Alveolar macrophages support interferon gamma-mediated viral clearance in RSV-infected neonatal mice

Abstract

Background: Poor interferon gamma (IFNγ) production during respiratory syncytial virus (RSV) is associated with prolonged viral clearance and increased disease severity in neonatal mice and humans. We previously showed that intra-nasal delivery of IFNγ significantly enhances RSV clearance from neonatal lungs prior to observed T-lymphocyte recruitment or activation, suggesting an innate immune mechanism of viral clearance. We further showed that alveolar macrophages dominate the RSV-infected neonatal airways relative to adults, consistent with human neonatal autopsy data. Therefore, the goal of this work was to determine the role of neonatal alveolar macrophages in IFNγ-mediated RSV clearance.

Methods: Clodronate liposomes, flow cytometry, viral plaque assays, and histology were used to examine the role of alveolar macrophages (AMs) and the effects of intra-nasal IFNγ in RSV infected neonatal Balb/c mice. The functional outcomes of AM depletion were determined quantitatively by viral titers using plaque assay. Illness was assessed by measuring reduced weight gain.

Results: AM activation during RSV infection was age-dependent and correlated tightly with IFNγ exposure. Higher doses of IFNγ more efficiently stimulated AM activation and expedited RSV clearance without significantly affecting weight gain. The presence of AMs were independently associated with improved RSV clearance, whereas AM depletion but not IFNγ exposure, significantly impaired weight gain in RSV-infected neonates.

Conclusion: We show here for the first time, that IFNγ is critical for neonatal RSV clearance and that it depends, in part, on alveolar macrophages (AMs) for efficient viral clearing effects. Early reductions in viral burden are likely to have profound short- and long-term immune effects in the vulnerable post-natally developing lung environment. Studies are ongoing to elucidate the pathologic effects associated with early versus delayed RSV clearance in developing neonatal airways.

Fig. 1

Inhaled IFNγ contributes to RSV clearance. Pup BALB/c or IFNγRKO mice were infected with RSV L19. Pups were treated with 16 ng/g of IFNγ or equal volumes of PBS on 1 and 3 dpi. Left lungs were harvested to quantify RSV using H&E plaque assays. * indicates p < 0.05 using a Kruskal-Wallis non-parametric analysis with Dunn’s multiple comparison test. Data represent the means and individual replicates for ≥ 3 mice per group and 2 separate experiments

Fig. 2

IFNγ exposure correlates with age-dependent AM activation. Neonatal Balb/c mice were infected with RSV L19 at 2 days (a) or 7 days (b) of age. Some mice were treated with 16 ng/g of i.n. IFNγ on 1, 3, and 5 dpi (a). BALF was harvested to quantify AM activation by flow cytometry (a-b) and IFNγ concentrations were measured and reported as the change from uninfected, age-matched controls (c). Data represent ≥ 5 mice per group and 2 separate experiments. * Indicates significant differences between RSV+ (+/− IFNγ) and RSV- groups and # between RSV+/IFNγ + and RSV+ groups based on a 2-way ANOVA with Bonferroni post-test; p < 0.05. Dot plots are representative samples from 7dpi, (the first time point in which MHC class II expression significantly increased in the RSV+/IFNγ + group) of at least five mice per group for RSV-/IFNγ- (d), RSV+/IFNγ- (f), and RSV+/IFNγ + (h). Each dot plot has a corresponding histogram representing MHC class II+ expression on CD11c + CD11b- cells with RSV-/IFNγ (e), RSV+/IFNγ- (g) and RSV+/IFNγ + (i) being presented

Fig. 3

Age-dependent IFNγ pharmacokinetics result in differential AM activation. AUCs were determined for uninfected pup and adult BALB/c mice following a single i.n. dose of IFNγ (16 ng/g) through intense sampling from LD over 48 h (a). Biologically, this translated to significantly greater activation of AMs (b) in adults beginning at 8 h and continuing through 48 and 24 h, respectively. Data represent ≥ 3 mice per group and 2 separate experiments. * Indicates significant differences based on a 2-way ANOVA with a Bonferroni post-test, between groups at the indicated time points; p < 0.5

