Dissecting cell-intrinsic roles of MyD88 and IFN-I signalling in pDC responses to a viral infection in vivo

Plasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN-I) during viral infections, in response to triggering of endosomal Toll Like Receptors (TLR) 7 or 9 by viral single-stranded RNA or unmethylated CpG DNA, respectively. IFN-I production in pDC occurs in specialized endosomes encompassing preformed signaling complexes of TLR7 or 9 with their adaptor molecule MyD88 and the transcription factor interferon regulatory factor 7 (IRF7). The triggering of TLR leads to IRF7 phosphorylation, nuclear translocation and binding to the promoters of the genes encoding IFN-I to initiate their transcription. pDC express uniquely high levels of IRF7 at steady state and this expression is further enhanced by positive IFN-I feedback signaling during viral infections. However, the specific cell-intrinsic roles of MyD88 versus IFN-I signaling in pDC responses to a viral infection have not been rigorously dissected. To achieve this aim, we generated mixed bone marrow chimera mice (MBMC) allowing to rigorously compare the gene expression profiles of WT versus Ifnar1-KO or MyD88-KO pDC isolated from the same animals at steady state or after infection with the mouse cytomegalovirus (MCMV). Our results indicate that, in vivo during MCMV infection, pDC undergo a major transcriptional reprogramming, under combined instruction of IFN-I, IFN-γ and direct TLR triggering. However, these different stimuli drive specific, largely distinct, gene expression programs. We rigorously determined which gene modules require cell-intrinsic IFN-I signaling for their induction in pDC during a physiological viral infection in vivo. We delineated non-redundant versus shared versus antagonistic responses with IFN-γ. We demonstrated that cell-intrinsic IFN-I responsiveness is dispensable for induction of the expression of all IFN-I/III genes and many cytokines or chemokines in pDC during MCMV infection, contrary to MyD88 signaling. Recipient CD45.1 mice were lethally irradiated and reconstituted with a 1:1 mixture of BM cells from WT CD45.1 and Ifnar1-KO or MyD88-KO CD45.2 donor animals, to generate test mice (TST MBMC). Congenic CD45.1+ mice differ from the CD45.2+ C57BL/6 strain for several gene polymorphisms and immune parameters (Waterstrat et al., 2010). Therefore, as controls, we included mice engrafted with a mixture of WT CD45.1 and WT CD45.2 BM cells (CTR MBMC). MBMC were infected with MCMV at least 8 weeks after BM graft. Their splenic pDC were isolated 1.5 days after infection, at the peak of IFN-I production, sorted into CD45.1+ versus CD45.2+ populations, and used for pangenomic gene expression profiling by microarrays. Cells from regular, uninfected or d1.5 MCMV-infected, C57BL/6 mice were added as controls to ensure lack of major impact on pDC responses to in vivo MCMV infection of the bone marrow graft.