Generation and transcriptional programming of intestinal dendritic cells: essential role of retinoic acid

Abstract

The vitamin A metabolite retinoic acid (RA) regulates adaptive immunity in the intestines, with well-characterized effects on IgA responses, Treg induction, and gut trafficking of T- and B-effector cells. It also controls the generation of conventional dendritic cell (cDC) precursors in the bone marrow and regulates cDC subset representation, but its roles in the specialization of intestinal cDC subsets are understudied. Here we show that RA acts cell intrinsically in developing gut-tropic pre-mucosal dendritic cell (pre-μDC) to effect the differentiation and drive the specialization of intestinal CD103(+)CD11b(-) (cDC1) and of CD103(+)CD11b(+) (cDC2). Systemic deficiency or DC-restricted antagonism of RA signaling resulted in altered phenotypes of intestinal cDC1 and cDC2, and reduced numbers of cDC2. Effects of dietary deficiency were most apparent in the proximal small intestine and were rapidly reversed by reintroducing vitamin A. In cultures of pre-μDC with Flt3L and granulocyte-macrophage colony-stimulating factor (GM-CSF), RA induced cDC with characteristic phenotypes of intestinal cDC1 and cDC2 by controlling subset-defining cell surface receptors, regulating subset-specific transcriptional programs, and suppressing proinflammatory nuclear factor-κB-dependent gene expression. Thus, RA is required for transcriptional programming and maturation of intestinal cDC, and with GM-CSF and Flt3L provides a minimal environment for in vitro generation of intestinal cDC1- and cDC2-like cDC from specialized precursors.

Figure 1. Vitamin A deficiency decreases cDC2 in GALT

a) FACS plots show subset composition of CD11c⁺MHCII⁺ cDC in SI LP and MLN from control and long-term AD mice. The number in each gate indicates percent of cells. b) Ratio of cDC2 to cDC1 in MLN and SI LP, numbers of cDC1 and cDC2 recovered from SI LP of control and VAD mice. Paired 2-tailed t test. Error bars show SEM. c) TLR3, CD207 and CD101 expression on SI cDC1 and cDC2 from control and VAD mice. d) Effects of VAD on cDC subsets in different segments of the SI. SI from control and VAD animals were divided into three equal segments. N=4 independent experiments, 3 mice each. Paired 1-tailed t test. e) Long-term VAD BALB/c mice were put on vitamin A sufficient diet for 7 or 14 days. Results for 7 days were from 2 animals in each group from 1 experiment; results for 14 days were pooled from 3 independent experiments with 2–3 animals per condition in each experiment. f) 0.5–1 millionsorted pre-μDC were adoptively transferred intravenously into control or VAD recipients and mice were sacrificed 6 or 7 days later. Histograms show ratios of host or pre-μDC derived SI cDC2 to cDC1..Unpaired 1-tailed t test. g) Pharmacological agonists of RAR, atRA and AM580, increased and antagonist BMS493 decreased the cDC2/cDC1 ratio in the SI of BALB/c mice. Fold change (FC) of the ratio of cDC2 to cDC1 in SI LP from treated vs. control animals. Results for each condition are pooled from 2 independent experiments with 3 animals each. Unpaired 1-tailed t-test.

Figure 2. Effects of DC-restricted deficiency in RA signaling on SI DC

a) Ratio of cDC2 to cDC1 in SI LP and MLN and number of cDC2 in SI LP from DC-RAR403^−/− (WT), DC-RAR403^fl/− (Heterozygous) and DC-RAR403^fl/fl (homozygous) mice. b) Aberrant expression of CD207 on SI cDC1 from DC-RAR403 mice. Shown are representative plots illustrating CD207 expression cells by SI cDC1 from DC-RAR^fl/fl mice (cells inside CD207⁺ gates are displayed as large dots for illustration). Scatter plot shows the percentage of cDC1 in SI that express CD207. Unpaired 1-tailed t-test.

Figure 3. RA directs generation of intestinal cDC-like DC subsets in vitro

a–c) Pre-μDC were sorted from bone marrow of Flt3L-injected mice and cultured in complete IMDM media with 10% delipidated serum supplemented with Flt3L and GM-CSF without or with 100 nM of RA (or AM580 in c) unless otherwise specified. Cultures were harvested on day 4 and analyzed by flow cytometry. a) Surface markers and RALDH activity indicated by AldeFluor staining on in vivo intestinal and in vitro-derived cDC1 (dotted line) and cDC2 (solid line) are shown. Representative of at least 3 independent experiments. b) Expression of Clec9a on in vitro derived cDC1 and of CD101 on cDC2 with varying RA concentration. Representative of 2 independent experiments. c) Ratio of cDC2 to cDC1, numbers of total progeny cells, and numbers of cDC1 and cDC2 in cultures treated with indicated concentrations of RA or AM580 are shown. Data are pooled from 3 independent experiments.

Figure 4. RA drives the transcriptomes of in vitro derived cDC1 and cDC2 towards patterns of physiologic gene expression

a) PCA of pre-μDC derived cDC1 and cDC2 generated in vitro in the presence or absence of RA, and spleen and SI cDC using 1200 genes with EV>120 that differed at least 2-fold between SI and/or spleen cDC1 and cDC2. b) Pair-wise comparison of the indicated cDC subsets and BM pre-μDC using the same genes as in a). c) Pairwise correlation of gene expression by in vitro derived cDC1 and cDC2 or spleen cDC1 and cDC2 replicates with the mean gene-expression profiles of SI cDC1 or cDC2, using the same genes as in a). Error bars are 95% CL. d) Heatmap of genes that are differentially expressed (>3X FC) between SI or spleen cDC1 and cDC2 and regulated by RA (>3X FC between in vitro cDC1 derived with and without RA OR cDC2 with and without RA). Clustering of genes is based on correlation. e) Transcription factor expression. Shown is expression of TF implicated previously in DC development, or regulated (2 FC) by RA in vitro and also differently and coordinately expressed by spleen and SI cDC1 vs. cDC2 (at least 1.5FC in both SI and spleen). TF implicated in cDC1 or cDC2 development are highlighted in green or red respectively.

Figure 4. RA drives the transcriptomes of in vitro derived cDC1 and cDC2 towards patterns of physiologic gene expression

a) PCA of pre-μDC derived cDC1 and cDC2 generated in vitro in the presence or absence of RA, and spleen and SI cDC using 1200 genes with EV>120 that differed at least 2-fold between SI and/or spleen cDC1 and cDC2. b) Pair-wise comparison of the indicated cDC subsets and BM pre-μDC using the same genes as in a). c) Pairwise correlation of gene expression by in vitro derived cDC1 and cDC2 or spleen cDC1 and cDC2 replicates with the mean gene-expression profiles of SI cDC1 or cDC2, using the same genes as in a). Error bars are 95% CL. d) Heatmap of genes that are differentially expressed (>3X FC) between SI or spleen cDC1 and cDC2 and regulated by RA (>3X FC between in vitro cDC1 derived with and without RA OR cDC2 with and without RA). Clustering of genes is based on correlation. e) Transcription factor expression. Shown is expression of TF implicated previously in DC development, or regulated (2 FC) by RA in vitro and also differently and coordinately expressed by spleen and SI cDC1 vs. cDC2 (at least 1.5FC in both SI and spleen). TF implicated in cDC1 or cDC2 development are highlighted in green or red respectively.