Critical Role of Airway Macrophages in Modulating Disease Severity during Influenza Virus Infection of Mice ▿

  1. Patrick C. Reading1,3,*
  1. 1Department of Microbiology and Immunology, University of Melbourne, Parkville 3010 Victoria, Australia
  2. 2Department of Molecular Cell Biology, Faculty of Medicine, Vrije Universiteit of Amsterdam, Amsterdam, Netherlands
  3. 3WHO Collaborating Centre for Reference and Research on Influenza, North Melbourne 3051, Victoria, Australia
  1. FIG. 1.
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    FIG. 1.

    Influenza viruses BJx109 and PR8 differ in their abilities to infect Mφ, but not epithelial cells. (A) Monolayers of mouse PEC Mφ, BAL Mφ, MDCK cells, and LA-4 epithelial cells were infected in chamber slides with 106 PFU of BJx109 or PR8, as described in Materials and Methods. At 6 to 8 h postinfection, cells were fixed and stained via immunofluorescence for expression of influenza A virus nucleoprotein (NP). The mean percent infection (plus 1 standard deviation [SD]) from a minimum of 4 independent fields per chamber is shown for a representative experiment, and infection of Mφ by PR8 was significantly reduced compared to BJx109 in 4 independent experiments (P < 0.01; one-way ANOVA). (B) BAL Mφ or LA-4 epithelial cells were infected with 106 PFU of BJx109 or PR8 or with reverse-engineered viruses expressing 7 genes derived from PR8 with the HA (PR8-BJx109 HA) or the NA (PR8-BJx109 NA) of BJx109 and stained by immunofluorescence at 8 h postinfection. The data from a representative experiment are shown, and infection of Mφ with PR8 or PR8-BJx109 NA was significantly different from that with BJx109 in 3 independent experiments (P < 0.01; one-way ANOVA). (C) Numbers of adherent PEC Mφ and LA-4 cells 24 h after exposure to influenza viruses. Monolayers of PEC Mφ and LA-4 cells were infected in chamber slides with 106 PFU of BJx109 or PR8 or mock infected with medium alone. At 24 h postinfection, cells were fixed and stained via immunofluorescence for nucleic acids using PI. Nuclear morphology was assessed, and intact nuclei were counted in 4 independent fields. The data are expressed as a percentage of the number of nuclei counted in mock-infected controls. The error bars represent 1 SD. The data from a representative experiment are shown, and numbers of adherent Mφ exposed to BJx109 were significantly reduced compared to PR8 in 3 independent experiments (P < 0.01; one-way ANOVA). (D) Release of infectious virus from influenza virus-infected PEC Mφ and LA-4 cells. Monolayers of cells were infected with 106 PFU of BJx109 or PR8, and samples of supernatant were removed at 2 h (to detect residual virus inoculum) and 24 h (to detect newly synthesized virions released from infected cells) postinfection. The samples were assayed for infectious virus by plaque assay on MDCK cells, and the fold increase in infectious virus was calculated by dividing the viral titer obtained at 24 h by that obtained at 2 h. The data represent the mean (plus 1 SD) from 3 independent experiments.

  2. FIG. 2.
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    FIG. 2.

    Virulence of influenza viruses for C57BL/6 mice. Groups of 5 mice were infected with 105 PFU of BJx109 or PR8 (A) or reverse-genetics virus PR8-BJx109 HA or PR8-BJx109 NA (B) via the intranasal route. Control mice received an equivalent volume of PBS. (Top) Mice were weighed daily, and the results are expressed as the mean percent weight change of each group ± standard error of the mean (SEM) compared to the weight immediately prior to infection. Animals displaying evidence of pneumonia and/or having lost >25% of their original body weights were euthanized. (Bottom) Survival of mice following intranasal infection with influenza viruses. The data shown are from one experiment and are representative of two or more independent experiments. The P values for survival proportions were obtained using the Mantel-Cox log rank test. BJx109 versus PR8, P < 0.01, and PR8-BJx109 HA versus PR8-BJx109 NA, P < 0.01, in 2 independent experiments.

  3. FIG. 3.
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    FIG. 3.

    Depletion of lung macrophages via treatment with clodronate-loaded liposomes. Groups of 5 naïve mice were treated with 100 μl of CL-LIP via the intranasal route. Control mice received an equivalent volume of PBS or SL-LIP. At 48 h posttreatment, the mice were killed, and the cell types present in BAL fluid and lungs were determined. (A) Representative dot plots of BAL cells from mice treated with PBS, SL-LIP, or CL-LIP. AM were identified as CD11c+ MHC class IIint and pulDC as CD11c+ MHC class IIhigh by flow cytometry. Total numbers of AM (B) and pulDC (C) in the BAL fluid and lung were determined by flow cytometry. *, CL-LIP-treated mice were significantly different from SL-LIP-treated controls (P < 0.05; one-way ANOVA).

  4. FIG. 4.
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    FIG. 4.

    Effect of macrophage depletion on the course of disease following intranasal infection with influenza viruses. Groups of 5 B6 mice were depleted of macrophages via i.n. treatment with CL-LIP 48 h prior to infection with 105 PFU of BJx109 (A), Phil/82-X (B), or PR8 (C) and every 3 days thereafter. Control groups received an equivalent volume of SL-LIP or PBS alone. (Top) The mice were weighed daily, and the results are expressed as the mean percent weight change of each group (±SEM) compared to the original body weight. (Bottom) Animals that had lost ≥25% of their original body weight and/or presented with evidence of pneumonia were killed. The P values for survival proportions were obtained from 2 independent experiments using the Mantel-Cox log rank test. BJx109, CL-LIP versus SL-LIP, P < 0.01; Phil/82-X, CL-LIP versus SL-LIP, P < 0.01; and PR8, CL-LIP versus SL-LIP, P > 0.05.

