Probiotics promote gut health through stimulation of epithelial innate immunity

Abstract

Probiotic formulations are widely available and have a variety of proposed beneficial effects, including promotion of gut health. The mechanisms of action of probiotic bacteria in the intestine are still unclear but are generally attributed to an antiinflammatory effect. Here, we demonstrate that the multiple probiotic formulation VSL#3 prevents the onset of intestinal inflammation by local stimulation of epithelial innate immune responses (i.e., increased production of epithelial-derived TNF-alpha and restoration of epithelial barrier function in vivo). We also demonstrate that probiotic bacteria stimulate epithelial production of TNF-alpha and activate NF-kappaB in vitro. Our results support the hypothesis that probiotics promote gut health through stimulation, rather than suppression, of the innate immune system. Furthermore, our findings provide the perspective that defects in innate immunity may play a critical role in the pathogenesis and progression of intestinal disorders, such as inflammatory bowel disease.

Fig. 1.

VSL#3 administration prevents the onset of ileitis in SAMP mice. Three-week-old mice were administered high- or low-dose VSL#3 for 6 weeks. (A) The total inflammatory score was markedly decreased in SAMP mice administered high-dose VSL#3. (B) Representative photomicrographs of H&E-stained sections, 10× and 20× original magnification: (i) control untreated mice (n = 12) display severe distortion of the villi, with intense leukocyte infiltration of the lamina propria and thickening of the muscularis mucosa; (ii) SAMP mice administered low-dose VSL#3 (n = 12) show less distortion of tissue architecture but a high level of cellular infiltration and thickening of the muscularis mucosa; and (iii) SAMP mice administered high-dose VSL#3 (n = 11) show almost complete prevention of mucosal damage, with preservation of the normal villi morphology and minimal inflammatory infiltrate. Data are expressed as mean ± SEM. *P < 0.01; **P < 0.005.

Fig. 2.

VSL#3 alters the fecal probiotic DNA composition of SAMP mice. RT-PCR with primers specific for VSL#3 probiotic bacteria was performed weekly on fecal extracts to assess the extent of bacterial colonization. Mice administered high-dose VSL#3 (n = 6) showed a relative increase in probiotic DNA for all three strains at each posttreatment time point compared with baseline (A–C). At week 6, mice given high-dose VSL#3 had significantly increased relative increases in probiotic DNA for each of the three strains, compared with mice given low-dose VSL#3 (E–G). Data are expressed as mean ± SEM. *P < 0.01 vs. low-dose VSL#3.

Fig. 3.

VSL#3 differentially alters the ileal probiotic DNA composition of SAMP mice. At the end of the study period (6 weeks), terminal ilea were collected from SAMP mice and DNA isolated. (A) RT-PCR with primers specific for probiotic bacteria showed a significant increase of B. infantis and S. thermophilus compared with untreated mice. (B) Visualization of PCR products resolved on a 2% agarose gel. Data are expressed as mean ± SEM. *P < 0.005 vs. control.

Fig. 4.

VSL#3 pretreatment restores small intestinal epithelial barrier function in SAMP mice. In vivo paracellullar permeability was determined by measuring the fractional urinary excretion of orally administered, region-specific (small intestinal) sugar probes (lactulose/mannitol ratio). Three-week-old SAMP mice were treated with VSL#3 for 2 weeks (n = 4) or 6 weeks (n = 6) and then compared with age-matched SAMP mice fed a normal diet. VSL#3 administration significantly decreased small intestinal epithelial permeability by the end of the treatment period. Data are expressed as mean ± SEM. *P < 0.05 vs. control.

Fig. 5.

VSL#3 administration stimulates epithelial cells in vivo. (A and B) Increased expression of TNF-α and IκBα mRNA in epithelial cells after VSL#3 administration. mRNA was extracted from freshly isolated epithelial cells from SAMP mice at the end of the study period. RT-PCR was performed with specific primers and data normalized to β-actin. All values are expressed as mean ± SEM. *P < 0.05. (C) Representative photomicrographs of immunohistochemical staining demonstrated that TNF-α immunolocalized to the epithelium and was markedly increased in high-dose (Bottom Right) compared with low-dose (Bottom Left) VSL#3-treated mice, whereas untreated controls (Top Right) showed no TNF-α staining. Absence of primary detecting antibody confirmed specificity of TNF-α staining (Top Left). ×40 original magnification.

Fig. 6.

VSL#3 CM stimulates TNF-α production and activates NF-κB in small intestinal organ cultures. Ilea from SAMP mice were harvested and cultured for 24 h with either VSL#3 CM, FE, or combination CM+FE and compared with controls (media alone). (A) Secreted TNF-α production was measured from resulting supernatants by ELISA and showed significantly increased levels in CM and CM+FE compared with controls. (B) Cytoplasmic and nuclear extracts analyzed by Western blot showed increased nuclear p65 (RelA) and decreased cytoplasmic IκBα levels in CM and CM+FE compared with controls, indicating activation of the NF-κB pathway by VSL#3. Data are representative of three independent experiments with similar results. *P < 0.001.