Effects of Synbiotic Preparation Containing Lactobacillus gasseri BNR17 on Body Fat in Obese Dogs: A Pilot Study.

Obesity is an important health concern in humans and dogs. It can cause a variety of secondary problems, including low bacterial diversity. Several approaches have been tried to solve this problem; one of them is probiotic supplementation. Lactobacillus gasseri BNR17 is derived from breast milk and has been proven to be effective for obesity in humans. However, there have been no studies using a synbiotic preparation containing L. gasseri BNR17 for obesity management in dogs. Therefore, the present study evaluated the effectiveness of a synbiotic preparation containing L. gasseri BNR17 in reducing body fat in obese dogs. A group of obese dogs were fed a synbiotic preparation for 10 weeks. Obesity variables included body weight, body condition score, subcutaneous fat thickness, subcutaneous fat mass and proportion of the fat mass. In addition, feces collected at 0-week and 10-week time points were analyzed for the intestinal microbiome. Results showed a significant decrease in body weight, body condition score, and subcutaneous fat mass and proportion at the level of the third lumbar vertebra. Diversity and functional analysis of the microbiota in obese dogs showed increased microbial diversity, and increased abundance of metabolism of carbohydrate, and lipid after supplementation with a synbiotic preparation. This study was conducted as a pilot study, and the results demonstrated that a synbiotic preparation containing L. gasseri BNR17 may play a role in reducing body fat and resolving the obesity in dogs.

Lipoteichoic Acid from Staphylococcus aureus Activates the Complement System via C3 Induction and CD55 Inhibition.

Staphylococcus aureus inhibits complement activity by secreting a variety of toxins. However, the underlying mechanism of complement component regulation by lipoteichoic acid (LTA), a cell wall component of S. aureus, has not been elucidated. In this study, we observed that aLTA (LTA of S. aureus) increased C3 expression in THP-1 cells. The mechanism of aLTA-mediated C3 induction includes an aLTA-toll-like receptor (TLR) 2 interaction, interleukin 1 receptor associated kinase (IRAK) 2 recruitment, and nuclear factor kappa B (NF-kB) activation. In HepG2 cells, C3 protein production begins to increase from 3 h and increases steadily until 48 h. On the other hand, CD55 levels increased up to 6 h after aLTA treatment and started to decrease after 24 h and levels were decreased at 48 h by more than 50% compared to untreated cells. The expression of CD55 in HepG2 cells was shown to be regulated by IRAK-M induced by aLTA. Serum C3 levels increased in mice injected with aLTA, which resulted in an increase in the amount and activity of the membrane attack complex (MAC). We also observed that CD55 mRNA was increased in the liver 24 h after aLTA injection, but was decreased 48 h after injection. These results suggest that aLTA increases complement levels via induction of C3 and inhibition of CD55, which may cause associated MAC-mediated liver damage.

Differential Cytokine Regulatory Effect of Three Lactobacillus Strains Isolated from Fermented Foods.

Lactic acid bacteria (LAB) isolated from fermented foods have potential as a treatment for immune-related disorders and the use of LAB has been increasing worldwide. In this study, the differential cytokine regulatory effect was examined with three isolates of lactobacilli strains; namely, Lactobacillus plantarum K55-5 isolated from dairy product, and L. sakei K101 and L. plantarum K8 previously isolated from kimchi (a Korean traditional fermented vegetable). Production of cytokines such as IL-10, IL-12, IFN-γ, and TNF-α was significantly increased in L. sakei K101- and L. plantarum K55-5-treated splenocytes as compared with controls. The oral administration of L. sakei K101 and L. plantarum K55-5 increased cytokine production in the immunosuppressed mouse splenocytes and blood. NK cell cytotoxic activity was also increased in L. sakei K101- and L. plantarum K55-5-fed mice. On the other hand, L. plantarum K8 did not affect cytokine induction in all the experiments performed in this study. The cytokine-inducing effect of L. plantarum K55-5 was significantly increased by lysates of heat-killed bacteria as compared with live, heat-killed, or supernatant of cell lysates. TNF-α production by lipoteichoic acids (LTAs) isolated from the three isolates of lactobacilli was compared, and it was found that K55-5 LTA had a highest cytokine-inducing ability, which was mediated by TLR2-mediated NF-κB and ERK activation. Taken together, our study suggests that L. plantarum K55-5 and L. sakei K101 can be used for the treatment of immunosuppressed disorders.

Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation.

BACKGROUND: Ultraviolet (UV) irradiation from the sun is the primary environmental factor that causes human skin aging. UV irradiation induces the expressions of matrix metalloproteinases (MMPs) and extracellular matrix degrading enzymes. Among the members of MMP family, MMP-1 is an interstitial collagenase that degrades the collagen triple helix. We investigated the effect of Lactobacillus plantarum, well known as useful microorganism, on UV-induced-MMP-1 expression in human dermal fibroblasts. METHODS: Human dermal fibroblasts (HDF) was pre-stimulated with lipoteichoic acid isolated from L. plantarum followed by UV irradiation. Secreted protein level of MMP-1 was evaluated by Western blot analysis. The phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) from the cell lysates was also examined by western blotting. Electrophoretic mobility-shift assay (EMSA) was used to detect the activated transcription factor, AP-1 and NF-κB. The detection of type 1 procollagen was carried with Procollagen type 1 C-peptide (PIP) EIA kit. The generation of reactive oxygen species (ROS) by LTA and UV irradiation was examined by Griess reagent assay and fluorescence microscope. RESULTS: We found that lipoteichoic acid (LTA), a cell-wall component of Gram-positive bacteria, isolated from L. plantarum, inhibited MMP-1 expression. Pretreatment with LTA from L. plantarum (pLTA) reduced MMP-1 expression in a dose-dependent manner and inhibited activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK). It also led to the inhibition of DNA binding activity of activator protein-1 (AP-1) and of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB). Furthermore, LTA promoted type 1 procollagen synthesis and reduced the generation of ROS induced by UV irradiation. CONCLUSION: Our study demonstrates that pLTA inhibits degradation of collagen and promotes its synthesis and that pLTA contributes to a decrease in ROS production. Therefore, pLTA from L. plantarum has potential abilities to prevent and treat skin photo-aging.

Lipoteichoic acid isolated from Lactobacillus plantarum inhibits melanogenesis in B16F10 mouse melanoma cells.

Lipoteichoic acid (LTA) is a major component of the cell wall of Gram-positive bacteria. Its effects on living organisms are different from those of lipopolysaccharide (LPS) found in Gram-negative bacteria. LTA contributes to immune regulatory effects including anti-aging. In this study, we showed that LTA isolated from Lactobacillus plantarum (pLTA) inhibited melanogenesis in B16F10 mouse melanoma cells. pLTA reduced the cellular activity of tyrosinase and the expression of tyrosinase family members in a dose-dependent manner. The expression of microphthalmia-associated transcription factor (MITF), a key factor in the synthesis of melanin, was also decreased by pLTA. Further, we showed that pLTA activated melanogenesis signaling, such as extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinse (PI3K)/AKT. In addition, the expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and HuR, which are important RNA-binding proteins (RBPs), was reduced. pLTA likely degrades MITF via regulation of melanogenic signaling and RNA stability of melanogenic proteins, resulting in the reduction of melanin. Thus, our data suggest that pLTA has therapeutic potential for treating hyperpigmentation disorders and can also be used as a cosmetic whitening agent.

Differential immune-stimulatory effects of LTAs from different lactic acid bacteria via MAPK signaling pathway in RAW 264.7 cells.

