Chronic Alcohol Exposure Renders Epithelial Cells Vulnerable to Bacterial Infection

Abstract

Despite two centuries of reports linking alcohol consumption with enhanced susceptibility to bacterial infections and in particular gut-derived bacteria, there have been no studies or model systems to assess the impact of long-term alcohol exposure on the ability of the epithelial barrier to withstand bacterial infection. It is well established that acute alcohol exposure leads to reduction in tight and adherens junctions, which in turn leads to increases in epithelial cellular permeability to bacterial products, leading to endotoxemia and a variety of deleterious effects in both rodents and human. We hypothesized that reduced fortification at junctional structures should also reduce the epithelial barrier’s capacity to maintain its integrity in the face of bacterial challenge thus rendering epithelial cells more vulnerable to infection. In this study, we established a cell-culture based model system for long-term alcohol exposure to assess the impact of chronic alcohol exposure on the ability of Caco-2 intestinal epithelial cells to withstand infection when facing pathogenic bacteria under the intact or wounded conditions. We report that daily treatment with 0.2% ethanol for two months rendered Caco-2 cells far more susceptible to wound damage and cytotoxicity caused by most but not all bacterial pathogens tested in our studies. Consistent with acute alcohol exposure, long-term ethanol exposure also adversely impacted tight junction structures, but in contrast, it did not affect the adherens junction. Finally, alcohol-treated cells partially regained their ability to withstand infection when ethanol treatment was ceased for two weeks, indicating that alcohol’s deleterious effects on cells may be reversible.

Pseudomonas aeruginosa ExoT Induces Mitochondrial Apoptosis in Target Host Cells in a Manner That Depends on Its GTPase-activating Protein (GAP) Domain Activity

“…3A, infec- tion with GAP-expressing ⌬U/T(G ϩ A Ϫ ) resulted in increased expression of JNK1/2 and c-Jun and their activation (phospho-JNK1/2 and phospho-c-Jun) at 5 h after infection. To further evaluate the role of JNK in mediating GAP-induced apoptosis, we repeated the aforementioned infection studies in the presence or absence of JNK inhibitor SP600125 (added 2 h prior to infection) and evaluated GAP-induced cytotoxicity by timelapse videomicroscopy using PI uptake as a marker for cell death, as we described previously (13,22). JNK inhibition by SP600125 partially protected HeLa cells against the GAP-induced apoptosis (Fig.…”

“…Cytotoxicity Measurement by Time-lapse VideomicroscopyCytotoxicity measurement by time-lapse videomicroscopy was performed as described previously (21,22). Briefly, HeLa cells were grown in DMEM without phenol red with (for transfection studies) or without antibiotics (for infection studies) for 24 h. These cells were then transfected with the indicated expression vectors as described (11) or infected with the indicated strains as described (13).…”

“…Time-lapse videos were taken using AxioVision 4.2.8 software. Cytotoxicity was determined from each frame, at 15-min interval, using the propidium iodide (PI) channel and analyzed in ImageJ v1.46 (National Institutes of Health) software by setting an appropriate threshold to isolate PI-positive cells, and image stacks were then analyzed for total positive pixels per frame, as we described previously (12,22).…”

“…Western Blot-Protein samples were assessed by Western blot as described previously (22,23). Briefly, cells were lysed following infection with 1% Triton X-100 containing a protease inhibitor mixture (Roche Diagnostics), 100 mM PMSF, and 100 mM Na 3 VO 4 .…”

Pseudomonas aeruginosa exotoxin T induces potent cytotoxicity against a variety of murine and human cancer cell lines

“…ExoT was delivered into cancer cells either by P. aeruginosa bacteria or by transient transfection using pIRES2 mammalian expression vector and cytotoxicity was assessed as described previously (Shafikhani et al, 2008;Wood et al, 2013). Briefly, bacteria were grown overnight in LB media, adjusted to OD 600 0.05, corresponding to m.o.i.~10.…”