The Innate Immune Response to Herpes Simplex Virus 1 Infection Is Dampened in the Newborn Brain and Can Be Modulated by Exogenous Interferon Beta To Improve Survival

Abstract

Newborns are particularly susceptible to severe forms of herpes simplex virus 1 (HSV-1) infection, including encephalitis and multisystemic disseminated disease. The underlying age-dependent differences in the immune response that explain this increased susceptibility relative to the adult population remain largely understudied. Using a murine model of HSV-1 infection, we found that newborn mice are largely susceptible to intracranial and intraperitoneal challenge while adult mice are highly resistant. This age-dependent difference correlated with differential basal-level expression of components of innate immune signaling pathways, which resulted in dampened interferon (IFN) signaling in the newborn brain. To explore the possibility of modulating the IFN response in the newborn brain to recapitulate the adult phenotype, we administered exogenous IFN-β in the context of disseminated HSV-1 infection. IFN-β treatment resulted in significantly increased survival and delayed viral neuroinvasion in the newborn. These effects were associated with changes in the type I IFN response in the brain, reduced viral replication in the periphery, and the stabilization of the blood-brain barrier (BBB). Our study reveals important age-dependent differences in the innate immune response to HSV-1 infection and suggests a contribution of the BBB and the brain parenchyma in mediating the increased susceptibility to HSV-1 infection observed in the newborn. These results could provide the basis for potential new therapeutic strategies for life-threatening HSV-1 infection in newborns.IMPORTANCE Herpes simplex virus (HSV) is a ubiquitous human pathogen affecting 50 to 80% of the population in North America and Europe. HSV infection is commonly asymptomatic in the adult population but can result in fatal encephalitis in the newborn. Current treatment with acyclovir has improved mortality in the newborn; however, severe neurologic sequelae are still a major concern following HSV encephalitis. For this reason, there is a critical need to better understand the underlying differences in the immune response between the two age groups that could be used to develop more effective treatments. In this study, we investigated differences in the innate immune response to viral infection in the brains of newborn and adult mice. We found that, similar to humans, newborn mice are more susceptible to HSV infection than the adult. Increased susceptibility was associated with dampened innate immune responses in the newborn brain that could be rescued by administering interferon beta.

Keywords: herpes simplex virus; interferon; newborn; viral encephalitis.

FIG 1

IFNAR protects against mortality and decreases viral replication during HSV-1 CNS infection in the adult but only prolongs survival in the newborn. (A and B) Survival (A) and viral titer of brains at mortality or experimental endpoint at day 14 (B) of adult WT or IFNARKO mice inoculated i.c. with 10⁴ PFU of HSV-1 KOS. d.p.i, days postinfection. (C and D) Survival (C) and viral titer of brains at mortality or day 14 (D) of 7-day-old (P7) WT or IFNARKO mice inoculated i.c. with 10⁴ PFU of HSV-1 KOS. (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant. All error bars represent SEM.)

FIG 2

Type I IFN response components are differentially expressed in the brain throughout development. (A) Representative immunoblots (left) and densitometry (right) of whole-brain homogenates from uninfected 7-day-old (P7), 14-day-old (P14), 20-day-old (P20), and adult (Ad; 8- to 10-week-old) mice. Protein levels of IFNAR1 and PKR were significantly lower in 7-day-old mice and gradually increased during the first weeks after birth to adult levels. TLR-3 and STAT1 (STAT1α [91 kDa] and STAT1β [84 kDa]) protein levels were higher in 7-day-old mice than in other age groups. (B and C) IFN-β mRNA levels quantified by qPCR following i.c. inoculation with 10⁴ PFU HSV-1 (KOS) of P7 newborn (B) and adult (C) mice at different time points. Samples were normalized to GAPDH gene expression for each age group. (N = 3 to 6 for each experiment. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. All error bars represent SEM.)

FIG 3

Type I IFN response components are rapidly upregulated in the adult but not in the newborn brain following HSV-1 infection. (A to H) Representative immunoblots (left) and densitometry (right) of IFNAR1 (A and B), STAT1 (C and D), cGAS (E and F), and PKR (G and H) in the brains of newborn and adult mice following i.c. inoculation with 10⁴ PFU HSV-1 analyzed by Western blotting at different time points. Samples were normalized to GAPDH levels for each age group at each time point. Significance denotes comparison of normalized values to the corresponding mock infection for each time point and age group. (N = 3 to 4 for each experimental condition. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. All error bars represent SEM.)

FIG 4

Recombinant murine IFN-β treatment protects the newborn from disseminated HSV-1 infection in an IFNAR-dependent manner. (A and B) Survival (A) and titer at mortality (B) of newborn WT or IFNARKO mice inoculated i.p. with 10⁵ PFU of HSV-1 KOS. IFNAR expression prolonged survival of newborn mice but failed to protect against mortality or control viral replication in the CNS. (C and D) Survival (C) and titer at mortality (D) of newborn WT mice inoculated i.p. with 10⁵ PFU of HSV-1 KOS and given daily doses of 10⁴ IU of recombinant murine IFN-β or vehicle control (PBS). (E and F) Survival (E) and titer at mortality (F) of newborn IFNARKO mice inoculated i.p. with 10⁵ PFU of HSV-1 KOS and given daily doses of 10⁴ IU of recombinant murine IFN-β or vehicle control (PBS). (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. All error bars represent SEM.)

FIG 5

IFN-β treatment increases levels of cGAS in the newborn brain in an IFNAR-dependent manner and delays neuroinvasion during disseminated HSV-1 infection. Representative immunoblots (left) and densitometry (right) of whole-brain homogenates from uninfected WT (A) or IFNARKO (B) newborn mice treated for 3 days with either 10⁴ IU of mouse IFN or PBS (vehicle-only) control (N = 4 to 8 in each group). Newborn WT mice were inoculated i.p. with 10⁵ PFU of HSV-1 (KOS) and given daily doses of 10⁴ IU of recombinant murine IFN or vehicle control (PBS). (C to F) Liver (C), lungs (D), spleen (E), and brain (F) were collected on days 1, 3, and 5 postinfection, and viral loads were determined by standard plaque assay. (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. All error bars represent SEM.)

FIG 6

HSV-1-induced breakdown of the blood-brain barrier during disseminated disease in the newborn is prevented by IFN-β treatment. (A) Newborn WT mice were inoculated i.p. with 10⁵ PFU of HSV-1 (KOS) or mock infected. Serum and brain were collected 1 h after 2 mM NaF i.p. injection at the indicated time points, and the blood-brain barrier permeability index was determined as the normalized ratio of brain fluorescence and serum fluorescence. (B) Newborn WT mice were inoculated i.p. with 10⁵ PFU of HSV-1 (KOS) and given daily doses of 10⁴ IU of recombinant murine IFN-β or vehicle control (PBS) starting the day before infection. The blood-brain barrier permeability index was determined using the same procedure. (C) Model for IFN-dependent increased susceptibility to HSV-1 infection in the newborn. (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. All error bars represent SEM.)