradiated and, 24 h later, were administered i.v. with BM cells purified from littermate Cre mice. The epidermis of the chimeric mice was analyzed 7, 14, and 21 d after BM transplantation to quantify the frequencies of YFP+ LCs. Representative flow cytometry plots and graph present the frequencies of YFP+ LCs among total LCs (n = 3). Data of one of two independent experiments are provided and present the mean values ± SD. Ctrl, control. (Scale bars:, 50 μm.)
Fig. 8.
Fig. 8.
Differentiation of LCs from circulating BM precursors is augmented in LysM/Pros1fl/flEyfp mice. (A) Lethally irradiated langerin-DTR mice were adoptively transferred with BM cells purified from LysM/Pros1fl/flEyfp mice or LysM/Pros1+/+Eyfp mice as a control. Eight weeks later, LCs were ablated by a single injection of DT, and the repopulation of LCs was examined 7 and 11 wk after the depletion. (B) The frequencies and numbers of repopulating LCs in the epidermis of the chimeric mice are presented (n = 3). (C) FACS plots present the percentages of YFP+ cells among the repopulating LCs (n = 3). Results of one of two independent experiments are provided and present the mean values ± SD. (D) Representative flow cytometry plots demonstrate the percentages of YFP+ cells among Ly6Clow, Ly6Chigh, and neutrophils in the blood of LysM/Pros1fl/flEyfp or LysM/Pros1+/+Eyfp mice before the transplantation. Data are representative of two independent experiments, and each experiment included at least three separately analyzed mice. (E) BM cells were purified from LysM/Pros1+/+Eyfp, LysM/Pros1fl/+Eyfp, or LysM/Pros1fl/flEyfp mice and then cultured for 5 d in serum containing media supplemented with GM-CSF and TGF-β1 to drive differentiation of LC-like cells. Representative flow cytometry plots demonstrate the percentages of MHCII+CD11c+ cells and further expression of CD205+EpCAM+, representing LC-like cells. The graph presents the frequencies of LC-like cells, as well as MHCII+CD11c+CD205EpCAM+ cells, which are also known to differentiate to LC-like cells (n = 3). Data of one of four independent experiments are provided and present the mean values ± SD. (F) LCs were differentiated in vitro from BM cells of WT mice and stained for PROS1 (red), AXL (green), and DAPI (blue) for nuclear visualization at 5 d in culture. Representative immunofluorescence images taken from one of two independent experiments are shown. **P < 0.01.

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