Abstract
Campylobacter jejuni is a widespread zoonotic pathogen and the leading bacterial cause of foodborne gastroenteritis in humans. Previous infection studies showed disruption of intercellular contacts, induction of epithelial apoptosis, and immune activation, all three contributing to intestinal barrier dysfunction leading to diarrhea. The present study aims to determine the impact of subepithelial immune cells on intestinal barrier dysfunction during Campylobacter jejuni infection and the underlying pathological mechanisms. Infection was performed in a co-culture of confluent monolayers of the human colon cell line HT-29/B6-GR/MR and THP-1 immune cells. Twenty-two hours after infection, transepithelial electrical resistance (TER) was decreased by 58 ± 6% compared to controls. The infection resulted in an increase in permeability for fluorescein (332 Da; 4.5-fold) and for FITC-dextran (4 kDa; 3.5-fold), respectively. In contrast, incubation of the co-culture with the pan-caspase inhibitor Q-VD-OPh during the infection resulted in a complete recovery of the decrease in TER and a normalization of flux values. Fluorescence microscopy showed apoptotic fragmentation in infected cell monolayers resulting in a 5-fold increase of the apoptotic ratio, accompanied by an increased caspase-3 cleavage and caspase-3/7 activity, which both were not present after Q-VD-OPh treatment. Western blot analysis revealed increased claudin-1 and claudin-2 protein expression. Inhibition of apoptosis induction did not normalize these tight junction changes. TNFα concentration was increased during the infection in the co-culture. In conclusion, Campylobacter jejuni infection and the consequent subepithelial immune activation cause intestinal barrier dysfunction mainly through caspase-3-dependent epithelial apoptosis. Concomitant tight junction changes were caspase-independent. Anti-apoptotic and immune-modulatory substances appear to be promising agents for treatment of campylobacteriosis.
Highlights
Diarrheal disease is a major cause of morbidity and mortality worldwide
The apoptosis inhibitor Q-VD-OPh did not result in any significant alteration of the transepithelial electrical resistance (TER) in the HT-29/B6-GR/MR cell culture on the first day of incubation
Since the mucosal immune activation in campylobacteriosis is a key feature of the disease, a co-culture of HT-29/B6/GR-MR and activated THP-1 immune cells was used for the infection assay
Summary
Diarrheal disease is a major cause of morbidity and mortality worldwide. Campylobacter jejuni (C. jejuni) is a frequent commensal bacterium in poultry and wild birds and the leading cause of bacterial diarrhea in humans. As a zoonotic pathogen being highly contagious via the fecal-oral route, C. jejuni infection occurs by consumption of raw or undercooked meat, raw dairy products or contaminated water. The symptoms of the campylobacteriosis vary from fever, aches, and dizziness to severe manifestations with abdominal cramps and bloody diarrhea. The disease is self-limiting and antibiotic treatment is only recommended in chronic or severe cases. C. jejuni infection result in very large health costs (Hoffmann et al, 2012; Tam and O’Brien, 2016) and can lead to complications such as post-infectious reactive arthritis and Guillain-Barré syndrome
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