Gut-derived cholecystokinin contributes to visceral hypersensitivity via nerve growth factor-dependent neurite outgrowth.

BACKGROUND AND AIM: Irritable bowel syndrome is characterized by abdominal pain and altered bowel habits and may occur following stressful events or infectious gastroenteritis such as giardiasis. Recent findings revealed a link between cholecystokinin (CCK), neurotrophin synthesis, and intestinal hyperalgesia. The aim was to investigate the role of CCK in visceral hypersensitivity using mouse models challenged with a bout of infection with Giardia lamblia or psychological stress, either alone or in combination. METHODS: Abdominal pain was evaluated by visceromoter response to colorectal distension. Nerve fibers in intestinal tissues were stained using immunohistochemistry (PGP9.5). Human neuroblastoma SH-SY5Y cells incubated with bacterial-free mouse gut supernatant or recombinant CCK-8S were assessed for neurite outgrowth and nerve growth factor (NGF) production. RESULTS: Intestinal hypersensitivity was induced by either stress or Giardia infection, and a trend of increased pain was seen following dual stimuli. Increased CCK levels and PGP9.5 immunoreactivity were found in colonic mucosa of mice following stress and/or infection. Inhibitors to the CCK-A receptor (L-364718) or CCK-B receptor (L-365260) blocked visceral hypersensitivity caused by stress, but not when induced by giardiasis. Nerve fiber elongation and NGF synthesis were observed in SH-SY5Y cells after incubation with colonic supernatants from mice given the dual stimuli, or after treatment with CCK-8S. Increased nerve fiber length by colonic supernatant and CCK-8S was attenuated by L-365260 or neutralizing anti-NGF. CONCLUSIONS: This new model successfully recapitulates intestinal hypernociception induced by stress or Giardia. Colonic CCK contributes to visceral hypersensitivity caused by stress, but not by Giardia, partly via NGF-dependent neurite outgrowth.

Persistent gut barrier damage and commensal bacterial influx following eradication of Giardia infection in mice.

BACKGROUND: Recent studies of Giardia lamblia outbreaks have indicated that 40-80% of infected patients experience long-lasting functional gastrointestinal disorders after parasitic clearance. Our aim was to assess changes in the intestinal barrier and spatial distribution of commensal bacteria in the post-clearance phase of Giardia infection. METHODS: Mice were orogastrically inoculated with G. lamblia trophozoites (strain GS/M) or pair-fed with saline and were sacrificed on post-infective (PI) days 7 (colonization phase) and 35 (post-clearance phase). Gut epithelial barrier function was assessed by Western blotting for occludin cleavage and luminal-to-serosal macromolecular permeability. Gut-associated, superficial adherent, and mucosal endocytosed bacteria were measured by agar culturing and were examined by fluorescence in situ hybridization. Intracellular bacteria cultured from isolated mucosal cells were characterized by 16S rDNA sequencing. Neutrophil-specific esterase staining, a myeloperoxidase activity assay, and enzyme-linked immunosorbent assays for cytokine concentrations were used to verify intestinal tissue inflammation. RESULTS: Tight junctional damage was detected in the intestinal mucosa of Giardia-infected mice on PI days 7 and 35. Although intestinal bacterial overgrowth was evident only during parasite colonization (PI day 7), enhanced mucosal adherence and endocytosis of bacteria were observed on PI days 7 and 35. Multiple bacterial strains, including Bacillus, Lactobacillus, Staphylococcus, and Phenylobacterium, penetrated the gut mucosa in the post-infective phase. The mucosal influx of bacteria coincided with increases in neutrophil infiltration and myeloperoxidase activity on PI days 7 and 35. Elevated intestinal IFNγ, TNFα, and IL-1ß levels also were detected on PI day 35. CONCLUSIONS: Giardia-infected mice showed persistent tight junctional damage and bacterial penetration, accompanied by mucosal inflammation, after parasite clearance. These novel findings suggest that the host's unresolved immune reactions toward its own microbiota, due to an impaired epithelial barrier, may partly contribute to the development of post-infective gut disorders.