The transcription factor NR4A3 (also known as NOR-1) is a member of the Nr4a family of nuclear receptors and is expressed in myeloid and lymphoid cells. Here, we have shown that Nr4a3 is essential for the migration of CD103+ dendritic cells (DCs) to lymph nodes (LNs). Nr4a3-deficient mice had very few CD103+ migratory DCs (mDCs) present in LNs, and mixed-chimera studies revealed that this migratory defect was cell intrinsic. We further found that CD103+ DCs from Nr4a3-deficient mice displayed a marked loss of surface expression of the chemokine CCR7. This defect in CCR7 expression was confined to CD103+ DCs, as CCR7 expression on T lymphocytes was unaffected. Moreover, CCR7 was not induced on CD103+ DCs from Nr4a3-deficient mice in response to either administration of the TLR7 agonist R848 or infection with Citrobacterrodentium in vivo. The transcription factor FOXO1 has been shown to regulate CCR7 expression. We found that FOXO1 protein was reduced in Nr4a3-deficient DCs through an AKT-dependent mechanism. Further, we found a requirement for NR4A3 in the maintenance of homeostatic mitochondrial function in CD103+ DCs, although this is likely independent of the NR4A3/FOXO1/CCR7 axis in the regulation of DC migration. Thus, NR4A3 plays an important role in the regulation of CD103+ mDCs by regulating CCR7-dependent cell migration.
Authors
Kiwon Park, Zbigniew Mikulski, Goo-Young Seo, Aleksander Y. Andreyev, Paola Marcovecchio, Amy Blatchley, Mitchell Kronenberg, Catherine C. Hedrick
(A) Ccr7 mRNA expression. Plasmacytoid DCs (CD11cmed, MHC-IImed, PDCA-1+, CD3–, CD19-), LN-resident DCs (CD11c+, MHC-IImed, CD3–, CD19–), and mDCs (CD11c+, MHC-IIhi, CD3–, CD19–) from WT and Nr4a3–/– mice were sorted by FACS. Data represent pools of 5 mice per group. Results represent 1 of 2 independent experiments. (B) Histogram of CCR7 surface expression on mDCs. (C) MFI of CCR7 on mDCs (n = 6 mice per group). Results represent 1 of 3 independent experiments. (D) CD11cYFP WT or CD11cYFPNr4a3–/– mice were treated with R848 for 2 hours in vivo. Confocal whole-mount images of mesenteric lymphatic vessels from R848-treated CD11cYFP WT or CD11cYFPNr4a3–/– mice were analyzed using Imaris software. Lymphatics (blue) were isosurfaced, and distance transformation was applied to create a new channel that encoded the 3D distance from the lymphatic vessels. CD11cYFP cells (green) were isosurfaced and classified as being associated with lymphatics (red) when the distance from the lymphatics was less than 15 μm. To normalize between the different data sets, the number of cells associated with lymphatics was divided by the volume of lymphatic vessels (expressed in μm3 and divided by 100,000). (E) Numbers of YFP+ cells associated with lymphatic vessels (red). (F) Numbers of YFP+ cells away from lymphatic vessels (green). (G) Day-7 BMDCs were stimulated with 0.5 μg/ml LPS for 36 hours. CD11c+MHC-II++ cells were gated for CCR7 expression. (H) MFI of CCR7 expression on CD11c+MHC-II++ cells was analyzed by flow cytometry. Representative histogram (top) and dot plot of individual mouse samples (bottom). Max, maximum. (I) Expression of Foxo1 mRNA and FOXO1, p-AKT, AKT, and β-actin proteins from CD11c+ BMDCs, stimulated with or without LPS. Results from G and I represent 1 of 3 to 4 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test.