Effects of Influenza on Alveolar Macrophage Viability Are Dependent on Mouse Genetic Strain

Abstract

Secondary bacterial coinfections following influenza virus pose a serious threat to human health. Therefore, it is of significant clinical relevance to understand the immunological causes of this increased susceptibility. Influenza-induced alterations in alveolar macrophages (AMs) have been shown to be a major underlying cause of the increased susceptibility to bacterial superinfection. However, the mechanisms responsible for this remain under debate, specifically in terms of whether AMs are depleted in response to influenza infection or are maintained postinfection, but with disrupted phagocytic activity. The data presented in this article resolves this issue by showing that either mechanism can differentially occur in individual mouse strains. BALB/c mice exhibited a dramatic IFN-γ-dependent reduction in levels of AMs following infection with influenza A, whereas AM levels in C57BL/6 mice were maintained throughout the course of influenza infection, although the cells displayed an altered phenotype, namely an upregulation in CD11b expression. These strain differences were observed regardless of whether infection was performed with low or high doses of influenza virus. Furthermore, infection with either the H1N1 A/California/04/2009 (CA04) or H1N1 A/PR8/1934 (PR8) virus strain yielded similar results. Regardless of AM viability, both BALB/c and C57BL/6 mice showed a high level of susceptibility to postinfluenza bacterial infection. These findings resolve the apparent inconsistencies in the literature, identify mouse strain-dependent differences in the AM response to influenza infection, and ultimately may facilitate translation of the mouse model to clinical application.

Figure 1

Influenza A virus infection induces depletion of AMs in BALB/c mice. (A) Flow cytometry analysis of AMs and IMs in the lung (A) and BAL (B) of naive and CA04-infected BALB/c mice 7 days post-infection. Representative plots show frequency and expression of PKH26 and CD11b on AMs (CD11c^hiF4/80⁺) and CD11c^loMCs (CD11c^loF4/80⁺). Fluorescence minus one (FMO) control represents negative control as solid gray peak. (C–D) Total numbers of AMs and PKH26⁺AMs in the lung (C) and BAL (D) of BALB/c mice on day 7 post-CA04 infection. (E–F) Total numbers of AMs and PKH26⁺AMs in the lung (E) and BAL (F) of BALB/c mice on day 9 post-CA04 infection. Statistical analyses were performed by two-way ANOVA; *** p<0.001; **** p<0.0001. (G–H) MFI of PKH26 on AMs and IMs in the lung (G) and BAL (H) of naïve and influenza-infected BALB/c mice. Statistical analyses were performed by Mann-Whitney U test; ** p<0.01. Data shown are representative of 2 independent experiments with 5 mice per group.

Figure 2

Lung resident AMs are maintained in post-influenza infected C57Bl/6 mice. (A) Flow cytometry analysis of AMs and IMs macrophages in the lung (A) and BAL (B) of naive and CA04-infected C57Bl/6 mice 7 days post-infection. Representative plots show frequency and expression of PKH26 and CD11b on AMs (CD11c^hiF4/80⁺) and CD11c^loMCs (CD11c^loF4/80⁺). Fluorescence minus one (FMO) control represents negative control as solid gray peak. (C–D) Total numbers of AMs and PKH26⁺AMs in the lung (C) and BAL (D) of C57Bl/6 mice on day 7 post-CA04 infection. (E–F) Total numbers of AMs and PKH26⁺AMs in the lung (E) and BAL (F) of C57Bl/6 mice on day 9 post-CA04 infection. Statistical analyses were performed by two-way ANOVA; ** p<0.01; *** p<0.001. (G–H) MFI of PKH26 on AMs and IMs in the lung (G) and BAL (H) of naïve and influenza-infected C57Bl/6 mice. Statistical analyses were performed by Mann-Whitney U test; ** p<0.01. Data shown are representative of 2 independent experiments with 5 mice per group.

Figure 3

AMs are reduced in C57Bl/6 mice following i.n., but not i.v., administration of clodronate liposomes. (A) Naïve and CA04 infected mice were treated with clodronate- or PBS-containing liposomes by i.n. or i.v. injection on days 7 and 8 post-influenza infection. Representative dot plots show the gating strategy for AMs and CD11c^loMCs in the BAL of naïve and CA04-infected C57Bl/6 mice on day 9 following CA04 infection. (B–E) Total numbers of AMs (B–C) and IMs (D–E) in the lung (B and D) and BAL (C and E). Data shown are representative of 2 independent experiments with 5 mice per group. Statistical analyses were performed by two-way ANOVA; * p<0.05; ** p<0.01.

Figure 4

IFN-γ deficiency prevents AM depletion in influenza-infected BALB/c mice. (A and B) Total number of AMs in the lung (A) and BAL (B) of BALB/c WT and IFN-γ^−/− mice on Day 9 after infection with 10 PFU of the CA04 virus. (C and D) Representative plots show frequency and expression of Siglec F and CD11b on AMs in the lungs (C) and BAL (D) of naïve and influenza infected BALB/c WT and IFN-γ^−/− mice. Data shown are representative of 2 independent experiments with 5 mice per group. Statistical analyses were performed by two-way ANOVA; * p<0.05; ** p<0.01.

Figure 5

BALB/c and C57Bl/6 mice are highly susceptible to synergistic viral-bacterial coinfection. BALB/c and C57Bl/6 mice were challenged with PBS or 10 PFU of CA04 virus. At day 8 post-influenza infection, mice were inoculated i.n. with PBS or 500 CFU of S. pneumoniae A66.1. (A and B) Survival (A) and weight loss (B) were monitored daily for 10 days following S. pneumoniae A66.1 infection. Survival data were analyzed using the log-rank Mantel-cox test and weight loss data were analyzed by the Mann-Whitney U test; **** p<0.0001; ns = not significant. Data shown were pooled from 2 independent experiments with 8 mice per group. (C–F) BALB/c and C57Bl/6 were i.n. inoculated with 250 CFU (C and D) or 30 CFU (E and F) of S. pneumoniae A66.1 eight days after influenza infection. Bacterial burden was evaluated in total lung (C and E) and BAL (D and F) 24 h post-bacterial i.n. challenge. Data shown were pooled from 2 independent experiments with 5 mice per group.

Figure 6

BALB/c and C57Bl/6 both exhibit similar mortality to influenza-pneumococcal infection superinfection, even at low bacterial challenge doses. BALB/c and C57Bl/6 mice were challenged with PBS or 10 PFU of CA04 virus. At day 8 post-influenza infection, the mice were inoculated i.n. with 250 (A), 30 (B), 15 (C), 7.5 (D) or 3 (E) CFU of S. pneumoniae A66.1. Survival was monitored daily for 10 days following S. pneumoniae A66.1 infection. Data shown were pooled from 2 independent experiments with 8 mice per group. (F) Viral titers were measured in whole lung homogenate of BALB/c and C57Bl/6 mice at day 8 post-influenza infection. Data shown are representative of 2 independent experiments with 5 mice per group.