Contribution of Epithelial Apoptosis and Subepithelial Immune Responses in Campylobacter jejuni-Induced Barrier Disruption

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

Curcumin Mitigates Immune-Induced Epithelial Barrier Dysfunction by Campylobacter jejuni

Campylobacter jejuni (C. jejuni) is the most common cause of foodborne gastroenteritis worldwide. The bacteria induce diarrhea and inflammation by invading the intestinal epithelium. Curcumin is a natural polyphenol from turmeric rhizome of Curcuma longa, a medical plant, and is commonly used in curry powder. The aim of this study was the investigation of the protective effects of curcumin against immune-induced epithelial barrier dysfunction in C. jejuni infection. The indirect C. jejuni-induced barrier defects and its protection by curcumin were analyzed in co-cultures with HT-29/B6-GR/MR epithelial cells together with differentiated THP-1 immune cells. Electrophysiological measurements revealed a reduction in transepithelial electrical resistance (TER) in infected co-cultures. An increase in fluorescein (332 Da) permeability in co-cultures as well as in the germ-free IL-10-/- mouse model after C. jejuni infection was shown. Curcumin treatment attenuated the C. jejuni-induced increase in fluorescein permeability in both models. Moreover, apoptosis induction, tight junction redistribution, and an increased inflammatory response-represented by TNF-α, IL-1β, and IL-6 secretion-was observed in co-cultures after infection and reversed by curcumin. In conclusion, curcumin protects against indirect C. jejuni-triggered immune-induced barrier defects and might be a therapeutic and protective agent in patients

Campylobacter concisus Impairs Sodium Absorption in Colonic Epithelium via ENaC Dysfunction and Claudin-8 Disruption

The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (-55%, n = 15, p < 0.001). This occurred via down-regulation of β- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction-both of which contribute to Na+ malabsorption and diarrhea

Campylobacter concisus stört die Funktion des epithelialen Natriumkanals im Kolon und verschlechtert die Barrierefunktion des Epithels durch mukosale Immunaktivierung

Campylobacter concisus impaired colonic epithelial sodium channel (ENaC) function with the transcriptional downregulation of regulatory ENaC subunits (-β and -γ). The activation of intracellular mitogen-activated protein kinase (MAPK) enzyme extracellular signal-regulated kinase (ERK) by C. concisus is ascertained as an intracellular cell signaling pathway in inducing ENaC dysfunction. The transcriptional upregulation of interleukin (IL)-32 plays a pivotal role in C. concisus-induced ENaC dysfunction as the bioinformatics prediction from ingenuity pathway analysis (IPA) identified IL-32 as a potential activator of the ERK signaling pathway. The increase in expression of tight junction (TJ) protein claudin-8 following steroid stimulation prevents paracellular back-leakage of sodium (Na+) ions absorbed by ENaC in colonic epithelium. Claudin-8 protein expression was decreased along with the subcellular redistribution off the tight junction (TJ) domain by C. concisus infection in the colonic epithelial cells stimulated by steroids. Therefore, claudin-8 disruption in the colonic epithelium is another cellular pathomechanism of C. concisus in the context of Na+-malabsorptive diarrhea. The epithelial barrier dysfunction induced by C. concisus was more pronounced under the influence of immune response in the co-culture model of colonic epithelial cells and macrophages. The macrophage activation by C. concisus in the co-culture setup was accompanied by an increase in the release of proinflammatory cytokines, tumor necrosis factor (TNF)-α, IL-1β and IL-6. Concomitantly, the expression of TJ proteins occludin and tricellulin was decreased along with the subcellular redistribution of these proteins from the TJ domains. Moreover, C. concisus infection increased the flux of fluorescein (332 Da) in the monoculture and co-culture cell monolayers. In contrast, the flux of the macromolecule fluorescein isothiocyanate (FITC)-dextran (4 kDa) was increased only in the co-culture monolayers after infection. Furthermore, C. concisus induced epithelial apoptosis in the co-culture monolayers. However, the quantification data implied no significant difference in the apoptotic cell numbers between the infected monolayers in monoculture and co-culture conditions. Therefore, the epithelial apoptotic leaks and the functional loss of occludin by C. concisus infection contribute to the increase in the flux of small molecules as fluorescein (332 Da). However, under the regulatory influence of macrophage immune response, C. concisus additionally enhances the flux of macromolecules as dextran 4 kDa by the opening of the leak pathway through tricellulin disruption in the tTJ.Campylobacter concisus beeinträchtigte die Funktion des epithelialen Natriumkanals (ENaC) im Colon durch die transkriptionelle Herabregulation der regulatorischen ENaC-Untereinheiten (-β und -γ). Die Aktivierung der intrazellulären mitogen-aktivierten Proteinkinase (MAPK) in Form des Enzyms Extracellular Signal-regulated Kinase (ERK) durch C. concisus wurde als intrazellulärer Zellsignalweg bei der Induktion der ENaC-Dysfunktion festgestellt. Die transkriptionelle Hochregulierung von Interleukin (IL)-32 spielt eine zentrale Rolle bei der C. concisus-induzierten ENaC-Dysfunktion, da die bioinformatische Vorhersage aus der Ingenuity Pathway Analysis (IPA) IL-32 als potenziellen Aktivator des ERK-Signalwegs identifizierte. Die Zunahme der Expression des Tight Junction (TJ)-Proteins Claudin-8 nach Steroidstimulation verhindert den parazellulären Rückstrom von Natrium Na+-Ionen, die vom ENaC im Kolonepithel resorbiert werden. Die Claudin-8-Proteinexpression wurde zusammen mit der subzellulären Umverteilung von Claudin-8 aus der Tight Junction (TJ)-Domäne der Epithelzelle heraus durch die C. concisus-Infektion vermindert. Daher ist die Claudin-8-Umverteilung im Kolonepithel ein weiterer zellulärer Pathomechanismus von C. concisus im Kontext der durch die Na+-Resorptionsstörung bedingten malabsorptiven Diarrhoe. Die durch C. concisus induzierte epitheliale Barrieredysfunktion war unter dem Einfluss der Immunantwort im Co-Kulturmodell von Kolonepithelzellen und Makrophagen stärker ausgeprägt. Die Makrophagenaktivierung durch C. concisus im Co-Kultur-Setup ging mit einer erhöhten Freisetzung der pro-inflammatorischen Zytokine Tumor-Nekrose-Faktor (TNF)-α, IL-1β und IL-6 einher. Gleichzeitig war die Expression der TJ-Proteine Occludin und Tricellulin vermindert, zusammen mit einer subzellulären Umverteilung dieser Proteine aus den TJ-Domänen heraus. Darüber hinaus erhöhte die C. concisus-Infektion die epitheliale Permeabilität für Fluorescein (332 Da) in den Zellmonolayern der Monokultur und der Co-Kultur. Im Gegensatz dazu war die epitheliale Permeabilität für das Makromolekül Fluorescein Isothiocyanat (FITC)-Dextran (4 kDa) nur in den Co-Kultur-Monolayern nach der Infektion erhöht. Darüber hinaus induzierte C. concisus in den Co-Kultur-Monolayern die epitheliale Apoptose. Die Quantifizierungsdaten ergaben jedoch keinen signifikanten Unterschied in der Anzahl der apoptotischen Zellen zwischen den infizierten Monolayern in Monokultur und Co-Kultur. Daher tragen die epithelialen apoptotischen Lecks und der Funktionsverlust von Occludin durch die C. concisus-Infektion zur Erhöhung der epithelialen Permeabilität für kleine Makromoleküle bei. Unter dem regulierenden Einfluss der Makrophagen-Immunantwort erhöht C. concisus darüber hinaus jedoch die epitheliale Permeabilität für Makromoleküle wie Dextran 4 kDa durch Öffnung der tTJ (Leak pathway) infolge einer Störung der Tricellulin-Verteilung

