Regulation of plasmacytoid dendritic cell development in mice by aryl hydrocarbon receptor

Abstract

Aryl hydrocarbon receptor (AhR) has an important role in the regulation of cell responses to different environmental stimuli, as well as to various endogenous ligands. Although AhR was previously implicated in the regulation of dendritic cell (DC) activation, very little is known about its potential role in the development of these cells. Here we report our unexpected findings that AhR may regulate the differentiation of plasmacytoid DCs (pDCs). Agonist of AhR markedly decreased the generation of pDCs in vitro, whereas the AhR antagonist had an opposite effect. The differentiation of conventional DCs (cDCs) was not affected. AhR-knockout mice had a substantial accumulation of pDCs in peripheral lymphoid organs; whereas no changes in cDCs were seen. Thus, this study has identified AhR as a transcription factor involved in the development of one population of DCs-pDCs.

Figure 1. AhR KO mice show the selective accumulation of pDCs in peripheral lymphoid organs

A. Typical example of staining the splenocytes from AhR KO and WT mice. The cells in the circle gate represent pDCs. B. The absolute number of cells in lymph nodes and spleens. Data are presented as Mean ± SEM (n=3 mice). C, D. The percentage and total cell number of different myeloid cells in spleens (C) and LNs (D) from AhR KO and control WT mice. Data are presented as Mean ± SEM (n=3 mice). *, p<0.05, **, p<0.01, compared to the WT mice. E. Enriched BM HPCs from AhR KO or WT mice were cultured with 100 ng/ml Flt3L for 9 days. The representative FACS plots from 3 independent experiments show the expression of B220 vs CD11c among live CD11b^- cells. Data are presented as Mean ± SEM (left). **, p<0.01, compared to WT mice. F. 50,000 of Siglec H⁺ cells from 9 days' BM cultures of HPC with Flt3L were stimulated with 20ng/ml CpG ODN type A for 18 hr. Data show the production of IFN-α measured in ELISA. Data are presented as Mean ± SEM of three independent experiments.

Figure 2. AhR negatively regulates pDC development in vitro

A. The relative mRNA levels of ahr, ahrr, arnt in HPC culture with Flt3L on days 3, 6, and 9. The ratio of ahr to ahrr was present. Data are presented as Mean ± SEM of three independent experiments. B-D. HPCs were cultured with 100 ng/ml Flt3L for 9 days in the presence of 10nM TCDD or vehicle. B. The total number of cells generated from HPC; C. Typical FACS plots of cells gated on CD11b^- cells from three independent experiments. The percentage of pDCs from the all live cells is shown. D. The proportions of pDCs (top panel) and cDCs (bottom panel) using different combination of antibodies. Data are presented as Mean ± SEM (n=3 replicates). E. HPCs were cultured with Flt3L in the presence of 1μM CH223191 or vehicle for 9 days. Left panel - representative FACS plots of cells gated on CD11b^- cells from 3 independent experiments. The percentage of pDCs from all live cells is shown. Right panel – proportion of cells. Data are presented as Mean ± SEM. F. Apoptosis was measured using staining with DAPI and Annexin-V within the population of pDCs generated from BM progenitors in the presence of TCDD and CH223191 as described in Fig. 2D,E. Mean and SEM are shown (n=3). G. HPCs cultured with 20ng/ml GM-CSF for 5 days in the presence of 10nM TCDD, 1μM CH223191, or vehicle. Data are presented as Mean ± SEM from 3 independent experiments.