Role of Serotonin in the Maintenance of Inflammatory State in Crohn's Disease

Abstract

Crohn's disease (CD) is a chronic intestinal inflammation considered to be a major entity of inflammatory bowel diseases (IBDs), affecting different segments of the whole gastrointestinal tract. Peripheral serotonin (5-HT), a bioactive amine predominantly produced by gut enterochromaffin cells (ECs), is crucial in gastrointestinal functions, including motility, sensitivity, secretion, and the inflammatory response. These actions are mediated by a large family of serotonin receptors and specialized serotonin transporter (SERT) located on a variety of cell types in the gut. Several studies indicate that intestinal 5-HT signaling is altered in patients with inflammatory bowel disease. Paraformaldehyde-fixed intestinal tissues, obtained from fifteen patients with Crohn's disease were analyzed by immunostaining for serotonin, Langerin/CD207, and alpha-Smooth Muscle Actin (α-SMA). As controls, unaffected (normal) intestinal specimens of seven individuals were investigated. This study aimed to show the expression of serotonin in dendritic cells (DCs) and myofibroblast which have been characterized with Langerin/CD207 and α-SMA, respectively; furthermore, for the first time, we have found the presence of serotonin in goblet cells. Our results show the correlation between different types of intestinal cells in the maintenance of the inflammatory state in CD linked to the recall of myofibroblasts.

Keywords: Crohn’s disease; dendritic cells; goblet cells; myofibroblast; serotonin.

Figure 1

H/E and AB/PAS staining. 40×, scale bar: 40 μm. In the control samples, the intestinal epithelium appeared regular with columnar cells along the villi covering the luminal surface, consisting mainly of enterocytes, and goblet cells with rounded calyxes. The altered epithelial architecture was observed in the CD sections. Rarefied enterocytes were present among goblet cells with an elongated calyx. Infiltration of inflammatory cells, collagen deposition, and accumulation of mesenchymal cells were observed in the lamina propria of CD samples. The control samples stained with AB/PAS showed purple goblet cells, while in the CD samples, the goblet cells were highlighted in blue, with an evident layer of acidophilic mucus.

Figure 2

Human intestine. 5-HT and α-SMA, 20×, scale bar 20 nm. In healthy intestine sections, red fluorescence evidenced serotonergic enteroendocrine cells (arrows) and the goblet cell cytoplasm (*). The positivity to α-SMA was observed in stromal cells (fibroblasts and mesenchymal cells) (large arrows). In CD samples, enterochromaffin cells (ECs), positive to 5-HT, were evident in the epithelium (arrows). Lamina propria cells with a high positivity to 5-HT were detectable (big arrows). The goblet cell cytoplasm was 5-HT negative (*). α-SMA positive cells in the lamina propria below the epithelium (large arrows). Blood vessels (arrowhead). The micrographs are also equipped with transmitted light (TL) to visualize the organ morphology.

Figure 3

Human intestine, 5-HT/α-SMA colocalization, 20×, scale bar 20 nm. Merge photomicrographs indicate the double localization of two antibodies in myofibroblasts, in which it is evident that the overlap (yellow fluorescence) concerns the CD samples rather than in the healthy intestine. In the control section 5-HT highlighted the goblet cells cytoplasm (*). A cluster of subepithelial myofibroblasts in the lamina propria colocalized with 5-HT and α-SMA (large arrows), blood vessels (arrowheads). The graph represents the “display profile” function of the laser scanning microscope to show the intensity profile of detected antibodies.

Figure 4

Human intestine. 5-HT and Langerin/CD207, 20×, scale bar 20 nm. In healthy intestine sections, ECs that were serotonin positive between epithelial cells (arrow) were highlighted with red fluorescence. 5-HT positivity was observed in the goblet cell cytoplasm (*). DCs that were Langerin positive (green fluorescence) were shown in the thickness of the epithelium and the lamina propria (double arrow). In CD samples, 5-HT positivity in goblet cells disappeared and it was more evident in stromal cells (arrows). DCs in the epithelium and lamina propria (double arrows) were marked with Langerin in green fluorescence. The micrographs are also equipped with transmitted light (TL) to visualize the organ morphology.

Figure 5

Human intestine. 5-HT/Langerin DC/207 colocalization, 20×, scale bar 20 nm. The merged photomicrograph indicates the double localization of two antibodies in DCs. In the healthy intestine, 5-HT and Langerin showed overlap, which was more evident in CD samples. Stromal cells are highlighted with arrows. The graph represents the “display profile” function of the laser scanning microscope to show the intensity profile of detected antibodies.