OBJECTIVE: Lipoteichoic acid (LTA) is an immune-stimulatory component found in the cell wall of lactic acid bacteria, which are a major group of Gram-positive bacteria known to have beneficial health effects in humans. In this study, we evaluated the stimulatory effects of LTAs isolated from different lactobacilli species with mouse macrophage RAW 264.7 cells. METHODS: RAW 264.7 cells were stimulated with pLTA (isolated from Lactobacillus plantarum K8), rLTA (isolated from Lactobacillus rhamnosus), dLTA (isolated from Lactobacillus delbreukii), and sLTA (isolated from Lactobacillus sakei K101). Tumor necrosis factor (TNF)-α and interleukin (IL)-10 production were examined by ELISA, and nitric oxide (NO) production was assayed using Griess reaction. The mRNA and protein expression levels of inducible nitric oxide synthase (iNOS) was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Signaling molecules were also examined by Western blotting. RESULTS: pLTA and rLTA moderately induced TNF-α, IL-10, and NO production compared with stimulation of RAW 264.7 cells with dLTA and sLTA. Similar results were obtained for the mRNA and protein expression levels of iNOS. Western blot analysis showed that treatment of cells with pLTA or rLTA resulted in minimal phosphorylation of ERK, JNK and p38 MAPK while, dLTA and sLTA were strong activators of MAPK signaling. In addition, the glycolipid structure of LTAs was found to be composed of different fatty acid chain groups and lengths. Taken together, these results suggest that the differential immuno-stimulatory effects of LTAs isolated from different lactobacillus species may be related to their different ability to activate the MAPK signaling pathway, which are modulated by a unique glycolipid structure of LTA.

Lipoteichoic acid from Lactobacillus plantarum inhibits the expression of platelet-activating factor receptor induced by Staphylococcus aureus lipoteichoic acid or Escherichia coli lipopolysaccharide in human monocyte-like cells.

Platelet-activating factor receptor (PAFR) plays an important role in bacterial infection and inflammation. We examined the effect of the bacterial cell wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from Lactobacillus plantarum (pLTA) and Staphylococcus aureus (aLTA) on PAFR expression in THP-1, a monocyte-like cell line. LPS and aLTA, but not pLTA, significantly increased PAFR expression, whereas priming with pLTA inhibited LPSmediated or aLTA-mediated PAFR expression. Expression of Toll-like receptor (TLR) 2 and 4, and CD14 increased with LPS and aLTA treatments, but was inhibited by pLTA pretreatment. Neutralizing antibodies against TLR2, TLR4, and CD14 showed that these receptors were important in LPS-mediated or aLTA-mediated PAFR expression. PAFR expression is mainly regulated by the nuclear factor kappa B signaling pathway. Blocking PAF binding to PAFR using a PAFR inhibitor indicated that LPS-mediated or aLTA-mediated PAF expression affected TNF-α production. In the mouse small intestine, pLTA inhibited PAFR, TLR2, and TLR4 expression that was induced by heat-labile toxin. Our data suggested that pLTA has an anti-inflammatory effect by inhibiting the expression of PAFR that was induced by pathogenic ligands.

Differential effects of low and high doses of lipoteichoic acid on lipopolysaccharide-induced interleukin-6 production.

OBJECTIVE: Interleukin-6 (IL-6), which is increased in patients who are suffering from septic shock, is an important mediator of the inflammatory response. Here, we examined the priming effect of lipoteichoic acid (LTA) and lipopolysaccharide (LPS) on IL-6 production in a monocyte-like cell line. METHODS: THP-1 cells were primed by treatingwith a low or high dose of LTA isolated from Staphylococcus aureus (aLTA) and then re-treated with LPS. IL-6 production, receptor expression, and the variation of signaling molecules were examined by ELISA, reverse transcriptase polymerase chain reaction, and western blotting, respectively. RESULTS: LPS-mediated IL-6 production was dramatically increased in THP-1 cells pretreated with a low dose aLTA, while it was significantly decreased when a high dose of aLTA was given along with LPS. LPS-induced IL-6 production in low dose aLTA priming cells mediated by NF-κB and MAPKs pathways, and Akt functioned as a negative regulator of IL-6 production. Together, the results of this study suggest that different doses of bacterial cell surface components can mediate a diverse range of responses with respect to inflammatory cytokine production.

