CD is a lifelong disease with unknown etiology, characterized by a chronic alteration of relapse-remission. Current medications may relieve clinical symptoms and control intestinal inflammation, but is not able to cure CD [16]. Patients with CD in the active phase usually required either a single steroid administration or a combination therapy with immunosuppressive agents, or even biologic therapy to induce remission. Non-steroidal immunosuppressants were often required to maintain remission. Recent studies, however, demonstrated that some drugs used to treat CD and gut inflammation, such as steroid hormones, might increase the risk of heart disease including coronary heart disease and heart failure, especially in women and young adults at increased risk for acute myocardial infarction and stroke [17]. Surgical intervention would be considered when clinical symptoms were difficult to control by medications available. Complications such as perianal disease, stenosis and intraperitoneal fistula will increase the surgical risk of CD patients. Furthermore, the treatment of these complications was difficult, which requires close cooperation between gastrointestinal surgeons and gastroenterologists [18]. All of those contributed to high morbidity and mortality of CD. Therefore, early diagnosis, appropriate treatment and control of inflammation were of great importance to improve the quality of life and prognosis of patients.
Enteric serotonin (5-HT) not only plays vital roles as a growth factor, a hormone, a paracrine factor and a neurotransmitter in gut, but also is an important mediator involved in a range of gastrointestinal inflammatory diseases. Enterochromaffin (EC) cells secrete 5-HT.and those cells are located throughout the intestinal tract from the stomach to the colon [19]. Alterations in the number of EC cells and in the quantity of 5-HT that EC cells produced could be observed in both IBD patients and animal models of colitis. It was reported that increased mucosal 5-HT signaling or increased 5-HT utilization may contribute to the inflammation in CD patients [20].
5-HT perform its biological functions by activating specific receptors, which consist of 7 different types based on structural and functional characteristics. Five members of the receptor family (5-HT1R, 5-HT2R, 5-HT3R, 5-HT4R and 5-HT7R) were expressed in intestinal neurons and participated in the regulation of gastrointestinal movement [21]. Initially 5-HT7R was considered to be able to regulate the motility of ileum, stomach and colon by regulating smooth muscle activity. Therefore, the biological function of 5-HT7R in IBD was related to the number of receptors expressed on smooth muscle and intestinal neurons, and participated in the regulation of the circular muscle adaptation in the early stage of ileum periperiasis, leading to abdominal distension. Recent studies also suggested that 5-HT7R might play an important role in the pathogenesis of IBD inflammation [22].
Previous studies in animal models found that 5-HT functions in intestinal inflammation by binding to 5-HT7R on the surface of DCs [23]. An additional observation by Guseva et al. [24] demonstrated that in gut 5-HT7R expressed by the intestinal CD11c/CD86 double-positive cell subsets, which suggest those CD11c+/CD86+ cells may play a key role in the onset and the development of intestinal inflammation. Pharmacological blockage or genetic deletion of 5-HT7R was able to aggravate intestinal inflammation; in contrast, activation of 5-HT7Rs promoted anti-inflammatory responses.
Furthermore, it was found 5-HT7R expression in DCs was significantly up-regulated in the lamina propria in inflammatory mouse model [21]. The current study demonstrated that the 5-HT7R positive cells were mainly located in both the neuronal intermuscular plexus and submucosal plexus, especially in the annular muscularis and intermuscular plexus (Fig. 3). This is consistent with the data shown in animal studies by Prause et al. [25]. Our histological analysis showed that CD patients presented with inflammatory infiltration of colonic mucosa and musculoskeletal, epithelial injury and crypt shape disorder. When 5-HT7R expression was significantly elevated, pathological damages in the same tissue were more significant. In contrast, there was little change in the morphology of the intestinal wall in the control group (Fig. 4). The biological function of 5-HT7R in IBD was similar to the mechanism of a series of symptoms caused by ileum peristalsis in functional bowel disease [7]. The role of 5-HT7R in IBD was thought to be mainly related to smooth muscle and receptors expressed on enteric neurons, as well as to the adaptation of circular muscles in the early stage of ileal peristalsis, which may lead to some similar symptoms to functional intestines disease.
In the current study, intestinal biopsies and blood samples from the same CD patients were used to analyze the role of 5-HT7R in CD. The results showed that 5-HT7R expressed by the intestinal CD11c+/CD86+ cell subsets is likely played a key role in the onset and clinical process of intestinal inflammation. Since the activation of 5HT7R may promote the anti-inflammatory response in the gut, expression of 5-HT7R could aggravate enteritis in patients with CD. The increased expression of 5-HT7R in the intestinal immune cells of CD patients further suggested the role of 5-HT7R in inflammation. Therefore, activation of 5-HT7R signaling pathway might play an important role in the progression of inflammation.
