Age predicts cytokine kinetics and innate immune cell activation following intranasal delivery of IFNγ and GM-CSF in a mouse model of RSV infection

Abstract

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children and is further associated with increased healthcare utilization and cost of care in the first years of life. Severe RSV disease during infancy has also been linked to the later development of allergic asthma, yet there remains no licensed RSV vaccine or effective treatment. Pre-clinical and clinical studies have shown that disease severity and development of allergic asthma are associated with differences in cytokine production. As a result, stimulation of the innate host immune response with immune potentiators is gaining attention for their prospective application in populations with limited immune responses to antigenic stimuli or against pathogens for which vaccines do not exist. Specifically, macrophage-activating cytokines such as interferon gamma (IFNγ) and granulocyte colony-stimulating factor (GM-CSF) are commercially available immune potentiators used to prevent infections in patients with chronic granulomatous disease and febrile neutropenia, respectively. Moreover, an increasing number of reports describe the protective function of IFNγ and GM-CSF as vaccine adjuvants. Although a positive correlation between cytokine production and age has previously been reported, little is known about age-dependent cytokine metabolism or immune activating responses in infant compared to adult lungs. Here we use a non-compartmental pharmacokinetic model in naïve and RSV-infected infant and adult BALB/c mice to determine the effect of age on IFNγ and GM-CSF elimination and innate cell activation following intranasal delivery.

Keywords: Age-dependent; Cytokine kinetics; Granulocyte macrophage-colony stimulating factor; Infant; Interferon gamma; Respiratory syncytial virus.

Figure 1. The pharmacokinetic profile of intranasal IFN-γ depends largely on age at the time of administration

Adult and infant BALB/c mice were treated with a single intranasal dose of IFNγ (16ng/g). IFNγ concentrations were subsequently measured in homogenized lung tissue and serum using a multi-plex Luminex platform at the indicated times. (A) Adult and (B) infant IFNγ concentrations in the lung (y-axis) and blood (z-axis) were reported on discriminate scales to accommodate differential concentration ranges; insets provide a detailed depiction of the kinetic changes during the early time points from 0–4 hours. Data was transformed on a log scale to identify the terminal elimination phase of IFNγ in adult and infant (C) lungs and (D) blood. Each time point represents the mean and SEM of three separate mice.

Figure 2. The pharmacokinetic profile of intranasal GM-CSF is largely dependent on age

Adult and infant BALB/c mice were treated with a single intranasal dose of GM-CSF (50ng/g). GM-CSF concentrations were subsequently measured in homogenized lung tissue and serum using a multi-plex Luminex platform at the indicated times. (A) Adult and (B) infant GM-CSF concentrations in the lung (y-axis) and blood (z-axis) were reported on discriminate scales to accommodate differential concentration ranges; insets provide a detailed depiction of the kinetic changes during the early time points from 0–4 hours. Data was transformed on a log scale to identify the terminal elimination phase of IFNγ in adult and infant (C) lungs and (D) blood. Each time point represents the mean and SEM of three separate mice.

Figure 3. GM-CSF and IFNγ differentially induce expression of activation markers in adults and infants

Adult and infant BALB/c mice were given a single intranasal dose of IFNγ (16ng/g) (shaded), GM-CSF (50ng/g) (dashed line), or PBS only (solid line). At the indicated time points lungs were harvested for flow cytometry to compare the frequency of CD11b (A and D), MHC class II (B and E), and CD86 (C and F) on CD11c+ large cells between the two different intranasal cytokines in both infants and adults.

