Alveolar macrophages contribute to the pathogenesis of human metapneumovirus infection while protecting against respiratory syncytial virus infection

Abstract

Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are leading causes of upper and lower respiratory tract infections in young children and among elderly and immunocompromised patients. The pathogenesis of hMPV-induced lung disease is poorly understood. The lung macrophage population consists of alveolar macrophages (AMs) residing at the luminal surface of alveoli and interstitial macrophages present within the parenchymal lung interstitium. The involvement of AMs in innate immune responses to virus infections remains elusive. In this study, BALB/c mice depleted of AMs by intranasal instillation of dichloromethylene bisphosphonate (L-CL2MBP) liposomes were examined for disease, lung inflammation, and viral replication after infection with hMPV or RSV. hMPV-infected mice lacking AMs exhibited improved disease in terms of body weight loss, lung inflammation, airway obstruction, and hyperresponsiveness compared with AM-competent mice. AM depletion was associated with significantly reduced hMPV titers in the lungs, suggesting that hMPV required AMs for early entry and replication in the lung. In contrast, AM depletion in the context of RSV infection was characterized by an increase in viral replication, worsened disease, and inflammation, with increased airway neutrophils and inflammatory dendritic cells. Overall, lack of AMs resulted in a broad-spectrum disruption in type I IFN and certain inflammatory cytokine production, including TNF and IL-6, while causing a virus-specific alteration in the profile of several immunomodulatory cytokines, chemokines, and growth factors. Our study demonstrates that AMs have distinct roles in the context of human infections caused by members of the Paramyxoviridae family.

Keywords: alveolar macrophages; human metapneumovirus; pathogenesis; respiratory syncytial virus; viral infection.

Figure 1.

Effect of alveolar macrophage (AM) depletion on human metapneumovirus (hMPV)- and respiratory syncytial virus (RSV)-induced disease. (A) Dichloromethylene bisphosphonate (L-CL₂MBP)-treated and untreated mice were infected with hMPV or RSV, and change in body weight was measured over a period of 14 days after infection. (B) Lungs were collected at Days 5 and 7 after infection, and pathology score was assessed by hematoxylin and eosin staining. Airway obstruction represented by baseline Penh for hMPV infection and airway hyperresponsiveness after methacholine challenge at Day 5 after infection after RSV infection (C) and airway hyperresponsiveness after methacholine challenge at Day 21 after hMPV infection and Day 14 after RSV infection (D) were determined by unrestrained plethysmography. Data are expressed as mean ± SEM (n = 4 mice/group) and are representative of three independent experiments. *P < 0.05 and ***P < 0.001 when comparing L-CL₂MBP–treated versus untreated infected mice.

Figure 2.

Effect of AM depletion on bronchoalveolar lavage (BAL) and lung cell recruitment after infection. L-CL₂MBP–treated and untreated mice were infected with hMPV or RSV, and BAL and lungs were collected at different time points after infection to determine differential cell counts by hematoxylin and eosin staining in BAL (A) and CD4 and CD8 T lymphocyte recruitment to the lung (B) as well as neutrophils (CD11b⁺Gr1⁺) and dendritic cells (CD11c^hiCD11b⁺) (C) by flow cytometry analysis after staining with specific antibodies. Data are expressed as mean ± SEM (n = 5 mice/group) and are representative of three independent experiments. *P < 0.001 when comparing L-CL₂MBP–treated versus untreated infected mice.

Figure 3.

Effect of AM depletion on cytokines and growth factors after infection. L-CL₂MBP–treated and untreated mice were infected with hMPV or RSV, and BAL was collected at different time points after infection to measure cytokines and growth factors by Luminex-based assay. (A) IL-1α, IL-1β, TNF-α, and IL-6. (B) IL-12 p(40), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF). Data are expressed as mean ± SEM (n = 5 mice/group) and are representative of three independent experiments. *P < 0.001 when comparing L-CL₂MBP–treated versus untreated infected mice.

Figure 3.

Effect of AM depletion on cytokines and growth factors after infection. L-CL₂MBP–treated and untreated mice were infected with hMPV or RSV, and BAL was collected at different time points after infection to measure cytokines and growth factors by Luminex-based assay. (A) IL-1α, IL-1β, TNF-α, and IL-6. (B) IL-12 p(40), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF). Data are expressed as mean ± SEM (n = 5 mice/group) and are representative of three independent experiments. *P < 0.001 when comparing L-CL₂MBP–treated versus untreated infected mice.

Figure 4.

Effect of AM depletion on chemokines and type I IFNs after infection. L-CL₂MBP–treated and untreated mice were infected with hMPV or RSV, and BAL was collected at different time points after infection to measure chemokines and type I IFN by Luminex-based assay and ELISA, respectively. (A) CCL3, CCL4, and CCL5. (B) IFN-α and IFN-β. Data are expressed as mean ± SEM (n = 5 mice/group) and are representative of three independent experiments. *P < 0.001 when comparing L-CL₂MBP–treated versus untreated infected mice.

Figure 5.

Effect of AM depletion on RSV/hMPV replication in the lung. (A) L-CL₂MBP–treated and untreated mice were infected with hMPV and harvested at Days 3, 5, and 7 after infection to determine viral titers by TCID₅₀ assay. (B) Mice were first infected with hMPV and were treated with L-CL₂MBP 48 hours later. At Day 5 after infection, lungs were collected, and viral titer was determined by TCID₅₀ assay. (C) L-CL₂MBP–treated and untreated mice were infected with RSV and harvested at Days 3, 5, and 7 after infection to determine viral titers by plaque assay. Mice were first infected with RSV and were treated with L-CL₂MBP 48 hours later. (D) At Day 5 after infection lungs were collected, and viral titer was determined by plaque assay. Data are expressed as mean ± SEM (n = 5 animals/group) and are representative of three independent experiments. nd = not detected. *P < 0.05 and **P < 0.01 when comparing L-CL₂MBP–treated versus untreated infected mice.

Figure 6.

Cytokine production and viral replication by murine AMs. AMs were isolated from BALB/c mice and infected ex vivo with hMPV or RSV (multiplicity of infection [MOI] of 5). (A) At 24 hours after infection, cell supernatant was collected, and cytokines, chemokines, and IFN-α and -β levels were measured. (B) AMs were infected with hMPV or RSV (MOI of 0.1), and the viral titer was measured at different days after infection by immunostaining for hMPV and plaque assay for RSV. Data are expressed as mean ± SEM and are representative of two to three independent experiments. *P < 0.001 when comparing with hMPV infected cells. nd, not determined.

Figure 7.

Fate of AMs after infection with hMPV and RSV in vivo and ex vivo. Mice were infected with hMPV or RSV or were mock inoculated. BAL was collected at different times after infection, and cells were stained with anti-CD11c antibody and analyzed by flow cytometry. AMs were identified on the basis of the side scatter (SSC) and expression of CD11c. (A) The percentage of AMs is indicated in the gates. Data are representative of two independent experiments. (B) AMs isolated from BALB/c mice were infected ex vivo with hMPV or RSV (MOI of 5). At different times after infection, cell supernatant was harvested to measure cell death using a cell death detection ELISA kit. Data are expressed as mean ± SEM (n = 6 animals/group) and are representative of two independent experiments. *P < 0.05.