The present study provides insight into how poxviruses are sensed by murine pDCs, a specialized subset of DCs that are important for type I IFN induction and antiviral immunity. We show that myxoma virus induces type I IFN, TNF, and IL-12p70 in murine pDCs and that the induction is dependent on TLR9/MyD88. The transcription factors IRF5 and IRF7 are required for this induction. Furthermore, the type I IFN feedback loop is critical for the induction, as IFNAR1-deficient pDCs fail to produce type I IFN in response to myxoma virus infection. In contrast, vaccinia virus infection of murine pDCs fails to induce type I IFN. Our results demonstrate that myxoma virus infection can be sensed in murine pDCs by endosomal TLR9 and induces type I IFN and IL-12p70 production through a PI3K/Akt-dependent signaling pathway. These findings may partly explain why myxoma virus is nonpathogenic in mice, whereas systemic infection with vaccinia virus can be lethal in either immunocompetent or immunodeficient mice.
Wang et al. (49
) demonstrated that type I IFN induction by myxoma virus in primary mouse embryonic fibroblasts (MEFs) mediates cellular restriction of myxoma virus replication in these cells. Myxoma virus infection becomes permissive in cultured WT MEFs in the presence of neutralizing antibodies to type I IFNs or in IFNAR1- or Stat1-deficient MEFs. Furthermore, myxoma virus causes lethality in Stat1−/−
mice in an intracranial infection model, highlighting the importance of the IFN-Stat1 pathway in host defense against poxvirus infection. How myxoma virus is initially sensed by primary MEFs to induce type I IFN production is currently unknown. It has been reported that myxoma virus infection in primary human macrophages induces type I IFN through a cytosolic RNA sensing pathway mediated by RIG-I (50
), but the inherent diversity of operational pathogen sensors that exist between cells of different lineages suggests caution in extrapolating these results to other cell types.
In this study, we focused our attention on murine pDCs because of their potency in the induction of type I IFN in response to virus infection. Both human and murine pDCs utilize endosomal TLR7 and TLR9 to sense ssRNA and viral DNAs. TLR9/MyD88 is important for the induction of type I IFN in pDCs in response to herpes simplex virus 1 and 2 (HSV-1 and HSV-2) (24
). TLR9/MyD88 is also required for the induction of cytokine responses in pDCs and NK cell activation in response to mouse cytomegalovirus (MCMV) infection, and it is essential for host defenses against MCMV infection (25
). Ectromelia virus (the causative agent of mousepox) activates murine pDCs through TLR9 (35
). Mice with TLR9 deficiency are more susceptible to ectromelia virus infection (35
). Here we show that the induction of type I IFN in murine pDCs by myxoma virus requires TLR9/MyD88. TLR7 is critical for sensing RNA viruses such as vesicular stomatitis virus and influenza virus in pDCs (13
). It also plays a compensatory role in host defense against MCMV in vivo
). Our results demonstrate that TLR7 plays a minor role in sensing myxoma virus in murine pDCs in vitro
Type I IFN is a critical mediator of antiviral innate immunity. The interferon regulator factors IRF3, IRF5, and IRF7 play important roles in the regulation of type I IFN genes. IRF3 is constitutively expressed in all cell types, whereas the constitutive expression of IRF7 is restricted to cells of lymphoid origin but can be induced in most cell types by type I IFN (3
). IRF3 is required for type I IFN induction triggered by TLR3/TLR4, cytosolic RNA sensing mechanisms mediated by RIG-I/MDA5/MAVS, or cytosolic DNA sensing pathway in many cell types, including cDCs, but it is not required for type I IFN induction in pDCs (8
). It has been proposed that IRF3 plays essential roles in both early and late phases of IFN-α/β gene induction, whereas IRF7 is more important for the late induction phase (28
). IRF7 has been shown to form complexes with MyD88 and TRAF6 upon TLR7/TLR9 stimulation (23
IRF5 can be activated in response to viral infections, including Newcastle disease virus, VSV, and HSV-1, but not Sendai virus (4
). In the initial report on IRF5−/−
mice by Takaoka et al. (46
), IFN-α induction was not affected in the pDCs from IRF5−/−