Figure 4. Adult cells are more responsive than infant cells to IFNγ and GM-CSF

Adult and infant BALB/c mice were given a single intranasal dose of (A–C) IFNγ (16ng/g) or (D–F) GM-CSF (50ng/g). At the indicated time points lungs were harvested for flow cytometry to determine the frequency of CD11b (A and D), MHC class II (B and E), and CD86 (C and F) on CD11c+ large cells. A Kruskal-Wallis, 1-way ANOVA with Dunn’s correction for multiple comparisons was used to identify differences within groups over time; dashed (infants) and solid (adults) lines indicate significant differences; p<0.05. A 2-way ANOVA with Bonferroni correction for multiple comparisons was used to determine differences between groups over time; ** p<0.01; *** p<0.001; **** p<0.0001

Figure 5. IFNγ differentially induced cytokine production in adult and infant lungs

Adult and infant BALB/c mice were given a single intranasal dose of IFNγ (16ng/g). At the indicated time points lungs were harvested and processed for a multi-panel Luminex cytokine array (A–F). A Kruskal-Wallis, 1-way ANOVA with Dunn’s correction for multiple comparisons was used to identify differences within groups over time; asterisks indicate significant differences from the respective infant or adult time “0” control. Lines indicate differences between Infants and adults at the same time point; *p<0.05. **p<0.01; ***p<0.001, ****p<0.0001.

Figure 6. GM-CSF differentially induced cytokine production in adult and infant lungs

Adult and infant BALB/c mice were given a single intranasal dose of GM-CSF (50ng/g). At the indicated time points lungs were harvested and processed for a multi-panel Luminex cytokine array following protein normalization by BCA (A–F). A Kruskal-Wallis, 1-way ANOVA with Dunn’s correction for multiple comparisons was used to identify differences within groups over time; asterisks indicate significant differences from the respective infant or adult time “0” control. Lines indicate differences between Infants and adults at the same time point; *p<0.05. **p<0.01; ***p<0.001.

Figure 7. IFNγ-induced activation of innate immune cells correlate with reduced viral clearance

Infant BALB/c mice were infected with 5×10⁵ PFU/g of body weight, then intranasal IFNγ (16ng/g) or PBS was delivered on 1, 3, and 5 dpi. At 4 or 7 dpi, lungs were harvested for flow cytometry to determine the frequency of CD11b (A and E), MHC class II (B and F), and CD86 (C and G) on CD11c+ large cells. RSV plaque assays were performed on homogenized lung to determine differences in plaque forming units (PFU)/g of lung weight at (D) 4 dpi and (H) 7dpi. A Kruskal-Wallis, 1-way ANOVA with Dunn’s correction for multiple comparisons was used to identify differences in marker expression between groups at 4 or 7 dpi. p<0.001. An unpaired t-test was used to determine differences in RSV (PFU/g of lung) at 4dpi or 7 dpi. * p<0.05; ** p<0.01; ***

Figure 8. GM-CSF fails to increase activation markers or reduce viral titers in infant lungs

Infant BALB/c mice were infected with 5×10⁵ PFU/g of body weight. The mice were then given intranasal IFNγ (16ng/g) or PBS on 1, 3, and 5 dpi or intranasal GM-CSF (50ng/g) or PBS daily. At 4 or 7 dpi, lungs were harvested for flow cytometry to determine the frequency of CD11b (A and E), MHC class II (B and F), and CD86 (C and G) on CD11c+ large cells. RSV plaque assays were performed to determine differences in plaque forming units (PFU)/g of lung weight at (D) 4 dpi and (H) 7dpi. A Kruskal-Wallis, 1-way ANOVA with Dunn’s correction for multiple comparisons was used to identify differences in marker expression between groups at 4 or 7 dpi. p<0.001. An unpaired t-test was used to determine differences in RSV (PFU/g of lung) at 4dpi or 7 dpi. * p<0.05; ** p<0.01; ***

Figure 9. No delay in weight gain was observed following intranasal delivery of IFNγ or GM-CSF in RSV-infected infant mice

Infant BALB/c mice were infected with 5×10⁵ PFU/g of body weight. The mice were then given intranasal IFNγ (16ng/g) or PBS on 1, 3, and 5 dpi (A) or intranasal GM-CSF (50ng/g) or PBS daily (B). Weights were recorded daily through 10 dpi for all groups. A 2-way ANOVA with Bonferroni correction for multiple comparisons was used to identify differences in marker expression between cytokine-treated groups and their respective controls over time. *p<0.05.