Aging Impairs Alveolar Macrophage Phagocytosis and Increases Influenza-Induced Mortality in Mice

Abstract

Influenza viral infections often lead to increased mortality in older people. However, the mechanisms by which aging impacts immunity to influenza lung infection remain unclear. We employed a murine model of influenza infection to identify these mechanisms. With aging, we found reduced numbers of alveolar macrophages, cells essential for lung homeostasis. We also determined that these macrophages are critical for influenza-induced mortality with aging. Furthermore, aging vastly alters the transcriptional profile and specifically downregulates cell cycling pathways in alveolar macrophages. Aging impairs the ability of alveolar macrophages to limit lung damage during influenza infection. Moreover, aging decreases alveolar macrophage phagocytosis of apoptotic neutrophils, downregulates the scavenging receptor CD204, and induces retention of neutrophils during influenza infection. Thus, aging induces defective phagocytosis by alveolar macrophages and increases lung damage. These findings indicate that therapies that enhance the function of alveolar macrophages may improve outcomes in older people infected with respiratory viruses.

Figure 1. Aging increases mortality, morbidity and lung inflammation during influenza viral lung infection

A: Young (n = 71), aged (n = 24) and advanced aged mice (n = 69) were infected intra-nasally (i.n) with 1×10⁴ pfu of influenza virus and mortality was recorded. Groups of mice that were given PBS control are shown (young n=12, aged n=7 and advanced aged n=12). DPI= days post infection. * = p<0.05; ** = p<0.01 (Log rank) B–C: Body weight (B) and clinical score (C) for young and advanced aged mice are shown during influenza viral infection described in A. * = p<0.05;**** p=<0.0001 D–F: Young and advanced aged mice were infected with influenza virus as stated in (A), LDH in BAL (D), protein concentration in BAL (E), and viral load as measured by plaque assay (F). G: Histological assessment (H and E staining) of lungs from young and advanced aged mice. The lungs from non-infected mice treated with PBS control are shown. The lungs from mice at 6 DPI are shown and were assessed for histological inflammation. The lungs from advanced aged mice exhibited increased cellular infiltration in contrast to lungs from young mice at 6 DPI. Images obtained at 10× magnification, scale bar = 200 microns. H: Survival of advanced aged mice with various dose of PR8 infection. PBS: n = 3, 10² pfu, n = 5, 10³ pfu, n = 15, 10⁴/10⁶ pfu n = 10, * = p<0.05; ** = p<0.001 (Log rank) A–F: Samples sizes= 4–5mice/time point and data shown are representative of 5 independent experiments.

Figure 2. AM are critical for mortality in both young and advanced aged mice

A–B: Survival of young (A) at 1×10⁴ pfu influenza virus and advanced aged mice at 1×10³ pfu influenza virus (B) with clodronate liposomes or control liposomes treatment during infection with. * = p<0.05; ** = p<0.001 (Log rank) C:Lungs were obtained from non-infected young (2–4 months) and advanced aged (22–24 months) C57BL/6 mice. The BAL was harvested and a single suspension was obtained. The cells were stained with relevant fluorescently tagged monoclonal antibodies and analyzed via flow cytometry. Quantification of number and frequency of AM is based on staining of frequency of F4/80⁺ CD11c^hi cells in Siglec F⁺ cells. Plots were gated on single CD45⁺ population from young and advanced aged mice D: Lungs were obtained from non-infected young (2–4 months of age) and advanced aged (i.e., 22–24 months of age) BALB/c mice and quantified as above. C–D: representative of an experiment repeated five times, * = p<0.05 E: Quantification of the absolute numbers of AM in BAL at day 6 post influenza infection. Absolute number of AM are normalized to gram of lung tissue. N = 4–5 mice / time point. *** = p<0.001 F: Lungs were obtained from young (2–4 months of age) and advanced aged (i.e., 22–24 months of age) C57BL/6 mice before (open symbols) or day 3 post infection (closed symbols). The lung homogenates were harvested and a single suspension was obtained as described in the Materials and Methods section. Flow cytometric plots were gated on AM (i.e., CD45⁺SiglecF⁺F4/80⁺CD11c^hi) and were stained with Annexin V, an apoptosis marker.

