Enterochromaffin Cells Are Gut Chemosensors That Couple To

Article Enterochromaffin Cells Are Gut Chemosensors that Couple to Sensory Neural Pathways Graphical Abstract Authors Nicholas W. Bellono, James R. Bayrer, Duncan B. Leitch, ..., Stuart M. Brierley, Holly A. Ingraham, David Julius Correspondence [email protected] (H.A.I.), [email protected] (D.J.) In Brief Organoid cultures are exploited to characterize rare chemosensory cells in the gut, revealing their receptive and signaling properties and demonstrating direct communication with neural sensory pathways. Highlights d Enterochromaffin (EC) cells are excitable and express voltage-gated ion channels d EC cells use sensory receptors to detect irritants, metabolites, and catecholamines d EC cell activation leads to voltage-gated Ca2+ channel- dependent serotonin release d EC cells modulate sensory nerves via serotonin receptors and synaptic connections Bellono et al., 2017, Cell 170, 185–198 June 29, 2017 ª 2017 Elsevier Inc. http://dx.doi.org/10.1016/j.cell.2017.05.034 Article Enterochromaffin Cells Are Gut Chemosensors that Couple to Sensory Neural Pathways Nicholas W. Bellono,1,6 James R. Bayrer,2,3,6 Duncan B. Leitch,1 Joel Castro,4,5 Chuchu Zhang,1 Tracey A. O’Donnell,4,5 Stuart M. Brierley,4,5 Holly A. Ingraham,3,* and David Julius1,7,* 1Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA 2Department of Pediatrics, Division of Gastroenterology, University of California, San Francisco, San Francisco, CA 94143, USA 3Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA 4Visceral Pain Group, Flinders University, Bedford Park, SA 5042, Australia 5Centre for Nutrition and Gastrointestinal Diseases, Discipline of Medicine, University of Adelaide, South Australian Health and Medical Research Institute (SAHMRI), North Terrace, Adelaide, SA 5000, Australia 6These authors contributed equally 7Lead Contact *Correspondence: [email protected] (H.A.I.), [email protected] (D.J.) http://dx.doi.org/10.1016/j.cell.2017.05.034 SUMMARY responses (Gribble and Reimann, 2016). Indeed, enteroendocrine cells form anatomical connections with neurons (Boho´ r- Dietary, microbial, and inflammatory factors modulate quez et al., 2015), consistent with the idea that the epithelium the gut-brain axis and influence physiological pro- participates in neural monitoring of the gut environment. Despite cesses ranging from metabolism to cognition. The growing interest in the gut-neural axis, relatively little is known gut epithelium is a principal site for detecting such about molecular mechanisms underlying chemosensory trans- agents, but precisely how it communicates with neural duction by the gut epithelium, or how this information is trans- elements is poorly understood. Serotonergic entero- mitted to the nervous system. The enterochromaffin (EC) cell is an enteroendocrine cell chromaffin (EC) cells are proposed to fulfill this role subtype that represents one of the major proposed epithelial by acting as chemosensors, but understanding how chemosensors. Although relatively rare (<1% of total intestinal these rare and unique cell types transduce chemosen- epithelia), these unique cells produce >90% of the body’s se- sory information to the nervous system has been rotonin and have been suggested to affect a variety of physio- hampered by their paucity and inaccessibility to sin- logical and pathophysiological states, such as gastrointestinal gle-cell measurements. Here, we circumvent this (GI) motility and secretion, nausea, and visceral hypersensitiv- limitation by exploiting cultured intestinal organoids ity (Gershon, 2013; Mawe and Hoffman, 2013). Previous together with single-cell measurements to elucidate studies suggest that EC cells express sensory receptors intrinsic biophysical, pharmacological, and genetic whose activation promotes release of 5-HT with consequent properties of EC cells. We show that EC cells express stimulation of gut contractility. While informative, these initial specific chemosensory receptors, are electrically insights have generally been gleaned from model endocrine tu- mor cell lines or ex vivo whole tissue preparations (Braun et al., excitable, and modulate serotonin-sensitive primary 2007; Doihara et al., 2009; Fukumoto et al., 2003; Hagbom afferent nerve fibers via synaptic connections, et al., 2011; Kim et al., 2001; Nozawa et al., 2009), leaving enabling them to detect and transduce environmental, fundamental questions unanswered. For example, what stimuli metabolic, and homeostatic information from the gut or downstream signaling pathways promote serotonin release directly to the nervous system. from EC cells, and how do EC cells communicate with the nervous system? INTRODUCTION To address such questions it is necessary to directly interro- gate