Differential Roles of Chemokines CCL2 and CCL7 in Monocytosis and Leukocyte Migration during West Nile Virus Infection

Abstract

West Nile virus (WNV) is a re-emerging pathogen and the leading cause of epidemic encephalitis in the United States. Inflammatory monocytes are a critical component of the cellular infiltrate found in the CNS during WNV encephalitis, although the molecular cues involved in their migration are not fully understood. In mice, we previously showed that WNV infection induces a CCR2-dependent monocytosis that precedes monocyte migration into the CNS. Currently, the relative contribution of the CCR2 ligands, chemokines CCL2 and CCL7, in directing monocyte mobilization and leukocyte migration into the CNS is unclear. In this study, we demonstrate that, although both CCL2 and CCL7 are required for efficient monocytosis and monocyte accumulation in the CNS, only CCL7 deficiency resulted in increased viral burden in the brain and enhanced mortality. The enhanced susceptibility in the absence of CCL7 was associated with the delayed migration of neutrophils and CD8(+) T cells into the CNS compared with WT or Ccl2(-/-) mice. To determine whether CCL7 reconstitution could therapeutically alter the survival outcome of WNV infection, we administered exogenous CCL7 i.v. to WNV-infected Ccl7(-/-) mice and observed a significant increase in monocytes and neutrophils, but not CD8(+) T cells, within the CNS, as well as an enhancement in survival compared with Ccl7(-/-) mice treated with a linear CCL7 control peptide. Our experiments suggest that CCL7 is an important protective signal involved in leukocyte trafficking during WNV infection, and it may have therapeutic potential for the treatment of acute viral infections of the CNS.

FIGURE 1. CCL7, but not CCL2, is critical for viral clearance and survival during WNV infection

(A) WT, Ccl2^−/−, and Ccl7^−/− mice were infected with 10² FFU WNV and evaluated for survival (n = 34 for WT; n = 29 for Ccl2^−/−; n = 23 for Ccl7^−/−,). Data shown are pooled from two independent experiments. (B) CNS viral titers were measured on days 8, 10, and 13 p.i. by FFU assay. Virus titers in blood (C) and spleen (D) were quantified by quantitative real-time PCR and FFU assay, respectively. Data are mean 6 SEM for n = 3–6 mice/ genotype/time point and are representative of two independent experiments. (E) Splenocytes from day-6 WNV-infected mice were stimulated ex vivo for 8 h with an immunodominant Db-restricted NS4B peptide. Cells were gated on CD3⁺CD8⁺ populations for analysis of intracellular IFN-γ and/ or TNF-α expression. Data are mean 6 SEM for n = 3–6 mice/genotype/time point and are representative of two independent experiments. (F) Levels of IFN-γ were measured in the brains of uninfected and infected mice by multiplex ELISA. Data are mean 6 SEM for n = 5–15 mice/genotype/time point and are representative of two independent experiments. Dashed lines indicate the assay’s limit of detection. *p < 0.05, **p < 0.01.

Figure 2. Monocyte trafficking into the CNS during WNV encephalitis is impaired in CCL2- and CCL7-deficient mice

(A) Flow cytometric analysis of total monocytes (CD45⁺Ly6C^hi CD11b⁺) isolated from the brains of WNV-infected WT (black), Ccl2^−/− (green), and Ccl7^−/− (blue) mice on days 8 and 13 is shown. Data are shown as mean ± SEM for n=5–15 mice and are representative of two independent experiments. (B) Representative flow cytometry dot plots for day 13 post infection are shown for CD45⁺Ly6C^hiCD11b⁺ monocytes (gated population). (C) Immunohistochemical analyses of the indicated brain sections of WNV-infected WT, Ccl2^−/−, and Ccl7^−/− mice for the presence of Mac-2 antigen at day 13 post infection are shown. Representative images are shown at 20× magnification.

Figure 3. Delayed recruitment of neutrophils and CD8⁺ T cells into the brains of WNV-infected CCL7-deficient mice

The total numbers of CNS neutrophils (CD45⁺Ly6C^intCD11b⁺Ly6G⁺) (A) and CD8⁺ T cells (CD45⁺CD3⁺CD8⁺) (C) were assessed by flow cytometry on days 8 and 13 post infection of WNV-infected WT (black), Ccl2^−/− (green), and Ccl7^−/− (blue) mice. Data are shown as mean ± SEM for n=5–15 mice per condition per time point and are representative of two separate experiments. Representative flow cytometry plots on day 8 for neutrophils (CD45⁺Ly6C^intCD11b⁺) (B) and (CD45⁺CD3⁺CD8⁺) CD8⁺ T cells (D) are shown. Immunohistological analysis of cerebral cortex for myeloperoxidase (E) and corpus callosum for CD3⁺ T cells (F) from WNV-infected WT, Ccl2^−/−, and Ccl7^−/− mice on day 8 and 13 post infection is shown. Representative images of brain sections are shown at 20× magnification. The total numbers of CD45⁺NK1.1⁺CD3⁻ NK cells (G), CD45⁺CD3⁺CD4⁺ T cells (H), and CD45^lo/intCD11b⁺ microglia (I) harvested from WNV-infected brains were assessed at the indicated time points in WNV-infected WT (black), Ccl2^−/− (green), and Ccl7^−/− (blue) mice. Data are shown as mean ± SEM for n=5–15 mice per condition per time point and are representative of two separate experiments.

