A Potential Role for Substance P in West Nile Virus Neuropathogenesis

Abstract

Of individuals who develop West Nile neuroinvasive disease (WNND), ~10% will die and >40% will develop long-term complications. Current treatment recommendations solely focus on supportive care; therefore, we urgently need to identify novel and effective therapeutic options. We observed a correlation between substance P (SP), a key player in neuroinflammation, and its receptor Neurokinin-1 (NK1R). Our study in a wild-type BL6 mouse model found that SP is upregulated in the brain during infection, which correlated with neuroinvasion and damage to the blood−brain barrier. Blocking the SP/NK1R interaction beginning at disease onset modestly improved survival and prolonged time to death in a small pilot study. Although SP is significantly increased in the brain of untreated WNND mice when compared to mock-infected animals, levels of WNV are unchanged, indicating that SP likely does not play a role in viral replication but may mediate the immune response to infection. Additional studies are necessary to define if SP plays a mechanistic role or if it represents other mechanistic pathways.

Keywords: West Nile encephalitis; West Nile virus; neuroinflammation; neurokinin-1 receptor; substance P.

Figure 1

RNA levels of SP as detected by qPCR against a standard curve in whole blood and brain. Top: Whole blood. Bottom: Whole brain tissue homogenate. Error bars represent the standard deviations. Significant differences between WNV-infected and mock mice were detected within 3 DPI (p = 0.0092) and remained significant at 4 DPI (p = 0.0007), 5 DPI (p = 0.0009), and 6 DPI (0.0012), while SP copy numbers’ increases in the brain are not detectable until 6 DPI (p = 0.0031). N = 10 per group per day, and N = 18 WNV-infected mice are included in the analyses of D6 and D7 due to early attrition. *: p < 0.01.

Figure 2

SP as detected by ELISA against a standard curve in whole brain homogenate. Error bars represent the standard deviations. Significantly higher mean protein levels were detected in WNV-infected mice when compared to mock at both 7 DPI (p = 0.017) and 8 DPI (p = 0.047). N = 10 per group per day, and N = 18 WNV-infected mice are included in the analyses of D6 and D7 due to early attrition. *: p < 0.05.

Figure 3

Blood–brain barrier permeability over time. These images represent one control mouse unexposed to WNV at 5 DPI and two mice infected with WNV at 5 and 7 DPI. An increase in color is indicative of higher signal intensity.

Figure 4

Kaplan–Meier analysis in treated and untreated mice exposed to WNV-NY99. Red: WNV-infected + 10mg/kg daily dose beginning at symptom onset (N = 10); Purple: WNV-infected + 10 mg/kg single dose at symptom onset (N = 5); Black: WNV-infected with no treatment (N = 8).

Figure 5

Comparison of SP levels between treatment groups at time of euthanasia. Means are expressed with the standard error. SP levels correspond to the number of SP RNA copies per µL of RNA. For these reactions, 2 µL of RNA was used.

Figure 6

Comparison of SP levels between survivors, non-survivors, and mock-infected animals at time of euthanasia. Means are expressed with the standard error. SP levels correspond to the number of SP RNA copies per µL of RNA. For these reactions, 2 µL of RNA was used.

Figure 7

WNV amplification from whole blood at 7 DPI. Levels of WNV RNA were determined and normalized to ACT to calculate the ∆CT for each treatment group. No significant differences in the levels of WNV RNA were found between each treatment group when compared using a One-Way ANOVA.