Dual Function of Ccr5 during Langat Virus Encephalitis: Reduction in Neutrophil-Mediated Central Nervous System Inflammation and Increase in T Cell-Mediated Viral Clearance

Abstract

Tick-borne encephalitis virus (TBEV) is a vector-transmitted flavivirus that causes potentially fatal neurologic infection. There are thousands of cases reported annually, and despite the availability of an effective vaccine, the incidence of TBEV is increasing worldwide. Importantly, up to 30% of affected individuals develop long-term neurologic sequelae. We investigated the role of chemokine receptor Ccr5 in a mouse model of TBEV infection using the naturally attenuated tick-borne flavivirus Langat virus (LGTV). Ccr5-deficient mice presented with an increase in viral replication within the CNS and decreased survival during LGTV encephalitis compared with wild-type controls. This enhanced susceptibility was due to the temporal lag in lymphocyte migration into the CNS. Adoptive transfer of wild-type T cells, but not Ccr5-deficient T cells, significantly improved survival outcome in LGTV-infected Ccr5-deficient mice. Concomitantly, a significant increase in neutrophil migration into the CNS in LGTV-infected Ccr5(-/-) mice was documented at the late stage of infection. Ab-mediated depletion of neutrophils in Ccr5(-/-) mice resulted in a significant improvement in mortality, a decrease in viral load, and a decrease in overall tissue damage in the CNS compared with isotype control-treated mice. Ccr5 is crucial in directing T cells toward the LGTV-infected brain, as well as in suppressing neutrophil-mediated inflammation within the CNS.

Figure 1. Ccr5 is critical for survival and viral clearance in the CNS during LGTV infection

(A) Kaplan-Meier survival analysis of LGTV-infected WT (black circles) and Ccr5^−/− (grey squares) mice. (B) Body weight was measured for LGTV-infected WT and Ccr5^−/− mice. All data shown (A–B) are pooled from three independent experiments (31 WT mice and 32 Ccr5^−/− mice). Viral titers were quantified in (C) the blood and (D) spleen by qRT-PCR, and in (E) the brain and (F) spinal cord by FFU assay. Dotted lines indicate the limit of detection. All data (C–F) are shown as mean ± SD for 3–8 mice per genotype and time point from two independent experiments. * p<0.05 and *** p<0.001.

Figure 2. Peripheral leukocytes and inflammatory cytokine production following LGTV infection

(A) Total cell numbers of monocytes (CD45^hiLy6C^hiCD11b⁺Ly6G⁻), neutrophils (CD45^hiLy6C^intCD11b⁺Ly6G⁺), NK cells (CD45^hiNK1.1⁺CD3⁻), CD4⁺ T cells (CD45^hiCD3⁺ CD4⁺), CD8⁺ T cells (CD45^hiCD3⁺CD8⁺) and B cells (CD45^hiCD19⁺) were assessed by flow cytometry from the blood of WT and Ccr5^−/− mice on days 0, 1, 3, and 5 post infection. Cells were gated on live singlet CD45^hi cells. (B) Levels of Ccl2, Cxcl9, Cxcl10, IL-10, Ccl4, and Ccl5 in the blood of WT and Ccr5^−/− mice were measured by multiplex ELISA. The dotted line represents the assay limit of detection. All data are shown as mean ± SD for 4–11 mice per genotype and time point from three independent experiments. * p<0.05, ** p<0.01 and *** p<0.001.

Figure 3. T and NK cell infiltration into the CNS is impaired in the absence of Ccr5 during LGTV infection

The total number of (A) CD4⁺ T cells (CD45^hiCD3⁺CD4⁺), (C) CD8⁺ T cells (CD45^hiCD3⁺CD8⁺) and (E) NK cells (CD45^hiNK1.1⁺CD3⁻) were assessed by flow cytometry in the brains of WT and Ccr5^−/− mice. Data are shown as mean ±SD for 3–9 mice per genotype and time point from two independent experiments. Representative flow cytometry plots on day 8 post infection for (B) CD4⁺ T cells, (D) CD8⁺ T cells, and (F) NK cells are displayed. Cells were gated on live singlet CD45^hi cells. (G) Paraffin-embedded brain sections from WT and Ccr5^−/− mice were stained for CD3, and positive cells (brown cells, indicated by arrows) are shown within the cerebellum of LGTV-infected mice on days 8 and 12 post infection for 3-5 mice per genotype from two independent experiments. Representative images are shown at 20× magnification. * p<0.05 and *** p<0.001.

