Dissecting the molecular programs governing interferon production by plasmacytoid dendritic cells during a viral infection in vivo

Plasmacytoid dendritic cells (pDC) are the major source of type I and type III interferons (IFN-I/III) during viral infections, in response to triggering of endosomal Toll Like Receptors (TLRs) 7 or 9 by viral single-stranded RNA or unmethylated CpG DNA, respectively. Interestingly, this function is restricted to a minor fraction of pDC (Zucchini et al. Int. Immunol. 2008). In this project, we aimed at identifying the molecular pathways involved in inducing IFN-I/III production in this minor faction of pDC during in vivo infection by the mouse cytomegalovirus (MCMV). To achive this goal, we infected with MCMV Ifnb1Eyfp mice, in which IFN-producing pDC can be detected by YFP expression (Scheu et al. PNAS 2008). Thanks to this model, we were able to sort three distinct subsets of pDC: CD86-YFP- (not activated, non IFN-producing), CD86+YFP- (activated, non IFN-producing) and CD86+YFP+ (activated, IFN-producing) and to perform microarray analysis. This allowed us to select genes differentially expressed among these three subsets and to mine these data in order to identify the related signaling pathways. Ifnb1Eyfp mice were infected or not with MCMV. Splenic pDC were isolated 36 hours after infection. The following pDC subsets were sorted: CD86-YFP-; CD86+YFP- and CD86+YFP+. The last subset was sorted exclusively from infected mice, while the other two were sorted from both uninfected and infected mice. We performed two independent sortings by pooling splenocytes of 4 (uninfected) or 10-12 (infected) Ifnb1Eyfp mice for each sorting. For each subset we sorted 10,000 cells.