RARα supports the development of Langerhans cells and langerin-expressing conventional dendritic cells

Abstract

Langerhans cells (LC) are the prototype langerin-expressing dendritic cells (DC) that reside specifically in the epidermis, but langerin-expressing conventional DCs also reside in the dermis and other tissues, yet the factors that regulate their development are unclear. Because retinoic acid receptor alpha (RARα) is highly expressed by LCs, we investigate the functions of RARα and retinoic acid (RA) in regulating the langerin-expressing DCs. Here we show that the development of LCs from embryonic and bone marrow-derived progenitors and langerin⁺ conventional DCs is profoundly regulated by the RARα-RA axis. During LC differentiation, RARα is required for the expression of a LC-promoting transcription factor Runx3, but suppresses that of LC-inhibiting C/EBPβ. RARα promotes the development of LCs and langerin⁺ conventional DCs only in hypo-RA conditions, a function effectively suppressed at systemic RA levels. Our findings identify positive and negative regulatory mechanisms to tightly regulate the development of the specialized DC populations.

Fig. 1

LC development requires RARα. a Frequency of langerin⁺ cells in the epidermis of WT and ∆Rara^CD11c mice. b Immunofluorescence detection of langerin⁺ DCs in cross-sections of the ear skin of WT and ∆Rara^CD11c mice. The white dashed lines indicate the border between the epidermis and dermis. c Immunofluorescence detection of langerin⁺ DCs in epidermal sheets of the ear skin of WT and ∆Rara^CD11c mice. Representative and combined data are shown. d Langerin expression by dermal DCs from WT vs. ∆Rara^CD11c mice. As WT control mice, C57BL/6 or CD11C-Cre-GFP mice were used. Representative and combined data (n = 4–8) from at least 3 experiments are shown. *Significant differences from control mice by Mann-Whitney U test (p < 0.05, unpaired, 2-sided). Error bars are defined as s.e.m

Fig. 2

LC development in newborn mice and following BM transfer requires RARα. a Kinetics of langerin up-regulation on CD11c⁺ MHC-II⁺ cells in the epidermis of WT and ∆Rara^CD11c mice determined at postnatal day 1, 3, and 9. b Phenotype of epidermal DCs in new born (1-day old) mice. Trunk skin of WT and ∆Rara^CD11c mice was examined for CD11c⁺ MHC-II⁺ cells. c A BM reconstitution study from WT or ∆Rara^CD11c mice into lethally irradiated ∆Rara^CD11c mice. As WT control mice, we used either C57BL/6 or CD11C-Cre-GFP mice. The frequency of total CD11c⁺ MHC-II⁺ cells and epidermal langerin⁺ cells (% of CD11c⁺ MHC-II⁺) were examined 12–15 weeks post-BM transfer. Representative and combined data (n = 4–8) from at least 3 experiments are shown. *Significant differences from control mice by Mann–Whitney U test (p < 0.05, unpaired, 2-sided). Error bars are defined as s.e.m

Fig. 3

RA negatively regulates LC development. a RA suppresses langerin⁺ cell (BM-LC) generation in vitro, while the RAR antagonist BMS493 enhanced BM-LC generation. For BM-LC culture, BM cells were cultured in GM-CSF and TGFβ1 for 3 days in the presence of At-RA (0.01, 0.1, 1 and 10 nM) or BMS493 (100 nM) in media containing charcoal-treated or regular FBS. Frequencies of indicated CD11c⁺ cells are shown in graphs. b Defective BM-LC generation from BM cells of ∆Rara^CD11c mice. BM cells were cultured in a medium containing charcoal-treated FBS. c Confocal fluorescent microscopy of langerin expression by CD11c⁺ BM cells cultured in the LC-inducing condition without and with RA (1 nM). d Expression of Rara mRNA by CD11c⁺ BM cells cultured in the LC-induction condition without or with RA (1 nM). Normalized values for a housekeeping gene (GAPDH) are shown. Representative and combined data (n = 3–8) from at least 3 experiments are shown. *Significant differences from control or between indicated groups by one-way ANOVA with Bonferroni multiple comparisons (a) or Mann–Whitney U test (p < 0.05, unpaired, 2-sided, b, d). Error bars are defined as s.e.m

