Han Geun Kim

Lipoteichoic Acid Isolated from Lactobacillus plantarum Inhibits Lipopolysaccharide-Induced TNF-α 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-κB activation, and the expression of TNF-α 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 IκBα and IκBβ, as well as the activation of the LPS-induced TNF-α 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-α 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-α after LPS restimulation. In addition, the i.p. injection of pLTA suppressed fatality, and decreased the level of TNF-α 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.

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

Bacterial peptidoglycans (PGNs) are recognized by the hosts 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 Crohns 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-1β 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.

Mechanical stress induces tumor necrosis factor-α 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.

Identification and Functional Analysis of Salmon Annexin 1 Induced by a Virus Infection in a Fish Cell Line

ABSTRACT 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.

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.

pTOC-KR: a positive selection cloning vector based on the ParE toxin

Several prokaryotic cloning vectors have been developed to clone foreign DNA in bacteria. The insertion inactivation of β-galactosidase activity, for instance, is a common screening method of identifying recombinant DNA molecules in many vectors (1,2). However, this system poses several problems. For one, many vectors can self-ligate and give false transformants. Although β-galactosidase is widely used as a screening marker, it is limited to use with specially mutated Escherichia coli hosts for α-complementation. The color of the colonies is also hard to distinguish when the blue/white selection system is used. This system also requires expensive reagents: 5-bromo4-chloro-3-indolyl-β-D-galactosidase (X-Gal) and isopropylthio-β-D-thiogalactopyranoside (IPTG). To address these problems, several positive selection vector systems have been developed based on the toxinantitoxin systems parD (Kis/Kid) of plasmid R1 (3) and on the CcdAB system of plasmid F (4). Similar positive selection cloning vectors using the transcriptional factor GATA-1 (5) or a cellulase gene (CelA) as screening marker (6) have also been developed. Nonetheless, the application of these systems is also limited; in most cases, they are host-limited and their cloning

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, DH5α, JM109, and JM110.

Differential gene expression profiles in human THP-1 monocytes treated with Lactobacillus plantarum or Staphylococcus aureus lipoteichoic acid

The lipoteichoic acid (LTA) of Staphylococcus aureus (aLTA) and Lactobacillus plantarum (pLTA) engage the same toll-like receptor 2 (TLR2) signaling pathway but exert different effects on innate immunity and inflammation. The mechanisms underlying these differential effects are not yet clear. Human oligonucleotide microarrays were used to investigate the transcriptome of human THP-1 monocytes upon exposure to aLTA or pLTA, and differential gene expression profiles were observed between the aLTA- and pLTA-treated cells. The expression level of 1,302 genes in aLTAtreated cells increased more than 2-fold; some of which have been implicated in immune or inflammatory responses, cell adhesion, cell signal transduction, transcription factors, anion transport, proteolysis, and oxidative processes. Particularly, a variety of genes that encode cytokines and chemokines, and TLR signaling-related molecules belonging to the tumor necrosis factor receptor-associated factor (TRAF), nuclear factor-kappa B, and signal transducer and activator of transcription families were remarkably up-regulated by aLTA stimulation. In contrast, pLTA treatment altered the expression of only 90 genes by more than 1.5-fold, and these genes were not correlated with innate immunity, inflammation or other related processes. The different effects mediated by aLTA and pLTA were further verified and compared by analysis of the expression of a selected group of genes, including TRAFs and some cytokines and chemokines, using real time-polymerase chain reaction and ELISA. These data suggest that aLTA and pLTA have different immunomodulatory potentials. Compared with pLTA, aLTA is a stronger stimulator and impacts the expression of many innate immunity- and/or inflammation-related genes.