Optogenetic activation of cholinergic enteric neurons reduces inflammation in experimental colitis

Intestinal inflammation is associated with loss of enteric cholinergic neurons. Given the systemic anti-inflammatory role of cholinergic innervation, we hypothesized that enteric cholinergic neurons similarly possess anti-inflammatory properties and may represent a novel target to treat inflammatory bowel disease (IBD).Mice were fed 2.5% dextran sodium sulfate (DSS) for seven days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the mid-colon of ChAT::Cre;R26-iDTR (ChAT-iDTR) mice by local injection of diphtheria toxin prior to colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 (ChAT-ChR2) mice, in which ChAT+ neurons express channelrhodopsin-2 (ChR2), with daily blue light stimulation (BLS) delivered via an intracolonic probe during the 7 days of DSS treatment. Colitis severity, ENS structure, and smooth muscle contractility were assessed by histology, immunohistochemistry, qPCR, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages.Ablation of ChAT+ neurons in DSS-treated mice exacerbated colitis, as measured by weight loss, colon shortening, histologic inflammation, and CD45+ cell infiltration, and led to colonic dysmotility. Conversely, optogenetic activation of enteric cholinergic neurons improved colitis, preserved smooth muscle contractility, protected against loss of cholinergic neurons, and reduced proinflammatory cytokine production. Both ACh and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in LPS-stimulated muscularis macrophages.These findings demonstrate that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential IBD treatment.