Optogenetic stimulation of cholinergic neuronal endings in the celiac‐superior mesenteric ganglion complex suppresses inflammation in murine endotoxemia

Abstract

The vagus nerve plays a key role in the regulation of inflammation within the inflammatory reflex (Annu Rev Immunol, 2018, 36:783). In this major physiological neuroimmunoregulatory mechanism, efferent vagus nerve cholinergic axons interact with catecholaminergic neurons of the splenic nerve in the celiac‐superior mesenteric ganglion (CSMG) complex(Proc Natl Acad Sci USA, 2020, 117(47):29803).Previous studies utilizing electrical and optogenetic neuromodulation have demonstrated that vagus nerve cholinergic signalling suppresses pro‐inflammatory cytokine levels during endotoxemia and other inflammatory conditions. However, the specific role of cholinergic axonal terminals in the CSMG complex in this regulation remained enigmatic. To provide insight, we investigated the effect of selective optogenetic stimulation of cholinergic neuronal endings in the CSMG complex on cytokine and chemokine levels during murine endotoxemia. We used transgenic female 10–14‐week‐old mice expressing channelrhodopsin 2 coupled with yellow fluorescent protein on cholinergic, i.e., choline acetyltransferase positive neurons (ChAT‐ChR2‐eYFP). Anesthetized mice were subjected to abdominal surgery and the CSMG complex was localized medial to the upper pole left kidney and along the branch of the abdominal aorta, above the left renal vein. Blue laser light stimulation at 473nm was delivered in square pulses for 10mins at 20Hz, 25% duty cycle through an optic fiber cannula placed in contact with the CSMG complex. 30mins following optogenetic stimulation or sham stimulation, lipopolysaccharide (0.25mg/kg) was injected intraperitoneally and 90mins later mice were euthanized and blood, and spleen were collected and processed for cytokine and chemokines. Optogenetic stimulation of the CSMG complex in ChAT‐ChR2 mice (n=14) suppressed serum pro‐inflammatory cytokines and chemokines compared with sham stimulation (n=13), including TNF(401.7 ± 102.9 pg/ml vs 766.1 ± 207.2 pg/ml; P<0.0001) and MIP‐1α (6,523 ± 3,041 pg/ml vs 10,672 ± 3,915 pg/ml, P=0.0066), while the serum levels of the anti‐inflammatory cytokine IL‐10 were significantly increased (30,779 ± 10,035 pg/ml vs 22,979 ± 9,669 pg/ml, P=0.0427). There was also a significant reduction in splenic TNF levels in the stimulated vs the sham group(P=0.0023). Of note, optogenetic stimulation of the CSMG complex in littermate mice that do not express ChR2 on cholinergic neurons did not alter serum and splenic cytokine levels. These results provide the first experimental evidence that selective stimulation of cholinergic neuronal endings in the CSMG complex attenuates cytokine and chemokine levels. Future studies examining the specific contribution of vagal and sympathetic preganglionic cholinergic neurons in the CSMG complex in the regulation of immune responses will be instrumental for developing new targeted therapeutic anti‐inflammatory approaches.