Effects of Bacillus subtilis Natto Strains on Antiviral Responses in Resiquimod-Stimulated Human M1-Phenotype Macrophages.

Bacillus subtilis natto is used in the production of natto, a traditional fermented soy food, and has beneficial immunomodulatory effects in humans. Single-stranded RNA (ssRNA) viruses, including influenza and coronavirus, often cause global pandemics. We proposed a human cell culture model mimicking ssRNA viral infection and investigated the ability of B. subtilis natto to induce antiviral effects in the model. The gene expressions were analyzed using quantitative real-time reverse transcription PCR. M1-phenotype macrophages derived from THP-1 cells strongly express the Toll-like receptor 8 (76.2-hold), CD80 (64.2-hold), and CCR7 (45.7-hold) mRNA compared to M0 macrophages. One µg/mL of resiquimod (RSQ)-stimulation induced the expression of IRF3 (1.9-hold), CXCL10 (14.5-hold), IFNß1 (3.5-hold), ISG20 (4.4-hold), and MxA (1.7-hold) mRNA in the M1-phenotype macrophages. Based on these results, the RSQ-stimulated M1-phenotype macrophages were used as a cell culture model mimicking ssRNA viral infection. Moreover, the B. subtilis natto XF36 strain induced the expression of genes associated with antiviral activities (IFNß1, IFNλ1, ISG20, and RNase L) and anti-inflammatory activities (IL-10) in the cell culture model. Thus, it is suggested that the XF36 suppresses viral infections and excessive inflammation by inducing the expression of genes involved in antiviral and anti-inflammatory activities.

Lactobacillus crispatus Strain KT-11 S-Layer Protein Inhibits Rotavirus Infection.

S-layer proteins (SLPs), which are present in the external layer of certain strains of lactic acid bacteria isolated from the intestinal tract, are known to recognize and bind to specific proteins and glycan structures and contribute to adsorption to the host intestinal mucosa. The binding properties of certain SLPs are considered to exert a competitive inhibitory effect on infection because similar properties are involved in the infection mechanisms of several viruses. However, little is known regarding whether SLPs directly inhibit viral infection. In the present study, we investigated the effect of an SLP of the Lactobacillus crispatus KT-11 strain, a probiotic strain isolated from a healthy human infant, on human rotavirus infection. The impact of KT-11 lithium chloride extract (KT-11 LE), which contains SLP, on the infection of the P[4] genotype human rotavirus strain DS-1 was evaluated by monitoring the amplification of viral protein 6 (VP6) expression in human intestinal epithelial Caco-2 cells by quantitative reverse transcription-polymerase chain reaction assay after infection. KT-11 LE showed a significant suppressive effect on DS-1 infection in a dose-dependent manner with pre-infection treatment, whereas post-infection treatment was not effective. A 45 KDa protein isolated from KT-11 LE was investigated for homology using the BLAST database and was found to be a novel SLP. KT-11 SLP concentrate (KT-11 SLP) significantly inhibited the proliferative process of the DS-1 strain but not that of the P[8] genotype human rotavirus strain Wa. KT-11 SLP exerted significant inhibitory effect on DS-1 infection by pre-infection treatment even after digestion with gastric juice up to 2 h. Our results provided crucial evidence that SLPs from certain Lactobacillus strains can inhibit human rotavirus infection of intestinal epithelial cells.

Bacillus subtilis BN strain promotes Th1 response via Toll-like receptor 2 in polarized mouse M1 macrophage.

Bacillus subtilis BN strain (BN strain) was isolated from natto, a traditional Japanese fermented soybean food product. The present study investigated the Th1 responses of the BN strain on a mouse macrophage cell line, J774.1. In cell cultures, the BN strain (spore cell cultured in Schaeffer's sporulation media) significantly increased the production of interleukin (IL-)12 protein. The BN strain induced the mRNA expression of M1 polarization genes, such as inducible nitric oxide synthase and IL-12p40 mRNA, and suppressed the mRNA expression of intracellular marker genes of M2 polarization, such as arginase 1 mRNA. The BN strain downregulated the mRNA expression of Toll-like receptor 4 (TLR4), while it upregulated the mRNA expression of TLR2, MyD88, and nuclear factor kappa B (NF-κB). The production of IL-12 protein induced by the BN strain was decreased by inhibitors of MyD88, NF-κB, and IκB kinase. Moreover, the production of IL-12 was strongly suppressed by neutralizing antibody against TLR2. These results suggest that the BN strain promotes Th1 response via TLR2 signal in mouse M1 macrophage. PRACTICAL APPLICATIONS: Bacillus subtilis is known to have beneficial effects for the host. B. subtilis BN stain (BN strain) was isolated from natto, a traditional Japanese fermented soybean food product. The effects of the BN strain on the Th1 response in macrophage cell cultures were investigated in this work. We found that the spore cells of BN strain promoted the production of Th1-type cytokine, and induced macrophage M1 polarization via Toll-like receptor 2. This study can serve as a significant reference for the development of functional food and feed with immunostimulatory effects. Over time, new food and feed products containing the BN strain may emerge, such as Juice, powder, and tablet.