Immune-Mediated Aggravation of the Campylobacter concisus-Induced Epithelial Barrier Dysfunction

Campylobacter concisus is a human-pathogenic bacterium of the gastrointestinal tract. This study aimed at the contribution of the mucosal immune system in the context of intestinal epithelial barrier dysfunction induced by C. concisus. As an experimental leaky gut model, we used in vitro co-cultures of colonic epithelial cell monolayers (HT-29/B6-GR/MR) with M1-macrophage-like THP-1 cells on the basal side. Forty-eight hours after C. concisus infection, the decrease in the transepithelial electrical resistance in cell monolayers was more pronounced in co-culture condition and 22 ± 2% (p < 0.001) higher than the monoculture condition without THP-1 cells. Concomitantly, we observed a reduction in the expression of the tight junction proteins occludin and tricellulin. We also detected a profound increase in 4 kDa FITC-dextran permeability in C. concisus-infected cell monolayers only in co-culture conditions. This is explained by loss of tricellulin from tricellular tight junctions (tTJs) after C. concisus infection. As an underlying mechanism, we observed an inflammatory response after C. concisus infection through pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) released from THP-1 cells in the co-culture condition. In conclusion, the activation of subepithelial immune cells exacerbates colonic epithelial barrier dysfunction by C. concisus through tricellulin disruption in tTJs, leading to increased antigen permeability (leaky gut concept)

Curcumin Mitigates Immune-Induced Epithelial Barrier Dysfunction by Campylobacter jejuni

Campylobacter jejuni (C. jejuni) is the most common cause of foodborne gastroenteritis worldwide. The bacteria induce diarrhea and inflammation by invading the intestinal epithelium. Curcumin is a natural polyphenol from turmeric rhizome of Curcuma longa, a medical plant, and is commonly used in curry powder. The aim of this study was the investigation of the protective effects of curcumin against immune-induced epithelial barrier dysfunction in C. jejuni infection. The indirect C. jejuni-induced barrier defects and its protection by curcumin were analyzed in co-cultures with HT-29/B6-GR/MR epithelial cells together with differentiated THP-1 immune cells. Electrophysiological measurements revealed a reduction in transepithelial electrical resistance (TER) in infected co-cultures. An increase in fluorescein (332 Da) permeability in co-cultures as well as in the germ-free IL-10&minus;/&minus; mouse model after C. jejuni infection was shown. Curcumin treatment attenuated the C. jejuni-induced increase in fluorescein permeability in both models. Moreover, apoptosis induction, tight junction redistribution, and an increased inflammatory response&mdash;represented by TNF-&alpha;, IL-1&beta;, and IL-6 secretion&mdash;was observed in co-cultures after infection and reversed by curcumin. In conclusion, curcumin protects against indirect C. jejuni-triggered immune-induced barrier defects and might be a therapeutic and protective agent in patients