Seok-Seong Kang

Differential immunostimulatory effects of Gram-positive bacteria due to their lipoteichoic acids.

Lipoteichoic acid (LTA) is a major immunostimulating component in the cell wall of Gram-positive bacteria as lipopolysaccharide of Gram-negative bacteria. However, LTA is expressed on not only pathogenic but also nonpathogenic Gram-positive bacteria. In order to examine whether the immunostimulating potentials of Gram-positive bacteria are correlated with their LTAs, we prepared highly pure LTAs from Staphylococcus aureus (pathogenic), Bacillus subtilis (non-pathogenic), or Lactobacillus plantarum (beneficial). When a murine macrophage cell-line, RAW 264.7, was stimulated with heat-killed bacteria, both S. aureus and B. subtilis induced nitric oxide (NO) production in a dose-dependent manner while L. plantarum showed a minimal induction. Interestingly, purified LTAs from S. aureus and B. subtilis, but not from L. plantarum, were able to induce the production of NO. The differential inflammatory potentials of LTAs coincided with their abilities to activate Toll-like receptor 2 (TLR2), which is known to recognize Gram-positive bacteria and LTA, and transcription factors NF-kappaB and AP-1. Similar results were obtained with the expression of cytokines related to inflammation by RAW 264.7 and human peripheral blood mononuclear cells as well. The ability of LTA to induce TNF-alpha and NO production was abolished when the LTAs were treated with 0.2 N NaOH. Collectively, we suggest that the immunostimulating potentials of Gram-positive bacteria differ due to their LTAs with differential potencies in the stimulation of TLR2.

Lipoproteins are an important bacterial component responsible for bone destruction through the induction of osteoclast differentiation and activation

Bacterial infection can cause inflammatory bone diseases accompanied by the bone destruction resulting from excess generation of osteoclasts. Although lipoproteins are one of the major immunostimulating components of bacteria, little is known about their effects on bone metabolism. In this study, we investigated the role of lipoproteins in bacteria‐induced bone destruction using Staphylococcus aureus wild type, its lipoprotein‐deficient mutant, and synthetic lipopeptides Pam2CSK4 and Pam3CSK4 known to mimic bacterial lipoproteins. Formaldehyde‐inactivated S. aureus or the synthetic lipopeptides induced severe bone loss in the femurs of mice after intraperitoneal administration and in a calvarial bone implantation model, whereas the lipoprotein‐deficient S. aureus did not show such effects. Mechanism studies further identified three action mechanisms for the lipopeptide‐induced osteoclast differentiation and bone resorption via (i) enhancement of osteoclast differentiation through Toll‐like receptor 2 and MyD88‐dependent signaling pathways; (ii) induction of pro‐inflammatory cytokines, TNF‐α and IL‐6; and (iii) upregulation of RANKL expression with downregulation of osteoprotegerin expression in osteoblasts. Taken together, these results suggest that lipoprotein might be an important bacterial component responsible for bone destruction during bacterial infections through augmentation of osteoclast differentiation and activation.

Induction of IL-8 expression by bacterial flagellin is mediated through lipid raft formation and intracellular TLR5 activation in A549 cells.

We investigated the mechanism for the induction of a chemokine, IL-8, by bacterial flagellins in the human alveolar type II epithelial cell line, A549. Bacterial flagellin induced expression of IL-8 mRNA and protein in dose- and time-dependent manners. IL-8 expression was inhibited by nystatin (a lipid rafts inhibitor) but not by chlorpromazine (a clathrin-coated pits inhibitor). Interestingly, Toll-like receptor 5 (TLR5) recognizing flagellins was found in the intracellular compartment of A549 but rarely on the cell surface. Flagellin-induced IL-8 expression appears to be mediated through TLR5 as determined by in vitro transient transfection experiment in HEK-293 cells expressing TLR5 using a reporter gene construct containing IL-8 promoter. IL-8 expression was attenuated by inhibitors for protein kinase C (PKC) and mitogen-activated protein (MAP) kinases. Furthermore, NF-kappaB and NF-IL6 transcription factors played an important role in the flagellin-induced IL-8 gene expression in A549 cells. Collectively, these results suggest that flagellin-induced IL-8 expression requires formation of lipid rafts, intracellular TLR activation, and subsequent activation of PKC and MAP kinases leading to the activation of the transcription factors NF-kappaB and NF-IL6 in human alveolar type II epithelial cells.