Protein denature extracts of Lactobacillus crispatus KT-11 strain promote interleukin 12p40 production via Toll-like receptor 2 in J774.1 cell culture.

The objective of the present study is to investigate the mechanism and the cell components of Lactobacillus crispatus KT-11 strain (KT-11) that induce interleukin (IL)-12p40 production. IL-12p40 production induced by KT-11 was decreased in the presence of inhibitors of extracellular signal-regulated kinase or nuclear factor kappa B. Guanidine hydrochloride, urea or lithium chloride extract of KT-11 induced IL-12p40 production, but production was suppressed in the presence of Toll-like receptor 2-specific neutralizing antibody. These findings suggest that the protein denature extracts of KT-11 promote IL-12p40 production via Toll-like receptor 2 in J774.1 cells. PRACTICAL APPLICATIONS: Heat-treated lactic acid bacteria are added to some foods because it is easier to store and transport, and have less interference with other food ingredients compared with living lactic acid bacteria. Heat-treated Lactobacillus crispatus KT-11 strain (KT-11) is included in some foods because of good handling characteristics and good dispersibility in the food product. We have previously reported that the administration of KT-11 led to beneficial health effects through the regulation of the immune system in mice, but the mechanism is not clear. We found that protein denature extracts, which may include proteins such as SLP and SLAPs, of KT-11 cells promoted IL-12p40 production via TLR2 in the J774.1 cell culture. This result will contribute to providing more effective lactic acid bacteria functional food.

Specific vaginal lactobacilli suppress the inflammation induced by lipopolysaccharide stimulation through downregulation of toll-like receptor 4 expression in human embryonic intestinal epithelial cells.

Vaginal lactobacilli (VLB) spread from the mother to the infant during vaginal delivery. However, the effects of VLB on infant intestinal function remain unclear. We investigated the probiotic function and immune effects of VLB on the human embryonic intestinal epithelial cell line INT-407. VLB survived artificial gastric juice and adhered to INT-407 cells. Exposure of INT-407 cells to VLB attenuated both the lipopolysaccharide (LPS)-induced stimulation of interleukin-8 and tumor necrosis factor alpha production and the LPS-stimulated upregulation of TLR4 expression. These results suggest that specific VLB suppresses the inflammation induced by LPS stimulation through downregulation of TLR4 expression in human embryonic intestinal epithelial cells.

The decreased secretion of hyaluronan by older human fibroblasts under physiological conditions is mainly associated with the down-regulated expression of hyaluronan synthases but not with the expression levels of hyaluronidases.

Although it has been reported that levels of hyaluronan are decreased in the dermis of aged skin, little is known about the cellular mechanism(s) underlying that hyaluronan deficiency. Since hyaluronan is produced by dermal fibroblasts and is secreted into the surrounding dermal tissues, we examined the secretion of hyaluronan by dermal fibroblasts and characterized its cellular mechanism using real-time RT-PCR and western blotting for its synthesizing and degrading enzymes, hyaluronan synthase and hyaluronidase, respectively. The secretion of hyaluronan by dermal fibroblasts derived from differently aged human donors, was higher in the younger human fibroblasts tested (0 and 19 years old) compared to the older human fibroblasts tested (39, 56 and 77 years old). The relative secretion levels of hyaluronan by the different human fibroblasts tested were attributable to the relative expression of hyaluronan synthases 1, 2, 3 but not hyaluronidases 1, 2 enzymes at the gene and protein levels among those fibroblasts. These findings indicate that the deficiency of hyaluronan in the aged dermis might result from the down-regulation in the potential of older human fibroblasts to secrete hyaluronan and that decrease in secretory potential is mainly associated with the down-regulated expression of hyaluronan synthases, especially hyaluronan synthase 2, but not with the expression levels of hyaluronidases.

Anti-allergic effects of Lactobacillus crispatus KT-11 strain on ovalbumin-sensitized BALB/c mice.

In this study, we investigated the effects of oral ingestion of Lactobacillus crispatus KT-11 strain (KT-11) on the immune response in an allergic rhinitis mouse model, ovalbumin (OVA)-sensitized BALB/c mice. Sneezing activity in mice that were administered a KT-11-supplemented diet was significantly lower than that in mice administered a KT-11-free diet (control diet) at age 11 weeks. We found that serum OVA-specific immunoglobulin E (IgE) levels and total number of interleukin (IL)-4(+) CD4(+) spleen cells in mice that were administered a KT-11-supplemented diet were significantly lower than in mice administered a control diet. The ratio of spleen interferon-γ(+) CD4(+) /IL-4(+) CD4(+) cells was higher in the mice administered the KT-11-supplemented diet compared to that in mice administered the control or L. rhamnosus GG-supplemented diet. In contrast, the number of CD11b(+) CD80(+) and FcεRIα(+) CD117(+) cells was significantly lower in mice administered the KT-11-supplemented diet. These results suggested that KT-11 reduced OVA-induced allergic symptoms in BALB/c mice via the adjustment of the T helper type 1/T helper type 2 balance, and a decrease in the number of antigen-presenting cells and high affinity IgE receptor-positive mast cells.

