Activated Murine Thymic Plasmacytoid Dendritic Cells alter Thymopoiesis

Abstract

Pediatric human immunodeficiency virus-1 (HIV-1) infection is characterized by rapid disease progression and profound thymus insufficiency; however the pathogenesis of thymus dysfunction during HIV infection is complex and poorly defined. A recently proposed mechanism for HIV-associated thymus dysfunction is the inhibition of thymopoiesis due to aberrant expression of IFN-α and pro-inflammatory cytokines by activated plasmacytoid dendritic cells (pDC) in the thymic microenvironment. We tested the hypothesis that the products of virus-activated pDC would inhibit or alter thymopoiesis. Thymic pDC were enriched and isolated from 3 week old ICR mice using density gradient centrifugation and magnetic separation using an antibody recognizing m-PDCA-1, a unique marker for murine pDC. pDC represented 0.12% of total thymocytes and 12% of magnetically sorted cells. The phenotype of thymic pDC was found to be CD4+CD11c+CD123+m-PDCA-1+. Murine pDC and non-pDC cells were stimulated with the TLR9 ligand CpG for 6 and 24 hours. Supernatant and RNA were collected at each time point and analyzed for expression of IFN-α, TNF-α, and IL-6 using ELISA and RT-PCR. Enriched thymic pDC secreted higher levels of TNF-α, IFN-α and IL-6 than non-pDC at 24 hours post-stimulation. mRNA for TNF-α and IL-6 peaked at 6 hours. To assess the impact of pDC activation on thymopoiesis, murine thymus lobes were obtained at embryonic day 20 or at birth and established in thymus organ culture (TOC). Conditioned media from stimulated and unstimulated pDC were added to TOC; controls included organ cultures that received normal medium or CpG. Murine TOC that received conditioned media from stimulated pDC tended to have a reduced percentage of CD4+CD8+ thymocytes and had a significant increase in percentage of CD4+CD8- when compared with TOC grown in unconditioned medium or organ cultures directly stimulated with CpG. These data suggest that the products of activated pDC can induce changes in thymopoiesis that could contribute to thymus dysfunction during immunosuppressive lentivirus infections, even in the absence of virus replication.