Inflammatory Monocytes Drive Influenza A Virus-Mediated Lung Injury in Juvenile Mice

Abstract

Healthy children are more likely to die of influenza A virus (IAV) infection than healthy adults. However, little is known about the mechanisms underlying the impact of young age on the development of life-threatening IAV infection. We report increased mortality in juvenile mice compared with adult mice at each infectious dose of IAV. Juvenile mice had sustained elevation of type I IFNs and persistent NLRP3 inflammasome activation in the lungs, both of which were independent of viral titer. Juvenile mice, but not adult mice, had increased MCP-1 levels that remained high even after viral clearance. Importantly, continued production of MCP-1 was associated with persistent recruitment of monocytes to the lungs and prolonged elevation of inflammatory cytokines. Transcriptional signatures of recruited monocytes to the juvenile and adult IAV-infected lungs were assessed by RNA-seq. Genes associated with a proinflammatory signature were upregulated in the juvenile monocytes compared with adult monocytes. Depletion of monocytes with anti-CCR2 Ab decreased type I IFN secretion, NLRP3 inflammasome activation, and lung injury in juvenile mice. This suggests an exaggerated inflammatory response mediated by increased recruitment of monocytes to the lung, and not an inability to control viral replication, is responsible for severe IAV infection in juvenile mice. This study provides insight into severe IAV infection in juveniles and identifies key inflammatory monocytes that may be central to pediatric acute lung injury secondary to IAV.

Figure 1. IAV causes increased mortality in juvenile mice that is not explained by viral titer

Juvenile and adult mice were infected intratracheally (i.t.) with 25 PFU WSN and A) survival and B) weight loss were monitored (n=9–12 mice per group, combined data from 2 independent experiments; Mantel-Cox log-rank test p<0.0001). C) IAV titer of lung homogenates on days 3, 5, and 7 p.i. as measured by viral plaque assay. Juvenile and adult data for each day were compared and analyzed by the Student’s t-test and no statistically significant difference was found. D) Immunohistochemistry for IAV nonstructural protein 1 (NS1) in lung sections from adult and juvenile mice 5 days p.i. Images shown are representative of three mice from two independent experiments. Scale bar, 100 µm.

Figure 2. IAV causes increased lung injury in juvenile mice

A) Hematoxylin and eosin (H&E) stained lung sections from uninfected adult and juvenile mice and B) 5 days p.i. with IAV (WSN 25 PFU i.t.). Images shown are representative of three mice from two independent experiments. Scale bars for insets, 100 µm. C) Wet-to-dry weight ratio day 7 p.i. D) Protein leakage into BALF on day 7 p.i. E) Total number of cells in BALF on day 7 p.i. F) Specific compliance on day 7 p.i. G) Resistance on day 7 p.i. Data in C-G are from 2 independent experiments of n=5–10 mice per group and presented as mean +/− SD. (**)p<0.01, (***)p<0.001, (****)p<0.0001, PBS versus IAV or adult versus juvenile by two-way analysis of variance (ANOVA) with a correction provided by the Sidak multiple comparisons test.

Figure 3. IAV-infected juvenile mice produce more Type I IFNs and have elevated levels of MCP-1 in serum and BALF

Juvenile and adult mice were infected with IAV (25 PFU WSN i.t.). A) IFN-β in BALF on days 0, 3, 5, and 7 p.i. as measured by ELISA. B) MCP-1 in serum on days 0, 5, and 7 p.i. as measured by ELISA. C) MCP-1 in BALF on days 0, 5, and 7 p.i. as measured by ELISA. Data in A–C are from n=8–10 mice per group and presented as mean +/− SD. (**)p< 0.01, (****)p<0.0001, adult versus juvenile by two-way ANOVA with a correction provided by the Sidak multiple comparisons test.

Figure 4. IAV-infected juvenile mice have increased activation of the NLRP3 inflammasome compared to adult mice

Juvenile and adult mice were infected with IAV (25 PFU WSN i.t.). A–B) TNF-α and IL-6 levels in BALF were measured by ELISA on days 0, 3, 5, and 7 p.i. (**)p<0.01, (****)p<0.0001, adult versus juvenile by two-way ANOVA with a correction provided by the Sidak multiple comparisons test. Lung homogenates from IAV-infected adult and juvenile mice were assessed by Western blot for C) NLRP3 D) ASC and E) pro-caspase-1. (*)=p<0.05, (**)p<0.01, PBS versus IAV or juvenile versus adult by two-way ANOVA with a correction provided by the Sidak multiple comparisons test. F–G) Mature Caspase-1 (mCaspase-1) and Mature IL-18 (mIL-18) were measured by ELISA on days 0, 3, 5, and 7 p.i. Lung homogenates from IAV-infected adult and juvenile mice were assessed by Western blot for H) mCaspase-1 and I) mIL-18. Data in A-I are from 2 independent experiments of n=5–9 mice per group and presented as mean +/− SD. (*)=p<0.05, (***)p<0.001, (****)p<0.0001, adult versus juvenile by two-way ANOVA with a correction provided by the Sidak multiple comparisons test.

