Kuo-Feng Hua

Extract of Reishi Polysaccharides Induces Cytokine Expression via TLR4-Modulated Protein Kinase Signaling Pathways

We have demonstrated that an extract of Ganoderma lucidum (Reishi or Ling-Zhi) polysaccharides (EORP) exerts immunomodulating activities by stimulating the expression of inflammatory cytokines from mouse spleen cells. Interestingly, via responding to LPS in genetic variation of murine macrophage HeNC2 and GG2EE cell lines, and using TLR4 Ab blockage in human blood-derived monocytic macrophages, we have found that the TLR4, but not complement receptor type 3, is a putative receptor of EORP, mediating the consequent immunomodulating events associated with IL-1 gene expression. Based on our studies of reactive oxygen species production, polymyxin B inhibition, and protein tyrosine kinase (PTK) activity, we ruled out the possibility of LPS contamination in EORP. We have found that EORP differentially modulates the protein kinase (PK)-mediated signal transduction pathways associated with inflammatory cytokine IL-1. In human macrophages and murine macrophage J774A.1 cells, EORP was found to up-regulate IL-1 secretion and pro-IL-1 (precursor of IL-1) as well as IL-1-converting enzyme expression. Specifically, EORP rapidly stimulates PTK-mediated phosphorylation, followed by induction of PKs and activation of MAPKs: ERK, JNK, and p38. Using PK inhibitors in the kinase activity assays, Western blot analyses and IL-1 ELISA, we have extensively examined and dissected the role of individual PK in the regulation of pro-IL-1/IL-1. Our findings establish that EORP-mediated signaling pathways are involved in the pro-IL-1/IL-1 regulation: PTK/protein kinase C/MEK1/ERK and PTK/Rac1/p21-activated kinase/p38.

Osthole regulates inflammatory mediator expression through modulating NF-κB, mitogen-activated protein kinases, protein kinase C, and reactive oxygen species.

Osthole, a coumarin compound, has been reported to exhibit various biological activities; however the cellular mechanism of its immune modulating activity has not yet been fully addressed. In this study we isolated osthole from the seeds of Cnidium monnieri and demonstrated that osthole inhibited TNF-α, NO and COX-2 expression in LPS-stimulated macrophages, without reducing the expression of IL-6. Furthermore, the phosphorylation of p38, JNK1/2, PKC-α and PKC-ε induced by LPS was inhibited by osthole; however, the phosphorylation of ERK1/2 and PKC-δ was not reduced by osthole. Osthole also inhibited NF-κB activation and ROS release in LPS-stimulated macrophages. Our current results indicated that osthole is the major anti-inflammatory ingredient of Cnidium monnieri seed ethanol extract.

Anti-Inflammatory Bioactivities of Honokiol through Inhibition of Protein Kinase C, Mitogen-Activated Protein Kinase, and the NF-κB Pathway To Reduce LPS-induced TNFα and NO Expression

Much recent research has demonstrated that honokiol, a phenolic compound originally isolated from Magnolia officinalis, has potent anticancer activities; however, the detailed molecular mechanism of its anti-inflammatory activity has not yet been fully addressed. In this study we demonstrated that honokiol inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha secretion in macrophages, without affecting the activity of the tumor necrosis factor-alpha converting enzyme. At the same time, honokiol not only inhibited nitric oxide expression in LPS-stimulated murine macrophages but also inhibited the LPS-induced phosphorylation of ERK1/2, JNK1/2, and p38. By means of confocal microscope analysis we demonstrated that phosphorylation and membrane translocation of protein kinase C-alpha, as well as NF-kappaB activation, were inhibited by honokiol in LPS-stimulated macrophages. Furthermore, it was found that honokiol neither antagonizes the binding of LPS to cells nor alters the cell surface expression of toll-like receptor 4 and CD14. Our current results have exhaustively described the anti-inflammatory properties of honokiol, which could lead to the possibility of its future pharmaceutical application in the realm of immunomodulation.

