The recruitment of peripheral blood leukocytes to the brain is delayed in susceptible BALB/c compared to resistant C57BL/6 mice during herpes simplex virus encephalitis.

The cerebral immune response induced by herpes simplex virus (HSV) encephalitis (HSE) was evaluated in susceptible BALB/c and resistant C57BL/6 mice. BALB/c and C57BL/6 (named C57BL/6-high) mice were respectively infected intranasally with 1 × 103 and 5 × 105 plaque-forming units (PFUs) of HSV-1. C57BL/6 mice (named C57BL/6-low) infected with a low inoculum (1 × 103 PFUs) of HSV-1 were tested in parallel. Mice were monitored for weight loss, sickness signs, and survival for 21 days. The viral load, infectious titers, cytokine/chemokine levels, and peripheral leukocyte infiltration were determined in brain homogenates on days 0 (non-infected), 4, 6, and 8 post-infection (p.i.) by qPCR, plaque assay, ELISA/Luminex™, and flow cytometry, respectively. Our results showed that the mortality of BALB/c mice (67%) was higher compared to those of C57BL/6-low (0%; P ≤ 0.01) and C57BL/6-high (20%; P ≤ 0.05) animals. This higher mortality was associated with increased infectious titers and cytokine/chemokine levels in the brains of BALB/c compared to C57BL/6 mice. Recruitment of inflammatory monocytes, dendritic cells, natural killer, and natural killer T cells to the brain was higher in C57BL/6-high compared to BALB/c animals on day 4 p.i. Infiltration of inflammatory monocytes and T cells in the brain of BALB/c mice was seen on day 6 p.i. Our data suggest that a rapid, sustained, and coordinated recruitment of peripheral leukocytes to the brain of C57BL/6-high mice results in an effective control of viral replication and inflammation whereas the delayed infiltration of immune cells in the brain of BALB/c mice was associated with an exacerbated inflammatory response during HSE.

Both IRF3 and especially IRF7 play a key role to orchestrate an effective cerebral inflammatory response in a mouse model of herpes simplex virus encephalitis.

The impact of a deficiency in interferon regulatory factor (IRF)3 and IRF7 was evaluated in an herpes simplex virus encephalitis (HSE) model. Compared to wild type (WT), the mortality rates of infected IRF3-/- and IRF7-/- mice were higher and associated with increased brain viral titers. At a critical time post-infection, IRF7-/- mice exhibited a deficit in IFN-ß production. At a later time point, levels of type I IFNs and cytokines were increased in brains of both deficient mice compared to WT. Our results suggest that IRF3, and especially IRF7, are important for an effective control of inflammatory responses during HSE.

Protective role of CX3CR1 signalling in resident cells of the central nervous system during experimental herpes simplex virus encephalitis.

CX3CR1 is an important chemokine receptor expressed on the surface of microglia and blood leukocytes, including monocytes. Signalling through this receptor influences the immune activity of microglia and monocyte trafficking into the central nervous system (CNS) in several neurological diseases. During experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE), CX3CR1 deficiency has been reported to exacerbate the outcome of the disease. However, the precise contribution of CX3CR1 expressed in resident cells of the CNS or peripheral monocytes in protection against HSE remains unclear. To dissect the role of CX3CR1 during HSE, we reconstituted irradiated C57BL/6 WT and CX3CR1-/- mice with CX3CR1-/- (CX3CR1-/-→WT) and WT (WT→CX3CR1-/-) bone marrow cells, respectively. Our results showed that following intranasal infection with 1.2×106 p.f.u. of HSV-1, mortality rates were significantly higher in CX3CR1-/- (61.7 %) and WT→CX3CR1-/- (66.2 %) compared to WT (16.6 %; P=0.012 and P=0.016, respectively) and CX3CR1-/-→WT animals (20 %; P=0.013 and P=0.011, respectively). Higher mortality rates in CX3CR1-/- and WT→CX3CR1-/- mice were associated with increased infectious viral titres and wider HSV dissemination in brains, as well as an overproduction of inflammatory cytokines and chemokines including IL-1ß, IL-6, IFN-γ, C-C motif ligand 2 and C-C motif ligand 5. Furthermore, CX3CR1 deficiency in resident cells of the CNS resulted in excessive and sustained Ly6Chi inflammatory monocyte and neutrophil infiltration into the brain. These data suggest that CX3CR1 deficiency in resident cells of the CNS affects mouse survival, HSV-1 replication control and cerebral inflammatory response whereas its deficiency in the haematopoietic system does not appear to influence the outcome of HSE.

Both Cerebral and Hematopoietic Deficiencies in CCR2 Result in Uncontrolled Herpes Simplex Virus Infection of the Central Nervous System in Mice.