Fig. 4

Neonates demonstrate dose dependent LM activation and viral clearance. Balb/c mice were infected with RSV L19 at 5–7 days of age and treated with i.n. IFNγ (16 or 60 ng/g) or PBS on 1, 3, and 5 dpi. Luminex was used to quantify IFNγ in BALF (a) and % of original weight was calculated from baseline litter weights (b). Lungs were collected for flow cytometry to analyze the % of CD11c + CD11b- cells expressing MHC class II+ (c) and viral titers using H & E plaque assay (d). # and * represent significant differences between pups treated with 16 and 60 ng/g compared to PBS treated pups, respectively. ^ indicates significant differences between pups treated with 16 vs. 60 ng/g of IFNγ using a 2-way ANOVA with Bonferroni post-test; p < 0.05

Fig. 5

Local IFNγ reduces airway mucus production. WT and IFNγR KO pups were infected with RSV line 19 or cell lysate, then treated with i.n. IFNγ (16 ng/g or 60 ng/g) or PBS on 1, 3, and 5 dpi as described in the methods. The lungs were harvested at 8 dpi and sections compared by extent of PAS staining. Airways were scored 0 to 4 for PAS positivity according to previously published methods [23]. a Representative examples of airway mucin production from RSV/IFNγRKO (a), RSV (b), Cell lysate (c), RSV/16 ng/g IFNγ (d), and RSV/60 ng/g IFNγ (e). Pie charts are provided to numerically represent a percentage of the total airways ranked according to mucin scores. The ratio of IL-13 to IFNγ, detected in BALF, is given for RSV-infected pups treated with IFNγ compared to pups that received PBS only on days 2–7 post-infection (f). Lastly, Taqman RT-PCR, Gob5 expression was determined at 9 dpi in RSV-infected pups receiving i.n. IFNγ compared to PBS-treated pups (g). Data represent the means ± SD for 5 mice per group and 3 separate experiments

Fig. 6

i.n. IFNγ reduces apoptosis in neonatal lungs at 8 dpi. Balb/c mice (5–7 days old) were infected with RSV L19 and treated with i.n. IFNγ (16 or 60 ng/g) or PBS on 1, 3, and 5 dpi. At 8 dpi right lungs were harvested, fixed with formalin and TUNEL stained. Two individuals blinded to treatment groups quantified TUNEL positive cells on each slide for each group. (A1-A5) Represents a single lung section from each animal in group A (RSV-/IFNγ-); Group B (RSV+/IFNγ-); Group C (RSV+ IFNγ 16 ng/g); and Group D (RSV+ IFNγ 60 ng/g). Images were captured at 40X magnification and the average number of apoptotic cells per lung section were quantified and graphed (E); there were 5 mice per group. * indicates a significant difference compared to RSV+ using ANOVA with a Tukey post-test; p < 0.5

Fig. 7

CLip effectively depletes AMs from RSV-infected neonatal lungs. Balb/c mice (2 days old) were treated with i.n. CLip and/or IFNγ as outlined in the Methods section. BALF (a and b) and digested right lung lobes (c and d) were harvested at 4 (a and c) and 8 (b and d) dpi. Total AMs (CD11c + CD11b-) were determined via flow cytometry. * Indicates a significant difference between groups using ANOVA with Bonferroni post-test. p < 0.5

Fig. 8

AM depletion reduces RSV clearance and impairs weight gain. Balb/c mice (2 days old) were treated with i.n. CLip and/or IFNγ as outlined in the Methods section. Left lungs were collected on 4 (a) and 8 (b) dpi for RSV quantification using plaque assay. Litters were weighed daily and the percent change in weight (c) was calculated from their baseline weight prior to i.n. treatments. a and b Means and individual replicates are depicted and statistical differences were defined as p < 0.05 using ANOVA with a Tukey post-test. c Mean ± SEM are depicted; *indicates a statistical difference between control (CLip-/IFNγ-) and CLip treatment groups (CLip+/IFNγ + and CLip+/IFNγ-) using repeated measures ANOVA (p < 0.05)