Lactobacillus sakei lipoteichoic acid inhibits MMP-1 induced by UVA in normal dermal fibroblasts of human.

Human skin is continuously exposed to ultraviolet (UV)-induced photoaging. UVA increases the activity of MMP-1 in dermal fibroblasts through mitogen-activated protein kinase (MAPK), p38, signaling. The irradiation of keratinocytes by UVA results in the secretion of the inflammatory cytokine, tumor necrosis factor-α (TNF-α), and the stimulation of MMP-1 in normal human dermal fibroblasts (NHDFs). Lipoteichoic acid (LTA) is a component of the cell wall of gram-positive Lactobacillus spp. of bacteria. LTA is well known as an anti-inflammation molecule. LTA of the bacterium Lactobacillus plantarum has an anti-photoaging effect, but the potential anti-photoaging effect of the other bacteria has not been examined to date. The current study showed that L. sakei LTA (sLTA) has an immune modulating effect in human monocyte cells. Our object was whether inhibitory effects of sLTA on MMP-1 are caused from reducing the MAPK signal in NHDFs. It inhibits MMP-1 and MAPK signaling induced by UVA in NHDFs. We also confirmed effects of sLTA suppressing TNF-α inducing MMP-1 in NHDFs.

Lipoteichoic acid isolated from Lactobacillus plantarum suppresses LPS-mediated atherosclerotic plaque inflammation.

Chronic inflammation plays an important role in atherogenesis. Experimental studies have demonstrated the accumulation of monocytes/macrophages in atherosclerotic plaques caused by inflammation. Here, we report the inhibitory effects of lipoteichoic acid (LTA) from Lactobacillus plantarum (pLTA) on atherosclerotic inflammation. pLTA inhibited the production of proinflammatory cytokines and nitric oxide in lipopolysaccharide (LPS)-stimulated cells and alleviated THP-1 cell adhesion to HUVEC by down-regulation of adhesion molecules such as intracellular adhesion molecule-1 (ICAM-I), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. The inhibitory effect of pLTA was mediated by inhibition of NF-κB and activation of MAP kinases. Inhibition of monocyte/macrophage infiltration to the arterial lumen was shown in pLTA-injected ApoE(-/-) mice, which was concurrent with inhibition of MMP-9 and preservation of CD31 production. The antiinflammatory effect mediated by pLTA decreased expression of atherosclerotic markers such as COX-2, Bax, and HSP27 and also cell surface receptors such as TLR4 and CCR7. Together, these results underscore the role of pLTA in suppressing atherosclerotic plaque inflammation and will help in identifying targets with therapeutic potential against pathogen-mediated atherogenesis.

Lactobacillus plantarum lipoteichoic acid alleviates TNF-α-induced inflammation in the HT-29 intestinal epithelial cell line.

We recently observed that lipoteichoic acid (LTA) isolated from Lactobacillus plantarum inhibited endotoxin-mediated inflammation of the immune cells and septic shock in a mouse model. Here, we examined the inhibitory role of L. plantarum LTA (pLTA) on the inflammatory responses of intestinal epithelial cells (IEC). The human colon cell line, HT-29, increased interleukin (IL)-8 expression in response to recombinant human tumor necrosis factor (TNF)-alpha, but not in response to bacterial ligands and interferon (IFN)-gamma. TNF-α also increased the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and intercellular adhesion molecule 1 (ICAM-1) through activation of p38 mitogen-activated protein kinase (MAPK) from HT-29 cells. However, the inflammatory response of HT-29 on TNF-α stimulation was significantly inhibited by pLTA treatment. This pLTA-mediated inhibition accompanied the inhibition of nuclear factor (NF)-kappa B and MAPKs. Our data suggest that pLTA regulates cytokine-mediated immune responses and may be a good candidate for maintaining intestinal homeostasis against excessive inflammation.

