This study provides a detailed analysis of the molecular and cellular immune response in the brain during WNV infection and demonstrates that expression of the chemokine receptor CCR5 is crucial for viral clearance and survival in a mouse model of disease. WNV induced production of all three CCR5 ligands in the brain, with particularly high and durable induction of CCL5, which is consistent with local accumulation of CCR5-expressing NK cells, macrophages, and CD4+
T lymphocytes that we observed in the model. Several lines of evidence lead us to believe that trafficking of leukocytes expressing CCR5 to the WNV-infected mouse brain is critical for survival. First, genetic disruption of CCR5 markedly reduced accumulation of these cell types in brains of WNV-infected mice, without significantly affecting CNS expression of other immunoregulatory factors, including the CCR5 ligands CCL3, CCL4, and CCL5. Second, there was no defect in clearance of the virus from the spleen in CCR5−/−
mice. Third, WNV-induced production of IFN-γ by CD4+
T cells in the spleen was identical for CCR5+/+
mice. Fourth, when splenocytes from WNV-infected CCR5+/+
mice were adoptively transferred into WNV-infected CCR5−/−
mice, we found much greater leukocyte accumulation in the brain after CCR5+/+
than after CCR5−/−
cell transfer and detected CCR5+
leukocytes in the brains of CCR5−/−
recipients. This experiment, which involves direct injection of WNV-activated CCR5+/+
splenocytes into the blood, bypasses the splenic egress step, and suggests strongly, when coupled with unaltered splenic T cell or viral clearance responses in CCR5−/−
mice, that CCR5 functions by promoting trafficking of leukocytes from the blood to the brain in response to WNV infection, for the purpose of containing and clearing the virus. This is consistent with previously published work on WNV which demonstrated that RAG−/−
mice have dramatically increased CNS viral burden and succumb to infection within 12 d, the same time frame that we found for CCR5−/−
). Furthermore, mice lacking CD8+
T cells also have increased viral burden in the CNS and mortality when infected with WNV (13
). Shirato et al. have recently reported an increased expression of mRNA for CCR5 ligands when mice were infected i.p. with a lethal versus nonlethal strain of WNV (14
). Our data confirm and extend these results in a distinct model by providing mRNA and protein analysis for these and numerous other immunoregulatory molecules and by directly testing the importance and role of CCR5.
The critical role of CCR5 in WNV pathogenesis appears to be unique compared with other neurotropic viruses that have been tested to date. CNS infection with lymphocytic choriomeningitis virus results in equivalent viral burden and mortality in CCR5−/−
mice suggesting that this receptor does not play a role in pathogenesis in this model (26
). Likewise, infection with the neurovirulent retrovirus FR98 results in identical mortality rates and CNS viral burden in CCR5−/−
). Infection with a neurovirulent strain of mouse hepatitis virus (MHV) results in similar viral titers in the CNS of CCR5−/−
mice, but knockouts have reduced demyelination, the major manifestation of disease in this model. The mechanism involves CCR5-dependent recruitment of macrophages and CD4+
T cells to the CNS (28
). Thus, CCR5 promotes demyelinating disease in MHV infection, not antiviral host defense as it does in WNV infection. CCR5 has also been demonstrated to regulate leukocyte trafficking to the brain, but not the lung, after Cryptococcus neoformans
The importance of CCR5 also varies among noninfectious, immunologically mediated, CNS disease. CCR5 deficiency has no effect on experimental autoimmune encephalitis (EAE), despite the fact that CCR5 ligands are highly expressed in the CNS of mice with EAE (30
). In addition, wild-type and CCR5-deficient mice have similar outcomes in experimental autoimmune neuritis, including similar levels of leukocytes recruited to the cauda equina (31
). These examples show the potential for chemokine receptors to play specific and important roles or more redundant roles depending on the disease context, and the spatial, temporal, and quantitative details of cognate ligand expression.
Our data raise new questions regarding whether CCR5 may play a more general role in the immune response to other flaviviruses. In this regard, in a recent study of 10 cytokines in patients infected with Japanese Encephalitis virus (the prototype flavivirus), increased serum CCL5 alone was associated with a fatal outcome (32
). The mechanism was not established, but could conceivably involve desensitization of leukocyte CCR5 in the bloodstream, thereby preventing trafficking to the infected CNS.
We found that CCR1, CCR2, CXCR3, and CX3
CR1 and their ligands were also up-regulated in mouse brains by WNV. These systems could work cooperatively with CCR5 and could explain why leukocytes accumulate in small numbers in the CNS of WNV-infected CCR5−/−
mice and why many of the leukocytes in the WNV-infected brain are CCR5 negative. Induction of CXCR3 was particularly strong, and is consistent with induction of a Th1-polarized cytokine response in the brain (18
). However, our results using knockout mice do not support an important, nonredundant role for CCR1 or CX3
CR1 in this model. Our gene and protein expression data also suggest new and testable hypotheses regarding the role in control of WNV infection of TNF-α, the IL-12/IFN-γ axis, IFN-α, the leukocyte adhesion molecule ICAM-1, the matrix metalloproteinase MMP-9, and the metalloproteinase inhibitor TIMP-1 (33
). WNV has been reported to be very sensitive to both exogenous type I and II interferons in vitro (34
), but they are only able to prevent infection in vivo in mice when given before challenge (35
). IFN-α may be even less effective in humans because infection with WNV has been reported in patients actively being treated with IFN-α and ribavirin for hepatitis C (36
). Matrix metalloproteinases may be important in disrupting the blood brain barrier to allow hematogenous spread of WNV to the CNS, as has been previously suggested (19
). Although the role of TNF-α in the brain is unclear, recent work has clarified its importance in the periphery where it is produced in a TLR3-dependent manner and mediates permeabilization of the blood brain barrier to facilitate WNV entry (12
). The specific roles in pathogenesis of these and other factors induced in the brain by WNV are currently under investigation.
An important question raised by our findings is whether CCR5 is also protective in WNV infection of humans. In limited studies, T cells and macrophages have been reported to accumulate in WNV-infected human brains as they do in our mouse model (25
). Moreover, like humans, the mouse is easily infected with WNV, and the major clinical manifestations of disease are similar in both species: encephalitis and mortality in a subset of infected individuals. If CCR5 is protective in humans, individuals homozygous for the CCR5Δ32 mutation, who lack functional CCR5 and include 1% of North American Caucasians (38
), may be at greater risk for fatal encephalitis from WNV infection. A related issue is the safety of CCR5 blocking agents under development for patients with HIV/AIDS who become infected with WNV.
In conclusion, our data identify CCR5 as a critical protective factor in fatal encephalitis caused by WNV in a mouse model. The effect is highly specific relative to other neurotropic viruses and at least two other relevant chemokine receptors. Loss of CCR5 results in decreased ability to recruit/maintain leukocytes into the infected CNS where they may function to clear the virus. The data also raise important new questions about the potential roles of other immunoregulatory systems that are induced by WNV in this model and in man.