T. Chan1, E. Williams1, B. Eliceiri1, A. Baird1, T. Costantini1 1University Of California – San Diego,Division Of Trauma, Surgical Critical Care, Burns And Acute Care Surgery/Department Of Surgery,San Diego, CA, USA
Introduction: The α-7 nicotinic acetylcholine receptor (α7nAChR) is required for the anti-inflammatory activity of the vagus nerve and is thought to be essential to resolve inflammation after severe injury. CHRFAM7A is a uniquely-human gene that encodes a human-specific subunit of the α7nAChR and a putative negative inhibitor of ligand binding. We previously demonstrated that CHRFAM7A expression is highly variable in human leukocytes suggesting a source of human variability in vagal anti-inflammatory responsiveness. We therefore hypothesized that expression of CHRFAM7A would alter ligand binding to the α7nAChR and give insight into human variability in the anti-inflammatory response to injury and infection.
Methods: Transgenic mice were engineered to ubiquitously express the uniquely-human gene CHRFAM7A under control of the EF-1α promoter. CHRFAM7A gene expression was confirmed by PCR, quantitative RT-PCR and immunoblotting with an antibody raised to a peptide sequence unique to CHRFAM7A. Muscle tissue and peritoneal macrophages were harvested from these transgenic mice and ligand binding to α7nAChR compared to sibling-matched wild-type C57 mice. Immunostaining of the neuromuscular junction was performed using α-bungarotoxin (α-BTX), a ligand specific for the α7nAChR. Macrophage α-BTX binding was measured using flow cytometry and immunohistochemistry.
Results: Human CHRFAM7A gene and protein expression was measured in transgenic mice but was undetectable in cells from wild-type animals. α-BTX co-stained with neurofilament at the neuromuscular junction in wild-type mice, however, α-BTX staining was absent in muscle from CHRFAM7A transgenic mice, demonstrating a loss of α7nAChR function. Similarly, CHRFAM7A expression in macrophages decreased α-BTX binding.
Conclusion: CHRFAM7A prevents binding of α-BTX to the α7nAChR. The variability of CHRFAM7A expression seen in humans may, therefore, contribute to human variability in the α7nAChR-dependent anti-inflammatory response to injury and infection. By extension, human CHRFAM7A expression may be responsible for the limited clinical effectiveness of vagal therapeutics that rely on a functional α7nAChR.