In this paper, we have characterized the mechanisms of viral control in CHIKV infection and we demonstrate a critical role for type I IFNs. We provide evidence in infected human subjects that CHIKV infection triggers the production of type I IFNs (). It has previously been reported that CHIKV does not directly infect primary leukocytes (Sourisseau et al., 2007
). Nonetheless, we expected that an ssRNA virus would be capable of directly activating hematopoietic cells. Remarkably, our in vitro stimulation of human PBMC, as well as human and mouse DC subsets, indicates that CHIKV is not capable of directly engaging PRRs for the induction of type I IFNs ( and data not depicted). Instead, we suggest that infected fibroblasts are the source of type I IFN. This conclusion is based on in vitro experiments and investigation of the injection site of WT animals ( and ). The use of defined knockout mice allowed us to further characterize the production and target of type I IFNs in CHIKV pathogenesis. Specifically, we report that fibroblast production of type I IFNs is under the control of Cardif (). Furthermore, we demonstrate that IFN acts on nonhematopoietic cells to achieve viral clearance ().
Although these data suggest a central role for fibroblasts in the production and reception of IFN signals, the host response to CHIKV is more complex. Notably, adult Cardif−/− mice infected with CHIKV had only a subtle phenotype. In addition to a role for the RLR pathway, we find that mice lacking MyD88 also became viremic (). Alone, neither of the pathways account for the phenotype observed in the IFNAR−/− mice, suggesting that there is cooperation between RLR and TLR recognition, acting together to mediate rapid clearance of CHIKV.
Based on the tissue tropism (), we argue that Cardif is engaged as a result of fibroblast and stromal cell infection. We attempted to define the upstream sensor, namely RIG-I or Mda5. As reported for the Cardif−/−
, neither of the sensor-deficient animals had a strong phenotype, although both demonstrated increased viral load in selected tissues as compared with control animals (). One caveat is that the RIG-I and Mda5 knockouts are of a mixed ICR/C57BL/6/129 background as a result of problems of lethality (Kato et al., 2006
). In contrast to inbred animals used in our studies, the outbred WT controls showed CHIKV replication in the joint, making the data in the knockout strains harder to interpret. Additionally, MEFs isolated from these animals showed high variability, indicating that other genetic loci may be impinging on the Cardif-mediated sensor pathways (Fig. S1). Based on these observations, we suggest that there is a shared role for both RIG-I and Mda5, as has been reported for West Nile virus (Fredericksen et al., 2008
). In addition to these two sensors, it will also be important to explore a role for PKR and NOD2, both of which have now been implicated in the response to ssRNA viruses and may signal via Cardif (Ryman and Klimstra, 2008
; Barry et al., 2009
; Sabbah et al., 2009
Regarding the role for MyD88, two possible pathways may account for its role in the control of CHIKV infection. As previously stated, hematopoietic cells are not directly stimulated, suggesting that CHIKV does not engage TLRs in a conventional manner. There is, however, the possibility that endosomal TLRs are engaged as a result of hematopoietic cells phagocytosing infected cells, the latter being a source of viral PAMPs. For example, cells infected by SFV, also a member of the Alphavirus
genus, results in the generation of dsRNA that may engage TLR3 on CD8+
DCs upon engulfment (Schulz et al., 2005
). Although TLR3 utilizes TRIF and thus does not help explain the phenotype in MyD88−/−
mice, this mechanism of PAMP transfer may generalize for other host sensors, and future studies will explore a role for TLR7 or TLR8. A second pathway for engaging MyD88 concerns its role as an adaptor of the IL-1β and IL18 receptors (Muzio et al., 1997
). There has been a surge of new information regarding the role of the inflammasome, which is well recognized as critical for IL-1β production after bacterial infection. Indeed, this pathway also seems to be participating in the control of viruses. Specifically, IL-1R activation enhances survival in a model of influenza infection (Schmitz et al., 2005
). Supporting this observation, mice deficient in Caspase-1, ASC, and NALP3 have been shown to have a defect in the control of influenza (Ichinohe et al., 2009
). As such, IL-1β or IL18 produced by CHIKV-infected cells may participate in viral control by stimulating noninfected cells in a MyD88-dependant manner.
Regarding the role of type I IFNs, we provide evidence that IFNAR signaling in radioresistant stromal cells is critical for clearance of CHIKV. This was demonstrated using WT→IFNAR−/−
bone marrow chimeras, which died with a similar kinetic as the IFNAR−/−
animals (). Using the IFNAR−/−
→WT chimeras, we also demonstrated that the IFN amplification loop in hematopoietic cells is not required for the control of CHIKV dissemination. To our knowledge, there is only one prior study that has taken a similar approach. In the case of reovirus infection, it is known that infection kills IFNAR−/−
, and the gut epithelium, but not hematopoietic, cells are the target of infection (Fleeton et al., 2004
). Distinguishing our findings with CHIKV, it is interesting that Johansson et al. (2007)
find that IFNAR−/−
→WT succumb to infection, whereas WT→IFNAR−/−
chimeras control reovirus. Although we fully acknowledge that IFN may be acting as both a direct antiviral and a proimmune cytokine in both cases, the former seems to be critical in CHIKV infection, whereas the latter is required for clearance of reovirus.
In addition to the importance of studying CHIKV pathogenesis because of the threat to public health, from a fundamental level, we have identified features that make CHIKV unique as compared with other alphaviruses. Notably, Ross River, Sindbis, SFV, and Venezuelan Equine Encephalitis are known to infect and stimulate DCs (Hidmark et al., 2005
; Ryman et al., 2005
; Shabman et al., 2007
). It should be noted that human and mouse DCs are not similar in their sensitivity to these viruses (Shabman et al., 2007
); however, in the case of CHIKV, neither human nor mouse DCs are directly infected. Recent data would thus indicate that in this regard, CHIKV is more similar to Eastern Equine Encephalitis Virus (Gardner et al., 2008
). Extending it to other arboviruses, we also note that infection in the WT mice failed to demonstrate evidence for direct infection of CD45+
cells, including Langerhans cells, thus distinguishing CHIKV from other vector borne infections such as Dengue (Wu et al., 2000
). This is relevant, as it indicates that the viral sensor as well as the host response in CHIKV pathogenesis may be distinct from other alphaviruses (MacDonald and Johnston, 2000
; Ryman et al., 2005
; Shabman et al., 2007
). Also unique may be the ISG responsible for antiviral activity. For SFV, MxA confers resistance (Landis et al., 1998
), whereas ISG15 appears critical for controlling Sindbis virus (Lenschow et al., 2007
). Future work will be aimed at defining the ISG in fibroblasts that actively controls CHIKV replication.
In sum, we believe our study provides important insight into developing preventive measures and therapeutic strategies for controlling CHIKV infection. Our results also suggest a role for nonhematopoietic cells as important players for IFN production and viral control.