The antiallergic effects and acute toxicity of Lactobacillus crispatus KT-11 cultured in food grade medium.

We synthesized a medium consisting of commercial food supplements (food grade medium) that could be used to cultivate Lactobacillus crispatus KT-11 (KT-11), and investigated the antiallergic effects and acute toxicity of KT-11 cultured in this medium. We found that the growth of KT-11 in the food grade medium was comparable to that in DeMan-Rogosa-Sharpe (MRS) medium. Sneezing event was reduced in ovalbumin (OVA)-sensitized BALB/c mice given a diet supplemented with KT-11 grown in the food grade medium (FG-KT-11 group) when compared to mice given a diet supplemented with KT-11 grown in MRS medium (MRS-KT-11 group). The number of CD80(+)CD11b(+) Peyer's patch cells was significantly lower in the FG-KT-11 group than in the MRS-KT-11 group, while IL-12(+)CD11b(+) Peyer's patch cells were higher in the FG-KT-11 group. Only minimal acute toxicity was observed in ICR mice given 1000 or 2000 mg of FG-KT-11/kg body weight. These results suggest that FG-KT-11 represents a safe antiallergic food material.

Lactobacillus crispatus KT-11 enhances intestinal immune functions in C3H/HeN mice.

We investigated the effect of Lactobacillus crispatus KT-11 (KT-11) on intestinal immune systems in C3H/HeN mice. The level of intestinal total immunoglobulin (Ig) A was significantly higher in mice given KT-11 than in mice not given KT-11. Gene expression relating to antibody production and innate immune response increased more than 2-fold in the former compared with the later. Moreover, the number of IL-6(+)CD11b(+) cells was significantly higher in Peyer's patch cells cultured with KT-11 than in those cultured without KT-11, although the number of CD4(+) cells and the cell ratio of CD4(+)/CD8(+) were remarkably lower in the culture with KT-11. These results indicate that KT-11 enhances intestinal IgA production and innate immune response in C3H/HeN mice.

Heat-treated Lactobacillus crispatus KT strains reduce allergic symptoms in mice.

In the current study, we investigated the effects of heat-treated Lactobacillus crispatus KT strains on allergic response in mice. We found that the number of interferon (IFN)-gamma(+)CD4(+) cells was higher in C3H/HeN mouse spleen cultures incubated with L. crispatus KT strains than in those cultured with Lactobacillus JCM type cultures. The serum immunoglobulin E levels in NC/Nga mice that were administered KT strains were lower than those in the mice that were not given any bacterium. The ratio of spleen IFN-gamma(+)CD4(+)/interleukin-4(+)CD4(+) was highest in mice given L. crispatus KT-11. L. crispatus KT-11 also increased the expression of Toll-like receptor (TLR) 2, nucleotide-binding oligomerization domain (NOD) 1, and NOD2 in C3H/HeN mouse Peyer's patch cells. These results suggest that the L. crispatus KT-11 strain reduces allergic symptoms in NC/Nga mice via the adjustment of the type 1 helper T cell and type 2 helper T cell balance via TLR2, NOD1, and NOD2.

Bovine beta-casein (1-28), a casein phosphopeptide, enhances proliferation and IL-6 expression of mouse CD19+ cells via Toll-like receptor 4.

This study was conducted to elucidate the target cells and receptors which participate in the mitogenic and interleukin (IL)-6-enhancing effect of bovine beta-casein (1-28), a casein phosphopeptide. When the spleen lymphocyte subset (CD4+, CD8+, and CD19+ cells) from C3H/HeN mice was cultured with the beta-casein (1-28), it exerted a dose-dependent mitogenic effect on CD19+ cells. The effect of beta-casein (1-28) was not apparent in the case of CD19+ cells from C3H/HeJ mouse. In addition, the effect was significantly inhibited by treating the C3H/HeN mouse-derived CD19+ cells with neutralizing antibody for toll-like receptor 4 (TLR4). Reverse transcription-polymerase chain reaction analysis showed that the beta-casein (1-28) exerted an IL-6-enhancing effect on the CD19+ cells. The effect was also abrogated in either C3H/HeJ mouse-derived CD19+ cell culture or the anti-TLR4 antibody-added culture. These results suggest that the beta-casein (1-28) stimulates both proliferation and IL-6 expression of CD19+ cells via TLR4.