Hyperresponsiveness to inhaled but not intravenous methacholine during acute respiratory syncytial virus infection in mice

Background: To characterise the acute physiological and inflammatory changes induced by low-dose RSV infection in mice. Methods: BALB/c mice were infected as adults (8 wk) or weanlings (3 wk) with 1x10(5) pfu of RSV A2 or vehicle (intranasal, 30 mu l). Inflammation, cytokines and inflammatory markers in bronchoalveolar lavage fluid (BALF) and airway and tissue responses to inhaled methacholine (MCh; 0.001-30 mg/ml) were measured 5, 7, 10 and 21 days post infection. Responsiveness to iv MCh (6-96 mu g/min/kg) in vivo and to electrical field stimulation (EFS) and MCh in vitro were measured at 7 d. Epithelial permeability was measured by Evans Blue dye leakage into BALF at 7 d. Respiratory mechanics were measured using low frequency forced oscillation in tracheostomised and ventilated (450 bpm, flexiVent) mice. Low frequency impedance spectra were calculated (0.5-20 Hz) and a model, consisting of an airway compartment [airway resistance (Raw) and inertance (law)] and a constant-phase tissue compartment [coefficients of tissue damping (G) and elastance (H)] was fitted to the data. Results: Inflammation in adult mouse BALF peaked at 7 d (RSV 15.6 (4.7 SE) vs. control 3.7 (0.7)x10(4) cells/ml; p<0.001), resolving by 21 d, with no increase in weanlings at any timepoint. RSV-infected mice were hyperresponsive to aerosolised MCh at 5 and 7 d (PC200 Raw adults: RSV 0.02 (0.005) vs. control 1.1 (0.41) mg/ml; p=0.003) (PC200 Raw weanlings: RSV 0.19 (0.12) vs. control 10.2 (6.0) mg/ml MCh; p=0.001). Increased responsiveness to aerosolised MCh was matched by elevated levels of cysLT at 5 d and elevated VEGF and PGE(2) at 7 d in BALF from both adult and weanling mice. Responsiveness was not increased in response to iv MCh in vivo or EFS or MCh challenge in vitro. Increased epithelial permeability was not detected at 7 d. Conclusion: Infection with 1x10(5) pfu RSV induced extreme hyperresponsiveness to aerosolised MCh during the acute phase of infection in adult and weanling mice. The route-specificity of hyperresponsiveness suggests that epithelial mechanisms were important in determining the physiological effects. Inflammatory changes were dissociated from physiological changes, particularly in weanling mice.