Jintaek Im

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.

Lipoteichoic Acid Partially Contributes to the Inflammatory Responses to Enterococcus faecalis

Enterococcus faecalis, a pathogenic gram-positive bacterium, is closely related to refractory apical periodontitis. Because lipoteichoic acid (LTA) is considered a major virulence factor of gram-positive bacteria, in the present study, highly pure LTA from E. faecalis was prepared, and its ability to stimulate murine macrophages was investigated in comparison with those of the killed whole cells. Upon exposure to E. faecalis LTA, RAW 264.7 (a murine macrophage cell line) produced a significantly (p < 0.05) high level of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in a concentration-dependent manner. It is to note that the LTA was able to stimulate Toll-like receptor 2 (TLR2) but not TLR4. Concomitantly, LTA enhanced the DNA-binding activity of a transcription factor, nuclear factor-kappa B (NF-kappaB), which plays an important role in the transcriptional activation of genes encoding inflammatory mediators. In contrast, heat-killed E. faecalis stimulated both TLR2 and TLR4, whereas the killed E. faecalis whole cells induced significant (p < 0.05) levels of TNF-alpha and NO in RAW 264.7 cells as their LTA did. These results suggest that LTA partially contributes to E. faecalis-induced inflammatory responses.

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.

Staphylococcus aureus induces IL-1β expression through the activation of MAP kinases and AP-1, CRE and NF-κB transcription factors in the bovine mammary gland epithelial cells.

Although mastitis caused by Staphylococcus aureus is a problematic inflammatory disease in lactating cows, the innate immunity to S. aureus in the mammary gland is poorly understood. In the present study, we observed that heat-killed S. aureus (HKS) induced IL-1β expression at both the mRNA and protein levels in the mammary gland epithelial cell-line, MAC-T. IL-1β production was suppressed by inhibitors of lipid rafts, ERK, JNK, and p38 kinases. Furthermore, HKS augmented the activities of the AP-1, CRE, and NF-κB transcription factors that regulate IL-1β gene expression. Among staphylococcal cell-wall components with inflammatory potential, Pam2CSK4 (a representative model for diacylated lipoproteins) enhanced IL-1β mRNA expression, while lipoteichoic acid and peptidoglycan did not. Collectively, we suggest that S. aureus-induced IL-1β production requires lipid raft formation, activation of MAP kinases, and activation of transcription factors AP-1, CRE, and NF-κB. Lipoprotein seems to be a major cell-wall component for the S. aureus-induced IL-1β production in bovine mammary gland epithelial cells.

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.

Induction of IL-8 expression by Cordyceps militaris grown on germinated soybeans through lipid rafts formation and signaling pathways via ERK and JNK in A549 cells.

AIM OF THE STUDY In order to elucidate immunoregulatory mechanisms of Cordyceps militaris, a methanol extract of Cordyceps militaris grown on germinated soybeans was prepared and its immunoregulatory effect in the human lung epithelial cells was investigated by examining its ability to induce IL-8 expression. MATERIALS AND METHODS Cordyceps militaris grown on germinated soybeans was extracted with 80% methanol (GSC4M) and used for stimulation of a human lung epithelial cell-line, A549. An enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction were performed to examine the production of IL-8 protein and its mRNA, respectively. For the analysis of transcription factors regulating IL-8 transcriptional activation, the nuclear fraction was extracted from GSC4M-treated A549 cells and subjected to electrophoretic mobility shift assay. RESULTS GSC4M induced IL-8 protein secretion and its mRNA expression from A549 cells in a dose- and time-dependent manner. GSC4M-induced IL-8 expression was inhibited by an inhibitor for lipid rafts formation but not by that for clathrin-coated pits. In addition, signaling pathways for GSC4M-induced IL-8 expression were mediated through ERK and JNK but hardly through p38 kinase. Furthermore, GSC4M augmented the DNA-binding activity of the transcription factors AP-1, NF-IL6, and NF-kappaB, all of which are involved in the transcriptional activation of the IL-8 gene. CONCLUSION Cordyceps militaris grown on germinated soybeans stimulates lung epithelial cells to produce IL-8 through lipid rafts formation and signaling pathways via ERK and JNK.

Armillariella mellea induces maturation of human dendritic cells without induction of cytokine expression.