single EC cells, but this is technically challenging given their The gut epithelium forms one of the largest exposed surfaces of relatively small size (<10 mm) and paucity. We therefore gener- the human body, representing a unique interface for integrating ated intestinal organoids in which EC cells are genetically environmental information with physiologic signals from ner- tagged to facilitate their detailed physiologic, pharmacologic, vous, immune, and vascular systems (Furness et al., 2013; and molecular characterization in a ‘‘native’’ environment. We O¨ hman et al., 2015). Dietary nutrients and irritants, microbiota show that EC cells are electrically excitable because they products, and inflammatory agents have been proposed to act express functional voltage-gated sodium (Na+) and calcium on the gut epithelium to modulate downstream signaling path- (Ca2+) channels, akin to other primary sensory cells. Moreover, ways controlling digestion, immunity, metabolism, and pain EC cells use specific receptors and signal transduction path- (Brierley and Linden, 2014; Furness et al., 2013). Diverse enter- ways to detect relevant stimuli: the transient receptor potential oendocrine cells within the gut epithelium respond to such A1 (TRPA1) ion channel serves as an irritant receptor, olfactory stimuli by releasing a variety of hormones to mediate physiologic receptor 558 (Olfr558) serves as a microbial metabolite sensor, Cell 170, 185–198, June 29, 2017 ª 2017 Elsevier Inc. 185 and an a2A adrendoreceptor (Adra2A)-TRPC4 channel signaling sponses were inhibited by u-agatoxin IVA, which blocks presyn- cascade detects stress response-related catecholamines. aptic P/Q-type CaV channels (Catterall et al., 2005b)(Figures 1H These sensory transduction pathways stimulate P/Q-type and 1I). A subset of EC cells were basally active, exhibiting spon- voltage-gated Ca2+ channels to control serotonin release onto taneous Ca2+ bursts that were attenuated by tetrodotoxin or 5HT3 receptor-expressing primary afferent nerve fibers that blocked by u-agatoxin IVA (Figure S2D). Consistent with this extend into intestinal villi and engage in synaptic-like contacts pharmacological profile, P/Q-type CaV2.1 channel transcripts with EC cells. Our findings establish EC cells as specialized, pol- were selectively expressed in EC cells (Figure 1J). Transcripts ymodal stimulus detectors that constitute a direct line of for the low voltage threshold T-type channel, CaV3.2, were communication between the gut epithelium and specific primary also highly enriched in EC cells. Perhaps not surprisingly, a afferent nerve fibers. This signaling system represents a key T-type CaV channel inhibitor did not significantly affect pathway for monitoring GI commensal or infectious microbes, K+-induced responses (Figure 1I), consistent with the fact that injury, or other changes to the luminal environment that stimu- CaV3.2 often functions in amplifying signals from low threshold late physiologic responses such as emesis, motility, and visceral stimuli or regulating membrane potential (Catterall et al., pain. 2005b). In summary, our results show that EC cells express voltage-gated ion channels, rendering them electrically excit- RESULTS able—a hallmark of sensory cell types. Indeed, our findings with serotonergic EC cells are similar to what has been observed EC Cells Are Electrically Excitable with hormone-secreting L-type enteroendocrine cells, which We used mice expressing a genetically encoded fluorescent pro- also express Nav and Cav channels that render them electrically tein (hrGFP) under control of the chromogranin A (ChgA)promoter excitable (Rogers et al., 2011). to selectively label EC cells for single-cell characterization (Fig- ure 1A). Consistent with previous results (Engelstoft et al., 2015), EC Cells Are Polymodal Chemosensors we found that ChgA-GFP co-localized with serotonin, but not To ask if EC cells serve a specific chemosensory role, we with lysozyme or GLP-1, which mark Paneth or peptide hor- screened 30 potential agonists, focusing on compounds known mone-producing enteroendocrine cells, respectively (Figures 1A to be present in the gut, including microbial products, irritants and S1A–S1C). Furthermore, transcriptional profiling of ChgA- and inflammatory agents, and neurotransmitters. Among these, GFP+ cells revealed enrichment of EC cell markers but not markers only allyl isothiocyanate (AITC), isovalerate, and the catechol- for hormone-producing enteroendocrine cells (Figure S1D). We amines dopamine, epinephrine, or norepinephrine specifically used whole-cell patch-clamp recording to analyze the electro- and consistently activated EC cells (Figure 2A). Additionally, iso- physiological properties

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