Figure 5. IAV-infected Juvenile mice secrete more MCP-1 and recruit more inflammatory monocytes to the lungs

Juvenile and adult mice were infected with IAV (25 PFU WSN i.t.). CD45+ cells were isolated from lung homogenates 7 days p.i. and intrapulmonary immune cells were identified via flow cytometry. A) Total number of neutrophils (Ly6G+, CD11b+, CD24+), eosinophils (CD11b+, CD24+, Siglec F+, CD11c–, CD64–), recruited monocytes (CD11b+, Ly6C+, CD64^+/−), and alveolar macrophages (CD64+, CD11c+, Siglec F+) per dry lung weight as determined by flow cytometry of lung homogenates. Data are from 3 independent experiments of n=4–8 mice per group and presented as mean +/− SD. (*) p<0.05 juvenile versus adult as assessed by multiple T-tests with a correction provided by the Holm-Sidak multiple comparisons test. B–E) Intracellular immunofluorescent antibody staining for caspase-1 and NLRP3 in Ly6C+CD64^+/− cells analyzed by flow cytometry. Data represented by histogram with fluorescence minus one (FMO) control (B and D) or the median fluorescent intensity (MFI) (C and E). Data in B-E are from 2 independent experiments of n=3–4 mice per group and presented as mean +/− SD. (*) p<0.05, adult versus juvenile (A) or PBS versus IAV and adult versus juvenile (C and E) by two-way ANOVA with a correction provided by the Sidak multiple comparisons test. Neut = neutrophils, eos = eosinophils, MoDCs = recruited monocytes, AMs = alveolar macrophages, FMO = fluorescence minus one.

Figure 6. Juvenile monocytes recruited to lungs differ from adult monocytes in their transcriptomic response to IAV

Recruited monocytes were isolated from juvenile and adult mice on days 0, 3, and 5 p.i. were analyzed by RNA-Seq. A) Volcano plots of pairwise comparisons on days 0, 3, and 5 p.i. reveal differences between juvenile and adult responses to IAV. Log₂FC cut-off was 1.0 B) Normalized counts data (FPKM) were analyzed by ANOVA between groups with BH adjustment, resulting in a list of 5,776 genes (FDR 0.05). K-means clustering of the 5,776 genes identified 5 clusters. Selected genes from each cluster are shown. C) Functional enrichment analysis of the 5 clusters using GOrilla revealed top GO terms related to immune response functions in four of five clusters. D) Normalized gene expression data (FPKM) of representative genes identified in (B). Data are displayed as mean FPKM +/− STD.

Figure 7. Recruited monocytes in juvenile murine lung display a more pro-inflammatory transcriptome following IAV infection

A) Heat maps demonstrate RNA transcript expression from recruited monocytes isolated from the lungs of juvenile and adult IAV-infected mice on 0, 3, and 5 days p.i. Genes of interest were separated into functional markers: pro-inflammation and reparative. B) Expresion data (FPKM) from selected genes identified following k-means clusterin in 6B. Data are displayed as mean FPKM +/− STD.

Figure 8. Inhibition of monocyte recruitment with anti-CCR2 protects Juvenile mice from IAV-induced mortality and lung injury without impacting viral clearance

Juvenile mice were infected intratracheally with 25 pfu WSN IAV. On day 2 p.i., mice were treated with either anti-CCR2 antibody (50 µL retro-orbital) or PBS control to deplete classical monocytes and prevent Ly6C⁺CD64^+/− monocyte recruitment. Blood was collected from the submandibular vein on days 3, 5, and 7 p.i. and A) CD115⁺ blood monocytes were measured by flow cytometry in uninfected/PBS-treated, IAV-infected/anti-CCR2-treated, and IAV-infected/PBS control-treated mice. Lungs were harvested from IAV-infected/PBS-treated and IAV-infected/anti-CCR2 treated mice on days 3, 5, and 7 p.i. and B) Ly6C+CD64^+/− cells were assessed by flow cytometry. Juvenile and adult mice were infected i.t. with 25 PFU WSN and treated with anti-CCR2 antibody (50 µL retro-orbital) on day 2 p.i. and C) survival and D) weight loss were monitored (n=8–10 mice per group, combined data from 2 independent experiments; Mantel-Cox log-rank test p<0.0001). E) Viral titer on days 3, 5, and 7 p.i. measure by plaque assay. F) IFNα in BALF on days 3, 5, and 7 p.i. measured by ELISA. G) Protein in BALF on day 7 p.i. H) Total cells in BALF on day 7 p.i. I–J) mCaspase-1 and mIL-18 in BALF on day 7 p.i. measured by ELISA. Data in E–J are from 2 independent experiments of n=5–10 mice per group and presented as mean +/− SD. (*)=p<0.05, (**)p<0.01, (***)p<0.001, (****)p<0.0001, IAV-infected/PBS control-treated versus IAV-infected/anti-CCR2 treated mice by two-way ANOVA with a correction provided by the Sidak multiple comparisons test.

Figure 9. A model depicting how the juvenile innate immune response to IAV predisposes to IAV-induced lung injury

Juvenile mice infected with IAV produce more IFN-αβ, secrete more MCP-1, recruit more inflammatory monocytes, and have increased activation of the NLRP3 inflammasome compared to adult mice. These differences contribute to increased IAV-induced lung injury in juvenile mice.