Figure 4. WNV induces an early monocytosis that is dependent on CCL2 and CCL7

Blood was collected from WT, Ccl2^−/−, and Ccl7^−/− mice before and after WNV infection. The total numbers of Ly6C^hiCD11b⁺ monocytes in WT versus Ccl2^−/− (A) or WT versus Ccl7^−/− (B) mice per ml of blood for the first 5 days post infection were determined by flow cytometry. Representative flow cytometry plots from day 1 (C) and day 3 (D) post infection are shown for CD45⁺Ly6C^hiCD11b⁺ monocytes (gated population). Each data point represents the mean ± SEM of 3–13 mice from two independent experiments. Total numbers of CD45⁺Ly6C^intCD11b⁺Ly6G⁺ neutrophils (E), CD45⁺CD3⁺CD8⁺ T cells (F), CD45⁺CD3⁺CD4⁺ T cells (G), and CD45⁺NK1.1⁺CD3⁻ cells (H) per ml of blood are shown. Each data point represents the mean ± SEM of 3–13 mice from two independent experiments.

FIGURE 5. CCL2 and CCL7 are induced in the blood and brain during WNV infection

Protein levels of CCL2 (A) and CCL7 (B) were measured in the plasma of the indicated mouse strains following WNV infection. Various regions of the brains of WT mice were tested for CCL2 (C) and CCL7 (E) expression via quantitative real-time PCR on day 8 p.i. Data are shown as fold change over uninfected tissue. Each data point represents the mean 6 SEM of n = 5 mice. Brain homogenates were measured for protein levels of CCL2 (D) and CCL7 (F) in the indicated mouse strains following WNV infection by multiplex ELISA. Dashed lines indicate the assay’s limit of detection. Each data point represents the mean 6 SEM of n = 3–15 mice from two independent experiments.

FIGURE 6. i.v. injection of CCL7 induces monocytosis and promotes survival during WNV infection

(A) A total of 1000 ng of active CCL7 was injected i.v. into WT mice, and the total number of CD45⁺Ly6C^hiCD11b⁺ monocytes in the blood was assessed over time compared with uninjected WT mice. Each data point represents the mean 6 SEM of six mice from two independent experiments. (B) Active CCL7 (at the indicated dose) or PBS was injected i.v. into WT mice, and the total number of CD45⁺Ly6C^hiCD11b⁺ monocytes in the blood was assessed at 30 min postinjection. Each data point represents the mean 6 SEM of n = 6 mice from two independent experiments. A total of 500 ng of active CCL7, linear CCL7, or PBS was injected in WT mice, and the number of CD45⁺Ly6C^hiCD11b⁺ monocytes (C), CD45⁺Ly6C^intCD11b⁺Ly6G⁺ neutrophils (D), and CD45⁺CD3⁺CD8⁺ T cells (E) in the blood was assessed at 30 min postinjection. Each data point represents the mean 6 SEM of n = 7–11 mice from two independent experiments. (F) Schematic diagram of the experimental layout for the in vivo therapeutic experiment; each arrow indicates an i.v. injection of active CCL7, linear CCL7, or PBS. Active CCL7 or linear CCL7 was administered i.v. into WNV-infected Ccl7^−/− mice every 12 h between days 6 and 3 p.i.; WT mice were administered PBS on the same schedule. Mice were evaluated daily for survival (G) and weight loss (H) for 18 d. Data shown are pooled from two independent experiments (n = 34 for WT, n = 25 for Ccl2^−/−, n = 26 for Ccl7^−/−). Flow cytometric analysis of CD45⁺Ly6C^hiCD11b⁺ monocytes (I), CD45⁺Ly6C^intCD11b⁺Ly6G⁺ neutrophils (J), and CD45⁺CD3⁺CD8⁺ T cells (K) isolated from the brains of WT mice receiving PBS, Ccl7^−/− mice receiving active CCL7, and Ccl7^−/− mice receiving linear CCL7 on day 8 post-WNV infection. Data are mean 6 SEM for n = 5 mice/condition/time point. (L) CNS viral titers were measured on day 10 p.i. by FFU assay. Dashed line indicates the assay’s limit of detection. Data are mean 6 SEM for n = 5–10 mice/genotype/time point. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.