Leflunomide Prevents Alveolar Fluid Clearance Inhibition by Respiratory Syncytial Virus

Rationale: Previously, we demonstrated that intranasal infection of BALB/c mice with respiratory syncytial virus (RSV) resulted in an early 40% reduction in alveolar fluid clearance (AFC), an effect mediated via P2Y purinergic receptors

Post-Infection A77-1726 Blocks Pathophysiologic Sequelae of Respiratory Syncytial Virus Infection

Despite respiratory syncytial virus (RSV) bronchiolitis remaining the most common cause of lower respiratory tract disease in infants worldwide, treatment has progressed little in the past 30 years. The aim of our study was to determine whether post-infection administration of de novo pyrimidine synthesis inhibitors could prevent the reduction in alveolar fluid clearance (AFC) and hypoxemia that occurs at Day 2 after intranasal infection of BALB/c mice with RSV. BALB/c mice were infected intranasally with RSV strain A2. AFC was measured in anesthetized, ventilated mice after instillation of 5% bovine serum albumin into the dependent lung. Post-infection systemic treatment with leflunomide has no effect on AFC. However, when added to the AFC instillate, leflunomide's active metabolite, A77-1726, blocks RSV-mediated inhibition of AFC at Day 2. This block is reversed by uridine (which allows pyrimidine synthesis via the scavenger pathway) and not recapitulated by genistein (which mimics the tyrosine kinase inhibitor effects of A77-1726), indicating that the effect is specific for the de novo pyrimidine synthesis pathway. More importantly, when administered intranasally at Day 1, A77-1726, but not its vehicle dimethyl sulfoxide, maintains its beneficial effect on AFC and lung water content until Day 2. Intranasal instillation of A77-1726 at Day 1 also reduces bronchoalveolar lavage nucleotide levels, lung inflammation, and hypoxemia at Day 2 without impairing viral replication at Day 2 or viral clearance at Day 8. Post-infection intranasal or aerosolized treatment with pyrimidine synthesis inhibitors may provide symptomatic relief from the pathophysiologic sequelae of impaired AFC in children with RSV bronchiolitis

Reactive Species Mediate Inhibition of Alveolar Type II Sodium Transport during Mycoplasma Infection

Rationale: Mycoplasma pneumoniae is a significant cause of pneumonia in humans

Cyclophosphamide Decreases Nitrotyrosine Formation and Inhibits Nitric Oxide Production by Alveolar Macrophages in Mycoplasmosis

We previously reported that congenic C57BL/6 inducible nitric oxide synthase(−/−) (iNOS(−/−)) mice infected with Mycoplasma pulmonis developed higher bacterial numbers and lung lesion scores than C57BL/6 iNOS(+/+) controls but had similar lung nitrotyrosine levels. The present studies investigated the role of inflammatory cells in nitrotyrosine formation during mycoplasmal infection. iNOS(+/+) and iNOS(−/−) mice were injected with cyclophosphamide (CYP) and inoculated with 10(7) CFU of M. pulmonis. CYP pretreatment of M. pulmonis-infected iNOS(+/+) and iNOS(−/−) mice reduced polymorphonuclear cells (PMNs) within bronchoalveolar lavages (BALs) by 88 and 72%, respectively, and whole-lung myeloperoxidase levels by 80 and 78%, respectively, at 72 h postinfection but did not alter the number of alveolar macrophages (AMs) in BALs. CYP treatment also significantly decreased nitrate and nitrite (NOx) levels in BALs and plasma of infected iNOS(+/+) mice, whereas neither CYP nor mycoplasmal infection altered NOx in iNOS(−/−) mice. CYP reduced lung nitrotyrosine levels in both iNOS(+/+) and iNOS(−/−) mice to uninfected-control levels as shown by immunohistochemical staining and enzyme-linked immunosorbent assay and inhibited mycoplasmal killing by iNOS(+/+) mice in vivo. CYP inhibited the production of gamma interferon-inducible NOx by iNOS(+/+) AMs in vitro but did not alter the number of iNOS-positive AMs, as detected by immunocytochemistry. In addition, AMs from CYP-treated iNOS(+/+) mice had significantly decreased ability to kill mycoplasmas in vitro. These results demonstrate that reactive species generated by inflammatory cells as well as PMN myeloperoxidase are important contributors to nitrotyrosine formation during mycoplasmal infection and that treatment with CYP decreases NO(⋅) production by AMs and inhibits mycoplasmal killing

