Absence of CCR5 increases neutrophil recruitment in severe herpetic encephalitis

Abstract

Background: The neuroinflammatory response aimed at clearance of herpes simplex virus-1 (HSV-1) plays a key role in the pathogenesis of neuroaxonal damage in herpetic encephalitis. Leukocytes activated in an adaptive immune response access brain tissue by passing through the blood-brain barrier. The chemokine CCL5/RANTES is involved in recruitment of these cells to the brain acting via the receptors CCR1, CCR3 and mainly CCR5. Here, we evaluated the role of CCR5 on traffic of leukocytes in the brain microvasculature, cellular and cytokines profile in a severe form of herpetic encephalitis.

Results: Wild type and mice lacking CCR5 (CCR5-/-) were inoculated intracerebrally with 104 PFU of neurotropic HSV-1. We evaluated the traffic of leukocytes in the brain microvasculature using intravital microscopy and the profile of cytokines by Enzyme-Linked Immunosorbent Assay at 1 day post infection. Flow cytometry and histopathological analyses were also carried out in brain tissue. Absence of CCR5 leads to lower viral load and an increased leukocyte adhesion in brain microvasculature, predominantly of neutrophils (CD11+ Ly6G+ cells). Moreover, there was a significant increase in the levels of MIP-1/CCL2, RANTES/CCL5, KC/CXCL1 and MIG/CXCL9 in the brain of infected CCR5-/- mice.

Conclusions: These results suggest that the absence of CCR5 may boost the immune response with a high neutrophil recruitment which most likely helps in viral clearance. Nonetheless, the elevated immune response may be detrimental to the host.

Figure 1

Course of infection: WT and CCR5^-/- mice were intracerebrally inoculated with 10⁴ PFU of virus HSV-1, and (A) survival was assessed daily. (B) Viral load in brain at 1 dpi was assessed in WT (n = 6) and CCR5^-/- (n = 10). After sacrifice, brains were collected, macerated, and inoculated into Vero cell cultures to perform the titration procedure in triplicate. Survival curve was created using the product limit method of Kaplan and Meier and survival curves were compared using logrank test. Statistical analysis used to evaluate the viral load was Student’s t-test. Statistically significant results were indicated by ***p < 0.001, **p < 0.01 and *p < 0.05.

Figure 2

WT and CCR5^-/- mice were infected with 10⁴ PFU of HSV-1 by intracranial route and brain removed at 1dpi (n = 4). Brain-sequestered cells were counted and then stained with specific antibodies. Neutrophils were characterized as CD11⁺ Ly6G⁺ cells and T lymphocytes were characterized as CD3⁺ TCD4⁺ or CD3⁺ TCD8⁺. Flow cytometry, according to size and granularity, was performed as analysis. Number of leukocyte (A), neutrophils (B), T lymphocytes CD8⁺ (C) and CD4⁺ (D) were evaluated in WT and CCR5^-/- mice. Results are expressed as mean ± SD and *p < 0.05, ***p < 0.001 when compared to non-infected mice. Representative gating strategy utilized for analysis of lymphocytes and granulocytes in the brain. Granulocytes population was isolated and collected for analysis. At this region, neutrophils were characterized as CD11⁺ Ly6G⁺ cells. Lymphocyte population was isolated and the cell population positive for CD3⁺ was collected for analysis. T lymphocytes were defined as CD3⁺ TCD4⁺ or CD3⁺ TCD8⁺. Flow cytometry data from a CCR5^-/- infected mice (E). Statistical analysis used: one-way ANOVA with Tukey correction.

Figure 3

Brain histopathological changes after intracerebral inoculation of 10⁴ PFU of HSV-1 in WT and CCR5^-/- mice, at 1 dpi. H&E-stained sections of meninges and cerebral cortex. Meninges of non-infected WT mouse (A) and CCR5^-/- animal (D) with normal brain tissue (n = 3 in each group); WT infected animal showing infiltration of immune cells in the meninges (B-C); Intense meningitis characterized by infiltration of polimorphonuclear and mononuclear in CCR5^-/- mouse (E-F). A-B: ×200; D-E: ×100; C-F: ×400.

Figure 4

Visualization of leukocyte-endothelium interaction at 1 day post infection with HSV-1. WT (n = 6) and CCR5^-/- (n = 6) were intracerebrally inoculated with 10⁴ PFU of HSV-1. Intravital microscopy was used to assess rolling (A) and adhesion (B) of leukocytes on the brain microvasculature, at 1 dpi. Data indicate mean ± SD of cells per minutes (A) and 100 mm (B). Intravital microscopy revealed a significant increase in leukocyte adhesion in CCR5^-/- infected mice at 1 dpi (***p < 0.001). Statistical analysis used: one-way ANOVA with Tukey correction.

Figure 5

Chemokine levels in the CNS after intracerebral inoculation with 10⁴ PFU of HSV-1 in WT (n = 10) and CCR5^-/- mice (n = 13). Cytokines were measured in brain extracts by ELISA at 1 dpi. The infection in CCR5^-/- mice was also followed by a significant increase in chemokine levels, including CCL2, CCL5, CXCL1 and CXCL9 and TNF-α level when compared with infected WT mice. Data indicate mean ± SD. Statistically significant results were indicated by ***p < 0.001, **p < 0.01 and *p < 0.05. Statistical analysis used: one-way ANOVA with Tukey correction.