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

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.

www.nature.com/scientificreports/ T, SIN and N/R) is effective in the treatment of pediatric AGE 13 . General antimicrobial and immunomodulatory properties of these B. clausii strains have been previously described 17 , but specific mechanisms of action against AGE are still largely undefined. The current study aimed to investigate the protective activities of a mixture of four B. clausii strains (O/C, T, SIN and N/R) and their metabolites, on human enterocytes in basal conditions and in a model of RV infection. The effects of B. clausii on indicators of mucosal barrier integrity and innate immune function were also examined.

Proliferation, cell cycle and apoptosis analysis by flow cytometry. After 24 h of treatment with B.
clausii probiotic strains or B. clausii supernatant, cell proliferation was comparable to that of the untreated cells, whereas RV significantly reduced cell growth ( Fig. 2A). The combination of RV with B. clausii probiotic strains or B. clausii supernatant partially restored the proliferation rate (P < 0.05 vs RV alone). Rotavirus exposure blocked proliferation, with almost 70% of cells arrested in G0/G1 phase (Fig. 2B). We observed a G1/S transition block with RV compared with untreated cells and compared with infected cells stimulated with B. clausii probiotic strains or B. clausii supernatant for 24 h (P < 0.05). Compared with RV alone, greater proportions of cells exposed to a combination of RV and B. clausii probiotic strains or B. clausii supernatant were in the G2/M phase. Double staining with Annexin V and PI to evaluate apoptosis induction showed a toxic effect of RV stimulation (Fig. 2C), as demonstrated by an increase in necrotic cells (positive only for PI) and late apoptotic cells (positive for both PI and Annexin V) relative to untreated cells and uninfected cells treated with B. clausii probiotic strains or B. clausii supernatant. Treatment of RV-infected cells with B. clausii strains or B. clausii supernatant reduced the proportion of necrotic and apoptotic cells.
Transepithelial electrical resistance. Treatment of uninfected cells with B. clausii probiotic strains or with B. clausii supernatant did not affect TEER, but RV-infected cells had decreased TEER (P < 0.05 vs untreated cells from 8 to 72 h; Fig. 3). Stimulation with B. clausii probiotic strains or B. clausii supernatant protected against a RV-induced decrease in TEER (P < 0.05 vs RV alone from 8 to 72 h).

Analysis of interleukin-8 and interferon-β production.
There was a significant increase in IL-8 and IFN-β production in RV-infected enterocytes (Fig. 6A,B). These effects were blunted by B. clausii probiotic strains and B. clausii supernatant (P < 0.05 vs RV alone).
Toll-like receptor 3 pathway analysis. TLR3, NF-κB1, MyD88 and TRAF6 expression were all significantly up-regulated in cells infected with RV compared with untreated cells (Fig. 7). Down-regulation of proinflammatory TLR3 pathway gene expression was observed in RV-infected cells treated with B. clausii probiotic strains and B. clausii supernatant (P < 0.05 vs RV alone). The respective proteins of these genes showed the same trend of mRNA expression (Fig. S2A,B).

