Xanomeline suppresses excessive pro-inflammatory cytokine responses through neural signal-mediated pathways and improves survival in lethal inflammation

Abstract

Inflammatory conditions characterized by excessive immune cell activation and cytokine release, are associated with bidirectional immune system-brain communication, underlying sickness behavior and other physiological responses. The vagus nerve has an important role in this communication by conveying sensory information to the brain, and brain-derived immunoregulatory signals that suppress peripheral cytokine levels and inflammation. Brain muscarinic acetylcholine receptor (mAChR)-mediated cholinergic signaling has been implicated in this regulation. However, the possibility of controlling inflammation by peripheral administration of centrally-acting mAChR agonists is unexplored. To provide insight we used the centrally-acting M1 mAChR agonist xanomeline, previously developed in the context of Alzheimer's disease and schizophrenia. Intraperitoneal administration of xanomeline significantly suppressed serum and splenic TNF levels, alleviated sickness behavior, and increased survival during lethal murine endotoxemia. The anti-inflammatory effects of xanomeline were brain mAChR-mediated and required intact vagus nerve and splenic nerve signaling. The anti-inflammatory efficacy of xanomeline was retained for at least 20h, associated with alterations in splenic lymphocyte, and dendritic cell proportions, and decreased splenocyte responsiveness to endotoxin. These results highlight an important role of the M1 mAChR in a neural circuitry to spleen in which brain cholinergic activation lowers peripheral pro-inflammatory cytokines to levels favoring survival. The therapeutic efficacy of xanomeline was also manifested by significantly improved survival in preclinical settings of severe sepsis. These findings are of interest for strategizing novel therapeutic approaches in inflammatory diseases.

Keywords: Brain muscarinic acetylcholine receptors; Cytokines; Endotoxemia; Inflammation; Sepsis; Sickness behavior; Splenic nerve; Vagus nerve; Xanomeline.

Copyright © 2014 Elsevier Inc. All rights reserved.

Figures

Figure 1. Xanomeline suppresses serum TNF levels and improves survival in endotoxemia through a CNS mAChR-mediated signaling

(A) Xanomeline (X) (5, 10, 20 mg/kg, i.p.) treatment 1h prior to endotoxin (6 mg/kg, i.p.) administration dose-dependently reduces serum TNF as compare to saline (S) treated controls (n=5–7 per group, *P<0.001, **P<0.0001) (B) Xanomeline (X) (5, 20 mg/kg, i.p.) dose-dependently improves survival in murine endotoxemia (n=17–20 per group, *P<0.0001) (C) Atropine sulfate (AS), but not atropine methyl nitrate (AMN) abolishes xanomeline (X) suppressive effect on serum TNF (n=8–10 per group, *P<0.0001) (D) Atropine sulfate (AS), but not atropine methyl nitrate (AMN) abolishes survival improvement by xanomeline (X) in endotoxemia (n=10 per group, *P<0.0001). Mice were pre-treated with AS (4 mg/kg, i.p.) or AMN (4 mg/kg, i.p.) 15 min prior to xanomeline (20 mg/kg, i.p.).

Figure 2. Anti-inflammatory effects of xanomeline during endotoxemia are vagus nerve- and splenic nerve-dependent

(A) Xanomeline (X) reduces splenic TNF (n=8–10 per group, *P<0.003) in endotoxemic mice as compared to saline (S) treated controls. (B) Xanomeline reduces splenic TNF in sham-operated mice, but not in mice with vagotomy (n=8–10 per group, *P<0.001) (C) Xanomeline i.p. administration in rats suppresses serum TNF levels during endotoxemia (n=5–7 per group, *P<0.03, **P<0.003). (D) Xanomeline i.p. administration in rats suppresses splenic TNF levels (n=5–7 per group, *P<0.03). (E) Xanomeline (X) i.p. administration suppresses serum TNF in sham-operated animals, but not in rats with splenic nerve transection (SNT) (n=4–6 per group, *P<0.02) (F) Xanomeline (X) i.p. administration suppresses splenic TNF in shamoperated animals, but not in rats with splenic nerve transection (SNT) (n=4–6 per group, *P<0.03).

Figure 3. Xanomeline treatment provides a long-lasting anti-inflammatory protection associated with specific changes in the proportions of spleen cell subpopulations and cytokine responsiveness

(A) Xanomeline (X, 20 mg/kg) i.p. injection in mice 20h prior to endotoxin (6 mg/kg, i.p.) lowers serum TNF levels as compared to saline (S) (n=8 per group, *P<0.005) (B) Xanomeline (X, 20 mg/kg) i.p. injection in mice 20h prior to endotoxin (6 mg/kg, i.p.) improves survival as compared to saline (S) (n=10 per group, *P<0.005) (C) Xanomeline (X, 20 mg/kg) i.p. injection in mice 30h prior to endotoxin (6 mg/kg, i.p.) does not alter survival as compared to saline (S) (n=10 per group) (D) Xanomeline (X, 20 mg/kg) i.p. injection in mice 20h prior to euthanasia alters the proportions of splenocyte subpopulations as compared to saline (S) injection (n=6 per group, *P<0.04, **P<0.02, ***P<0.01) (E) Xanomeline (X, 20 mg/kg) i.p. injection in mice 20h prior to euthanasia alters splenocyte cytokine production in response to endotoxin (n=6 per group, *P<0.03, **P<0.003, ***P<0.0005, ****P<0.0001)

Figure 4. Delayed xanomeline treatment improves survival in CLP-induced severe sepsis

(A) Xanomeline (X, 20 mg/kg) i.p. injection in mice 20h prior to cecal ligation and puncture (CLP) worsens survival as compared to saline (S) (n=15 per group, *P<0.05) (B) Xanomeline (X, 2 or 5 mg/kg) i.p. injection in mice 20h prior to CLP does not alter survival as compared to saline (S) (n=22–24 per group) (C) Xanomeline (X, 1, 2, or 3 mg/kg) i.p. injection, initiated 24h following CLP and continued twice daily for 3 consecutive days improves survival as compared to saline (n=26–28 per group, *P<0.04) (S) (D) Xanomeline (X, 1 or 2 mg/kg) injected i.p. at 24h, 30h and 46h after CLP dose-dependently improves survival as compared to saline (n=27 per group, *P<0.04)