ETHNOPHARMACOLOGICAL RELEVANCE Armillariella mellea, an edible and medicinal mushroom possessing immuno-modulating potential, has been frequently used for the treatment of infectious diseases or cancers. AIM OF THE STUDY In order to elucidate immune-regulatory mechanisms of Armillariella mellea, we investigated the effect of water-soluble components from Armillariella mellea (AME) on the regulation of human dendritic cell (DC) maturation and activation. MATERIALS AND METHODS Immature DCs (iDCs) were prepared by differentiating human peripheral blood CD14-positive cells with GM-CSF and IL-4. Then, iDCs were treated with AME at 2-20 microg/ml for 48 h and subjected to flow cytometry to analyze the expression of DC markers. Dextran-FITC uptake assay and enzyme-linked immunosorbent assay were performed to examine the endocytic capacity of AME-stimulated DC and their production of cytokines, respectively. RESULTS iDCs stimulated with AME showed representative features during DC maturation such as up-regulated expression of CD80, CD83, CD86, both MHC class I and II molecules, and CD205, with a simultaneous decrease in the expression of CD206 and the endocytic capacity. Interestingly, AME was not able to induce the production of TNF-alpha, IL-12p40, or IL-10, whereas lipopolysaccharides induced a substantial increase of all of the cytokines. CONCLUSION Armillariella mellea induces maturation of human DCs through a unique mechanism without inducing cytokine expression.

Bacterial flagellin induces IL-6 expression in human basophils

Binding of allergen to IgE on basophils positively affects allergic inflammation by releasing inflammatory mediators. Recently, basophils were shown to express pattern-recognition receptors, such as toll-like receptors (TLRs), for recognizing microbe-associated molecular patterns (MAMPs) that are independent of allergen-IgE binding. In this study, we investigated whether MAMP alone can induce IL-6 production in a human basophil cell line, KU812. Stimulation with flagellin in the absence of allergen-IgE association induced IL-6 expression in KU812 cells, while stimulation with lipoteichoic acid, peptidoglycan, or poly I:C did not under the same condition. Flagellin-induced IL-6 expression was also observed in human primary basophils. Flow cytometric analysis showed that KU812 cells expressed flagellin-recognizing TLR5 both on the cell surface and in the cytoplasm while TLR2 and TLR3 were observed only in the cytoplasm. We further demonstrated that although flagellin augmented the phosphorylation of mitogen-activated protein kinases including p38 kinase, ERK, and JNK, flagellin-induced IL-6 production was attenuated by inhibitors for p38 kinase and ERK, but not by JNK inhibitors. In addition, flagellin enhanced phosphorylation of signaling molecules including CREB, PKCδ, and AKT. The inhibitors for PKA and PKC also showed inhibitory effects. Interestingly, flagellin-induced IL-6 production was further enhanced by pretreatment with inhibitors for PI3K, implying that PI3K negatively affects the flagellin-induced IL-6 production. Furthermore, DNA binding activities of NF-κB, AP-1, and CREB, which play pivotal roles in the induction of IL-6 gene expression, were increased by flagellin. These results suggest that flagellin alone is sufficient to induce IL-6 gene expression via TLR5 signaling pathways in human basophils.

Enterococcus faecalis lipoteichoic acid suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in human periodontal ligament cells.

Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.

Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus.

A Gram-positive bacterium, Staphylococcus aureus is known to be one of the major pathogenic bacteria responsible for causing bovine mastitis. Among the various cell wall components of S. aureus, lipoteichoic acid (LTA) and peptidoglycan (PGN) are closely associated with inflammatory responses. However, the role of LTA and PGN derived from S. aureus in bovine mastitis has not been clearly elucidated. In this study, we characterized the gene expression profile of a bovine mammary gland epithelial cell line, MAC-T cells, in the presence of LTA and PGN from S. aureus. LTA plus PGN, but not LTA or PGN alone, activated MAC-T cells. The analysis of transcriptional profiles using an Affymetrix genechip microarray showed that stimulation with LTA plus PGN produced a total of 2019 (fold change >1.2) differentially expressed genes (DEGs), with 801 up-regulated genes and 1218 down-regulated genes. Of the up-regulated genes, 14 inflammatory mediator-related DEGs, 22 intra-cellular signaling molecule-related DEGs, and 15 transcription factor-related DEGs were observed, whereas among the down-regulated DEGs 17 inflammation-related DEGs were found. The microarray results were confirmed using real-time RT-PCR of 18 genes with substantial changes in expression (9 each from the up-regulated and down-regulated DEGs). These results provide a comprehensive analysis of gene-expression profiles elicited by S. aureus LTA and PGN in MAC-T cells, contributing to an understanding of the pathogenesis for S. aureus-induced bovine mastitis.