CpGs induce differentiation of Atlantic salmon mononuclear phagocytes into cells with dendritic morphology and a proinflammatory transcriptional profile but an exhausted allostimulatory activity

Due to their ability to present foreign antigens and prime naïve T cells, macrophages and dendritic cells (DCs) are referred to as professional antigen-presenting cells (APCs). Although activated macrophages may function as APCs, these cells are particularly effective at directly engaging pathogens through phagocytosis, and production of antimicrobial compounds. On the other hand, DCs possess superb antigen-presenting and costimulatory capacity and they are essential for commencement and regulation of adaptive immune responses. In in vitro models, development of mature mammalian DCs from monocytes requires sequential exposure to growth factors (including GM-CSF and IL-4) and proinflammatory stimuli such as toll-like receptor (TLR) ligands. Currently, except for IL-4/13, neither orthologues nor functional analogues of the growth factors which are essential for the differentiation of mammalian DCs (including GM-CSF and FLT3) have been identified in teleosts and data about differentiation of piscine APCs is scant. In the present study, primary salmon mononuclear phagocytes (MPs) stimulated in vitro for 5-7 days with a B-class CpG oligodeoxynucleotides (ODN 2006PS) underwent morphological differentiation and developed “dendritic” morphology, characterized by long, branching pseudopodia. Transcriptional profiling showed that these cells expressed high levels of proinflammatory mediators characteristic for M1 polarized MPs. However, the cells treated with CpGs for 7 days downregulated their surface MHCII molecules as well as their capacity to endocytose ovalbumin and exhibited attenuated allostimulatory activity. This concurred with transcriptional downregulation of costimulatory CD80/86 and upregulation of inhibitory CD274 (B7-H1) genes. Despite their exhausted allostimulatory activity, these cells were still responsive to re-stimulation with gardiquimod (a TLR7/8 ligand) and further upregulated a wide array of immune genes including proinflammatory mediators such as intereukin-1 beta and tumor necrosis factor. Overall, the presented data highlight the disparate effects TLR ligands may have on the proinflammatory status of APCs, on one side, and their antigen-presenting/costimulatory functions, on the other. These findings also indicate that despite the poor phylogenetic conservation of the growth factors involved in the differentiation of DCs, some of the processes that orchestrate the development and the differentiation of professional APCs are conserved between teleosts in mammals. Primary salmon mononuclear phagocytes (MPs) were isolated from head kidney and cultured in vitro for up to 7 days. RNA samples were harvested from control (non-stimulated) cells after 24 hours and 7 days of in vitro culture. Stimulated samples included: MPs stimulated for 24 h with 20 μg/ml of poly IC; MPs stimulated with 20μg/ml of polyIC or 2 uM CpG (2006PS) for 7 days and control cells as well as MPs stimulated with polyIC and CpGs for 6 days and restimulated for another 24 h with Gardiquimod. The Gardiquimod-stimulated samples were hybridized to a common reference – the day 7 non-stimulated samples. The other sampels were compared with the control sample harvested after 24 hours of culture.