Bactericidal Function of Alveolar Macrophages in Mechanically Ventilated Rabbits

Protective ventilation strategies have been universally embraced because of reduced mortality. We tested the hypothesis that tidal volume (VT) in an in vivo model of mechanical ventilation would modulate bactericidal function of alveolar macrophages (AMs). Adult New Zealand White rabbits were mechanically ventilated for 4 h with a VT of 6 ml/kg (low) or a VT of 12 ml/kg (traditional), with each group receiving 3 cm H2O positive end-expiratory pressure with and without intratracheal lipopolysaccharide (LPS) instillation (20 mg/kg). AMs were isolated from bronchoalveolar lavage fluid taken from the whole left lung and used for bacterial killing assays. There were no significant differences in steady-state levels of nitrite or AM phagocytosis and killing of Klebsiella pneumoniae, although these values trended to be slightly higher in the traditional VT group. However, bronchoalveolar lavage fluid protein concentrations were significantly increased in traditional VT groups receiving LPS compared with animals ventilated with a low VT (1,407.8 ± 121.4 versus 934.7 ± 118.2; P < 0.001). Lung wet:dry weight ratio in the traditional VT group was increased when compared with the low VT group without LPS (7.3 ± 0.4 versus 6.1 ± 0.3, respectively; P < 0.05). Additionally, IL-8 expression was significantly greater under conditions of LPS treatment and mechanical ventilation at VT of 12 ml/kg. These results suggest that the traditional ventilator approach (12 ml/kg VT) in a model of in vivo mechanical ventilation results in lung pathology without affecting AM antibacterial function

Leflunomide Prevents Alveolar Fluid Clearance Inhibition by Respiratory Syncytial Virus

Rationale: Previously, we demonstrated that intranasal infection of BALB/c mice with respiratory syncytial virus (RSV) resulted in an early 40% reduction in alveolar fluid clearance (AFC), an effect mediated via P2Y purinergic receptors. Objectives: To confirm that RSV-induced inhibition of AFC is mediated by uridine triphosphate (UTP), and to demonstrate that inhibition of de novo pyrimidine synthesis with leflunomide prevents increased UTP release after RSV infection, and thereby also prevents inhibition of AFC by RSV. Methods: BALB/c mice were infected intranasally with RSV strain A2. AFC was measured in anesthetized, ventilated mice by instillation of 5% bovine serum albumin into the dependent lung. Some mice were pretreated with leflunomide or 6-mercaptopurine. Measurements and Main Results: RSV-mediated inhibition of AFC is associated temporally with a 20-nM increase in UTP and ATP content of bronchoalveolar lavage fluid, hypoxemia, and altered nasal potential difference. RSV-mediated nucleotide release, AFC inhibition, and physiologic sequelae thereof can be prevented by pretreatment of mice with the de novo pyrimidine synthesis inhibitor leflunomide, which is not toxic to the mice, and which does not affect RSV replication in the lungs. In contrast, pretreatment of mice with 6-mercaptopurine, an inhibitor of de novo purine synthesis, has no beneficial effect on AFC or other indicators of disease progression. Finally, RSV-mediated inhibition of AFC is prevented by volume-regulated anion channel inhibitors. Conclusion: Pyrimidine synthesis or release pathways may provide novel therapeutic targets to counter the pathophysiologic sequelae of impaired AFC in RSV disease