Rami T. El-Sharkawy

CD4+ T cell-mediated immunological control of enterochromaffin cell hyperplasia and 5-hydroxytryptamine production in enteric infection

Background: Enterochromaffin (EC) cells are dispersed throughout the gastrointestinal (GI) mucosa and are the main source of 5-hydroxytryptamine (5-HT) in the gut. 5-HT has been implicated in the pathophysiology of several GI disorders, but the mechanisms regulating 5-HT production in the gut are unknown. Aim: To investigate the role of CD4+ T cells in the production of 5-HT using a model of enteric parasitic infection. Methods and results: Severe combined immunodeficient (SCID) mice and their wild-type controls were infected with the nematode Trichuris muris and killed on various days after infection to study colonic EC cells and 5-HT production. The number of EC cells and the amount of 5-HT produced were significantly higher in infected wild-type mice than in non-infected mice. The number of EC cells and the amount of 5-HT after infection were significantly lower in SCID mice after infection than in wild-type mice. The number of EC cells and the amount of 5-HT was significantly increased after reconstitution of SCID mice with CD4+ T cells from infected mice and this was accompanied by an upregulation of colonic CD3 T cells and T helper 2 (Th2) cytokines. Laser capture microdissection-based molecular and immunofluorescence techniques revealed the presence of interleukin 13 receptor α1-chain on EC cells. Conclusion: These results show an important immunoendocrine axis in the gut, where secretory products from CD4+ T cells interact with EC cells to enhance the production of 5-HT in the gut via Th2-based mechanisms. These results show new insights into the mechanisms of gut function, which may ultimately lead to improved therapeutic strategies in functional and inflammatory disorders of the GI tract.

Helminth antigen-based strategy to ameliorate inflammation in an experimental model of colitis

Inflammatory bowel disease (IBD) is the most common and serious chronic inflammatory condition of the gut. Among the distinct T helper (Th) cell subsets, a Th1 type response is associated predominantly with Crohns disease (CD) while helminth infections generate a strong Th2 type response. IBD is most prevalent in developed countries but rare in countries where infections with helminths are common. Thus, it has been hypothesized that infection with helminth infection influence the development of CD and recent clinical and experimental studies suggest strongly a beneficial role of helminth infection in IBD. In the present study we examined the effects of rectal submucosal administration of helminth antigens on subsequent experimental colitis. Mice were treated with Trichinella spiralis antigens prior to the induction of dinitrobenzenesulphonic acid (DNBS)‐induced colitis and were killed 3 days post‐DNBS to assess colonic damage macroscopically, histologically and by myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOS) and cytokine levels. Previous treatment with T. spiralis antigens reduced the severity of colitis significantly, as assessed macroscopically and histologically, and reduced the mortality rate. This benefit was correlated with a down‐regulation of MPO activity, interleukin (IL)‐1β production and iNOS expression and an up‐regulation of IL‐13 and transforming growth factor‐β production in colon. These results clearly show a beneficial role of local treatment with helminth antigens for experimental colitis and prompt consideration of helminth antigen‐based therapy for IBD instead of infection with live parasites.

Enterochromaffin cell and 5-hydroxytryptamine responses to the same infectious agent differ in Th1 and Th2 dominant environments

Background/aim: 5-Hydroxytryptamine (5-HT) released from enterochromaffin cells influences intestinal homeostasis by altering gut physiology and is implicated in the pathophysiology of various gut disorders. The mechanisms regulating 5-HT production in the gut remain unclear. This study investigated the T helper (Th) 1/Th2-based immunoregulation of enterochromaffin cell function and 5-HT production in a model of enteric infection. Methods and results: Trichuris muris-infected AKR (susceptible to infection and generates Th1 response), BALB/c (resistant to infection and generates Th2 response), Stat4-deficient (impaired in Th1 response) and Stat6-deficient (impaired in Th2 response) mice were investigated to assess enterochromaffin cells, 5-HT and cytokines. In association with the generation of a Th2 response we observed higher enterochromaffin cell numbers and 5-HT content in the colon of BALB/c mice compared with AKR mice. Numbers of enterochromaffin cells and amount of 5-HT were significantly lower in Stat6-deficient mice after infection compared with Stat4-deficient mice. In addition, enterochromaffin cell numbers and 5-HT content were significantly higher after reconstitution of severe combined immunodeficient mice with in-vitro polarised Th2 cells. Conclusion: The study demonstrated that enterochromaffin cell and 5-HT responses to the same infectious agent are influenced by Th1 or Th2 cytokine predominance and suggests that the immunological profile of the inflammatory response is important in the regulation of enterochromaffin cell biology in the gut. In addition to new data on enterochromaffin cell function in enteric infection and inflammation, this study provides important information on the immuno–endocrine axis in the gut, which may ultimately lead to improved strategies against gut disorders.

