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.

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.

Inhibitory effects of Lactobacillus plantarum lipoteichoic acid (LTA) on Staphylococcus aureus LTA-induced tumor necrosis factor-alpha production.

Staphylococcus aureus is a common etiologic agent for Gram-positive sepsis, and its lipoteichoic acid (LTA) may be important in causing Gram-positive bacterial septic shock. Here, we demonstrate that highly purified LTA (pLTA) isolated from Lactobacillus plantarum inhibited aureus LTA (aLTA)-induced TNF-alpha production in THP- cells. Whereas pLTA scarcely induced TNF-alpha production, aLTA induced excessive TNF-alpha production. Interestingly, aLTA-induced TNF-alpha production was inhibited by pLTA pretreatment. Compared with pLTA, aLTA induced strong signal transduction through the MyD88, NF-kappaB, and MAP kinases. This signaling, however, was reduced by a pLTA pretreatment, and resulted in the inhibition of aLTA-induced TNF-alpha production. Whereas dealanylated LTAs, as well as native LTAs, contributed to TNF- induction or TNF-alpha reduction, deacylated LTAs did not, indicating that the acyl chain of LTA played an important role in the LTA-mediated immune regulation. These results suggest that pLTA may act as an antagonist for aLTA, and that an antagonistic pLTA may be a useful agent for suppressing the septic shock caused by Gram-positive bacteria.

Mechanical stress induces tumor necrosis factor-{alpha} production through Ca2+ release-dependent TLR2 signaling.

We studied centrifugation-mediated mechanical stress-induced tumor necrosis factor-alpha (TNF-alpha) production in the monocyte-like cell line THP-1. The induction of TNF-alpha by mechanical stress was dependent on the centrifugation speed and produced the highest level of TNF-alpha after 1 h of stimulation. TNF-alpha production returned to normal levels after 24 h of stimulation. Mechanical stress also induced Toll-like receptor-2 (TLR2) mRNA in proportion to the expression of TNF-alpha. The inhibition of TLR2 signaling by dominant negative myeloid differentiation factor 88 (MyD88) blocked TNF-alpha expression response to mechanical stress. After transient overexpression of TLR2 in HEK-293 cells, mechanical stress induced TNF-alpha mRNA production. Interestingly, mechanical stress activated the c-Src-dependent TLR2 phosphorylation, which is necessary to induce Ca(2+) fluxes. When THP-1 cells were pretreated with BAPTA-AM, thapsigargin, and NiCl(2).6H(2)O, followed by mechanical stimulation, both TLR2 and TNF-alpha production were inhibited, indicating that centrifugation-mediated mechanical stress induces both TLR2 and TNF-alpha production through Ca(2+) releases from intracellular Ca(2+) stores following TLR2 phosphorylation. In addition, TNF-alpha treatment in THP-1 cells induced TLR2 production in response to mechanical stress, whereas the preincubation of anti-TNF-alpha antibody scarcely induced the mechanical stress-mediated production of TLR2, indicating that TNF-alpha produced by mechanically stimulated THP-1 cells affected TLR2 production. We concluded that TNF-alpha production induced by centrifugation-mediated mechanical stress is dependent on MyD88-dependent TLR2 signaling that is associated with Ca(2+) release and that TNF-alpha production induced by mechanical stress affects TLR2 production.

Lipoteichoic acid isolated from Lactobacillus plantarum inhibits lipopolysaccharide-induced TNF-alpha production in THP-1 cells and endotoxin shock in mice.

In this study, the effect of Lactobacillus plantarum lipoteichoic acid (pLTA) on LPS-induced MAPK activation, NF-kappaB activation, and the expression of TNF-alpha and IL-1R-associated kinase M (IRAK-M) was examined. The expression of the pattern recognition receptor and the survival rate of mice were also examined. pLTA pretreatment inhibited the phosphorylation of ERK, JNK, and p38 kinase. It also inhibited the degradation of IkappaBalpha and IkappaBbeta, as well as the activation of the LPS-induced TNF-alpha factor in response to subsequent LPS stimulation. These changes were accompanied by the suppression of the LPS-induced expression of TLR4, NOD1, and NOD2, and the induction of IRAK-M, with a concurrent reduction of TNF-alpha secretion. Furthermore, the overexpression of pattern recognition receptors such as TLR4, NOD1, and NOD2 and the degradation of IRAK-M by transient transfection were found to reinstate the production of TNF-alpha after LPS restimulation. In addition, the i.p. injection of pLTA suppressed fatality, and decreased the level of TNF-alpha in the blood, in LPS-induced endotoxin shock mice. In conclusion, these data extend our understanding of the pLTA tolerance mechanism, which is related to the inhibition of LPS-induced endotoxin shock, and suggest that pLTA may have promise as a new therapeutic agent for LPS-induced septic shock.

