Macrophages in lung and innervating ganglia express TLR7 and release nitric oxide

Abstract

Background: Toll-like receptor 7 (TLR7) detects RNA viruses and triggers an innate immune response. TLR7 imidazoquinoline agonists bronchodilate airways via release of nitric oxide. TLR7 expression has been reported in airway epithelium and nerves, however lack of antibody specificity questioned these results.

Objective: To determine TLR7 expression in airways using immunohistochemistry and RNAscope in situ hybridization and to determine the source of TLR7-induced nitric oxide.

Methods: A published TLR7 antibody and species specific TLR7 RNAscope probes (Advanced Cell Diagnostics) were tested in wild type (WT) mice, TLR7 knock out (KO) mice, and guinea pigs. The identity of TLR7-positive cells was further determined by immunohistochemistry. Cultured sensory ganglia were loaded with a fluorescent nitric oxide indicator and treated with the TLR7 agonist R837.

Results: TLR7 RNA was detected in WT mouse and guinea pig alveolar macrophages and in macrophages adjacent to neurons in sensory ganglia and parasympathetic ganglia. TLR7 RNA was absent in neurons. RNAscope probes did not detect TLR7 RNA in TLR7 KO mice. In contrast, TLR7 antibody labeled airway nerves and epithelium in WT and TLR7 KO mice and in guinea pigs. Notably, TLR7 RNAscope probes and antibody labeled distinctly different cells. In cultured ganglia, TLR7 agonist R837 significantly increased nitric oxide in macrophages and support cells, but not in neurons.

Conclusion: TLR7 is expressed by lung macrophages, supporting their antiviral role. Macrophages are a source of TLR7-induced nitric oxide. The difference in TLR7 expression using RNAscope probes versus the antibody challenges previous reports of airway TLR7 expression.