( F) Primary immature human DCs were incubated overnight with DMSO (vehicle only VO) or 5 μM GW3965 and 1 μM 9-cis-retinoic acid (G+9). Shown is mean ± SD for a representative experiment assayed in triplicate. Results denote induction relative to vehicle-treated samples (set as 1). Precipitated DNA was amplified using CCR7 primers located between –800 to –350 bp upstream of transcription start (LXR-responsive region LXRR) and –3.8 kb (arrows), normalized to input chromatin. ( E) DC2.4 cells were incubated with vehicle or 5 μM T0901317 for 2 hours, and chromatin was prepared. Shown is mean ± SD luciferase activity normalized to β-galactosidase (relative luciferase activity RLU). HEK293T cells transfected with the indicated constructs and with Nr1h3 and Nr1h2 expression plasmids were incubated with DMSO (–) or 1 μM T0901317 for 24 hours. ( D) CCR7 promoter luciferase reporter constructs (left). * P < 0.05 versus respective 2-hour or mock control. y axes denote fold change of Ccr7 mRNA relative to Ppia. ( A– C) DC2.4 cells were ( A) treated with 1 μM T0901317 for the indicated times, ( B) preincubated with actinomycin D, or ( C) transfected with control siRNA (mock) or with siRNA specific for Nr1h3, Nr1h2, or both, then treated with T0901317. We conclude that LXR is required for maximal effects on plaque CD68+ cell expression of CCR7 and monocyte-derived cell egress during atherosclerosis regression in mice. Moreover, LXR agonist treatment of primary human immature DCs resulted in functionally significant upregulation of CCR7. This increase was blunted when LXRalpha and LXRbeta levels were reduced by siRNAs. Using an immature DC line, we found that LXR agonist treatment increased Ccr7 mRNA levels. In addition, the CD68+ cells displayed reduced emigration and CCR7 expression. Plaques from both LXRalpha and LXRbeta-deficient Apoe-/- mice exhibited impaired regression. To test the requirement for LXR for CCR7-dependent regression, we transplanted aortic arches from atherosclerotic Apoe-/- mice, or from Apoe-/- mice with BM deficiency of LXRalpha or LXRbeta, into WT recipients. To extend these results, atherosclerotic Apoe-/- mice sufficient or deficient in CCR7 were treated with an LXR agonist, resulting in a CCR7-dependent decrease in plaque CD68+ cells. Concurrent with regression, mRNA levels of the gene encoding LXRalpha are increased in plaque CD68+ cells, suggestive of a functional relationship between LXR and CCR7. We have previously shown that mouse atherosclerosis regression involves monocyte-derived (CD68+) cell emigration from plaques and is dependent on the chemokine receptor CCR7.
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