C99-06 Parainfluenza Virus Causes Muc5ac-dependent Nerve-mediated Airway Hyperreactivity

Abstract Rationale Respiratory viruses cause airway hyperreactivity. Muc5ac, a component of airway mucus, is upregulated during infection. Aim To test whether Muc5ac is required for parainfluenza virus-induced airway hyperreactivity. Methods Wild-type and Muc5ac knock-out C57BL/6 mice were intratracheally inoculated with type 1 parainfluenza virus (1.4x105 TCID50 /ml) or mock (RPMI). Four days later, anesthetized mice were ventilated, and airway response to aerosolized methacholine (10 - 1000 µM) or to electric stimulation of the vagus nerves (16V, 2ms pulse duration, 5-20Hz, 10 second train at one minute intervals) were tested. Lungs were fixed using methacarn to preserve mucus and stained using Alcian Blue-Periodic Acid Schiff. Trachea and nodose-jugular ganglia were immunostained for substance P and the pan-neuronal marker PGP9.5, optically cleared, and whole mounted for confocal imaging. Viral RNA and inflammatory cytokines were quantified in lung homogenate using RT-PCR. Results Viral RNA was greater in Muc5ac knock-out mice versus wild-type mice (3.01*109 ±7.7*108 TCID50U/ml vs 1.74*107±8.92*106 TCID50U/ml, p 0.05), as were IL-6 (90.06±25.54ΔΔCT vs 29.90±5.126 ΔΔCT p 0.05), TNFα (12.13±2.60ΔΔCT vs 5.51±0.95 ΔΔCT, p 0.05), and INFγ (40.61±12.96ΔΔCT vs 8.72±1.96, p 0.05).In bronchoalveolar lavage, total leukocytes were increased by infection in Muc5ac knock out mice compared to wild-type (2.75*106±7.96*105 total cells vs 5.63*105±3.21*105 total cells, p 0.05). Neutrophils were the primary driver of this increase (1.76*106 ±4.56*105 neutrophils vs 6.85*104±2.92*104 neutrophils, p 0.05). Virus-infected wild-type mice developed airway hyperreactivity to electrical stimulation of the vagus nerves and to methacholine that were blocked by vagotomy (Figure 1A/B). In contrast, viral infection of Muc5ac knock-out mice did not cause airway hyperreactivity (Figure 1A). Mucus plugging was absent in both virus- and mock-infected mice regardless of genotype, while substance P-expressing neurons in nodose jugular ganglia were increased after virus infection (Figure 1C/D). Conclusion Deletion of Muc5ac increases viral content and inflammatory response, but prevents virus induced hyperreactivity. Although mucus plugging plays a role in house dust mite-induced hyperreactivity, here we show it is not important to virus induced airway hyperreactivity. This suggests Muc5ac contributes to virus induced hyperreactivity by a novel mechanism, possibly involving substance P. This abstract is funded by: None