Recognition of bacteria by intestinal epithelial cells and regulation of p53 signaling and cell death – interactions at the epithelial interface leading to spontaneous bacterial peritonitis in liver cirrhosis

Background:

Spontaneous bacterial peritonitis (SBP) is one of the most common complications in liver cirrhosis with a very high 1-year-mortality of up to 93%. SBP is defined as infection of ascitic fluid without detectable source of infection. Until today, distinct mechanisms regarding pathogenesis of SBP remain unclear, although some risk factors have been identified. These include genetic variations of TLR2 and NOD2, pattern recognition receptors (PRR) that play an important role in immune defense. The p53 family is known for its tumor-suppressive functions, but also has an impact on bacterial infections, e.g. by inhibiting inflammatory responses. To investigate how intestinal epithelial cells and bacteria can interact, PRR levels as well as p53 regulation of epithelial cells were examined upon bacterial stimulation.

Methods:

Human intestinal epithelial cell line HCT-116wt expressing wildtype p53 was coincubated with Escherichia coli (E. coli) at MOI0 to MOI5 for 2 or 4 hours. To compare effects of living bacteria to single bacterial components and bacterial metabolites cells were also incubated with lipopolysaccharide (LPS) from E.coli or separated from bacteria by a semi-permeable membrane in transwell experiments. Protein levels of p53 family members were analyzed by Western blot. Cell death was analyzed by flow cytometry after coincubation with E.coli, bacterialsupernatant or supernatant of cells that were pretreated with bacteria. To evaluate the mechanism by which bacteria can be recognized, surface TLR2 and TLR4 were stained after stimulation with LPS or PMA and analyzed by flow cytometry. NOD1 and NOD2 levels were examined by Western blot after treatment with different stimuli (TNF, MDP, iE-DAP).

Results:

Coincubation of HCT-116wt and E.coli resulted in a decrease of protein levels of p53 and p73 in a time- and dose-dependent manner. LPS stimulation of HCT-116wt cells resulted in similar diminishing effects on the p53-family. In contrast, p53 and p73 levels were unaffected when cells were separated from bacteria by a semi-permeable membrane or after coincubation with heat-inactivated bacteria. Despite reduced p53 levels, coincubation with E.coli induced high rates of cell death. Stimulation with supernatants of E.coli-pretreated HCT116wt cells also resulted in increased cell death, albeit to a lower extent. In contrast, bacterial supernatants had no effect on cell survival. TLR2 and TLR4 were not detected on the surface of HCT-116wt cells and could also not be induced by stimulation with LPS or PMA. However NOD1 and NOD2 were present on the cell surface, but levels were not further increased after specific stimulation.

Conclusion:

Direct contact of intestinal epithelial cells with living bacteria results in downregulation of p53 family members. Recognition of bacteria is TLR2- and TLR4-independent, but might occur via NOD receptors. Interestingly, despite reduced levels of the pro-apoptotic protein p53, E.coli causes cell death in HCT-116wt, leading to the suggestion that after contact between bacteria and cells either E.coli or HCT-116wt themselves produce self-killing substances. In the setting of liver cirrhosis, both effects might be part of a bacterial mechanism to protect themselves from host immune response and thus to overcome the intestinal barrier and cause SBP.