In the present work, we showed that CCR5-/- mice infected with HSV-1 virus present an increased inflammatory cell infiltrate in the brain, composed mainly of neutrophils. In comparison with infected WT animals, CCR5-/- mice also presented a reduction in HSV-1 load, while no changes were found in survival rate.
Previous studies have shown that C57BL/6 mice infected with HSV-1 strain EK did not develop encephalitis by peripheral (nasal) route . Therefore, we standardized a model of infection by inoculating the virus HSV-1 strain EK by intracranial route. This route is associated with a severe disease characterized by diffuse meningoencephalitis in contrast with focal lesions determined by nasal or ocular inoculation .
The inoculum of 104 PFU resulted in elevated rates of mortality in both WT and CCR5-/- mice, suggesting that this viral load may be lethal regardless of the immune or inflammatory pathways involved. This prompted us to use a lower inoculum but the mortality rate of both groups remained similar. Interestingly, CCR5-/- mice infected with HSV-1 virus by ocular route also showed no difference in mortality rate when compared to WT mice .
A lower HSV-1 load was observed in CCR5-/- infected mice. This might suggest either that the virus presented a diminished replication rate or that the inflammatory response could be enhancing viral clearance in the brain. Both situations may be ascribed to the change in the type of cells infiltrating the brain. Histopathology and flow cytometry analysis showed an early increase in the number of total cells in the brain of CCR5-/- mice, especially neutrophils. Neutrophils may contribute to virus clearance through the release of antiviral cytokines like TNF-α or oxygen and nitrogen reactive species .
Neutrophils are the first cells to infiltrate the cornea and they remain the predominant cell type during the development of HSV-1 keratitis [24–26]. This neutrophil response is known to both inhibit viral replication and induce corneal lesion [27, 28]. In contrast with the present results, Carr and coworkers  found decreased neutrophil recruitment into the cornea and trigeminal ganglia in the early stage of HSV-1 infection by corneal route in CCR5-/- mice. The number of recruited neutrophils in the cornea became similar in WT and CCR5-/- mice only at day 7 p.i. Interestingly, there was increase in viral load in CCR5-/- mice at day 7 p.i. which was probably secondary to defective neutrophil trafficking during the earlier stages of infection. At the same time, trigeminal ganglia and brainstems of CCR5-/- mice revealed an increase of CD4 and/or CD8 T cells .
In the present work, there was also a significant change in leukocyte recruitment during HSV-1 brain infection. However, in contrast with a previous work , CCR5-/- infected mice showed an increase of neutrophil recruitment in detriment of the CD4 and CD8 T cells recruitment in brain tissue. These conflicting results suggest that there is a significant difference between the immune response elicited in the periphery (i.e. cornea) and the response in the CNS in CCR5-/- mice infected with HSV-1. When the virus is inoculated in peripheral sites, such as cornea, an immune response against HSV-1 can develop in trigeminal ganglia and related lymph nodes. This response can modulate the immune response before HSV-1 reaches the CNS, differing from the current study in which the virus was directly inoculated in the brain. Previous studies involving other knock out mice have also demonstrated conflicting data depending on the route of HSV-1 inoculation. For instance, Lundberg and coworkers  demonstrated that TNF-α antiviral effects are independent of TNFR1 in mice infected with HSV-1 by corneal scarification. When the virus was inoculated in the periphery of C57BL/6 mice, immune response of the host efficiently controlled virus infection even without TNFR1 . Conversely, it was described a significant decrease in the survival rate of TNFR1−/− mice infected by intracranial route . The use of intracranial route is a way to elicit an immune response directly from the CNS, aiming to exclude immune activation in the periphery. In this regard, TNFR1 seemed to be relevant for the control of HSV-1 replication in CNS when there is no activation of the immune system in the periphery .
In contrast to neutrophil increase, the brain of CCR5-/- infected mice showed a reduction of CD8+ cells. CCR5 is a member of the CC family of chemokine receptors that is expressed on a variety of leukocytes, including T cells and macrophages . Accordingly, it would be expected that, without CCR5, recruitment of T cells might be reduced. The number of CD8+ T cells was also decreased in the brain of mouse hepatitis virus-infected CCR5-/- mice as compared to WT mice .