Probiotic genomic DNA reduces the production of pro-inflammatory cytokine tumor necrosis factor-alpha.

The effect of Lactobacillus plantarum genomic DNA on lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, nuclear factor-kappa B activation, and the expressions of tumor necrosis factor-alpha, interleukin-1 receptor-associated kinase M, and the pattern recognition receptor were examined. Pretreatment of p-gDNA inhibited the phosphorylation of MAPKs and nuclear factor-kappa B, and also inhibited LPS-induced TNF-α production in response to subsequent LPS stimulation. L. plantarum genomic DNA-mediated inhibition of signaling pathway and tumor necrosis factor-alpha was accompanied by the suppression of toll-like receptor (TLR) 2, TLR4, and TLR9 and the induction of interleukin-1 receptor-associated kinase M, a negative regulator of TLR. This study can extend our understanding of the biological function of probiotic genomic DNA as an anti-inflammatory agent.

Lactobacillus plantarum lipoteichoic acid down-regulated Shigella flexneri peptidoglycan-induced inflammation.

Bacterial peptidoglycans (PGNs) are recognized by the host's innate immune system. This process is mediated by the NOD/CARD family of proteins, which induces inflammation by activating nuclear factor (NF)-κB. Excessive activation of monocytes by Shigella flexneri PGN (flexPGN) leads to serious inflammatory diseases such as intestinal bowel diseases (IBD) and Crohn's disease. In this study, we examined whether Lactobacillus plantarum lipoteichoic acid (pLTA) could attenuate the pro-inflammatory signaling induced by flexPGN in human monocytic THP-1 cells. Compared to control THP-1 cells, pLTA-tolerant cells showed a significant reduction in TNF-α and IL-1ß production in response to flexPGN. We also examined the inhibition of NF-κB and the activation of mitogen-activated protein kinase (MAPK) in pLTA-tolerant cells. We found that the expression of NOD2 in pLTA-tolerant cells was down-regulated at the mRNA and protein levels, suggesting that pLTA is a potent modulator of the pro-inflammatory NOD2-related signaling pathways induced by flexPGN. Together, these data indicate that pLTA induces cross-tolerance against flexPGN. Notably, these effects are related not only to IL-1 signaling, which is known to play a role in LPS tolerance, but also to NOD-Rick signaling. This study provides insight into how commensal microflora may contribute to homeostasis of the host intestinal tract.

Protein expression changes in human monocytic THP-1 cells treated with lipoteichoic acid from Lactobacillus plantarum and Staphylococcus aureus.

Lipoteichoic acid (LTA) from Staphylococcus aureus (aLTA) and from Lactobacillus plantarum LTA (pLTA) are both recognized by Toll-like receptor 2 (TLR2), but cause different stimulatory effects on the innate immune and inflammatory responses, and their underlying cellular mechanisms are unknown. In this study, comparative proteome analysis was performed using two-dimensional gel electrophoresis and mass spectrometry on protein extracts from human monocyte THP-1 cells stimulated with either aLTA or pLTA. Differentially expressed proteins might be involved in innate immunity and inflammation. Cells treated with aLTA and with pLTA showed different protein expression profiles. Of 60 identified proteins, 10 were present only in treated cells (8 in aLTA-treated only, and 2 in pLTA-treated only), 1 protein (IMPDH2) was suppressed by pLTA, and 49 were up- or down-regulated more than three-fold by aLTA- or pLTA- stimulation. Several proteins involved in immunity or inflammation, antioxidation, or RNA processing were significantly changed in expression by aLTA- or pLTA-stimulation, including cyclophilin A, HLA-B27, D-dopachrome tautomerase, Mn- SOD, hnRNP-C, PSF and KSRP. These data demonstrated that aLTA and pLTA had different effects on the protein profile of THP-1 cells. Comparison of the proteome alterations will provide candidate biomarkers for further investigation of the immunomodulatory effects of aLTA and pLTA, and the involvement of aLTA in the pathogenesis of Staphylococcus aureus sepsis.