Discussion
Children are constantly being exposed to infectious agents in the gastrointestinal tract and are able to resist these infections due to the action of two main defense mechanisms: the non-immune mucosal barrier and immune response 18,19 . The results of our study provide evidence that a mixture of four B. clausii probiotic strains, namely O/C, T, SIN, and N/R, and their metabolites modulate a number of non-immune and immune defense mechanisms against RV-induced AGE.
To determine whether treatment with B. clausii probiotic strains and their supernatant induce a protective action against RV infection, we evaluated enterocyte proliferation and survival. Rotavirus reduced cell growth in association with a cytotoxic effect. We demonstrated that B. clausii probiotic strains and their metabolites (from the supernatant) were able to restore cell proliferation, inducing a restart in cell cycle progression and protecting against apoptosis, in RV-infected human enterocytes.
The non-immune mucosal barrier acts through a well-modulated network involving epithelial cell layers that express tight cell-cell contacts (tight-junctions regulated by several proteins including occludin and ZO-1), and the secreted mucus layer that overlays the epithelium in the intestinal tract 20 . The integrity of the non-immune mucosal barrier, which is of pivotal importance in protecting children against pathogens, is disrupted by RV 21 . We found that B. clausii probiotic strains and their metabolites had beneficial effects on markers of epithelial barrier damage and enterocyte monolayer permeability in cells infected by RV. Whereas B. clausii probiotic strains or supernatant did not affect epithelial integrity as assessed by TEER in uninfected cells, they protected infected cells against a RV-induced increase in TEER. B. clausii probiotic strains and supernatant also up-regulated the expression of mucin protein MUC5AC and occluding and ZO-1 tight junction proteins, all of which are important for effective mucosal barrier function 20 .
The intestinal epithelium is an integral component of innate immunity, which provides the initial host response to invading pathogens 22 . Soluble proteins and bioactive small molecules are involved in the innate www.nature.com/scientificreports/ immune response. These are either constitutively present at a systemic level in many biological fluids (e.g., complement proteins and defensins) or are released from activated cells resulting in inflammation (e.g., cytokines, reactive free radical species and bioactive amines) 22 . In experiments conducted in Caco-2 enterocytes under basal conditions, B. clausii probiotic strains and supernatant increased the synthesis of the innate immunity antimicrobial peptides HBD-2 and LL-37, which are responsible for effective defense mechanisms against several pathogens in the gastrointestinal tract 23 . B. clausii probiotic strains and supernatant also inhibited ROS production, and the release of pro-inflammatory cytokines (IL-8 and IFN-β) in RV-infected cells. The innate immune system also includes membrane-bound receptors and cytoplasmic proteins that bind invading microbes 22 . For example, RV double stranded RNA binds TLR3, expressed in intestinal epithelial cells, and this interaction elicits an upregulation of the expression of type I IFN (IFN-α, IFN-β) 24-26 that are crucial to limit RV infection 27 to block cell replication, and a secretion of various cytokines and chemokines, including IL-8 and IFN-β 22 . We showed that B. clausii probiotic strains were able to reduced secretion of IL-8 and IFN-β, and down-regulate the expression of pro-inflammatory TLR3 pathway genes (TLR3, NF-κB1, MyD88, TRAF6) activate by RV infection. Our results are consistent with clinical trials 13 , and help to explain the beneficial effects of the same mixture of four B. clausii probiotic strains (O/C, T, SIN, N/R) in children with AGE. In a metaanalysis of randomized controlled trials, the same B. clausii strains mixture reduced the duration of diarrhea by 9.12 h relative to control (oral rehydration solutions or placebo), and duration of hospitalization was reduced by 0.85 days 13 . There was also a trend towards a reduction in stool frequency. In line with the findings of the meta-analysis, data from a large observational study indicated a reduction in diarrhea duration (median duration 3 days) and stool frequency in children with viral or antibiotic-associated AGE who were treated with the O/C, T, SIN, N/R mix of B. clausii strains 28 . There was no significant difference in the mean duration of diarrhea between patients with viral diarrhea and those with antibiotic-associated diarrhea. The B. clausii O/C strain has previously been reported to produce bacteriocin and protease antimicrobials with activity against Gram-positive bacteria, which may be partially responsible for the protective effects of this probiotic in antibiotic-associated diarrhea 17,29 .
Although a specific recommendation for B. clausii in the treatment of childhood AGE has hitherto been missing because of limited data, there is now a growing body of clinical evidence in support of the O/C, T, SIN, N/R mix of B. clausii probiotic strains as an effective therapeutic option in this clinical setting 13,28,30 . Modulation of the immune response with this B. clausii strains mix may also have health benefits beyond the treatment of AGE, ranging from the prevention of recurrent respiratory infections in allergy-prone children to influencing outcomes in cancer patients 31,32 . In addition to its documented efficacy and safety in childhood AGE 13,28 , B. clausii www.nature.com/scientificreports/ has the practical advantage of being a spore-forming probiotic, making it heat stable and able to be transported and stored at room temperature without loss of viability 33 . Given that certain effects of probiotics, including immunomodulatory effects, are likely to be strain-specific 11 , a key strength of our study and the aforementioned clinical studies is that a clearly defined mix of four B. clausii strains was used. Just as the clinical effects of a particular strain of probiotic should not be extrapolated to other strains, intestinal mechanisms of action should not be ascribed to all strains 8,11 . Another strength of our study is the use of a validated in vitro model of enterocyte infection with RV 34 , which is the most relevant AGE-causing pathogen in the pediatric age worldwide 2,7 . Additionally, we assessed a wide range of variables that are indicative of intestinal mucosal barrier integrity and innate immune function. As well as testing a mixture of four B. clausii strains, we tested the effects of its supernatant on all variables. This is important to assess as it is often mainly the metabolites produced by probiotics could modulate intestinal epithelial cell functions 35 .
The main limitation of our study is related to the fact that we did not explore the potential protective effects of B. clausii against other viral and non-viral agents responsible for AGE in the pediatric age group. Evaluation of the efficacy and mechanisms of action of B. clausii probiotic strains on more complex systems, such as human biopsies and/or organoids exposed to different gastrointestinal pathogens, is advocated to further explore the potential of such therapeutic approach.