  5. FIG. 5.
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    FIG. 5.

    Depletion of airway macrophages during BJx109 infection is associated with enhanced virus replication in the airways. Groups of 5 B6 mice were depleted of macrophages via i.n. treatment with 100% CL-LIP or 30% CL-LIP 48 h prior to infection with 105 PFU of BJx109 and every 3 days thereafter. Control groups received an equivalent volume of SL-LIP or PBS alone. The mice were killed and analyzed at day 7 postinfection. (A) Lungs and nasal tissues were homogenized, and virus titers were determined by plaque assay on MDCK cells. The bars represent mean viral titers plus 1 SD. The detection limit for the plaque assays is indicated as a dotted line. *, virus titers from 100% CL-LIP- and 30% CL-LIP-treated mice that were significantly higher than those from SL-LIP-treated control animals (P < 0.01; one-way ANOVA). (B) Distribution of viral antigen in the lungs of macrophage-depleted or control mice. Representative images are shown at ×10 magnification. Cells positive for viral antigen are stained brown, and the arrows indicate representative areas of intense antigen staining.

  6. FIG. 6.
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    FIG. 6.

    Depletion of airway macrophages during BJx109 infection is associated with enhanced pulmonary inflammation. Groups of 5 B6 mice were depleted of airway macrophages via i.n. treatment with 100% or 30% CL-LIP 48 h prior to infection with 105 PFU of BJx109 and every 3 days thereafter. Control groups received an equivalent volume of SL-LIP or PBS alone, and all mice were killed and analyzed at day 7 postinfection. (A) Representative images of inflammation in lung sections following H&E staining. Mice infected with 105 PFU of PR8 and analyzed at day 5 postinfection are included for comparison. The images are shown at ×10 magnification. (B) Histopathological scores for lung sections from BJx109-infected mice. Lung sections were scored blind for alveolitis and peribronchiolar inflammation from 0 to 5. The data shown represent scores from individual mice (as indicated by circles) and median values (as indicated by bars) obtained from 1 of 3 independent readers. Samples were compared for statistical significance using the Kruskal-Wallis test (nonparametric). For each reader, significant differences were observed in immunopathology scores between CL-LIP-treated mice (100% or 30%) and SL-LIP-treated mice (P < 0.05). (C) Inflammatory cells present in BAL fluid of BJx109-infected mice. BAL cells recovered from the airspaces of the lung were examined by flow cytometry for total numbers of CD45+ inflammatory cells, as well as for leukocyte subsets, including neutrophils (Gr-1high), NK cells (NK1.1+ TCR-β−), CD4+ cells (CD4+), CD8+ cells (CD8+), and pulDC (CD11c+ MHC class IIhigh). A minimum of 50,000 living cells (PI−) were collected and analyzed for each mouse. *, cell numbers from 100% CL-LIP- and 30% CL-LIP-treated mice that were significantly higher than those from SL-LIP-treated control animals (P < 0.05; one-way ANOVA).

  7. FIG. 7.
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    FIG. 7.

    Vascular leakage and pulmonary edema in macrophage-depleted mice infected with BJx109. Groups of 5 B6 mice were depleted of macrophages via i.n. treatment with 100% or 30% CL-LIP 48 h prior to infection with 105 PFU of BJx109 and every 3 days thereafter. Control groups received an equivalent volume of SL-LIP or PBS alone, and all mice were killed and analyzed at day 7 postinfection. (A) Total protein concentrations in cell-free BAL supernatants. *, 100% CL-LIP- and 30% CL-LIP-treated mice were significantly different from SL-LIP control mice (P < 0.01; one-way ANOVA). (B) Lung wet-to-dry ratios as an assessment of pulmonary edema. The data are representative of at least 2 independent experiments. *, 100% CL-LIP- and 30% CL-LIP-treated mice were significantly different from SL-LIP-treated control mice (P < 0.01; one-way ANOVA).

  8. FIG. 8.
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    FIG. 8.

    Lymphopenia and thymic atrophy in macrophage-depleted mice infected with BJx109. Groups of 5 B6 mice were depleted of macrophages via i.n. treatment with 30% or 100% CL-LIP 48 h prior to infection with 105 PFU of BJx109 and every 3 days thereafter. Control groups received an equivalent volume of SL-LIP or PBS alone. All mice were killed and analyzed at day 7 postinfection. (A) Viable-cell counts were performed on whole blood to determine total leukocyte numbers, and flow cytometry was used to determine total numbers of T cells (TCR-β+), CD4+ T cells (CD4+ TCR-β+), and CD8+ T cells (CD8+ TCRβ+). *, cell numbers from 100% CL-LIP- and 30% CL-LIP-treated mice that were significantly lower than those from SL-LIP-treated control animals (P < 0.05; one-way ANOVA). (B) Viable-cell counts were performed on single-cell suspensions prepared from mouse thymi (total cells), and flow cytometry was used to determine the number of double-negative (CD4− CD8−) (DN), DP (CD4+ CD8+), and single-positive (CD4+ or CD8+) (SP) thymocytes present. *, cell numbers from macrophage-depleted mice (100% CL-LIP and 30% CL-LIP) that were significantly higher or lower than those from SL-LIP-treated controls (P < 0.05; one-way ANOVA).

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  1. Accepted manuscript posted online 26 May 2010, doi: 10.1128/JVI.00291-10 J. Virol. vol. 84 no. 15 7569-7580
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