CCR2 is a chemokine receptor expressed on the surface of blood leukocytes, particularly «Ly6Chi¼ inflammatory monocytes and microglia. Signaling through this receptor is thought to influence the immune activity of microglia as well as monocytes egress from the bone marrow (BM) and their trafficking into the central nervous system (CNS) in several neurological diseases. During experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE), CCR2 deficiency has been reported to exacerbate the outcome of the disease. However, the precise contribution of CCR2 expressed in cells of the CNS or peripheral monocytes in the protection against HSE remains unclear. To dissect the differential role of CCR2 during HSE, chimeric mice with receptor deficiency in the brain or blood cells were generated by transplanting wild-type (WT) C57BL/6 or CCR2-/- BM-derived cells in CCR2-/- (WT→CCR2-/-) and WT (CCR2-/-→WT) mice, respectively. Our results indicate that following intranasal infection with 1.2x106 plaque forming units of HSV-1, CCR2 deficiency in hematopoietic cells and, to a lesser extent, in CNS exacerbates the outcome of HSE. Mortality rates of CCR2-/- (71.4%) and CCR2-/-→WT (57.1%) mice were significantly higher than that of WT (15.3%; P<0.01 and P<0.05, respectively) but the difference did not reach statistical significance for WT→CCR2-/- animals (42.8%; P = 0.16). Both peripheral and CNS deficiencies in CCR2 resulted in increased infectious viral titers and wider dissemination of HSV antigens in the brain as well as an overproduction of inflammatory cytokines and chemokines including IL-1ß, IL-6, CCL2, CCL3 and CCL5. Furthermore, CCR2 deficiency in the hematopoietic system altered monocytes egress from the BM and their recruitment to the CNS, which may contribute to the failure in HSV-1 containment. Collectively, these data suggest that CCR2 expressed on cells of CNS and especially on peripheral monocytes is important for the control of HSV-1 replication and inflammatory environment during experimental HSE.

Infiltration Pattern of Blood Monocytes into the Central Nervous System during Experimental Herpes Simplex Virus Encephalitis.

The kinetics and distribution of infiltrating blood monocytes into the central nervous system and their involvement in the cerebral immune response together with resident macrophages, namely microglia, were evaluated in experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE). To distinguish microglia from blood monocyte-derived macrophages, chimeras were generated by conditioning C57BL/6 recipient mice with chemotherapy regimen followed by transplantation of bone morrow-derived cells that expressed the green fluorescent protein. Mice were infected intranasally with a sub-lethal dose of HSV-1 (1.2 x 10(6) plaque forming units). Brains were harvested prior to and on days 4, 6, 8 and 10 post-infection for flow cytometry and immunohistochemistry analysis. The amounts of neutrophils (P < 0.05) and "Ly6C hi" inflammatory monocytes (P < 0.001) significantly increased in the CNS compared to non-infected controls on day 6 post-infection, which corresponded to more severe clinical signs of HSE. Levels decreased on day 8 for both leukocytes subpopulations (P < 0.05 for inflammatory monocytes compared to non-infected controls) to reach baseline levels on day 10 following infection. The percentage of "Ly6C low" patrolling monocytes significantly increased (P < 0.01) at a later time point (day 8), which correlated with the resolution phase of HSE. Histological analysis demonstrated that blood leukocytes colonized mostly the olfactory bulb and the brainstem, which corresponded to regions where HSV-1 particles were detected. Furthermore, infiltrating cells from the monocytic lineage could differentiate into activated local tissue macrophages that express the microglia marker, ionized calcium-binding adaptor molecule 1. The lack of albumin detection in the brain parenchyma of infected mice showed that the infiltration of blood leukocytes was not necessarily related to a breakdown of the blood-brain barrier but could be the result of a functional recruitment. Thus, our findings suggest that blood monocyte-derived macrophages infiltrate the central nervous system and may contribute, with resident microglia, to the innate immune response seen during experimental HSE.

Valacyclovir combined with artesunate or rapamycin improves the outcome of herpes simplex virus encephalitis in mice compared to antiviral therapy alone.

Despite antiviral therapy, the mortality rate of herpes simplex virus encephalitis (HSE) remains high and many surviving patients harbor neurological sequelae. Although viral replication is responsible for substantial neurological damages, an exaggerated inflammatory response could also contribute to this process. Artesunate (ART) and rapamycin (RAPA) have shown some benefits in the treatment of herpes simplex virus infections. Herein, we evaluated the benefit of combining ART or RAPA with valacyclovir (VACV) in a murine model of HSE. Infected mice were treated with VACV (1mg/mL in drinking water) from day 3 post-infection (p.i.) combined or not with daily intraperitoneal administration of ART (30mg/kg) or RAPA (20mg/kg) from days 4 to 13 p.i. Viral load, infectious titers, cytokine and chemokine levels were measured in brain homogenates on days 5, 7 and 9. The survival rates of mice treated with VACV and ART or RAPA were higher than with VACV alone (71.9% versus 43.2% for ART and 66.7% versus 43.2% for RAPA; both P⩽0.05) but no significant difference was seen in the brain viral loads. Levels of IL-1ß, IL-2 (both P⩽0.05), IL-6, IFN-γ (both P⩽0.01), CCL2 (P⩽0.01), CCL3 and CCL4 (both P⩽0.05) were reduced in mice treated with VACV combined with ART versus VACV alone. Levels of IL-6, IL-1ß and IFN-γ slightly increased on day 7 in mice treated with VACV combined with RAPA compared to VACV alone and then decreased on day 9. Our results suggest that immunomodulatory compounds such as ART or RAPA could benefit antiviral therapy in HSE.