Protective action of Bacillus clausii probiotic strains in an in vitro model of Rotavirus infection

Abstract

Rotavirus is the most common cause of acute gastroenteritis (AGE) in young children. Bacillus clausii (B. clausii) is a spore-forming probiotic that is able to colonize the gut. A mixture of four B. clausii strains (O/C, T, SIN and N/R) is commonly used for the treatment of AGE, and it has been demonstrated that it can reduce the duration and severity of diarrhea in children with AGE. Few studies have sought to characterize the mechanisms responsible for such beneficial effects. Intestinal effects of probiotics are likely to be strain-specific. We conducted a series of in vitro experiments investigating the activities of this mixture of B. clausii strains on biomarkers of mucosal barrier integrity and immune function in a cellular model of Rotavirus infection. B. clausii protected enterocytes against Rotavirus-induced decrease in trans-epithelial electrical resistance, and up-regulated expression of mucin 5AC and tight junction proteins (occludin and zonula occludens-1), all of which are important for effective mucosal barrier function. B. clausii also inhibited reactive oxygen species production and release of pro-inflammatory cytokines (interleukin-8 and interferon-β) in Rotavirus-infected cells, and down-regulated pro-inflammatory Toll-like receptor 3 pathway gene expression. Such mechanisms likely contributed to the observed protective effects of B. clausii against reduced cell proliferation and increased apoptosis in Rotavirus-infected enterocytes.

Figure 1

B. clausii increases HBD-2 and LL-37 expression in human enterocytes. Cells were exposed to B. clausii probiotic strains mix at different concentrations; B. clausii supernatant (Sup, dilution 1:100) or E. coli K12 (1 × 10⁶ cells/mL) as control. Only the exposure to B. clausii strains was able to elicit a significant increase in HBD-2 (A) and LL-37 (B) production by human enterocytes. HBD 2, human beta defensin 2; LL-37, cathelicidin; NT, untreated. *p < 0.05 vs NT; **p < 0.001 vs NT.

Figure 2

B. clausii counteracts the Rotavirus effects on human enterocytes proliferation and viability. (A) Rotavirus (RV) (10 pfu/cell) reduced human enterocytes proliferation rate. B. clausii probiotic strains (3 × 10⁸ cells/mL) and B. clausii supernatant (Sup, dilution 1:100) were able to inhibit the RV effect. (B) Cell cycle analysis confirmed the reduction in proliferation and a block in G0/G1 phases induced by RV. Again, the effect was inhibited by the incubation with B. clausii probiotic strains (3 × 10⁸ cells/mL) and B. clausii supernatant (dilution 1:100). (C) Apoptosis analysis showed that the exposure to RV resulted in pro-apototic effect on human enterocytes. Again, both B. clausii and its supernatant were able to inhibit this effect. *p < 0.05 vs NT, ^#p < 0.05 vs RV.

Figure 3

B. clausii and its supernatant significantly inhibit Rotavirus-induced TEER reduction in human enterocytes. The incubation with Rotavirus (RV) (10 pfu/cell), but not with B. clausii probiotic strains (3 × 10⁸ cells/mL) or with B. clausii supernatant (Sup, dilution 1:100), elicited a significant reduction of TEER. B. clausii probiotic strains (3 × 10⁸ cells/mL) and B. clausii supernatant (Sup, dilution 1:100) significantly inhibited the RV-induced TEER decrease. *p < 0.05 vs NT; ^#p < 0.05 vs RV.

Figure 4

B. clausii and its supernatant significantly inhibit Rotavirus-induced ROS production in human enterocytes. Rotavirus (RV) (10 pfu/cell), but not with B. clausii probiotic strains (3 × 10⁸ cells/mL) or with B. clausii supernatant (Sup, dilution 1:100), induced a significant increase in ROS production in a time-dependent manner. B. clausii probiotic strains (3 × 10⁸ cells/mL) and B. clausii supernatant (Sup, dilution 1:100) significantly inhibited the RV-induced increase in ROS. H₂O₂ as a positive control. *p < 0.05 vs NT.

Figure 5

B. clausii and its supernatant significantly counteract the Rotavirus-induced alteration in MUC-5, ZO-1 and Occludin expression in human enterocytes. Rotavirus (RV) (10 pfu/cell) significantly reduced the expression of MUC5AC (A), ZO-1 (B) and Occludin (C) in human enterocytes, whereas both B. clausii probiotic strains (3 × 10⁸ cells/mL) and B. clausii supernatant (Sup, dilution 1:100) significantly increased the expression of these proteins in basal condition and blunted the RV effect. *p < 0.05 vs NT; ^#p < 0.05 vs RV.

Figure 6

B. clausii and its supernatant significantly reduce Rotavirus-induced expression of pro-inflammatory cytokines in human enterocytes. Rotavirus (RV) (10 pfu/cell) elicited a significant increase in IL-8 (A) and IFN-β (B) production. B. clausii probiotic strains (3 × 10⁸ cells/mL) and B. clausii supernatant (Sup, dilution 1:100) significantly inhibited the RV-induced increase in IL-8 and IFN-β production. *p < 0.05 vs NT; ^#p < 0.05 vs RV.

Figure 7

B. clausii and its supernatant contrast Rotavirus-mediated activation of Toll-like receptor-3 pathway in human enterocytes. Rotavirus (RV) (10 pfu/cell) significantly up-regulated TLR3, NF-κB1, MyD88 and TRAF6 expression. B. clausii probiotic strains (3 × 10⁸ cells/mL) and B. clausii supernatant (Sup, dilution 1:100) significantly inhibited such effects. *p < 0.05 vs NT; ^#p < 0.05 vs RV.