Lipoteichoic acid of Streptococcus mutans interacts with Toll-like receptor 2 through the lipid moiety for induction of inflammatory mediators in murine macrophages

Streptococcus mutans is a pathogenic Gram-positive bacterium that is closely associated with dental caries and subsequent pulpal inflammation. Although lipoteichoic acid (LTA) is considered a major virulence factor of Gram-positive bacteria, little is known about the innate immunity to S. mutans LTA. In this study, we purified LTA from S. mutans (Sm.LTA) through n-butanol extraction, hydrophobic interaction column chromatography, and ion-exchange column chromatography to investigate its immunological properties using murine macrophages. The Sm.LTA preparation had no detectable contamination with endotoxins, proteins, or nucleic acids. Upon exposure to Sm.LTA, the murine macrophage cell-line RAW 264.7 cells produced TNF-α and nitric oxide (NO) in a dose-dependent manner. Sm.LTA preferentially bound to and activated CHO/CD14/TLR2 cells rather than CHO/CD14/TLR4 cells, which are stable transfectants expressing CD14 and TLR2 or CD14 and TLR4, respectively. Sm.LTA could not induce TNF-α or NO production in macrophages derived from TLR2-deficient mice whereas it dose-dependently induced those inflammatory mediators in wild-type macrophages. TLR2-dependent induction of NO by Sm.LTA was also confirmed in RAW 264.7 cells using specific antibodies blocking TLR2. Furthermore, Sm.LTA deacylated by alkaline hydrolysis neither stimulated TLR2 nor induced TNF-α or NO production, suggesting that Sm.LTA lipid moieties are crucial for the immuno-stimulatory activity of Sm.LTA. Unlike Staphylococcus aureus LTA, which has potent immuno-stimulating activity, Sm.LTA showed a modest induction of NO production comparable to LTAs of other oral bacteria Enterococcus faecalis and Lactobacillus plantarum. In conclusion, our results suggest that the Sm.LTA interacts with TLR2 through the lipid moiety for the induction of inflammatory mediators in macrophages.

Lipoteichoic acid of Enterococcus faecalis induces the expression of chemokines via TLR2 and PAFR signaling pathways

Enterococcus faecalis is one of the most common opportunistic pathogens responsible for nosocomial infections, and its LTA is known as an important virulence factor causing inflammatory responses. As chemokines play a key role in inflammatory diseases by triggering leukocyte infiltration into the infection site, we purified EfLTA and investigated its effect on the expression of chemokines, IP‐10, MIP‐1α, and MCP‐1, in murine macrophages. EfLTA induced the expression of these chemokines at the mRNA and protein levels. TLR2, CD14, and MyD88 were involved in the EfLTA‐induced chemokine expression, as the expression was reduced remarkably in macrophages derived from TLR2‐, CD14‐, or MyD88‐deficient mice. EfLTA induced phosphorylation of MAPKs and enhanced the DNA‐binding activity of NF‐κB, AP‐1, and NF‐IL6 transcription factors. The induction of IP‐10 required ERK, JNK, p38 MAPK, PKC, PTK, PI3K, and ROS. We noticed that all of these signaling molecules, except p38 MAPK and ROS, were indispensable for the induction of MCP‐1 and MIP‐1α. Interestingly, the EfLTA‐induced chemokine expression was mediated through PAFR/JAK/STAT1 signaling pathways without IFN‐β involvement, which is different from LPS‐induced chemokine expression requiring IFN‐β/JAK/STAT1 signaling pathways. Furthermore, the culture supernatant of EfLTA‐treated RAW 264.7 cells promoted the platelet aggregation, and exogenous PAF induced the chemokine expression in macrophages derived from WT and TLR2‐deficient mice. These results suggest that EfLTA induces the expression of chemokines via signaling pathways requiring TLR2 and PAFR, which is distinct from that of LPS‐induced chemokine expression.