mice in response to CpG stimulation, whereas the induction of proinflammatory cytokines was impaired (46
). Subsequent studies from the same group showed that IRF5−/−
mice are more susceptible to VSV or HSV-1 infections. The IRF5−/−
mice had reduced serum levels of type I IFN and IL-6 after viral infections (54
). Paun et al. (33
) reported that IRF5−/−
mice are more susceptible to NDV infection, and the serum levels of IFN-α, TNF, and IL-6 were lower in IRF5−/−
mice than in WT mice in response to NDV infection. IRF5 has also been shown to be a central mediator of TLR7 signaling (38
). Here we show that both IRF5 and IRF7 are required for the induction of IFN-α/β and IL-12p70 in murine pDCs infected by myxoma virus, whereas IRF3 is dispensable for the IFN induction. This is consistent with our model that myxoma virus infection of pDCs leads to the detection of viral DNA by TLR9 and the activation of transcription factors IRF5 and IRF7 via MyD88, TRAF6, and other associated factors and results in the induction of type I IFN, TNF, and IL-12p70, which are the critical mediators of innate immunity.
The role of the type I IFN positive-feedback loop in the induction of type I IFN in pDCs in response to TLR stimulation or viral infection is not well understood. Sato et al. (36
) reported that the induction of IRF7 mRNA in mouse embryonic fibroblasts in response to Newcastle disease virus (NDV) was dependent on type I IFN receptor. Marié et al. (31
) reported that although an immediate-early response gene (IFNA4
) was induced by NDV in fibroblasts in the absence of the type I IFN positive-feedback loop, secondary induction of other IFN-α subtypes was impaired in the absence of Stat1 or IFNAR. They also reported that ectopic expression of IRF7 in fibroblasts led to the induction of the secondary IFN-α subtypes. Conventional DCs produced type I IFN and IL-12p70 in response to TLR stimulation in a Stat1- and IFNAR-dependent manner (15
). The type I IFN positive-feedback loop is also important for pDC activation in vivo
and IFN induction in vitro
in response to CpG stimulation or viral infection (2
). We observed that IFNAR1 is required for the induction of both IFN-α/β and IL-12p70 in pDCs by myxoma virus, supporting the important role of the IFN positive-feedback loop in the induction and sustaining of antiviral innate immune responses. Although IRF7 is expressed in pDCs, type I IFN might be necessary for the enhancement of IRF7 levels.
It is unclear how myxoma viral DNA might be sensed by endosomally localized TLRs in pDCs. Poxviruses enter host cells through fusion with the plasma membrane via a large entry/fusion complex or via a low-pH-dependent endosomal pathway and subsequent fusion with endosomal membrane to release the virion cores into the cytoplasm (39
). Because most of the viral entry studies were conducted with vaccinia virus in various transformed cell lines, little is known about how myxoma virus enters primary pDCs. If the basic mechanisms of viral entry are preserved among different poxviruses, we presume that myxoma virion cores are released into the cytoplasm shortly after viral entry. Some of the virion cores might then be transported to the endosomal/lysosomal compartments where viral DNAs are released and detected by TLR9. Alternatively, the cores are uncoated in the cytoplasm and some of the released viral DNA is then taken up and transported to the endosomes. Our findings that treatment of pDCs 1 h postinfection with an endosomal/lysosomal acidification inhibitor, chloroquine, blocks type I IFN and IL-12p70 induction indicate that endosomal/lysosomal processing of virions might be important for the detection of viral DNAs through TLR9. Alternatively, low pH in the endosomes/lysosomes might be important for optimal ligand and receptor interactions. It has been reported that during VSV infection of pDCs, autophagy plays an important role in the detection of cytosolic viral replication intermediates by TLR7 (27
). We also observed that treatment of pDCs 1 h postinfection with the PI3K inhibitors wortmannin and 3-MA blocked the induction of type I IFN and IL-12p70. Wortmannin and 3-MA have been used widely as inhibitors of autophagy. Further investigations on the role of autophagy in poxvirus sensing in pDCs is warranted.
