Background
Irritable bowel syndrome (IBS) is a highly prevalent functional gastrointestinal disorder characterized by abdominal pain and alterations in bowel habits [
1]. Between 3.7% and 36% of patients with acute gastrointestinal infection subsequently develop a form of IBS known as post-infectious IBS (PI-IBS) [
2].
Many pathogens can cause PI-IBS. Bacteria and parasites are often used in PI-IBS animal models; however, for bacterially-induced PI-IBS animal models, the major characteristics of IBS such as visceral hypersensitivity, alterations in motility and secretion are weak or sometimes absent, and it therefore remains controversial whether bacterial infection results in a valid model of PI-IBS.
Nippostrongylus brasiliensis and
Cryptosporidium parvum have been used in rat models of PI-IBS. However, it was found that models infected by
N. brasiliensis lacked visceral sensitivity. The features of IBS such as motility dysfunction and altered secretion have not been evaluated in the
C. parvum infection model [
3]. Infection by
Trichinella spiralis larvae induced changes in visceral sensitivity, alterations of intestinal smooth muscle function, and altered secretion. These abnormalities persisted after animals recovered from infection, suggesting that this is a suitable model of PI-IBS [
4,
5].
The pathophysiology of PI-IBS is not fully understood, but low-grade inflammation and chronic alteration of the immune system at the molecular level have been shown to be associated with mucosal secretory function, and with smooth muscle and enteric nervous fibers [
6-
8]. In particular, an imbalance of pro- and anti-inflammatory cytokines is seen, which may play a key role in the local intestinal inflammation.
A number of reports related to PI-IBS patients have found a clear increase in many immune cell types within the mucosa [
9-
15]. Various animal models have been developed to gain insight into the underlying mechanism of IBS. A large number of studies have demonstrated that certain indicators, such as visceral hypersensitivity and persistent dysfunction of the intestinal muscle, exist in mice infected with
T. spiralis [
4,
16,
17]. In fact, the inflammatory response to intestinal parasites has been regarded as a representative defense response against pathogens. For this reason, experimental infection with the parasite
Trichinella has been widely used to establish models for detecting the pathogenesis of intestinal dysfunction [
18,
19]. Activated immune cells continue to release various cytokines after an acute intestinal infection [
20], for example, T-helper (Th) cells produce interferon (IFN)-γ and interleukin (IL)-1β to promote the inflammatory response; T-regulatory cells release IL-10 to prevent autoimmunity; in contrast, IL-17, which is produced by Th17 cells, can induce autoimmunity [
20]. These cytokines may alter the physiology and immunity of the host gut to cause symptoms of PI-IBS: IL-1β activates nitric oxide synthase and enkephalin immunoreactivity on interneurons or motorneurons and suppresses presynaptic cholinergic neurotransmission [
21,
22]. Moreover, IL-1β can stimulate cecocolonic motor activity and cause a migrating myoelectric complex pattern in the small intestine [
23]. IFN-γ and IL-1β can disrupt the colonic epithelial barrier and increase intestinal tight junction permeability [
24-
26]; reduction of IL-10 may cause an imbalance between pro- and anti-inflammatory mechanisms, resulting in chronic intestinal inflammation [
27]. IL-17 and IFN-γ can act cooperatively in the promotion of inflammatory responses in the intestinal mucosa [
28]. Hence, in this study, we established a PI-IBS mouse model and assessed local expression levels of a range of cytokines in different intestinal segments in order to investigate the probable immune mechanisms of PI-IBS.
Discussion
Eight weeks after
T. spiralis infection, the GI system still had disturbed visceral hypersensitivity without any histological evidence of intestinal inflammation. This means that the
T. spiralis infection model is an acceptable model to represent PI-IBS [
33]. AWR scores were altered in response to low or medium pressures, but did not significantly differ from healthy mice at high pressure. This indicates that the low/medium threshold nerves, but not the high threshold nerves, are altered. This may be related to different mechanical stimulation activated by different pressure expansion [
34]. When distention volume was 0.25 ml, the pressure was too low to cause any visceral sensation. When the distention volume was 0.65 ml (high pressure), the level of stimulation was so high that it resulted in a very intense response in both groups of mice. When the distention volume was either 0.35 or 0.5 ml, the AWR scores in the model group were higher than those in the control group, and the pain threshold in the model group was lower than that in the control group at the same time point. This suggests an increase of the visceral sensitivity in mice after infection. Although the pathogenesis of PI-IBS is not well understood, increasing evidence suggests that low-grade inflammation and immune activation play a pivotal role in the occurrence and persistence of its symptoms [
35-
40]. Several reports have described high numbers of T cells in various lymphoid compartments of the small or large intestine in IBS patients [
9,
10,
41], and activated T cells produce many cytokines involved in the inflammatory process, including IL-1β, IL-10, IL-17, and IFN-γ. Further studies have shown that chronic alterations of inflammatory cytokines are found in the peripheral blood and intestinal mucosa, which is consistent with the development of IBS symptoms at the molecular level [
20,
37]. However, alterations of certain cytokines in different locations within the GI tract have not been systematically reported in PI-IBS.
