Abstract Background Acute pancreatitis is a common and serious inflammatory condition currently lacking disease modifying therapy. The cholinergic anti-inflammatory pathway (CAP) is a potent protective anti-inflammatory response activated by vagus nerve-dependent α7 nicotinic acetylcholine receptor (α7nAChR) signaling using splenic CD4+ T cells as an intermediate. Activating the CAP ameliorates experimental acute pancreatitis. Galantamine is an acetylcholinesterase inhibitor (AChEI) which amplifies the CAP via modulation of central muscarinic ACh receptors (mAChRs). However, as mAChRs also activate pancreatitis, it is currently unknown whether galantamine would be beneficial in acute pancreatitis. Methods The effect of galantamine (1–6 mg/kg-body weight) on caerulein-induced acute pancreatitis was evaluated in mice. Two hours following 6 hourly doses of caerulein (50 µg/kg-body weight), organ and serum analyses were performed with accompanying pancreatic histology. Experiments utilizing vagotomy, gene knock out (KO) technology and the use of nAChR antagonists were also performed. Results Galantamine attenuated pancreatic histologic injury which was mirrored by a reduction in serum amylase and pancreatic inflammatory cytokines and an increase the anti-inflammatory cytokine IL-10 in the serum. These beneficial effects were not altered by bilateral subdiaphragmatic vagotomy, KO of either choline acetyltransferase+ T cells or α7nAChR, or administration of the nAChR ganglionic blocker mecamylamine or the more selective α7nAChR antagonist methyllycaconitine. Conclusion Galantamine improves acute pancreatitis via a mechanism which does not involve previously established physiological and molecular components of the CAP. As galantamine is an approved drug in widespread clinical use with an excellent safety record, our findings are of interest for further evaluating the potential benefits of this drug in patients with acute pancreatitis.

BACKGROUND:Choline acetyltransferase (ChAT) is required for the biosynthesis of acetylcholine, the molecular mediator that inhibits cytokine production in the cholinergic anti-inflammatory pathway of the vagus nerve inflammatory reflex. Abundant work has established the biology of cytoplasmic ChAT in neurons, but much less is known about the potential presence and function of ChAT in the extracellular milieu. OBJECTIVES:We evaluated the hypothesis that extracellular ChAT activity responds to inflammation and serves to inhibit cytokine release and attenuate inflammation. METHODS:After developing novel methods for quantification of ChAT activity in plasma, we determined whether ChAT activity changes in response to inflammatory challenges. RESULTS:Active ChAT circulates within the plasma compartment of mice and responds to immunological perturbations. Following the administration of bacterial endotoxin, plasma ChAT activity increases for 12-48 h, a time period that coincides with declining tumor necrosis factor (TNF) levels. Further, a direct activation of the cholinergic anti-inflammatory pathway by vagus nerve stimulation significantly increases plasma ChAT activity, whereas the administration of bioactive recombinant ChAT (r-ChAT) inhibits endotoxin-stimulated TNF production and anti-ChAT antibodies exacerbate endotoxin-induced TNF levels, results of which suggest that ChAT activity regulates endogenous TNF production. Administration of r-ChAT significantly attenuates pro-inflammatory cytokine production and disease activity in the dextran sodium sulfate preclinical model of inflammatory bowel disease. Finally, plasma ChAT levels are also elevated in humans with sepsis, with the highest levels observed in a patient who succumbed to infection. CONCLUSION:As a group, these results support further investigation of ChAT as a counter-regulator of inflammation and potential therapeutic agent.

Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, affects up to 3 million people in the United States alone, significantly worsens a patient’s quality of life, and is associated with a high financial burden. Current treatments for IBD are mainly biologicals that have significant side effects. No efficient pharmacological treatments are currently available for IBD. Previous studies established that activation of the vagus nerve‐based inflammatory reflex inhibits cytokine production and regulates immune responses through cholinergic signaling. Acetylcholine released by a T cell subset expressing choline acetyltransferase (ChAT), the enzyme that catalyzes the biosynthesis of acetylcholine, inhibits cytokine production via signaling through α7 nicotinic acetylcholine receptors (α7nAChR). However, the possibility of controlling inflammation by systemic administration of ChAT is unexplored. Here we examined the efficacy of ChAT in improving disease activity in a preclinical model of colitis. To prolong the half‐life of circulating ChAT, the protein was modified by covalently attaching repeating units of polyethylene glycol (PEG), resulting in enzymatically active PEG‐ChAT. We utilized a well‐established model of colitis ‐ dextran sulfate sodium induced colitis. Wild type C57/black 6 mice were exposed to 4% DSS (M.W 36 000‐50 000) in drinking water for 7 days prior to administration of PEG‐ChAT. Starting on day 8, animals received PEG‐ChAT or vehicle intraperitoneally twice per day for 7 days. PEG‐ ChAT treated mice showed a significantly lower disease activity index as compared with vehicle treated controls with the disease (d11 p=0.006 ChAT mice: 0.17DAI±0.05, n=9 versus vehicle mice: 0.66DAI±0.13, n=10). The decreased severity of colitis in PEG‐ChAT‐treated mice as compared with vehicle‐treated controls is further demonstrated by a significant improvement in body weight gain (d14 p=0.01 ChAT mice: ‐5.1%±1.1, n=10 versus vehicle mice: ‐15.5%±2.9, n=10) and colon length (p=0.006 ChAT mice: 7.15cm±0.2, n=10 versus vehicle mice: 6.2cm±0.2, n=10). In conclusion, PEG‐ChAT reduces disease activity in a preclinical model of colitis. Together these studies provide new insights into the role of PEG‐ChAT as an experimental anti‐inflammatory therapeutic modality for the treatment of IBD.

Excessive immune cell activation and cytokine release leading to inflammatory conditions are associated with bidirectional immune system-brain communication and other physiological responses. The vagus nerve conveys sensory information to the brain and brain derived immunoregulatory signals suppressing peripheral cytokine levels and inflammation. Acetylcholine (A mediated cholinergic signaling has been implicated in this regulation. However, the possibility of controlling inflammation by peripheral administration of choline acetyl transferase (ChAT), enzyme that catalyzes biosynthesis of acetylcholine, is unexplored. We studied the administration of ChAT in endotoxemia, sepsis and DSS colitis models. Intraperitoneal administration of ChAT significantly (p=0.002) suppresses serum levels of TNF (ChAT 1062.0pg/mL ± 113.0 vs vehicle 1711.0pg/mL ± 210.2) during murine endotoxemia. In a murine CLP sepsis model, administration of pegylated ChAT (PEG-ChAT) significantly improves survival (p=0.015), with a 55.6% survival in PEG-ChAT treated mice compared to a 22.2% survival in vehicle mice. In a preclinical model of inflammatory bowel disease, ChAT administration significantly improves body weight gain (d14 p=0.01 ChAT mice -5.1%±1.1 vs vehicle mice -15.5%±2.9), disease score (d11 p=0.006 ChAT mice 0.17DAI±0.05, vs vehicle mice 0.66DAI±0.13), and colon length (p=0.006 ChAT mice 7.15cm±0.2 vs vehicle mice 6.2cm±0.2) compared to vehicle. These results indicate that administration of ChAT inhibits TNF levels in acute endotoxemia and attenuates disease severity in murine models of sepsis and DSS-induced colitis, suggesting that further study of ChAT as an experimental anti-inflammatory therapeutic is warranted. Supported by grant from NIH to KJT and SSC.