Heme oxygenase-1 mediates the anti-inflammatory effect of Curcumin within LPS-stimulated human monocytes†

Curcumin, a polyphenolic compound derived from plant, regulates heme oxygenase (HO‐1) expression within certain cell types; however, the Curcumin‐mediated signal transduction in the regulation of HO‐1 expression within human monocytes/macrophages is unclear. Herein, we show that Curcumin dose dependently induced HO‐1 expression and HO‐1 activity through the activation of PKCα, PKCδ/ERK1/2, p38α, and PI3‐kinase. In addition, H2O2 release is essential for Curcumin‐mediated ERK1/2 and p38 phosphorylation and HO‐1 expression. Further, Curcumin inhibited LPS‐induced IL‐1 and IL‐6 secretion and blockage of HO‐1 expression/activity by HO‐1 siRNA or HO‐1 inhibitor, SnPP reversed the inhibitory effects of Curcumin on cytokines secretion. HO‐1 over‐expression produced the same inhibitory effects of Curcumin on IL‐1 secretion. Collectively, our results suggest that Curcumin inhibits cytokines secretion within LPS‐stimulated monocytes through a mechanism that involves the action of HO‐1. J. Cell. Physiol. 215: 603–612, 2008.

Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression

We have previously reported that a well‐characterized glycoprotein fraction containing fucose residues in an extract of Ganoderma lucidum polysaccharides (EORP) exerts certain immuno‐modulation activity by stimulating the expression of inflammatory cytokines via TLR4. Continuing our studies, we have demonstrated that EORP increases the surface expression of CD14 and TLR4 within murine macrophages J774A.1 cells in vitro, and further promotes LPS binding and uptake by J774A.1 cells in a CD14‐dependent fashion. Moreover, we observed the co‐localization of internalized LPS with lysosome‐ and Golgi‐apparatus markers within 5 min after J774A.1 cells stimulated with LPS. In addition, EORP pretreatment of J774A.1 cells and human blood‐derived primary macrophages, followed by LPS stimulation, results in the super‐induction of interleukin‐1beta (IL‐1) expression. Endocytosis inhibitors: such as cytochalasin D and colchicine effectively block EORP‐enhanced LPS internalization by J774A.1 cells; yet they fail to decrease the LPS‐induced phosphorylation of certain mitogen‐activated protein kinases, and IL‐1 mRNA and proIL‐1 protein expression, indicating that LPS internalization by J774A.1 cells is not associated with LPS‐dependent activation. Our current results could provide a potential EORP‐associated protection mechanism for bacteria infection by enhancing IL‐1 expression and the clearance of contaminated LPS by macrophages. J. Cell. Physiol. 212: 537–550, 2007.

A novel exopolysaccharide from the biofilm of Thermus aquaticus YT-1 induces the immune response through Toll-like receptor 2.

Bacterial polysaccharides are known to induce the immune response in macrophages. Here we isolated a novel extracellular polysaccharide from the biofilm of Thermus aquaticus YT-1 and evaluated its structure and immunomodulatory effects. The size of this polysaccharide, TA-1, was deduced by size-exclusion chromatography as 500 kDa. GC-MS, high performance anion-exchange chromatography with pulsed amperometric detection, electrospray ionization-MS/MS, and NMR revealed the novel structure of TA-1. The polysaccharide is composed of tetrasaccharide-repeating units of galactofuranose, galactopyranose, and N-acetylgalactosamine (1:1:2) and lacked acidic sugars. TA-1 stimulated macrophage cells to produce the cytokines TNF-α and IL-6. Screening of Toll-like receptors and antibody-blocking experiments indicated that the natural receptor of TA-1 in its immunoactivity is TLR2. Recognition of TA-1 by TLR2 was confirmed by TA-1 induction of IL-6 production in peritoneal macrophages from wild-type mice but not from TLR2−/− mice. TA-1, as a TLR2 agonist, could possibly be used as an adjuvant and could enhance cytokine release, which increases the immune response. Furthermore, TA-1 induced cytokine release is dependent on MyD88/TIRAP.

Synthesis and Biological Evaluation of Polyenylpyrrole Derivatives as Anticancer Agents Acting through Caspases-Dependent Apoptosis

A class of polyenylpyrroles and their analogues were designed from a hit compound identified in a fungus. The compounds synthesized were evaluated for their cell cytotoxicity against human non-small-cell lung carcinoma cell lines A549. Two compounds were found to exhibit high cytotoxicity against A549 cells with IC(50) of 0.6 and 0.01 μM, respectively. The underlying mechanisms for the anticancer activity were demonstrated as caspases activation dependent apoptosis induction through loss of mitochondrial membrane potential, release of cytochrome c, increase in B-cell lymphoma-2-associated X protein (Bax) level, and decrease in B-cell lymphoma-2 (Bcl-2) level. The two compounds were nontoxic to normal human lung Beas-2b cells (IC(50) > 80 μM), indicating that they are highly selective in their cytotoxicity activities. Furthermore, one compound showed in vivo anticancer activity in human-lung-cancer-cell-bearing mice. These results open promising insights on how these conjugated polyenes mediate cytotoxicity and may provide a molecular rationale for future therapeutic interventions in carcinogenesis.