Calcium Hydroxide Inactivates Lipoteichoic Acid from Enterococcus faecalis through Deacylation of the Lipid Moiety

INTRODUCTION Lipoteichoic acid (LTA) is a major virulence factor of Enterococcus faecalis that is closely associated with refractory apical periodontitis. Recently, we have shown that calcium hydroxide, a commonly used intracanal medicament, abrogated the ability of LTA to stimulate the production of tumor necrosis factor α in a murine macrophage line, RAW 264.7. Because calcium hydroxide could potentially modify the glycolipid moiety of LTA, we examined if calcium hydroxide inactivates LTA through deacylation of the LTA. METHODS LTA was prepared from E. faecalis by organic solvent extraction followed by chromatography with the hydrophobic-interaction column and the ion-exchange column. RAW 264.7 cells were stimulated with intact LTA or calcium hydroxide-treated LTA for 24 hours, and the productions of nitric oxide (NO) and chemokines interferon-gamma-induced protein (IP-10) and macrophage inflammatory protein-1α (MIP-1α) were determined. The glycolipid structure of LTA was analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and thin layer chromatography (TLC). RESULTS The production of NO, IP-10, and MIP-1α was augmented in LTA-stimulated cells, whereas no such effect was observed upon stimulation with calcium hydroxide-pretreated LTA. Mass spectrometry showed that intact glycolipids of LTA yielded distinct mass peaks at 930 to 1,070 mass over charge (m/z) units, corresponding to dihexosyl-diacylglycerol consisting of two acyl chains with chain lengths of C(16) to C(22) and with one or two unsaturated double bonds. However, those peaks were not observed in the mass spectra of the calcium hydroxide-treated LTA. Furthermore, free fatty acids released from the calcium hydroxide-treated LTA were detected using TLC. CONCLUSION We suggest that calcium hydroxide attenuates the inflammatory activity of E. faecalis LTA through deacylation of the LTA.

Lipoteichoic acid from Lactobacillus plantarum induces nitric oxide production in the presence of interferon-γ in murine macrophages.

Lipoteichoic acid (LTA) is a major immuno-stimulating component of Gram-positive bacteria. LTA from the beneficial bacterium Lactobacillus plantarum induces weak nitric oxide (NO) production in murine macrophages. Currently, it is not clear if LTA from L. plantarum is able to stimulate the innate immune response, even in the presence of inflammation. In the present study, we prepared highly pure and structurally intact LTA from L. plantarum and investigated its ability to induce NO in the presence of interferon (IFN)-γ in the RAW 264.7 murine macrophage cell line and bone marrow-derived macrophages (BMMs) from mice. L. plantarum LTA alone was unable to induce NO production, even at 30μg/ml. However, LTA in the presence of IFN-γ significantly induced NO production in RAW 264.7 cells. The observed NO production was inhibited by a NO synthase (NOS) inhibitor l-NAME and an inducible NOS (iNOS) inhibitor l-NIL, suggesting that iNOS is specifically required for this action. Western blot analysis and reverse transcription and polymerase chain reaction further confirmed that L. plantarum LTA increased protein and mRNA levels of iNOS, respectively. However, such induction was substantially inhibited in BMMs from Toll-like receptor 2 (TLR2)-deficient mice and the macrophages treated with an inhibitor blocking platelet-activating factor receptor. In addition, L. plantarum LTA plus IFN-γ induced IFN-β expression and STAT1 phosphorylation, which are key pathways for inducing iNOS expression. Electrophoretic mobility shift assay demonstrated that L. plantarum LTA in the presence of IFN-γ remarkably increased the DNA-binding activity of NF-κB transcription factor, which is known to be involved in the iNOS gene expression. Collectively, these results suggest that LTA from L. plantarum alone has no inflammatory potential but does induce NO production under conditions of inflammation, such as the presence of IFN-γ.

