The neonatal anti-viral response fails to control measles virus spread in neurons despite interferon-gamma expression and a Th1-like cytokine profile

Abstract

Neonates are highly susceptible to viral infections in the periphery, potentially due to deviant cytokine responses. Here, we investigated the role of interferon-gamma (IFNγ), a key anti-viral in the neonatal brain. We found that (i) IFNγ, which is critical for viral control and survival in adults, delays mortality in neonates, (ii) IFNγ limits infiltration of macrophages, neutrophils, and T cells in the neonatal brain, (iii) neonates and adults differentially express pathogen recognition receptors and Type I interferons in response to the infection, (iv) both neonates and adults express IFNγ and other Th1-related factors, but expression of many cytokines/chemokines and IFNγ-responsive genes is age-dependent, and (v) administration of IFNγ extends survival and reduces CD4 T cell infiltration in the neonatal brain. Our findings suggest age-dependent expression of cytokine/chemokine profiles in the brain and distinct dynamic interplays between lymphocyte populations and cytokines/chemokines in MV-infected neonates.

Keywords: Interferon-gamma; Measles virus; Microglia; Natural killer cells; Neonatal; T cells.

Graphical abstract

Fig. 1

IFNγ delays, but does not prevent, mortality despite higher viral load in infected CD46+ neonates. A. Kaplan-Meier plot of CD46+ neonates on various knockout backgrounds. CD46+, CD46+/IFNγ-KO, and CD46+/RAG2-KO neonates were infected intracranially with measles virus (MV) (10^4 PFU/10 μl PBS) at 2 days of age. Mice were monitored for symptoms of illness and death for 35 days post-infection. Statistical analysis was applied by log rank test (p < 0.0001). Results from 4 to 6 separate litters were pooled (n = 30–50 mice per condition). Whole brain lysates from neonatal (B) and adult (C) MV-infected mice were collected at 4 and 6 dpi. RNA levels of measles virus nucleocapsid (N) transcript were quantified using qRT-PCR. Bars represent the average of mice from three independent experiments (n = 9–14) and error bars represent SD. Statistical analysis was applied by two-way ANOVA (#p < 0.001, *p < 0.05, NS = not significant) with Bonferroni post hoc test.

Fig. 2

CD46+ neonates lose body and brain weight during infection. The body weights (A, B) and brain weights (C, D) of CD46+ (A, C) and CD46+/IFNγ-KO neonates (B, D) at different time points post-infection were measured. Weights were recorded at the time of harvest for flow cytometry experiments. Mean values are represented by horizontal bars for each condition. Statistical analysis was applied by two-way ANOVA (*p < 0.05) with Bonferroni post hoc test.

Fig. 3

Infiltration of macrophages and activation of microglia/macrophages in the CNS occurs during MV-infection in an IFNγ-independent manner. Whole brain homogenates from CD46+ (A) and CD46+/IFNγ-KO (B) neonates were analyzed for microglia (A; CD45^intermediate) and macrophages (B; CD45^high) by flow cytometry. The horizontal line represents the mean number of cells for each condition. Results from 4 to 5 different litters were collected, and statistical analysis was applied by two-way ANOVA (#p < 0.001, *p < 0.05) with Bonferroni post hoc test. Whole brains from MV-infected and uninfected control CD46+ neonates (C) and CD46+/IFNγ-KO neonates (D) were collected at 7 dpi. Sagittal sections from the neocortex were immunostained for measles (Hemagglutinin and Matrix protein; red), microglia/macrophages (Iba1; green) and Hoechst 33,342 stain (blue) as a nuclear marker. Slides from 4 to 5 mice per condition were examined, and representative sections with MV + cells are shown. Scale bar = 200 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

IFNγ does not affect NK cell infiltration, but downregulates neutrophil infiltration, in neonates. Whole brain homogenates from CD46+ (A) and CD46+/IFNγ-KO (B) neonates were analyzed for total natural killer (NK) cell numbers at 2, 4, and 6 dpi by flow cytometry (CD3-/NK1.1+/CD49b+). The horizontal line represents the mean number of cells for each condition. Results from 4 to 5 different litters were collected, and statistical analysis was applied by two-way ANOVA (#p < 0.001, *p < 0.05) with Bonferroni post hoc test. Whole brain homogenates from MV-infected CD46+, CD46+/IFNγ-KO, and CD46+/RAG2-KO neonates (C) were analyzed for neutrophils numbers (CD45^hi/CD11b+/Ly6G+) at 4 dpi and 6 dpi by flow cytometry. Results from 3 different litters were collected, and statistical analysis was applied by one-way ANOVA (*p < 0.05) with Bonferroni post hoc test.

