Abrogation of macrophage migration inhibitory factor decreases West Nile virus lethality by limiting viral neuroinvasion

Abstract

The flavivirus West Nile virus (WNV) is an emerging pathogen that causes life-threatening encephalitis in susceptible individuals. We investigated the role of the proinflammatory cytokine macrophage migration inhibitory factor (MIF), which is an upstream mediator of innate immunity, in WNV immunopathogenesis. We found that patients suffering from acute WNV infection presented with increased MIF levels in plasma and in cerebrospinal fluid. MIF expression also was induced in WNV-infected mice. Remarkably, abrogation of MIF action by 3 distinct approaches (antibody blockade, small molecule pharmacologic inhibition, and genetic deletion) rendered mice more resistant to WNV lethality. Mif(-/-) mice showed a reduced viral load and inflammatory response in the brain when compared with wild-type mice. Our results also indicate that MIF favors viral neuroinvasion by compromising the integrity of the blood-brain barrier. In conclusion, the data obtained from this study provide direct evidence for the involvement of MIF in viral pathogenesis and suggest that pharmacotherapeutic approaches targeting MIF may hold promise for the treatment of WNV encephalitis.

Figure 1. WNV infection induces MIF expression.

(A) MIF plasma levels of patients suffering from acute WNV infection (n = 27) and control donors (n = 31). The distribution of data points in each group is represented by box-and-whisker plots, where horizontal lines mark (from the bottom up) the 25th, 50th (median), and 75th percentiles. The central vertical line in each box represents the range of values (5th–95th percentiles). (B) MIF levels in the CSF of WNV-infected patients (n = 7) and controls (n = 5). MIF mRNA levels were determined by Q-PCR in the brain (C) and spleen (D) of WT BALB/c mice at days 0, 2, 4, 6, and 8 after i.p. inoculation of WNV. Data are mean ± SEM of 3–6 mice per time point. (E) MIF serum levels determined by ELISA in WT BALB/c mice at days 0, 2, 4, 6, and 8 after i.p. inoculation of WNV. Data are mean ± SEM of 3–6 mice per time point. *P < 0.05, per Student’s t test. Data from mice were pooled from 2 independent experiments (3 mice per time point and experiment).

Figure 2. Abrogation of MIF renders mice more resistant to WNV-induced mortality.

(A) Mice were inoculated i.p. with WNV and administered ISO-1 or vehicle (10% DMSO) daily on days –1 through 7. (B) Mice were inoculated i.p. with WNV and treated with anti-MIF antibody or isotype control on days –1, 3, and 6. (C) Mif^–/– and matched wild-type mice were inoculated i.p. with WNV. Data were pooled from 2 to 3 independent experiments and represent a total of 20–30 mice per group. *P < 0.05, compared with the control group per χ² test.

Figure 3. Mif^–/– mice have a reduced viral load in the brain but not in the spleen.

WNV-E mRNA levels were determined by Q-PCR in the brain (A) and spleen (B) of WT and Mif^–/– mice at days 2, 4, 6, and 8 after i.p. inoculation of WNV. Data (mean ± SEM) were pooled from 2 independent experiments, yielding a total of 6–8 mice per group per time point. *P < 0.05, compared with day 2 per Student’s t test; ^#P < 0.05, compared with WT group per 2-way ANOVA. UD, undetectable.

Figure 4. Reduced infection and inflammatory cell infiltration in Mif^–/– brains when compared with WT brains.

Immunofluorescent confocal images from olfactory bulb, cerebellum, and brain stem double stained for WNV-E (WNV antigen) and CD45 at day 6 (A) and 8 (B) after i.p. inoculation of WNV. Original magnification, ×25.

Figure 5. Mif^–/– mice have reduced levels of inflammatory cytokines in the brain in response to WNV infection.

IFN-α (A), TNF-α (B), IL-6 (C), and IL-12 (D) levels were determined by Q-PCR in the brain of WT and Mif^–/– mice at days 4, 6, and 8 after i.p. inoculation of WNV. Data are mean ± SEM of 6–8 mice, pooled from 2 independent experiments. *P < 0.05, compared with day 4 per Student’s t test; ^#P < 0.05, compared with WT group per 2-way ANOVA.

Figure 6. Peripheral cytokine response to WNV infection in WT and Mif^–/– mice.

TNF-α mRNA levels in the spleen (A) and protein levels in serum (B), IL-12 mRNA levels (C), and IL-1β mRNA levels (D) in the spleen were determined at days 2, 4, and 6 after i.p. inoculation of WNV. Data are mean ± SEM of 6–8 mice per group per time point obtained from 2 independent experiments. *P < 0.05, compared with day 2 per Student’s t test; ^#P < 0.05, compared with WT group per 2-way ANOVA.

Figure 7. MIF facilitates WNV entry into the brain.

(A) BBB permeability is increased after 24 hours of poly(I:C) challenge or 4 days after WNV infection in WT but not in Mif^–/– mice. Representative photographs of Evans blue dye staining of whole brains. Original magnification, ×0.5. (B) Survival analysis did not reveal a statistically significant difference (P > 0.1) between WT and Mif^–/– mice after intracerebral inoculation of 200 PFU (left panel) or 20 PFU (right panel) of WNV (n = 10 mice per group).