Abstract / Summary
Background: The cholinergic anti-inflammatory pathway regulates inflammation through vagus signaling that activates the α7 nicotinic acetylcholine receptor (α7nAChR) on leukocytes. Suppression of macrophage inflammatory cytokine production through α7nAChR-mediated inhibition of NF-κB activation is considered a central mechanism of this pathway. However, the effects of cholinergic activation of α7nAChR on neutrophils remain poorly understood. Here, we investigated whether and how activation of α7nAChR in neutrophils regulates neutrophil migration, defined the underlying molecular mechanism, and evaluated its contribution to anti-inflammatory protection during endotoxemia. Results. The ex vivo swarming assays revealed that activation of α7nAChR with the selective agonists PNU-282987 and AR-R17779 significantly inhibited human and mouse neutrophil migration. The inhibitory effect of PNU-282987 was abolished in α7nAChR-deficient neutrophils, confirming receptor specificity. Mechanistically, α7nAChR activation reduced integrin αMβ2 activation, decreased F-actin polymerization, and impaired neutrophil polarization. These effects required αMβ2, as PNU-282987 failed to inhibit actin remodeling in αM-deficient neutrophils. Modulation of αMβ2 activation with an activating antibody (MEM48) demonstrated that α7nAChR regulates neutrophil migration by limiting excessive αMβ2 activation and maintaining an optimal adhesive state. In vivo, adoptively transferred fluorescently labeled PNU-282987-treated neutrophils exhibited reduced recruitment to the lungs compared with vehicle-treated neutrophils during LPS-induced endotoxemia. Furthermore, neutrophil depletion markedly diminished the protective effect of PNU-282987 on survival, demonstrating that neutrophils contribute to α7nAChR-mediated protection during systemic inflammation. Conclusions. Our findings identify neutrophils as an important cellular target of the cholinergic anti-inflammatory pathway and demonstrate that α7nAChR activation limits neutrophil migration by suppressing integrin αMβ2 activation and actin cytoskeletal remodeling. Together, these findings establish αMβ2 as a critical downstream effector of α7nAChR signaling in neutrophils and identify regulation of integrin activation as a previously unrecognized mechanism of the cholinergic anti-inflammatory pathway.