Fig. 4

The effects of RARα deficiency vs. RA on gene expression in BM-derived DCs cultured in a LC-inducing condition. For all panels in this figure, RNA-seq was performed on WT and ∆Rara^CD11c BM cells cultured in charcoal-treated FBS medium containing GM-CSF and TGFβ1 for 3 days. WT cells were cultured with or without At-RA (10 nM). 2–4 independent samples were examined for each group. a Scatter plot analysis of RPKM (Reads Per Kilobase of transcript per Million mapped reads) values for ∆Rara^CD11c vs. WT for X-axis and RA vs. WT for Y-axis. 2619 differentially expressed genes were selected based on T-test (P < 0.2) adjusted with Benjamini–Hochberg procedure of the Multiplot Studio (Version 1.5.29, GenePattern). Total 8 samples were examined by RNA-seq (4 WT, 2 ∆Rara^CD11c, and 2 At-RA). Genes highlighted in orange are the top 10 up- or down-regulated genes. b Principal component analysis (PCA) of the 2619 selected genes based on their RPKM values. c A Treeview showing hierarchical clustering of 2619 genes commonly or differentially regulated in BM-derived DCs cultured in a LC-inducing condition. Pearson correlation indices are shown. d A Treeview showing hierarchical clustering of manually selected genes from the differentially regulated 2619 genes. The green and red triangles respectively highlight Runx3 and Cebpb genes. Error bars are defined as s.e.m

Fig. 5

RA and RARα reciprocally regulate the expression of the positive and negative LC-regulating transcription factors, Runx3 and C/EBPβ. a Impact of RA and RARα deficiency on Runx3 expression at mRNA level. b Impact of RA and RARα deficiency on Runx3 expression at protein level. c Effect of enforced Runx3 expression on BM-derived LC differentiation in the presence and absence of RA. The data shown are gated for transduced Thy1.1⁺CD11c⁺ cells. d Impact of RA and RARα deficiency on expression of Cebpb mRNA. e Impact of RA and RARα deficiency on expression of C/EBPβ protein. f Effect of dnC/EBPβ on BM-derived LC differentiation in the presence and absence of RA. BM cells from WT or ∆Rara^CD11c mice were cultured with GM-CSF and TGFβ1 for 5 days (3 days following retroviral transduction) in the presence of At-RA (10 nM except in panels c and f where 0.1 nM was used) in a medium containing charcoal-treated FBS. Representative and combined data (n = 3–7) from at least 3 experiments are shown. Significant differences from controls by one-way ANOVA with Bonferroni adjustments (p < 0.05)* or between indicated groups by two-way ANOVA with Tukey adjustments (p < 0.05)**. Error bars are defined as s.e.m

Fig. 6

The DNA binding, but not ligand binding, function of RARα is required for BM-LC generation. Impact of RARα variants on BM-derived LC differentiation in vitro. BM cells were transduced with retroviral vectors harboring WT and variant RARα genes that lack ligand binding (ΔLB) or DNA binding (ΔDB) activity and differentiated with GM-CSF and TGFβ1 for 5 days in the presence of RA in medium containing charcoal-treated FBS. The data shown are for transduced Thy1.1⁺CD11c⁺ cells. Representative and combined data are shown. At-RA was used at 0.1 nM. Representative and combined data from 5 experiments are shown. *^,**Significant differences by two-way ANOVA with Tukey adjustments (p < 0.05). Error bars are defined as s.e.m

Fig. 7

RA regulates CEBPb and RUNX3 expression and human langerin⁺ cell differentiation from blood monocytes. a Human LC differentiation from blood monocytes in regular vs. charcoal FBS. b At-RA promotes C/EBPβ⁺ non-LC differentiation. c RA and RARα antagonists reciprocally regulate human LC differentiation. d Effects of At-RA and BMS493 on the expression of human CEBPb and RUNX3. Human blood CD14⁺ monocytes were cultured in GM-CSF and TGF-β1 for 5–7 days in media containing charcoal FBS except in panel A where regular FBS was also used. At-RA was used at 1 nM, and BMS493 and Ro4153 were used at 500 nM. Representative and combined data (n = 5–7) from at least 5 experiments are shown. *Significant differences from control by Mann–Whitney U test (p < 0.05, unpaired, 2-sided). Error bars are defined as s.e.m. Langerhans cells (LC) and langerin-expressing conventional dendritic cells are made from distinct progenitors and enriched in the distinct microenvironments of the skin. Here the authors show that these immune cells are regulated by RARα via simultaneous induction of LC-promoting Runx3 and repression of LC-inhibiting C/EBPβ