Vaccinia virus E3 is a key viral immunomodulator that inhibits type I IFN induction in host cells (12
). E3 has two distinct domains, the N-terminal Z-DNA/RNA binding domain and the C-terminal dsRNA binding domain. Infection of murine keratinocytes with ΔE3L or E3LΔ26C vaccinia virus but not with WT vaccinia virus or the E3LΔ83N mutant virus induces IFN-β and related cytokine and chemokine production in a MAVS/IRF3-dependent manner (12
; P. Dai and L. Deng, unpublished data). This induction effect is dependent on viral DNA replication, indicating that cytosolic dsRNA produced postreplicatively is the trigger for the induction of innate immune responses but is targeted by the dsRNA binding domain of E3 (12
). Here we show ΔE3L or E3LΔ26C vaccinia virus infection of murine pDCs fails to induce type I IFN, TNF, or IL-12p70 and is less capable of inhibiting type I IFN induction by myxoma virus infection or CpG stimulation than WT or E3LΔ83N vaccinia virus, implying that ΔE3L vaccinia virus infection fails to produce activators in pDCs and the Z-DNA/RNA binding domain of E3 mediates inhibition of type I IFN induction in pDCs by myxoma virus or CpG. These results indicate that both domains of vaccinia virus E3 function to block innate immune responses in a cell-type-specific fashion. Significantly, the E3 ortholog expressed by myxoma virus, M029, has an intact C-terminal dsRNA binding domain but lacks the N-terminal Z-DNA/RNA binding domain. This difference may partly explain the immune activating property of myxoma virus.
A recent study by Delaloye et al. (10
) investigated the immune sensing mechanism of modified vaccinia Ankara virus (MVA) in macrophages that revealed that multiple viral sensing pathways mediate the induction of type I IFN and proinflammatory cytokine induction (10
). MVA failed to induce type I IFN induction in purified murine or human pDCs but induced type I IFN and proinflammatory cytokine and chemokine secretion in cDCs (data not shown). It is possible that MVA produces an inhibitor(s) that blocks poxviral sensing in pDCs. The apparent differences in the induction of type I IFN in pDCs and cDCs between myxoma virus and MVA are interesting and warrant further investigation.
In conclusion, this study provides a molecular and genetic basis of how poxvirus sensing is mediated in pDCs. We demonstrate that induction of type I IFN and proinflammatory cytokines in murine pDCs by myxoma virus is mediated by TLR9/MyD88. In addition to IRF7, transcription factor IRF5 is also required for this induction. We also reveal an important role of the N-terminal Z-DNA/RNA binding domain of vaccinia virus E3 in attenuating poxvirus sensing and TLR9 signaling in pDCs. Our findings that drug inhibitors of PI3K/Akt signaling pathway block type I IFN and IL-12p70 induction suggest that PI3K/Akt might be involved in poxvirus sensing in pDCs. The availability of mice with the TLR and RIG-I-like receptor (RLR) sensing pathways knocked out and the feasibility of generating various primary cell types using bone marrow cells in the in vitro
cell culture system allow us to define the role of various sensors and adaptors in innate immune responses to poxvirus infection. Once these are clearly defined, the roles of these relevant innate immune sensors in host defense against poxvirus need to be examined using in vivo
infection models (21
). These studies will aid in developing the next generations of poxvirus platforms best suited for either oncolytic virotherapy, where the suppression of innate antiviral immune pathways is often desired in order to increase the lifetime of the oncolytic virus in tumors, or vaccine agendas, where the robust induction of early innate cytokines by DCs is most beneficial in obtaining maximal immunogenicity of the desired immunogen.