Sequential Activation of Two Pathogen-Sensing Pathways Required for Type I Interferon Expression and Resistance to an Acute DNA Virus Infection

Abstract

Summary Toll Like Receptor 9 (TLR9), its adapter MyD88, the downstream transcription factor interferon regulatory factor 7 (IRF7) and type I interferons (IFN-I) are all required for resistance to infection with ectromelia virus (ECTV). However, it is not known how or in which cells these effectors function to promote survival. Here, we showed that after infection with ECTV, the TLR9-MyD88-IRF7 pathway was necessary in CD11c+ cells for the expression of proinflammatory cytokines and the recruitment of inflammatory monocytes (iMo) to the draining lymph node (D-LN). In the D-LN, the major producers of IFN-I were infected iMo, which used the DNA sensor-adapter STING to activate IRF7 and nuclear factor κB (NF-κB) signaling to induce the expression of IFNα and IFNβ, respectively. Thus, in vivo, two pathways of DNA pathogen sensing act sequentially in two distinct cell types to orchestrate resistance to a viral disease.

Migratory Dendritic Cells, Group 1 Innate Lymphoid Cells, and Inflammatory Monocytes Collaborate to Recruit NK Cells to the Virus-Infected Lymph Node

“…When we sorted by flow cytometry at 2.5 dpi from pooled dLNs (Figure 5A), we found that iMo (CD3 − , B220 − , NK1.1 − MHC II + CD11c + CD11b + ) and to a lesser extent classical DC (CD3 − , B220 − , NK1.1 − MHC II + CD11c + CD11b − ) but not total lymphocytes (stained with a cocktail of cascade blue-labeled Abs to CD3e, B220 and NK1.1) transcribed CXCL9 (Figure 5B) and also CXCL10 (data not shown). Similarly, we found that iMo (CD11c + , CD11b + ) and to a lesser degree DC (CD11c + CD11b − ) specifically stained intracellularly with CXCL9 mAb in the dLN at 2.5 dpi but not in the uLN (Figure 5C and Figure S2A) (note that we classified the cells in the CD11c + , CD11b + gate in the dLN plots as iMo, based on our previous extensive analysis of this population (Xu et al, 2015) using markers for DC and monocytic lineages as described by publications from the Immunolgical Genomic consortium (Gautier et al, 2012; Miller et al, 2012) and by morphology of sorted cells at the microscope). Given the post-infection excess of iMo over DC in the dLN, we conclude that iMo are the major producers of CXCL9 in the dLN and to a lesser extent, DC.…”

“…Note that in this case, where we used pooled LNs, there were significant differences in the levels of Cxcl9 expression between ndLN and dLN (presort) from Tlr9 −/− and Itgax-Cre + Tlr9 fl/fl mice. This suggest that the absence of a significant increase in CXCL9 in the dLN of Tlr9 −/− and Myd88 −/− mice by RT-qPCR in Figures 3C was likely due to insufficient statistical power for an experiment where there was intra-group variability, in which the n (=4) was small, and where inter-group differences were minor likely due to the very small number of iMo present in the dLN of these mice, which is 10-20 times lower than in WT mice (Xu et al, 2015). Together, these experiments show that iMo do not need autonomous TLR9 or MyD88 to produce CXCL9, and that the low amount of CXCL9 in the dLN of mice with global or CD11c + cell-restricted MyD88/TLR9 deficiency is due to their inability to efficiently recruit iMo into the dLN.…”

“…We concluded that the recruitment of NK cells to the dLN requires MyD88 and TLR9 in CD11c + cells of hematopoietic origin, suggesting that TLR9 and MyD88 must be expressed by some type of dendritic cell (DC). Importantly, we have previously shown that Vav1-Cre Myd88 fl/fl or - Tlr9 fl/fl and Itgax-Cre Myd88 fl/fl or - tlr9 fl/fl mice, which succumb to mousepox due to unrestrained viral replication, also fail to recruit iMo to the dLN (Xu et al, 2015). This suggests that the recruitment of iMo and NK cells to the dLN could be mechanistically linked.…”

“…We have previously shown that ECTV infection targets iMo (Xu et al, 2015). When we used ECTV-GFP, we found that uninfected (GFP − ), rather than infected (GFP + ) iMo predominantly produced CXCL9 (Figure 5E).…”

“…CD11c + cells (presumably skin-derived mDC) require TLR9 and MyD88 to express CCR2-binding chemokines, which are necessary for the recruitment of inflammatory monocytes (iMo) to the dLN. These recruited iMo are major targets of ECTV infection and, when infected, produce Type I interferon (IFN-I) in the dLN (Xu et al, 2015). Given that both, NK cells and iMo are recruited to the dLN soon after infection and are critical for anti-viral defense, we investigated possible shared mechanisms for their recruitment and whether there is cross-talk between iMo, NK cells, and mDCs.…”