Bronchodilator Effects of Toll-Like Receptor-7 Agonists.

Our recent studies have demonstrated an entirely new and previously unrecognized effect of toll-like receptor 7 (TLR7) agonists in the airways, namely that they are potent bronchodilators, and prevent bronchoconstriction caused by methacholine, histamine, stimulation of parasympathetic nerves, and depolarization of smooth muscle by potassium chloride. Pharmacological studies using TLR7 antagonists, as well as studies in TLR7- deficient mice, demonstrate that part of the effect is TLR7 dependent and part is TLR7 independent. TLR7 dependent bronchodilation in guinea pigs and in human airways is mediated by production of nitric oxide. We have also shown that in human airways, stimulation of the other ssRNA receptor, TLR8, also causes bronchodilation, but that this pathway does not involve nitric oxide. In this application, we propose three specific aims: SPECIFIC AIM #1. To A) establish the role of the ssRNA receptors TLR7 and TLR8 in relaxation of human airway smooth muscle, B) test whether these receptors signal through nitric oxide, prostaglandins, and the large conductance, calcium activated potassium channel (BKCa) in human airways in vitro, and C) explore the role of nitric oxide and changes in intracellular calcium in rapid signaling by these receptors in primary cultures of human airway smooth muscle cells. SPECIFIC AIM #2. To identify which TLR7 mediated pathways are inhibited in antigen sensitized and in virus infected guinea pigs to cause the profound loss of TLR7 mediated bronchodilation that we identified in preliminary experiments (see figure 12). We will test whether a similar 2 log shift occurs in human airway tissues using passive sensitization and in vitro parainfluenza virus infection. SPECIFIC AIM #3: To determine the mechanisms of loss of TLR7/nitric oxide mediated bronchodilation after antigen sensitization or viral infection. The results of the experiments we propose will be important in establishing the potential of TLR7 agonists as treatments for asthma and other airway diseases. In addition, because these receptors respond to viral RNA, understanding the effects of stimulating TLR7 receptors in the airways, as well as the loss of these effects in models of asthma, will help us understand the pathophysiology of virus induced asthma attacks.