Conclusion
The mixture of four B. clausii probiotic strains investigated in this study has protective effects and stimulates various non-immune mucosal barrier and innate immune system defense mechanisms in a human enterocyte model of RV infection. These observations provide insights into the mechanisms that are potentially responsible for the beneficial effects of this B. clausii mixture in pediatric patients with AGE, and should encourage further study into the effects of this probiotic on AGE caused by other viral and non-viral pathogens. Cell line. All experiments were conducted using the Caco-2 cell line of human enterocytes (American Type Culture Collection, Middlesex, UK; accession number: HTB-37). Cells were grown to confluence in Dulbecco's modified Eagle's medium (Gibco, Berlin, Germany) supplemented with 20% fetal bovine serum (FBS; Lonza, Visp, Switzerland), 1% l-glutamine (Lonza), 1% non-essential amino acids, and 1% penicillin/streptomycin (Lonza). Cells were cultured at 37 °C in a water-saturated atmosphere consisting of 95% air and 5% CO 2 . The medium was changed every 2 days and Caco-2 cells were grown for 14 days after confluence and cultured in 6-well plates.

Rotavirus strain and infection protocol.
The simian RV strain SA11 was used as previously described 30 .
Briefly, the virus (10 pfu/cell) was activated with 20 µg/mL porcine trypsin for 1 h at 37 °C. The viral suspension was added to the apical side of Caco-2 cell monolayers. After 1 h, the cells were washed and incubated in FBSfree medium for the indicated time periods after infection. www.nature.com/scientificreports/ ness, stirring occasionally. The reaction was stopped by adding PBS with 20% FBS followed by centrifugation and repeated twice. Cells were then counted and 0.5 × 10 6 cells/well were plated in 6-well plates, stimulated and analyzed after 24 h. To perform cell cycle analysis, 0.5 × 10 6 un-differentiated Caco-2 cells were plated in 6-well plates. After stimulation for 24 h, cells were collected and stained with propidium iodide (PI) 50 μg/mL (Sigma-Aldrich, St. Louis, MO, USA) in the presence of RNase A 100 μg/mL (Serva, Heidelberg, Germany).

B. clausii cell stimulation protocol.
To analyze cell apoptosis rates, Annexin V Apoptosis Detection Kit APC was used (eBioscience; San Diego, CA, USA) according to the manufacturer's protocol. After 48 h of treatment, the cells were washed with PBS and incubated with 1 × Annexin V binding buffer, then 5 × 10 5 cells were stained with Annexin V-fluorescein isothiocyanate (FITC) for 10 min at room temperature in the dark. Before reading with a BD FACS Calibur flow cytometer flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), PI 5 μg/mL was added.
Quantitative real-time polymerase chain reaction. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the effect of intestinal exposure to B. clausii probiotic strains on gene expression of mucin 5AC (MUC5AC) and the tight junction proteins occludin and zonula occludens-1 (ZO-1), as well as toll-like receptor 3 (TLR3), nuclear factor κ B subunit 1 (NF-κB1), myeloid differentiation primary response 88 (MyD88), and tumor necrosis factor receptor-associated factor 6 (TRAF6), as previously described 37 . Briefly, qRT-PCR was performed with the TaqMan gene expression assay kit, (Applied Biosystems; Grand Island, NY, USA) according to the manufacturer's instructions. Samples were run in duplicate at 95 °C for 15 s and 60 °C for 1 min using an ABI Prism 7900 Sequence Detection System (Applied Biosystems). Data were analyzed using the comparative threshold cycle method. We used the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene to normalize the level of mRNA expression.

Statistical analysis.
All experiments were performed in triplicate and were repeated twice. The Kolmogorov-Smirnov test was used to determine whether variables were normally distributed. We used the t test to evaluate differences among continuous variables. The level of significance for all statistical tests was 2-sided, P < 0.05. All analyses were conducted in SPSS version 16.0 for Windows (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 (La Jolla, CA, USA) 37 .

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.