Macrophages, CD4+ and CD8+ T cells are present in perivascular infiltrates close to and in contact with HSV-infected cells in areas of massive myelin loss necrosis in the brainstem of mice with HSV encephalitis . CD8+ T lymphocytes are the first mononuclear cells that cross the blood brain barrier and move toward the infected tissue. The role played by CD8+ lymphocytes in HSV-1 encephalitis is still controversial. While one study reported no significant differences in viral load or viral reactivation when the virus was injected in cornea of mice lacking CD8+ T cells , another report showed persistent elevated viral titers in the brain of CD8-deficient, suggesting a role for CD8+ T cells in the control of HSV-1 replication . In the current study, we found no difference in the levels of CD8+ lymphocytes in CCR5-/- mice, thus, these cells cannot account for the decrease in viral load in the brain of CCR5-/- mice. Following CD8+ T lymphocytes, tissue-penetrating Th1 CD4+ T cells contact local antigen presenting cells. This may result in an up-regulation of MHC molecules and secretion of more chemotactic molecules . The inflammatory site during encephalitis involves the immune cells cited above and also B cells. Mice deficient for B cell had increased susceptibility to HSV-1 after peritoneal infection, presenting increased viral replication in the CNS. Thus, those results evidenced the importance of B cells in the control of viral replication.
Interestingly, treatment with anti-CCL5 or Met-RANTES, an antagonist of the CCL5 receptors CCR1 and CCR5, had no effect on viral titers but significantly decreased the number of leukocytes adherent to the pial microvasculature . CCL5/RANTES is able to bind other chemokine receptors like CCR1 and CCR3, possibly compensating the absence of CCR5 in the trafficking and migration of leukocytes . Using intravital microscopy, we found that CCR5-/- infected mice presented an increase of leukocyte adhesion to the pial microvasculature. Bearing in mind that CCR5 is one of the most important receptors in leukocyte migration, this result seems paradoxical. However, one limitation of this technique is the impossibility to identify the kind of leukocyte that roll and adhere in vessels . It is possible that those leukocytes observed in intravital microscopy are neutrophils.
Here we found an increase in cerebral chemokine levels, including KC/CXCL1, MIG/CXCL9, MCP-1/CCL2, RANTES/CCL5 and cytokine TNF-α in CCR5-/- infected mice. Blocking the action of CCL5 receptor may result in compensatory mechanisms leading to the overproduction of other chemokines . Thapa and coworkers  showed that mice deficient in CCR5 expressed higher levels of TNF-α, KC/CXCL1, MCP-1/CCL2, MIP1-α/CCL3 and RANTES/CCL5 in the brain after intravaginally HSV-2 infection. CCR5-/- mice showed a significant elevation in chemokines MCP-1/CCL2, RANTES/CCL5, MIG/CXCL9 and IP10/CXCL10 in the trigeminal ganglion and brainstem after ocular HSV-1 infection .
The increase in chemokine concentration within the brain was also found in a study addressing the role of CXCR3 in HSV-1 infection. This receptor is expressed predominantly on CD4+ and CD8+ T cells, subsets of NK cells, and peripheral blood B cells. CXCR3-/- mice infected with HSV-1 in the cornea had an increase in CCL5, CXCL10, and IFN-γ in the brainstem and IL-6 in the brain tissue. In addition, CXCR3-/- infected mice exhibited high levels of chemokines which were coincident with high viral titers in the brainstem. The authors suggested that the absence of CXCR3 expression would suppress the capacity of T cells to respond to HSV-1 infection through limited trafficking to the trigeminal ganglia .
In our study, the lack of CCR5 was associated with lower viral load, implicating this receptor in viral multiplication. However, it is not clear whether the virus depends on this receptor to enhance viral multiplication or the immune system mounts a different response in its absence, resulting in more efficient viral control.
Our study has several limitations. We did not measure CCL-3, which is another ligand for CCR5 and could be related to HSV-1 infection. We did not evaluate natural killer cells as well. These cells may be relevant for the control of viral infection as they are putative effectors in the response against virus. Also, future experiments for the evaluation of viral titers and chemokine levels each day after infection are warranted. There is a possibility that compensatory mechanisms could eventually impact the immune-associated phenotype of CCR5-/- mice. Nevertheless, no differences between non-infected WT and knock out mice were observed in the parameters assessed.