Figure 4. Neutrophil numbers are increased in the CNS in the absence of Ccr5

The total number of (A) monocytes (CD45^hiCD11b⁺Ly6C^hiLy6G⁻) and (E) neutrophils (CD45^hiCD11b⁺Ly6C^intLy6G⁺) in the brain of uninfected and LGTV-infected WT and Ccr5^−/− mice on days 8 and 12 post infection were assessed by flow cytometry. Representative flow cytometry plots from day 12 post infection are shown for (B) monocytes and (F) neutrophils. Cells were gated on live singlet CD45^hi cells. Neutrophils were additionally gated on CD11b⁺ and Ly6C^int cells. The protein levels of monocyte chemoattractants (C) Ccl2 and, (D) Ccl7, and neutrophil chemoattractants (G) Cxcl1 and (H) Cxcl2 were measured by multiplex ELISA. All data are shown as mean ± SD with 4–10 mice per time point and genotype and were pooled from two independent experiments. A Student's t-test was performed for statistical analysis with *p<0.05.

Figure 5. Enhanced neutrophil accumulation in the brain is accompanied by increased apoptosis in Ccr5^−/− mice during LGTV-induced encephalitis

Paraffin-embedded brain sections of LGTV-infected mice on days 8 and 12 post infection were stained for (A) myeloperoxidase and (B) apoptosis using TUNEL assay for 3-5 mice per genotype and condition from two independent experiments. Arrows indicate the positive cells (brown staining) within the cortex and meninges. Representative images are shown at a magnification of 20×.

Figure 6. Early recruitment of T cells and the absence of neutrophils in the CNS of LGTV-infected Ccr5-deficient mice result in decreased neuropathology

(A) A schematic diagram of the experimental layout for the T cell adoptive transfer experiment is displayed. Donor mice (WT or Ccr5^−/−) were infected with LGTV, and T cells were isolated from spleens harvested on day 8 post infection. 1×10⁶ T cells were transferred into LGTV-infected recipient mice (Ccr5^−/− mice) intravenously on day 5 post infection and evaluated for survival for 18 days (B). Kaplan-Meier analysis of recipient mice with 18–20 Ccr5^−/− mice per group. WT→Ccr5^−/− mice received T cells from WT mice and Ccr5^−/−→Ccr5^−/− received T cells from Ccr5^−/− mice. (C) The total number of CD3⁺ T cells (CD45^hiCD3⁺) was assessed by flow cytometry on day 8 post infection in the brains of Ccr5^−/− mice receiving either WT or Ccr5^−/− CD3⁺ T cells. Data are shown as mean ±SD for 4–6 mice per group. (D) Body weight was measured daily for isotype and anti-Ly6G (clone 1A8) treated LGTV-infected mice for 12 days post infection with 16-17 mice per group pooled from two independent experiments. (E) Ccr5^−/− mice were injected with either isotype or 1A8 antibody, and survival was assessed by a Kaplan-Meier analysis through day 18 post infection with 16-17 mice per group pooled from two independent experiments. The arrows in C and D indicate the days when the antibody was administered. (F) The viral load was quantified in the brains of isotype or 1A8 antibody-treated mice on day 12 post infection. Shown is the mean ±SD from 7–8 mice per group on day 12 post infection from two independent experiments. (G) Immunohistochemical analysis was performed on paraffin-embedded brain sections for myeloperoxidase (MPO) from mice treated with isotype or 1A8 antibody on day 12 post infection. Immunohistochemical analysis was performed on paraffin-embedded brain sections after staining for apoptosis (TUNEL stain, left panels) or LGTV (right panels) in the (H) meninges, (I) hippocampus, and (J) deep cortical layers for 3-5 mice per genotype and condition from two independent experiments. Representative images are shown at 20× magnification. * p<0.05.