Immunostimulatory bioactivity of algal polysaccharides from Chlorella pyrenoidosa activates macrophages via toll-like receptor 4.

Much research suggests that a dietary supplement of Chlorella pyrenoidosa may be helpful to human health, but the molecular mechanism involved remains unclear. The aim of this research was to investigate the effects of certain hot-water-soluble polysaccharides from Chlorella pyrenoidosa (CWSP) on cytokine production, human leukocyte antigen (HLA) expression, and costimulatory molecule expression in macrophages. We demonstrated that CWSP induced IL-1beta secretion in macrophages via Toll-like receptor 4 (TLR4) mediated protein kinase signaling pathways. In addition, CWSP also stimulated the cell surface expression of HLA-DA, -DB, and -DC, and HLA-DR, -DP, and -DQ as well as the expression of costimulatory family molecules such as CD80 and CD86 in macrophages. Furthermore, we demonstrated that preinjection of C57BL/6J mice with CWSP increased lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha and IL-1beta secretion into serum in vivo. This outcome was consistent with the corresponding outcome for cells treated with CWSP in vitro. Our current results provide support for the possible use of CWSP as a modulation agent of immune responses in humans and certain animal species. Finally, in using GC-MS to analyze the polysaccharides, we found that the major monosaccharides of CWSP were rhamnose (31.8%), glucose (20.42%), galactose (10.28%), mannose (5.23%), and xylose (1.27%). This study is the first to report the molecular mechanism of immune-modulated signal transduction in vitro from the polysaccharides of Chlorella pyrenoidosa.

Reishi immuno-modulation protein induces interleukin-2 expression via protein kinase-dependent signaling pathways within human T cells†

Ganoderma lucidum, a medicinal fungus is thought to possess and enhance a variety of human immune functions. An immuno‐modulatory protein, Ling Zhi‐8 (LZ‐8) isolated from G. lucidum exhibited potent mitogenic effects upon human peripheral blood lymphocytes (PBL). However, LZ‐8‐mediated signal transduction in the regulation of interleukin‐2 (IL‐2) gene expression within human T cells is largely unknown. Here we cloned the LZ‐8 gene of G. lucidum, and expressed the recombinant LZ‐8 protein (rLZ‐8) by means of a yeast Pichia pastoris protein expression system. We found that rLZ‐8 induces IL‐2 gene expression via the Src‐family protein tyrosine kinase (PTK), via reactive oxygen species (ROS), and differential protein kinase‐dependent pathways within human primary T cells and cultured Jurkat T cells. In essence, we have established the nature of the rLZ‐8‐mediated signal‐transduction pathways, such as PTK/protein kinase C (PKC)/ROS, PTK/PLC/PKCalpha/ERK1/2, and PTK/PLC/PKCalpha/p38 pathways in the regulation of IL‐2 gene expression within human T cells. Our current results of analyzing rLZ‐8‐mediated signal transduction in T cells might provide a potential application for rLZ‐8 as a pharmacological immune‐modulating agent. J. Cell. Physiol. 215: 15–26, 2008.

Structure and Immunological Characterization of the Capsular Polysaccharide of a Pyrogenic Liver Abscess Caused by Klebsiella pneumoniae ACTIVATION OF MACROPHAGES THROUGH TOLL-LIKE RECEPTOR 4

The active components of a primary pyrogenic liver abscess (PLA) Klebsiella pneumoniae in stimulating cytokine expression in macrophages are still unclear. The capsular polysaccharide (CPS) of PLA K. pneumoniae is important in determining clinical manifestations, and we have shown that it consists of repeating units of the trisaccharide (→3)-β-d-Glc-(1→4)-[2,3-(S)-pyruvate]-β-d-GlcA-(1→4)-α-l-Fuc-(1→) and has the unusual feature of extensive pyruvation of glucuronic acid and acetylation of C2-OH or C3-OH of fucose. We demonstrated that PLA K. pneumoniae CPS induces secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by macrophages through Toll-like receptor 4 (TLR4) and that this effect was lost when pyruvation and O-acetylation were chemically destroyed. Furthermore, expression of TNF-α and IL-6 in PLA K. pneumoniae CPS-stimulated macrophages was shown to be regulated by the TLR4/ROS/PKC-δ/NF-κB, TLR4/PI3-kinase/AKT/NF-κB, and TLR4/MAPK signaling pathways.