In the present study, we successfully established a PI-IBS mouse model induced by
Trichinella larvae and found that the levels of IFN-γ, as well as IL-17, were increased in the duodenum and ileum. IFN-γ is a classical pro-inflammatory cytokine, which can act cooperatively with IL-17 in the promotion and shaping of inflammatory responses in the intestinal mucosa [
28], and also can be regulated by IL-17 to drive neutrophil migration and mediate tissue injury [
42-
44]. Moreover, the high expression of IFN-γ could inhibit IL-10 production [
45], consistent with our results showing that IL-10 levels were decreased in jejunum and ileum. In addition, IFN-γ can disrupt the colonic epithelial barrier and increase intestinal tight junction permeability [
24-
26]. Many mechanisms have been proposed for this phenomenon, including dysbiosis and elimination of Paneth cells [
46]. Proliferation of intestinal epithelial cells is inhibited through suppression of β-catenin/T cell factor signaling [
47], and the AMPK signaling pathway is activated by phosphorylation [
48]. IL-17,which plays a protective role in infections, exhibits its inflammatory effects by activating NF-κB, MAPKs and C/EBP cascades to induce the production of multiple pro-inflammatory molecules and subsequent activation of macrophages and neutrophils [
49]. It also has a regulatory function limiting the accumulation and activity of neutrophils by attenuating the anti-apoptotic effect of inflammatory cytokines during the inflammatory process [
50]. In our PI-IBS model, by 56 days PI,
T. spiralis was completely absent from the intestinal mucosa but IL-17 still persisted, suggesting that increased IL-17 in the duodenum and ileum may be vital for maintaining intestinal low-grade inflammation. First, the IL-23/IL-17 signal pathway (which regulates IL-12/IFN-γ, and drives neutrophil migration to mediate inflammation injury) has been well described [
42-
44]. Second, uncontrolled IL-17 responses can augment production of inflammatory factors including IL-1, IL-6, IL-8, TNF-α, GM-CSF, and MIP
-2 [
44,
51]. These cytokines were reported to be increased in the peripheral blood and intestinal mucosa of PI-IBS patients [
20], which would alter the gut physiology and immunity of the host, causing clinical symptoms [
20,
36].
IL-10, as a classical anti-inflammatory cytokine, decreases the inflammatory reaction through a number of mechanisms. It can diminish the production of inflammatory mediators including IL-1β and IFN-γ in T cells and activate macrophages [
52-
54]. It also can reduce the expression of major histocompatibisblity complex class II, co-stimulating, and adhesion molecules on the surface of antigen-presenting cells [
55,
56]. Importantly, it can also suppress the development of mast cells [
57]. In our study, IL-10 levels were decreased in the jejunum, ileum and colon of PI-IBS model mice. Because of the decreased IL-10, on one hand pro-inflammatory cytokines were much more highly expressed, resulting in intestinal low-grade inflammation persisting. On the other hand, the antigen-presenting cells maintained their function, contributing to the adaptive immune response. Furthermore, hyperplasia of mast cells may be related to the decreased IL-10 levels [
57], and they can also alter homeostatic intestinal epithelial migration and barrier function [
57,
58].
In the present study, the expression levels of IL-1β were not significantly different between the two groups. Further research is required to explore whether IL-1β levels were suppressed by anti-inflammatory cytokines other than IL-10. Although changes of the four cytokines that we measured were not found in all intestinal segments, it is reasonable to deduce that the physiology and immunity of the host may be impaired by other cytokines [
47,
59].
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
The study was designed by CL and XZ. BY carried out the animal and molecular genetic studies, research, and data analysis and wrote the paper. All authors read and approved the final manuscript.