Anti-inflammatory Effects of Vagus Nerve Stimulation Are Mediated by the Notch Signaling Pathway during Sepsis-Associated AKI

Background: Activating the cholinergic anti-inflammatory pathway (CAP), a neuroimmune circuit, is a novel strategy for the treatment of acute kidney injury (AKI). The CAP is initiated by vagus nerve stimulation (VNS) and subsequent activation of α7-nicotinic acetylcholine receptor (α7nAChR) on macrophages (MΦ), leading to suppression of pro-inflammatory cytokine production. Our RNA-Seq analysis revealed that an increase in hairy and enhancer of split 1 (Hes1) in MΦ contributes to the kidney protective effects of CAP. Hes1 is a transcriptional factor and is involved in the Notch signaling pathway. However, a direct relationship between the CAP and Notch signaling has been unclear. In this study, we focused on Notch signaling in MΦ to unravel the detailed molecular mechanism of CAP and to determine the utility of Notch signaling in ameliorating AKI. Methods: We used a mouse lipopolysaccharide (LPS)-induced AKI model. MΦ-specific Notch2 KO (Cx3cr1Cre; Notch2fl/fl) or WT mice were subjected to VNS 24 hours before LPS administration. The expression of cytokines and Notch components in the spleen and kidney injury was assessed. Mouse peritoneal MΦ were also used for in vitro experiments to examine the relationship between α7nAChR and Notch signaling. Results: VNS suppressed an LPS-induced increase in spleen size and the levels of pro-inflammatory cytokines and chemokines such as Ccl2, M-CSF, and G-CSF (regulators of MΦ and neutrophil populations) in splenic MΦ. In the kidneys, LPS-induced inflammation including MΦ and neutrophil infiltration, and histological damage were also attenuated by VNS. These events were accompanied by an increase in Notch2 and Hes1 expression in splenic MΦ. On the other hand, in MΦ-specific Notch2 KO mice, the anti-inflammatory effects of VNS were attenuated in both the spleen and kidneys. We further found that during LPS treatment, chemical stimulation of α7nAChR by GTS-21 reduced Ccl2 release and upregulated Hes1 expression in peritoneal MΦ, indicating that Notch signaling might be activated downstream of α7nAChR to dampen inflammatory responses. Conclusion: Our findings suggest VNS protects the kidneys against sepsis-associated AKI via activation of the Notch2 signaling in MΦ. Funding: Government Support – Non-U.S.