Figure 3. Aging induces a downregulation of pathways enriched in cell cycle pathways in AM

FACS purified AM from the lungs of non-infected young or advanced aged C57BL/6 mice were obtained, RNA was harvested from the lungs, and a microarray was performed. A: An overall heatmap of the array profile in AM from young and advanced aged mice. 3,545 genes were significantly altered between cohorts. N=6 biological replicates per group. B–C: Heatmap of the two most downregulated pathways with aging: cell cycle metaphase checkpoint (B) and cell cycle initiation of mitosis (C). Gene names are shown adjacent to heatmaps. N = 6 biological replicates per group were employed. D: Lungs were obtained from young (2–4 months of age) and advanced aged (i.e., 22–24 months of age) C57BL/6 mice prior to infection. The lung homogenates were harvested and a single suspension was obtained as described in the Materials and Methods section. Flow cytometric plots were gated on AM (i.e., CD45⁺SiglecF⁺F4/80⁺CD11c^hi) and stained for KI67 (a proliferative marker) as described in the Materials and Methods section and analyzed via flow cytometry. ** = p<0.01. Data are representative of an independent experiment repeated three times. E–F: mRNA was harvested from FACS purified AM from young and advanced aged mice and gene expression was measured for genes that repress self-renewal Maf-b (E) and c-Maf (F) ** = p<0.01.

Figure 4. Adoptive transfer of young AM into advanced aged mice reduces tissue injury caused by influenza virus

A: Representative flow cytometry plot showing FACS sorted AM from young donor C57BL/6 CD45.1⁺ mice that were transferred intra-nasally (i.n.) into the lungs of advanced aged (non-infected) CD45.2⁺ mice. At 4 days post adoptive transfer, the lungs and BAL were obtained and stained with indicated fluorescent antibodies and analyzed via flow cytometry. B: FACS purified AM from CD45.2⁺ young and advanced aged mice were adoptively transferred via i.n. route into young, non-infected C57BL/6 CD45.1⁺ mice. At 4 days post transfer, the lungs were obtained and stained as described in (A). Gate shows frequency of donor cells. C FACS purified AM from young or advanced aged CD45.2⁺ mice were adoptively transferred into advanced aged mice 1 day prior to influenza infection. At 3 DPI, the BAL was obtained. The level of tissue injury was quantified by LDH level in the BAL. ** = p<0.01. Pooled data from two independent experiments. D: Total viral titer in the lung quantified by qPCR.

Figure 5

Aging impairs the ability of AM to bind and internalize particles in vivo. A–B: Young and advanced aged mice were instilled with 3 × 10⁶ apoptotic neutrophils i.n. The lung tissue was subsequently harvested to obtain a cellular suspension, which was analyzed via flow cytometry. Representative flow cytometry plots in young and advanced aged mice. Samples are gated on AM as per Figure 1. B: Quantification of data; (t-test). * = p<0.05. C: Young and advanced aged mice were instilled with Alexa488 fluorescent beads i.n. Lung tissue and gating as per A. **p<0.01 B–C: Pooled data from two independent experiments. D–E: Young and advanced aged mice were infected with influenza virus as per stated Figure 1, neutrophils were quantified (via enumeration via cytospin) (D) and MPO level in BAL via ELISA (E) F: Advanced aged mice were infected with 1 ×10³ pfu of influenza virus. They were then administered clodronate liposomes or control liposomes as outlined in Figure 2B. Day 6 post infection, BAL was obtained and MPO levels were measured. *P<0.05, **** p=<0.0001

Figure 6. Aging downregulates CD204 on AM

AM from young and advanced aged mice were stained with indicated fluorescently tagged monoclonal antibodies and data were acquired via flow cytometry. Fluorescent intensity level of each surface scavenger receptor shown as change median fluorescent intensity (MFI, arbitrary units) from that obtained from isotype control staining is shown as a histogram. Adjacent figure: representative flow plots: Y axis = % of max, x axis = fluorescence. * = p<0.05 FMO = fluorescence minus one control