Induction of BAFF expression by IFN‐γ via JAK/STAT signaling pathways in human intestinal epithelial cells

BAFF plays an important role in the development of B cells. Here, we investigated the effect of IFN‐γ on BAFF expression in human intestinal epithelial cells. IFN‐γ induced soluble and membrane‐bound BAFF production in a dose‐ and time‐dependent manner. IFN‐γ‐induced BAFF release from polarized intestinal epithelial cells was observed in apical and basolateral compartments. JAK I inhibitor suppressed IFN‐γ‐induced BAFF expression. Moreover, IFN‐γ enhanced STAT1 phosphorylation and expression of IRF‐1. Transient transfection and reporter gene assay showed that the BAFF promoter region spanning −750 to −500 bp from the translation initiation site was crucial for IFN‐γ‐induced BAFF expression. Nucleotide sequence analysis revealed a GAS element in the promoter region. ChIP assay confirmed the enhanced binding of phosphorylated STAT1 to the BAFF promoter region at −800 to −601 bp. Furthermore, IFN‐γ enhanced DNA binding to GAS and its transcriptional activation, as determined by the EMSA and reporter gene assay. Collectively, these results suggest that IFN‐γ induces BAFF expression in human intestinal epithelial cells through JAK/STAT signaling pathways that might activate the GAS and IRF‐1‐binding element in the BAFF promoter.

Staphylococcus aureus induces IL-8 expression through its lipoproteins in the human intestinal epithelial cell, Caco-2

Staphylococcus aureus can cause the intestinal inflammatory diseases. However, little is known about the molecular mechanism of S. aureus infection in the intestine. In the present study, we investigated whether S. aureus could stimulate human intestinal epithelial cells triggering inflammation. When the human intestinal epithelial cell-line, Caco-2, and the primary colon cells were stimulated with ethanol-inactivated S. aureus, IL-8 expression was induced in a dose-dependent manner. The inactivated S. aureus preferentially stimulated Toll-like receptor (TLR) 2 rather than TLR4. Lipoproteins, lipoteichoic acid (LTA), and peptidoglycan (PGN) are considered as potential TLR2 ligands of S. aureus. Interestingly, S aureus lipoproteins and Pam2CSK4 mimicking Gram-positive bacterial lipoproteins, but not LTA and PGN of S. aureus, significantly induced IL-8 expression in Caco-2 cells. Furthermore, lipoprotein-deficient S. aureus mutant strain failed to induce IL-8 production. Collectively, these results suggest that S. aureus stimulates the human intestinal epithelial cells to induce the chemokine IL-8 production through its lipoproteins, potentially contributing the development of intestinal inflammation.

Lipoteichoic acid from Lactobacillus plantarum inhibits Pam2CSK4-induced IL-8 production in human intestinal epithelial cells

Lactobacilli are probiotic bacteria that are considered to be beneficial in the gastrointestinal tract of humans. Although lactobacilli are well known to alleviate intestinal inflammation, the molecular basis of this phenomenon is poorly understood. In this study, we investigated the effect of Lactobacillus plantarum lipoteichoic acid (Lp.LTA), which is a major cell wall component of this species, on the production of interleukin (IL)-8 in human intestinal epithelial Caco-2 cells. Treatment with Pam2CSK4, a synthetic lipopeptide that is known to mimic Gram-positive bacterial lipoproteins as an important virulence factor, significantly induced IL-8 expression in Caco-2 cells. However, neither heat-inactivated L. plantarum nor L. plantarum peptidoglycan inhibited Pam2CSK4-induced IL-8 mRNA expression. In addition, both a deacylated form and a dealanylated form of Lp.LTA failed to inhibit Pam2CSK4-induced IL-8 expression, indicating that the lipid and D-alanine moieties are critical for Lp.LTA-mediated inhibition. Moreover, Lp.LTA inhibited Pam2CSK4-induced activation of p38 kinase, JNK, and NF-κB transcription factor by suppressing toll-like receptor 2 activation. Collectively, these results suggest that Lp.LTA exerts anti-inflammatory effects on human intestinal epithelial cells by blocking IL-8 production.