ABSTRACT
We have previously discovered a synergistically therapeutic combination of two Toll-like receptor ligands, an oligodeoxynucleotide (ODN) and Pam2CSK4. Aerosolization of these ligands stimulates innate immunity within the lungs to prevent pneumonia from bacterial and viral pathogens. Here we examined the safety and tolerability of this treatment in mice, and characterized the expression of biomarkers of innate immune activation. We found that neutrophils appeared in lung lavage fluid 4 h after treatment, reached a peak at 48 h, and resolved by 7 days. The peak of neutrophil influx was accompanied by a small increase in lung permeability. Despite the abundance of neutrophils in lung lavage fluid, only rare neutrophils were visible histopathologically in the interstitium surrounding bronchi and veins and none were visible in alveolar airspaces. The cytokines interleukin 6 (IL-6), tumour necrosis factor, and Chemokine (C-X-C motif) ligand 2 rose several hundred-fold in lung lavage fluid 4 h after treatment in a dose-dependent and synergistic manner, providing useful biomarkers of lung activation. IL-6 rose fivefold in serum with delayed kinetics compared to its rise in lavage fluid, and might serve as a systemic biomarker of immune activation of the lungs. The dose-response relationship of lavage fluid cytokines was preserved in mice that underwent myeloablative treatment with cytosine arabinoside to model the treatment of hematologic malignancy. There were no overt signs of distress in mice treated with ODN/Pam2CSK4 in doses up to eightfold the therapeutic dose, and no changes in temperature, respiratory rate, or behavioral signs of sickness including sugar water preference, food disappearance, cage exploration or social interaction, though there was a small degree of transient weight loss. We conclude that treatment with aerosolized ODN/Pam2CSK4 is well tolerated in mice, and that innate immune activation of the lungs can be monitored by the measurement of inflammatory cytokines in lung lavage fluid and serum.
ABSTRACT
Glucocorticosteroids are the mainstay treatment for chronic asthma; however, adverse effects can limit their usefulness. We previously determined in experimental asthma that chronic administration of ß2-adrenoceptor inverse agonists reduced airway hyperresponsiveness and indexes of inflammation. However, the effect of co-administration of glucocorticosteroids with ß2-adrenoceptor inverse agonists is unknown. Therefore, we evaluated the anti-inflammatory effect of co-administration of dexamethasone, a glucocorticosteroid, and nadolol, a ß2-inverse agonist, in a murine asthma model. We measured eosinophils and cytokines in bronchoalveolar lavage fluid and mucin content in epithelial cells after exposure to different concentrations of dexamethasone and nadolol. Dexamethasone was administered for 3 days and nadolol for 24 days prior to ovalbumin challenge. Both drugs were continued during five daily intranasal challenges with ovalbumin. Independent administration of dexamethasone (0.4 mg/kg/day) or nadolol (25 ppm) reduced bronchoalveolar lavage eosinophils by 58% and 36%, respectively (P < 0.05). Co-administration of both drugs yielded an additive reduction in eosinophils (81%, P < 0.05). Co-administration of both drugs (dexamethasone 0.4 mg/kg/day and nadolol 25 ppm) also yielded a greater reduction in mucin volume density (83%) than either drug alone (18% for dexamethasone and 62% for nadolol) and greater than high-dose dexamethasone (71%) alone (P < 0.05). Similarly, co-administration of both drugs (dexamethasone 0.4 mg/kg/day and nadolol 25 ppm) yielded an additive effect on the reduction of type 2 cytokines in bronchoalveolar lavage fluid equivalent to the administration of a 10-fold higher dose of dexamethasone. In Summary, the simultaneous administration of a glucocorticosteroid and a ß2-adrenoceptor inverse agonist was more effective at reducing indexes of airway inflammation than either drug given alone; suggesting nadolol may possess "glucocorticoid-sparing" properties.
