TRPA1 protects mice from pathogenic Citrobacter rodentium infection via maintaining the colonic epithelial barrier function.

Transient receptor potential ankyrin 1 (TRPA1) is expressed in gastrointestinal tract and plays important roles in intestinal motility and visceral hypersensitivity. However, the potential role of TRPA1 in host defense, particularly against intestinal pathogens, is unknown. Here, we show that Trpa1 knockout mice exhibited increased susceptibility to Citrobacter rodentium infection, associated with the increased severity of diarrhea and intestinal permeability associated with the disrupted tight junctions (TJs) in colonic epithelia. We further demonstrated the expression of TRPA1 in murine colonic epithelial cells (CECs) and human epithelial Caco-2 cells both at protein level and transcription level. Using calcium imaging, TRPA1 agonists allyl isothiocyanates (AITC) and hydrogen peroxide were observed to induce a transient Ca2+ response in Caco-2 cells, respectively. Moreover, TRPA1 knockdown in Caco-2 cells resulted in the decreased expression of TJ proteins, ZO-1 and Occludin, and in the increased paracellular permeabilities and the reduced TEER values of Caco-2 monolayers in vitro. Furthermore, inhibition of TRPA1 by HC-030031 in the confluent Caco-2 cells caused the altered distribution and expression of TJ proteins, ZO-1, Occludin, and Claudin-3, and exacerbated the bacterial endotoxin lipopolysaccharide (LPS)-induced damage to these TJ proteins and actin cytoskeleton. By contrast, AITC pretreatment restored the distribution and expression of these TJ proteins in the confluent Caco-2 cells upon LPS challenge. Our results identify an unrecognized protective role of TRPA1 in host defense against an enteric bacterial pathogen by maintaining colonic epithelium barrier function, at least in part, via preserving the distribution and expression of TJ proteins in CECs.

Identification of MrgprD expression in mouse enteric neurons.

Mas-related G protein-coupled receptor D (MrgprD) was first identified in small-diameter sensory neurons of mouse dorsal root ganglion (DRG). The role of MrgprD has been studied in somatosensation, especially in pain and itch response. We recently showed that MrgprD also participated in the modulation of murine intestinal motility. The treatment of MrgprD receptor agonist suppressed the spontaneous contractions in the isolated intestinal rings of mice, indicating the intrinsic expression of MrgprD in the murine gastrointestinal (GI) tract. Although the expression of Mrgprd in GI tract has been previously detected by the way of quantitative real-time PCR, the cell-type-specific expression of MrgprD in GI tract is no yet determined. Herein, we employed Mrgprd-tdTomato reporter mouse line and the whole-mount immunohistochemistry to observe the localization of MrgprD in the smooth muscle layers of ileum and colon. We show that tdTomato-positive cells colocalized with NeuN-immunostaining in the myenteric plexus in the whole-mount preparations of the ileum and the colon. Further immunohistochemistry using the commercially available MrgprD antibody revealed the expression of MrgprD in NeuN-labeled enteric neurons in the myenteric plexus. Our results demonstrate the expression of MrgprD in the enteric neurons in the murine GI tract, highlighting the implications of MrgprD in the physiology and pathophysiology of the GI tract.

Mas-related G protein-coupled receptor D is involved in modulation of murine gastrointestinal motility.

NEW FINDINGS: What is the central question of this study? The physiological function of Mas-related G protein-coupled receptor D (MrgprD) in gastrointestinal motility is unknown. The aim of this study was to assess the effects of MrgprD and its receptor agonists on murine gastrointestinal motility. What is the main finding and its importance? Mrgprd deficiency improved murine gastrointestinal motility in vivo but had no effects on the spontaneous contractions of murine intestinal rings ex vivo. Systemic administration of the MrgprD ligand, either ß-alanine or alamandine, delayed gastrointestinal transit in vivo and attenuated the spontaneous contractions of isolated intestinal rings ex vivo. ABSTRACT: Mas-related G protein-coupled receptor D (MrgprD) was first identified in sensory neurons of mouse dorsal root ganglion and has been demonstrated to be involved in sensations of pain and itch. Although expression of MrgprD has recently been found in the gastrointestinal (GI) tract, its physiological role in GI motility is unknown. To address this question, we used Mrgprd knockout (Mrgprd-/- ) mice and MrgprD agonists to examine the effects of Mrgprd gene deletion and MrgprD signalling activation, respectively, on murine intestinal motility, both in vivo and ex vivo. We observed that the deletion of Mrgprd accelerated the transmission of charcoal through the mouse GI tract. But Mrgprd deficiency did not affect the mean amplitudes and frequencies of spontaneous contractions in ileum ex vivo. Colonic motor complexes in the proximal and the distal colon were recorded from wild-type and Mrgprd-/- mice, but their control frequencies were not different. Moreover, in wild-type mice, systemic administration of an MrgprD agonist, either ß-alanine or alamandine, delayed GI transit in vivo and suppressed spontaneous contractions in the ileum and colonic motor complexes in the colon ex vivo. Our results suggest that MrgprD and its agonist are involved in the modulation of GI motility in mice.