Development of a monitoring vector for Leuconostoc mesenteroides using the green fluorescent protein gene.

The vector pCW5 with plasmid pC7, originally isolated in Lactobacillus paraplantarum C7 derived from kimchi, was constructed using a p32 strong promoter, the pC7 replicon, and green fluorescent protein (GFP) as the reporter. The constructed vector was transformed into E. coli and Leuconostoc mesenteroides, and GFP expression detected using a Western blot analysis. GFP fluorescence was recognized in E. coli and Leuconostoc mesenteroides using a confocal microscope. In addition, GFP fluorescence was also clearly detected in several industrially important lactic acid bacteria (LAB), including Lactobacillus bulgaricus, Lactobacillus paraplantarum, and Lactobacillus plantarum. Thus, pCW5 was shown to be effective for Leuconostoc mesenteroides when using GFP as the reporter, and it can also be used as a broad-host-range vector for other lactic acid bacteria.

Identification and functional analysis of salmon annexin 1 induced by a virus infection in a fish cell line.

In this study, we investigated changes in protein expression of fish cells induced by infection of infectious pancreatic necrosis virus (IPNV) using two-dimensional electrophoresis and matrix-assisted laser desorption-time of flight proton motive force analysis and identified a novel type of salmon annexin 1 that is induced in fish cells by infection with IPNV. Northern blotting showed that this annexin is overexpressed in IPNV-infected cells compared to control cells, and further analysis revealed that it has a 1,509-bp full-length cDNA sequence with an open reading frame encoding 339 amino acids (GenBank accession no. AY944135). Amino acid sequence analysis revealed that this protein belongs to the annexin 1 subfamily. By applying RNA interference, the mRNA levels of salmon annexin 1 were suppressed and, under these conditions, apoptosis of IPNV-infected cells was significantly increased. While small interfering RNA (siRNA) treatment did not affect the levels of the viral proteins significantly until 10 h postinfection, it reduced the titer of extracellular virus to 25% of that of a scrambled siRNA-treated control. These data provide evidence of an antiapoptotic function for salmon annexin 1 that is important for IPNV growth in cultured cells.

Lipoteichoic acid from Lactobacillus plantarum elicits both the production of interleukin-23p19 and suppression of pathogen-mediated interleukin-10 in THP-1 cells.

In this study, the stimulatory effects of different lactic acid bacteria strains, and their subcellular fractions, on the THP-1 cell line were evaluated. Lactobacillus plantarum was found in particular to induce high levels of IL-23p19 mRNA, but it moderately induced TNF-alpha production. IL-10 production was not entirely affected by L. plantarum stimulation. When subcellular fractions of L. plantarum were used to treat THP-1 cells, IL-23p19 mRNA expression was enhanced in a dose-responsive manner, specifically by lipoteichoic acid (LTA). The cotreatment of THP-1 cells by both L. plantarum and Staphylococcus aureus LTA resulted in decreased IL-10 production when compared with cells treated by S. aureus LTA alone. Taken together, these data suggest that LTA isolated from L. plantarum elicits stimulatory effects upon the expression of IL-23p19 and inhibitory effects on pathogen-mediated IL-10 production.

Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products.

Using linker insertion mutations, we determined the most stable region of the parE gene which encodes a toxic protein (ParE) that inhibits growth of Escherichia coli. The toxicity of ParE was sustained until a 144 bp linker was inserted into this region. We have developed a 3' T-overhang vector based on these characteristics of the GST-ParE toxin, and named pTOC-T. Because pTOC-T uses a post-segregational killing system, all transformants grown up on the plates can be considered as recombinants containing foreign DNA. pTOC-T not require X-Gal, IPTG or other substrates for selection. This T-vector using a positive selection system can be applied to various E. coli strains such as XL1-Blue, BL21, DH5alpha, JM109, and JM110.