Induction of a fibrogenic response in mouse colon by overexpression of monocyte chemoattractant protein 1.

Background and aims: Monocyte chemoattractant protein 1 (MCP-1) is increased transmurally in inflammatory bowel disease (IBD). Although MCP-1 is considered to play an important role in fibrotic disease in other organs, the role of MCP-1 in gut fibrosis is unknown. We investigated the fibrotic potential of MCP-1 in the gut by overexpressing this chemokine in the mouse colorectal wall. Methods: Intramural gene transfer by direct injection of adenovector into the mouse rectal wall was established. C57BL/6 and Rag2−/− (B and T cell deficient) mice received 2.5×109 plaque forming units of an adenovector encoding murine MCP-1 (AdMCP-1) or control virus (AdDL70) via intramural injection. Mice were killed at various time points and tissues were obtained for histopathological and biochemical analysis. Results: AdMCP-1 significantly increased collagen production in the colorectum and this was associated with significant elevation of transforming growth factor β (TGF-β) and tissue inhibitor of metalloproteinase (TIMP-1) protein. Transmural collagen deposition was observed after AdMCP-1 administration, and was accompanied by CD3+ T cells, F4/80+ macrophages, and vimentin+ cell infiltrates. Collagen was differentially distributed, with type I deposited in the muscularis mucosa and muscularis propria and type III in the submucosa and myenteric plexus. AdMCP-1 failed to induce collagen overproduction in immunodeficient Rag2−/− mice. Conclusion: These findings suggest that MCP-1 can induce fibrosis in the gut and that this process involves interaction between T cells and vimentin positive fibroblasts/myofibroblasts, as well as the subsequent upregulation of TGF-β and TIMP-1 production. This model provides a basis for considering MCP-1 in the pathogenesis of strictures in IBD.

Mechanisms underlying gut dysfunction in a murine model of chronic parasitic infection.

Irritable bowel syndrome (IBS) is common in countries where chronic parasitic infestations are endemic. However, the relationship between parasitic infection and IBS is not clear. The aim of this study was to examine whether chronic parasitic infection is accompanied by gut dysfunction and whether the continued presence of the parasite is required for the maintenance of the dysfunction. We used chronic Trichuris muris infection in Th1-biased susceptible AKR mice to evaluate this relationship. AKR mice were infected with T. muris and were euthanized on various days postinfection (pi) to examine worm burden, muscle function, and immune and inflammatory responses. Mice were treated with the anthelmintic oxantel pamoate to assess the effect of eradication of infection on muscle function. Infection resulted in persistence of the parasite, elevated IFN-γ, and increased MPO activity evident at 45 days pi. This was accompanied by a reduction in muscle contractility and excitatory innervation. Whereas parasite eradication at 7 days pi normalized IFN-γ and muscle contractility, eradication at 28 days pi failed to normalize muscle contractility. Administration of dexamethasone after parasite eradication normalized all parameters. Anthelmintic treatment improved histology except for eosinophils, which were normalized by subsequent dexamethasone therapy. Persistent gut dysfunction is independent of the continued presence of the parasite and is maintained by inflammatory process that includes eosinophils. Thus data in this preclinical model suggest that parasitic infection could be a cause of IBS, and the lack of symptomatic improvement following eradication is insufficient evidence to refute a causal relationship between the infection and IBS.

M1624 Role of Serotonin in Immune Activation and Inflammation in Experimental Colitis

mg tissue, p<0.01). MPO activity was reduced in DAMGO-treated I/R mice (0.29±0.07 mU/ mg) compared to saline I/R group (p<0.01). DAMGO effect was reversed by CTAP (1.62±0.65 mU/mg, p<0.01 vs. DAMGO). MPO activity was higher in CTAP-treated I/R mice (1.38±0.34 mU/mg) compared to saline I/R. DAMGO, DAMGO+CTAP, or CTAP did not affect MPO activity in SO mice. TNF-α mRNA was increased in I/R compared to SO (3.84±0.75 and 2.21±0.48 RQ-fold) and normal mice (1.11±0.38). There was a 30% reduction of TNF-α mRNA levels in DAMGO-treated I/R mice, which was abolished by CTAP. DAMGO did not affect TNF-α mRNA expression in SO mice. Conclusion: These data support a protective effect of μORs activation against intestinal inflammation, but not GIT delay induced by I/ R, which could be mediated by TNF-α. Peripheral μOR might be potential targets for therapeutic approaches for I/R-induced inflammatory injury.