Strain-specific effects of probiotics on gut barrier integrity following hemorrhagic shock

Abstract

Probiotic therapy modulates the composition of the intestinal flora and inhibits the inflammatory response. These properties may be of benefit in the preservation of gut barrier integrity after injury or stress. In this study, we examined the effect of two Lactobacillus strains selected for their pathogen exclusion properties on intestinal barrier integrity following hemorrhagic shock. Additionally, the responsiveness of the macrophage cell line RAW 264.7 to combined exposure to Lactobacillus DNA or oligodeoxynucleotides containing CpG motifs (CpG-ODN) and endotoxin was assessed by measuring tumor necrosis factor alpha (TNF-alpha) release. Rats were administered lactobacilli (5 x 10(9) CFU) or vehicle for 7 days and were subjected subsequently to hemorrhagic shock by withdrawal of 2.1 ml blood/100 g tissue. Levels of plasma endotoxin, bacterial translocation to distant organs, and filamentous actin (F-actin) in the ileum were determined 24 h later. Rats treated with Lactobacillus rhamnosus showed reduced levels of plasma endotoxin (8 +/- 2 pg/ml versus 24 +/- 4 pg/ml; P = 0.01), bacterial translocation (2 CFU/gram versus 369 CFU/gram; P < 0.01), and disruption of F-actin distribution following hemorrhagic shock compared with nontreated control rats. In contrast, pretreatment with Lactobacillus fermentum had no substantial effect on gut barrier integrity. Interestingly, DNA preparations from both lactobacilli reduced endotoxin-induced TNF-alpha release dose dependently, whereas CpG-ODN increased TNF-alpha release. In conclusion, the pathogen exclusion properties of both Lactobacillus strains and the reduction of endotoxin-induced inflammation by their DNA in vitro are not prerequisites for a beneficial effect of probiotic therapy on gut barrier function following hemorrhagic shock. Although pretreatment with Lactobacillus spp. may be useful to preserve gut barrier integrity following severe hypotension, a thorough assessment of specific strains seems to be essential.

FIG. 1.

Experimental design. Probiotics were administered daily by oral gavage 7 days prior to hemorrhagic shock with standard chow. Rats were fasted 18 h before induction of shock; a femoral artery catheter was inserted 45 min before shock, and rats were monitored for 50 min, after which the catheter was removed. Rats were allowed standard chow 6 h after hemorrhagic shock, and rats were sacrificed 24 h later.

FIG. 2.

L. rhamnosus LMG P-22799 and L. fermentum NumRes2 inhibit the adhesion of pathogens similarly. Lactobacilli and pathogens were added to cultured Caco-2 cells; after a 1-h incubation period, cells were washed, and adherent bacteria were determined by culture techniques. Both L. rhamnosus LMG P-22799 and L. fermentum NumRes2 inhibited the adhesion of E. coli, K. pneumoniae, P. aeruginosa, and S. flexneri, with no significant differences between probiotic strains. Data are presented as percentages of pathogen exclusion compared to a control situation (e.g., adhesion of pathogen without addition of probiotics).

FIG. 3.

Pretreatment with L. rhamnosus LMG P-22799 decreases plasma endotoxin levels in rats subjected to hemorrhagic shock. Plasma endotoxin was measured 24 h after sham and hemorrhagic shock. Endotoxin levels were near the detection level in all sham-treated groups and were markedly elevated by hemorrhagic (Hem.) shock in nontreated or vehicle-treated rats (nontreated, 24 ± 4 pg/ml, and vehicle treated, 21 ± 1 pg/ml). Pretreatment for 7 days with L. rhamnosus LMG P-22799 strongly reduced endotoxin levels compared with those of vehicle-treated controls (8 ± 2 pg/ml) (*, P < 0.01), whereas pretreatment with L. fermentum NumRes2 had no effect. Data are presented as means ± SEM.

FIG. 4.

Pretreatment with L. rhamnosus LMG P-22799 prevents disruption of F-actin filament organization in ileum segments by hemorrhagic shock. Immunolocalization of F-actin (green) at a ×600 magnification showed a regular organization throughout the cell in terminal ilea of nontreated and treated rats subjected to sham shock (A, D, and F). Hemorrhagic shock markedly disrupted the filament organization of F-actin in fasted animals and those that were pretreated with vehicle (B and C). Moreover, tissue was disrupted and disorganized, illustrated by an irregular distribution of nuclei (blue). In contrast, pretreatment for 7 days with L. rhamnosus LMG P-22799 prevented disruption of cytoarchitecture by hemorrhagic shock (E), whereas L. fermentum NumRes2 had no substantial effect (G). The histology shown is representative of all tissue samples studied (see Materials and Methods).

FIG. 5.

DNA from L. rhamnosus LMG P-22799 and L. fermentum NumRes2 suppresses endotoxin-induced TNF-α in RAW 264.7 cells. RAW 264.7 cells were incubated with CpG-ODN (open circles), nonCpG-ODN (filled squares), or DNA preparations from L. rhamnosus LMG P-22799 (filled circles) or L. fermentum NumRes2 (stars) at 0.5, 1.5, 4.5, and 13.5 μg/ml before exposure to 10 ng/ml endotoxin. Prestimulation with CpG-ODN increased TNF-α concentrations dose dependently in the supernatant with a maximum at 13.5 μg/ml DNA (18 ± 1.4 ng/ml). Compared with preincubation with medium (3.7 ± 0.5 ng/ml), preincubation with DNA from L. rhamnosus LMG P-22799 reduced TNF-α levels maximally at 13.5 μg/ml (2.1 ± 0.8 ng/ml; *, P < 0.001), and preincubation with DNA from L. fermentum NumRes2 reduced TNF-α levels maximally at 4.5 μg/ml (2.5 ± 0.5 ng/ml; †, P < 0.001). There were no significant differences between DNA preparations from both lactobacillus strains. Data are presented as means ± SEM on a log scale (y axis).