Fig. 5

Neonates show higher T cell infiltration at later stages of infection in the absence of IFNγ. Flow cytometry was performed on whole brain homogenates for CD4 T cells (CD3+/CD4+/CD19−) or CD8 T cells (CD3+/CD8+/CD19−). CD4 T-cells (left column; A, C, E) and CD8 T cells (right column; B, D, E) were quantified in uninfected and MV-infected CD46+ and CD46+/IFNγ-KO neonates at 4 (A, B), 7 (C, D), and 10 dpi (E, F). The black line represents the mean number of cells for each group. Results represent pups from 4 to 5 different litters. Statistical analysis was applied by two-way ANOVA (*p < 0.05, ^#p < 0.001) with Bonferroni post hoc test.

Fig. 6

CD4 T cell infiltration in the CNS is greater in MV-infected adults than in neonates. Whole brain homogenates from neonatal and adult CD46+ (A) and CD46+/IFNγ-KO (B) mice were analyzed for infiltrating T cells at 7 dpi. Flow cytometry was performed for CD4 T cells (CD3+/CD4+/CD19−) or CD8 T cells (CD3+/CD8+/CD19−). Mice from 4 to 5 different litters were compared for each condition. Statistical analysis was applied by two-way ANOVA (*p < 0.05) with Bonferroni post hoc test.

Fig. 7

MV-infection induces distinct expression of pattern recognition receptors in the neonatal and adult CNS. Brains of uninfected and MV-infected CD46+ mice were analyzed for the mRNA and protein expression of pattern recognition receptors (PRRs) at 7 dpi. CD46+, CD46+/IFNγ-KO, and CD46+/RAG2-KO neonates (first column) or CD46+ neonates and adults (second, third, and fourth columns) were compared. qRT-PCR analysis of the whole brain was performed for RIGI (A, B), TLR3 (E, F), and TLR7 (I, J). mRNA expression is shown as the fold-change normalized to the CD46+ uninfected controls (n = 4–5 mice/condition). Lysates of hippocampal (C, G, K) and cerebellar (D, H, L) tissue were analyzed by western blots for RIG-I (C, D), TLR3 (G, H), and TLR7 (K, L). Protein expression is shown as the fluorescence signal for each protein quantified and normalized to GAPDH (n = 3–4 mice/condition). Each bar represents the mean fold-change and SEM. Statistical differences were determined by two-way ANOVA (*p < 0.05, ^#p < 0.001) with Bonferroni post hoc test.

Fig. 8

Neonatal mice induce greater expression of Type I interferons during MV infection in comparison to adults. Brains of uninfected and MV-infected CD46+ mice were analyzed for the mRNA expression of Type I interferons at 3 dpi and 7 dpi. CD46+, CD46+/IFNγ-KO, and CD46+/RAG2-KO neonates (left column; A, C, E) and CD46+ neonates and adults (right column; B, D, F) were compared. qRT-PCR analysis was performed for IFNα4 (A, B), IFNβ (C, D) and MDA5 (E, F). Relative gene expression is shown as the fold-change normalized to the CD46+ uninfected controls (n = 4–5 mice/condition). Each bar represents the mean fold-change and SEM. Statistical differences were determined by three-way ANOVA (*p < 0.05, ^#p < 0.001) with Bonferroni post hoc test.

Fig. 9

Despite elevated IFNγ expression during infection, transcription of IFNγ-responsive genes is age-dependent. Brains of uninfected and MV-infected CD46+ mice were analyzed for the mRNA expression of IFNγ and CIITA at 7 dpi. CD46+, CD46+/IFNγ-KO, and CD46+/RAG2-KO neonates (left column; A, C) and CD46+ neonates and adults (right column, B, D) were compared. qRT-PCR analysis was performed for IFNγ (A, B) and CIITA (C, D). Relative gene expression is shown as the fold-change normalized to the CD46+ uninfected controls (n = 4–5 mice/condition). Each bar represents the mean fold-change and SEM. Statistical differences were determined by two-way ANOVA (*p < 0.05, ^#p < 0.001) with Bonferroni post hoc test.

Fig. 10

IFNγ delays mortality and reduces CD4 T cell infiltration in CD46+/IFNγ-KO neonates. Kaplan-Meier plot of CD46+/IFNγ-KO neonates infected intracranially with measles virus (MV) (10^4 PFU/10 μl PBS) at 2 days of age and treated with recombinant IFNγ (100 U in 10μl PBS at the start of infection and every three days post-infection). Mice were monitored for symptoms of illness and death until all mice had succumbed to infection. Statistical analysis was applied by log rank test (p < 0.01). Results from two separate litters were pooled (n = 7–10 mice per condition). Whole brain homogenates were analyzed by flow cytometry for CD4 T cells (A; CD3+/CD4+/CD19−) or CD8 T cells (B; CD3 +/CD8 +/CD19-) at 7 dpi from untreated (black bars) and IFNγ-treated (gray bars) CD46+/IFNγ-KO neonates infected with MV. Mice from 3 to 5 different litters were compared for each condition. Each bar represents the average number of T cells per 1 × 10⁵ events and the error bars represent SEM. Statistical differences were determined by student t-test (*p < 0.05, ^#p < 0.001).