Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation

Abstract

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations.

Figure 1

Live minor-group rhinovirus-1B, but not UV-inactivated rhinovirus-1B or major-group rhinovirus-16, induces airway and lung inflammation and mucin production in BALB/c mice. Mice were infected with 5 × 10⁶ TCID₅₀ of rhinovirus-1B (RV-1B), UV-inactivated rhinovirus-1B (UV–RV-1B) or rhinovirus-16 (RV-16). (a) Kinetic analysis of BAL cells showing neutrophils and lymphocytes at the indicated time points after infection, as assessed by differential cell counts. Representative of five experiments. Data are means for groups of four mice ± s.e.m., **P < 0.01 and ***P < 0.001 compared to both control groups. (b) Representative H&E-stained lung sections in naive mice (left column) or lungs taken at day 2 after infection with UV–RV-1B (center column) or RV-1B (right column). Perivascular (black arrow) and peribronchial (white arrow) inflammation are indicated. Scale bars, 50 μm. Images shown are typical of three mice. (c) Quantification of Muc5AC mRNA levels in lung tissue by quantitative RT-PCR. (d) Quantification of Muc5AC protein secretion in BAL by ELISA. The same analyses were carried out for Muc5B in e and f. Data are means ± s.e.m., **P < 0.01 and ***P < 0.001 compared with UV–RV-1B.

Figure 2

Rhinovirus-1B induces neutrophil, dendritic cell and lymphocyte chemoattractant chemokine production and proinflammatory cytokine production. (a–h) Groups of four BALB/c mice were infected with 5 × 10⁶ TCID₅₀ of RV-1B or UV–RV-1B. BAL was collected at 8 h and 1, 2, 4 and 7 days after infection, and cell-free BAL fluid was analyzed by quantitative ELISA for neutrophil chemoattractant chemokines MIP-2 (a) and KC (b); dendritic cell chemokine MIP-3α (c); lymphocyte chemokines IP-10 (d), RANTES (e) and I-TAC (f); and the proinflammatory cytokines IL-6 (g) and IL-1β (h). **P < 0.01 and ***P < 0.001 for RV-1B compared with UV-inactivated control. Results are expressed as means ± s.e.m.

Figure 3

RV-1B replication and induction of innate (antiviral IFN) and acquired virus-specific cellular and humoral immune responses in BALB/c mice. Groups of four mice were inoculated with 5 × 10⁶ TCID₅₀ of RV-1B or UV–RV-1B. (a) Quantification of rhinovirus RNA in lung homogenates by quantitative RT-PCR. (b) Quantification of rhinovirus viral load in total BAL, as measured by titration of infectious virus in HeLa cells. (c) In situ hybridization staining of lung sections from mice infected with RV-1B, RV-16 or UV–RV-1B. Top row, sections probed with antisense RNA probe to detect genomic viral RNA. Bottom row, sections probed with sense RNA probe to detect negative-strand replicative viral RNA. Sections probed with a respiratory syncytial virus M protein–specific probe were negative (data not shown). Sections are representative of three mice per group. (d) Induction of antiviral IFNs α, β and λ in BAL, as measured by quantitative ELISA. (e) Frequency of IFN-γ–producing lung leukocytes in mice infected with RV-1B assessed at day 4 (left) or day 7 (right) after infection, cultured with or without RV-1B stimulation ex vivo. ^###P < 0.001 comparing mice dosed with RV-1B to mice dosed with RV-16. At day 7, induction was virus-specific, ⁺⁺⁺P < 0.001 for leukocytes stimulated ex vivo with RV-1B compared to unstimulated cells. (f) Rhinovirus-specific IgG measured by ELISA in serum collected at the indicated day after infection. All data are expressed as means ± s.e.m., *P < 0.05, **P < 0.01 and ***P < 0.001 for RV-1B compared with UV-inactivated control.

Figure 4

Major group rhinovirus-16 infection of transgenic mice expressing a human/mouse ICAM-1 chimeric receptor. Groups of four human-mouse ICAM-1–expressing transgenic BALB/c mice were dosed intranasally with 5 × 10⁶ TCID₅₀/ml RV-16 (huICAM + RV-16) or UV–RV-16 (huICAM + UV–RV-16); in addition, transgene-negative wild-type BALB/c mice were infected with RV-16 (WT RV-16). (a) Kinetic analysis of BAL cells showing neutrophils and lymphocytes at the indicated time points after infection, as assessed by differential cell counts. (b) Muc5B protein secretion in BAL, as measured by ELISA. Left, RV-16–infected transgenic mice compared with transgene-negative mice inoculated with RV-16. Right, transgenic mice inoculated with live or UV–RV-16. (c) Viral RNA (vRNA) in lung homogenate, as measured by qRT-PCR. (d–g) Production of IFN-α (d, left), IFN-β (d, center), IFN-λ (d, right), MIP-2 (e), I-TAC (f) and IL-1β (g) in BAL, as measured by quantitative ELISA. All data are expressed as means (groups of seven for huICAM + RV-16 and groups of three or four for control groups) ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 for huICAM + RV-16 groups compared to huICAM + UV–RV-16 controls. ⁺P < 0.05, ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.001 for huICAM + RV-16 groups compared to transgene negative controls dosed with live rhinovirus-16.

Figure 5

Rhinovirus exacerbates airway inflammation, airway hyper-responsiveness, mucus production, and T_H1 and T_H2 cytokine responses in a model of acute allergic airway inflammation. OVA-sensitized mice were challenged intranasally with OVA or PBS control and infected with RV-1B (RV-OVA or RV-PBS) or UV-inactivated virus (UV-OVA or UV-PBS). (a) Kinetic analysis of BAL cells showing neutrophils (left) and lymphocytes (right) at the indicated time points after infection, as assessed by differential cell counts. (b) Airway hyper-responsiveness to increasing doses of methacholine (MCH), as assessed at 24 h after infection by Penh area under the curve (AUC) analysis (left). Penh results were confirmed by invasive measures of airway resistance (right). BL, baseline. (c) Muc5AC (left) and Muc5B (right) protein abundance in BAL, as determined by ELISA. Measurement of T_H2 cytokines IL-4 (d) and IL-13 (e) in BAL was performed by quantitative ELISA, and IFN-γ production by lung leukocytes (f) was assessed by ELISpot. All data are expressed as means ± s.e.m. for groups of four mice. *P < 0.05, **P < 0.01 and ***P < 0.001 RV-OVA compared to RV-PBS. ⁺P < 0.05, ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.001 for RV-OVA compared to UV-OVA. ^#P < 0.05 and ^###P < 0.001 for allergic groups (RV-OVA and UV-OVA) compared to controls (UV-PBS).