The immune profile associated with acute allergic asthma accelerates clearance of influenza virus

Abstract

Asthma was the most common comorbidity in hospitalized patients during the 2009 influenza pandemic. For unknown reasons, hospitalized asthmatics had less severe outcomes and were less likely to die from pandemic influenza. Our data with primary human bronchial cells indicate that changes intrinsic to epithelial cells in asthma may protect against cytopathology induced by influenza virus. To further study influenza virus pathogenesis in allergic hosts, we aimed to develop and characterize murine models of asthma and influenza comorbidity to determine structural, physiological and immunological changes induced by influenza in the context of asthma. Aspergillus fumigatus-sensitized and -challenged C57BL/6 mice were infected with pandemic H1N1 influenza virus, either during peak allergic inflammation or during airway remodeling to gain insight into disease pathogenesis. Mice infected with the influenza virus during peak allergic inflammation did not lose body weight and cleared the virus rapidly. These mice exhibited high eosinophilia, preserved airway epithelial cell integrity, increased mucus, reduced interferon response and increased insulin-like growth factor-1. In contrast, weight loss and viral replication kinetics in the mice that were infected during the late airway remodeling phase were equivalent to flu-only controls. These mice had neutrophils in the airways, damaged airway epithelial cells, less mucus production, increased interferons and decreased insulin-like growth factor-1. The state of the allergic airways at the time of influenza virus infection alters host responses against the virus. These murine models of asthma and influenza comorbidity may improve our understanding of the epidemiology and pathogenesis of viral infections in humans with asthma.

Figure 1

Human bronchial epithelial cells from healthy and asthmatic donors grown in the air–liquid interface infected with pH1N1 influenza virus at 0.01 m.o.i. Cells from both donors maintained a pseudostratified morphology and produced mucus under steady state. The thickness of the epithelial layer from the healthy donor was reduced and appeared squamous after pH1N1 infection, while cells from the asthmatic donor maintained normal morphology (a). Viral replication was comparable between cells (inset values, b). Cells from the healthy donor (solid bars) produced more mucus (b) and expressed more TLR3 and IL-1α after virus infection compared to asthmatic donor cells (checkered bars) (c). Data are presented as the mean and s.d. from a representative experiment, ^Δ,#P<0.05 compared with uninfected control and *P<0.05 by Student's t-test. Top panel hematoxylin and eosin (H&E) staining; bottom panel, periodic acid Schiff's (PAS) staining. Scale bar=10 μm.

Figure 2

The developmental stage of allergic asthma impacts influenza morbidity. Schema of comorbidity models (A): (a) AA+Flu and (b) CA+Flu. Images represent the level of airway remodeling in AA and CA lungs at the time of infection. Weight loss and viral titers were affected by the state of allergy in lungs (B, n=8–9 per group). Airway physiological parameters including Newtonian resistance (Rn), tissue damping (G) and tissue elastance (H) were reduced in the CA+Flu model compared with that of AA+Flu (C, n=4 per group). Data are representative of two independent experiments and presented as the mean and s.d., *P<0.05 by analysis of variance. Ctr, control; IH, inhalation; IN, intranasal; PBS, phosphate-buffered saline; SQ/IP, subcutaneous/intraperitoneal; Wk, weeks.

Figure 3

Inflammatory cell recruitment into the airways after pH1N1 infection. There were more cells recruited into the AA+Flu airways, particularly eosinophils and flu-specific CD8⁺ T cells (a). The influx of cells was reduced in the CA+Flu airways, wherein macrophages made up a large proportion (b). Data are representative of two independent experiments and presented as the mean and s.d., n=5 mice per group and *P<0.05 by analysis of variance. Ctr, control; T_H2, T helper type 2. N, naive.

Figure 4

Peribronchovascular inflammation after pH1N1 infection. Inflammation increased after viral infection in both models of comorbidity. There was less/no damage to the bronchial epithelium of AA+Flu compared with that of CA+Flu. Data are representative of two independent experiments. Representative images are at × 400 (first four columns) and × 1000 (last column) after hematoxylin and eosin staining. Ctr, control.

Figure 5

Presence of GCs in the airways of mice after pH1N1 infection. Virus infection alone did not induce GC metaplasia. More GCs were noted in the AA+Flu compared with CA+Flu. Representative images are at × 400 after periodic acid Schiff's staining. Although Muc5AC gene expression was reduced, the number of GCs that interspersed the airways remained elevated in the comorbidity groups. Data are representative of two independent experiments and presented as the mean and s.d., n=5 mice per group and *P<0.05 by analysis of variance. Ctr, control.

Figure 6

Host immune factors were altered in the lungs following pH1N1 infection. Viral infection results in reduced IFNs and their downstream factors in AA+Flu, while levels in CA+Flu were more equivalent to flu-only controls (a). An opposing trend was observed for IGF-1 between the AA+Flu model and flu-only controls. IGF-1 levels in the BAL fluid followed the same trend between the CA+Flu and flu-only controls (b). Data are representative of two independent experiments and presented as the mean and s.d., n=5 mice per group and *P<0.05 by analysis of variance. Schematic representation of our proposed mechanism of enhanced viral clearance and reduced lung damage in the AA+Flu model based on IGF-1 (c). Ctr, control.