It is interesting that 5-HT7R expression was significantly increased in the inflammatory regions of the intestinal tract in CD patients. 5-HT7R was mainly expressed on CD11c+/CD86+ cells, which suggests the important role of 5-HT7R in regulating the function of mature DCs in the mucosal immune response of patients with CD. In addition, the expression of 5-HT7R was positively correlated with CDAI, SESCD, and MaRIA scores, suggesting that 5-HT7R expression was consistent not only with the clinical manifestations of intestinal inflammation, but also with the mucosal inflammation of the intestinal wall and the alterations of inflammatory cells infiltrating the intestinal wall including mucosa, submucosa, and serosa.
Accurate determination of CD disease severity is imperative in personalized medication [26]. Evaluation of CD disease severity includes clinical severity evaluation, intestinal mucosal injury evaluation and intestinal penetrative inflammatory exudate evaluation. CDAI, SES-CD, and MaRIA scores were commonly used to assess clinical severity, endoscopic intestinal mucosal injury, and intestinal transmural inflammatory exudation, respectively. SES-CD score is used to judge CD activity directly and objectively. Pathology observations suggested that multiple inflammatory cell infiltration and non-caseous granuloma formation were of great significance for the diagnosis of CD [27]. Penetrative inflammatory exudation and parenteral complications might be identified by MaRIA. Changes in the severity of the lesions caused by treatment can be accurately detected by thickening of the intestinal wall, enhancement of relevant contrast, tooth comb or mesangial vessel dilation and tortuosity. Therefore, MaRIA may be used to evaluate mucosal healing [28]. According to numerous clinical studies, most of the laboratory indicators used to determine the activity of clinical diseases, such as ESR and CRP, were inconsistent with the extent of intestinal mucosal injury and MRI results. And there was no significant correlation between these indicators and SES-CD and MaRIA scores. Therefore, these indicators cannot truly reflect the severity of inflammation in the intestinal mucosa [29]. However, the CDAI score was based on the subjective feeling of the patients as the gold standard, and the symptoms and signs of abdominal pain, diarrhea, abdominal mass and complications of CD patients were scored, without the mucosal inflammation being included in the scoring range [30]. Thus, it is important for clinicians to find a laboratory indicator that can reflect the severity of CD disease.
Our study showed that 5-HT7R was differentially expressed between the CD active group and the remission group, indicating its significance in differentiating disease activity status. In addition, 5-HT7R expression was well correlated with MaRIA, SESCD, CDAI scores and inflammatory markers (WBC, CRP & ESR). Although it was reported that CRP, CDAI and WBC had a low correlation with SES-CD score (0.3 < r ≤ 0.5) [29, 30], our study showed that 5-HT7R expression was strongly correlated with SES-CD (r > 0.8). Endoscopy is an invasive procedure that leaves a painful experience to patients, and in addition the procedure has some risks of perforation, bleeding, and unsuccessful examination. In addition, 5-HT7R expression was also correlated with MaRIA scores (r = 0.8126, P < 0.001).
MRI played an irreplaceable role in the evaluation of CD condition. For example, thickening of the intestinal wall and high signal intensity of T2 were considered as effective markers of CD activity, but its disadvantages such as high price and high consumption of liquid before examination limited its wide application. Serological markers such as WBC, CRP and ESR may represent systemic inflammation status but tthey are lack of intestinal specificity, while CDAI scores were not reliable indicators for disease and severity. Therefore, 5-HT7R expression in gut could be a potentially non-invasive, convenient, economic and cross-check approach to evaluate inflammatory activity in CD patients.
In summary, the present study found that 5-HT7R expression was significantly increased in the inflammatory regions of human intestine in CD patients. 5-HT7R was mainly expressed by CD11c/CD86 double positive cells, indicating that 5-HT7R might play an important role in regulating the function of mature DCs in mucosal immune response. Additionally, 5-HT7R expression may be used as an indicator to evaluate CD intestinal inflammation in clinical practice, so as to accurately determine disease stage, inflammatory activity and prognosis, and then implement individualized treatment. Overall, our data suggested that 5-HT7R might serve as a novel therapeutic and evaluation target for CD mucosal inflammation.
The clinical manifestations of CD are complex and it usually is not straightforward to make diagnosis. At present, there are no reliable and clinically applicable biomarkers. We now provided evidence that the gene expression of 5-HT7R is correlated with the clinical, radiological, and endoscopic activity scores of CD patients, which suggests that 5-HT7R expression might be an applicable biomarker to evaluate the disease severity of CD. However, more research is needed to evaluate the 5-HT7R gene expression with the pathological features in the affected area of the bowel of the patients with CD, as well as with the status of disease recurrence and drug efficacy in patients with CD. Additional prospective studies with standardized experimental procedure could be carried out to establish a cut-off value to facilitate clinical application.