The present study demonstrates a critical role for chemokine receptor Ccr2 on Ly6chi
inflammatory monocytes for accumulation of these cells in the brain in a mouse model of WNV encephalitis. This extends to the molecular level previous work showing that monocytes accumulate in the brains of WNV-infected mice and humans and are important for survival in the mouse model (2
). Consistent with this, we found that Ccr2-deficiency resulted in markedly decreased survival and increased viral load in the brains after WNV infection of mice.
The detailed leukocyte dynamics that we defined in the blood and brain of WNV-infected mice allow important new insights into the role of Ccr2 in WNV pathogenesis, based on several unexpected findings. First, although WNV induces a multifocal mixed leukocyte encephalitis, leukocyte deficiency in the brain in WNV-infected Ccr2 knockout mice appears to be restricted exclusively to monocytes (~90% reduction). This suggests that Ccr2 is most important in monocyte trafficking in mouse brain, however additional work will be needed to define whether there is also a deficiency of T cell subsets and other leukocyte subsets known to express Ccr2, such as highly differentiated Th1 cells that are compensated by subsets that do not express the receptor.
Second, although Ccr2 ligands Ccl2, Ccl7 and Ccl12 are strongly induced by WNV in the brain at the RNA and protein levels, Ccr2 appears to be dispensable for trafficking of monocytes from the blood to the brain of WNV-infected mice. Instead, the driving force appears to be a highly selective, WNV-induced, and entirely Ccr2-dependent monocytosis that precedes accumulation of monocytes in the brain. This conclusion is based in part on competitive repopulation experiments performed using differentially-labeled Ccr2+/+ and Ccr2−/− donor monocytes transferred to monocytopenic WNV-infected Ccr2−/− mice. These studies also suggest that monocytes lacking Ccr2 may traffic back to the bone marrow and preferentially accumulate, unable to egress. Thus the mechanism of monocytopenia in uninfected Ccr2−/− mice may involve both decreased monocyte egress from bone marrow to blood and increased monocyte return from blood to bone marrow, among other possibilities.
Because CCR2 appears not to be involved in the migration of monocytes from blood into the CNS, this suggests that another chemokine receptor may be involved at this level. Our previous studies have shown a dominant role of chemokine receptor CCR5 in migration of monocytes, T cells, and NK cells into the CNS (REF GLASS). However, our data suggest that CCR2 is involved very early after infection with WNV, increasing monocyte numbers in the periphery. Once monocytes are in circulation, it appears that another receptor is critical for their migration into the infected CNS, perhaps CCR5. To address this question, a similar competitive repopulation analysis to the one we conducted in could be performed using monocytes derived from Ccr5+/+ and Ccr5−/− mice.
We are unaware of any other infectious agent that induces a selective monocytosis in any host. As with WNV, other infectious agents have been reported that fail to overcome the monocytopenia in Ccr2-deficient mice, but in these diseases a specific monocytosis has not been described as a typical feature. Additional work will be needed to define whether Ccr2 and its ligands are needed for trafficking, organization and/or activation of monocytes within the brain, once the cells have crossed the blood brain barrier. Although previous work has shown that both Ccl2 and Ccl7 are important determinants of the normal monocyte count in unstressed wild type mice, additional work will be needed to define which Ccr2 ligands induce monocytosis in WNV-infected mice.
Although previous studies have shown that monocytes play an important role in controlling WNV infection in mice, their precise mechanism of action has not been defined and may vary depending on the model. One study using a lethal intranasal model has suggested that monocytes may play a pathogenic role, since delaying migration of these cells prolonged survival. The mechanism may involve macrophage/microglial cell production of substances potentially toxic to tissue, such as superoxide anion, nitric oxide, and pro-inflammatory cytokines (26
). In contrast, a second study using a model very similar to ours showed that monocytes may be protective, since depletion using clodronate-loaded liposomes decreased survival of the infected mice (15
). This is also plausible, since a beneficial role of macrophages/microglia in CNS recovery has also been demonstrated through the production of neurotrophic factors, removal of debris, and axonal regeneration (28
). Future approaches for defining functional roles of monocytes in controlling WNV infection, once they reach the CNS, may include 1) histopathologic studies of the correspondence of virus, macrophages and neurons in vivo
, 2) ex vivo
studies of expression of pro-inflammatory cytokines and effector molecules in monocytes and monocyte-derived cells (macrophages and microglia) purified from infected brain, 3) immunophenotyping the cells that produce Ccr2 ligands after viral entry into the CNS, and 4) in vitro
studies of monocyte/macrophage interaction with virally-infected neurons.
At present, there are no data delineating leukocyte dynamics in patients with WNV infection. Our data suggest that factors limiting monocytosis could be associated with poor outcome, and that novel therapeutics able to induce monocytosis, such as GM-CSF and AMD3100 (a CXCR4 antagonist), could be useful in such patients. AMD3100 is currently approved for stem cell mobilization, and can also mobilize monocytes in both humans (32
) and in mice (33
). In this regard, a study by Klein and colleagues showed that AMD3100 can significantly improve survival from WNV infection in mice, although the mechanism of leukocytosis has not been defined (12
Monocytosis could also potentially be induced in humans using agonists to CCR2, since the ‘inflammatory’ monocyte subset in humans (CD14+
monocytes) that is analogous to the Ccr2+
mouse monocyte subset that we studied also uniformly expresses CCR2 (16
). At present, CCR2 agonists are not available clinically and are in general limited to chemokine ligands which have very short half-lives. Unless modified, CCR2 ligands may not be ideal for such an application. Conversely, CCR2 antagonists, which are currently being developed by the pharmaceutical industry with anticipated long-term administration for chronic inflammatory diseases, could potentially predispose to poor outcomes in WNV-infected patients.
In previous work, we have identified two genetic determinants of WNV susceptibility in humans, homozygous CCR5Δ32
) and OAS1 rs10774671
). These mutations were tested as candidates based on work we performed with the corresponding mouse genes in mouse models of WNV (35
). A similar genetic approach may also be available for translating our findings on Ccr2 to human WNV disease, since several common polymorphisms have been described for CCR2, CCL2 and CCL7 (37
). Understanding the mechanisms of Ccr2 action in monocyte migration during WNV infection in mice is an important first step towards these goals.
In conclusion, our data identify Ccr2 as a critical protective factor in encephalitis caused by WNV in a mouse model. Our data show for the first time that chemokine receptors may function at an entirely different migratory step than previously shown for WNV infection, and that early events following infection may affect outcome of WNV encephalitis. Further studies are needed to investigate the role of Ccr2 ligands, and to further understand the functional role of monocytes in the CNS.