Staphylococcus epidermidis polysaccharide intercellular adhesin activates complement

Staphylococcus epidermidis is a frequent cause of nosocomial infections. The central virulence factor of S. epidermidis is biofilm formation. Polysaccharide intercellular adhesin (PIA) constitutes the major biofilm matrix-component. PIA and biofilm have been implicated in S. epidermidis evasion of host immune defence. We examined the effects of S. epidermidis PIA on the inflammatory response with focus on complement activation. We used a human whole-blood ex vivo model of infection and compared the effects of a PIA-positive S. epidermidis strain (SE1457) and its PIA-negative isogenic mutant (M10). The independent effect of purified PIA on complement activation was investigated. In glucose-rich media, the mutant formed a proteinacious DNA-rich biofilm, whereas SE1457 formed a thick PIA-biofilm. In biofilm growth, SE1457 induced a stronger activation of the complement system compared with M10. We verified that purified PIA was independently responsible for a strong activation of the complement system. In contrast, M10 induced higher granulocyte activation by expression of CD11b and higher secretion of cytokines. We conclude that PIA has potent pro-inflammatory properties by activating the complement system. However, in a complex balance of the immune response, the decreased activation of granulocytes and cytokines by a PIA biofilm may limit host eradication of S. epidermidis.     

Dexamethasone regulation of the expression of cytokine mRNAs induced by interleukin-1 in the astrocytoma cell line U373MG

BSF-2/IL-6, GM-CSF and IL-1 beta mRNAs were induced by recombinant IL-1 in human astrocytoma cell line U373MG. The induction of BSF-2/IL-6 and IL-1 beta mRNAs did not require de novo protein synthesis while that of GM-CSF mRNA required a newly synthesized protein. Dexamethasone inhibited the induction of these cytokine mRNAs by IL-1. This process seems to require continued protein synthesis. These results suggest that the production of these cytokines are positively and negatively controlled by IL-1 and glucocorticoids, respectively, in astrocytes.     

葡萄糖类似物甲基葡萄糖对表皮葡萄球菌生物被膜形成的影响及调节机制的研究

生物被膜(Biofilm)是条件致病菌表皮葡萄球菌(Staphylococcusepidermidis)的主要致病因素,生物被膜的形成依赖多糖PIA的合成,PIA合成与细菌糖代谢相关。通过研究葡萄糖类似物甲基葡萄糖(MethylDglucoside,MG)对生物被膜的形成及相关基因表达的影响,考察生物被膜形成的调控机制并寻找抑制生物被膜形成的方法。甲基葡萄糖能抑制97337株生物被膜的形成,而且不同浓度的甲基葡萄糖对生物膜作用不同。甲基葡萄糖对97337株生物被膜形成的早期的粘附有较强的抑制作用;不同浓度的甲基葡萄糖处理后对ica和AtlE基因的mRNA表达水平影响不大,但能诱导agr基因的表达,这与甲基葡萄糖处理不同时间后的结果一致;而且甲基葡萄糖处理后97337的表面相关蛋白的组成明显改变。甲基葡萄糖对生物膜的抑制并不直接由于它对生长的抑制,它对细菌生长和生物被膜形成的抑制与其在细菌糖代谢中的竞争性相关;甲基葡萄糖能通过调控agr基因的表达改变细菌表面从而抑制97337的早期粘附和生物被膜的形成,但没有通过调控icaADBC、icaR的表达抑制生物膜的形成,可能与其对合成PIA相关糖基转移酶的竞争性抑制相关。     

Suppressed production of pro-inflammatory cytokines by LPS-activated macrophages after treatment with Toxoplasma gondii lysate

During Toxoplasma gondii infection, macrophages, dendritic cells, and neutrophils are important sources of pro-inflammatory cytokines from the host. To counteract the pro-inflammatory activities, T. gondii is known to have several mechanisms inducing down-regulation of the host immunity. In the present study, we analyzed the production of proand anti-inflammatory cytokines from a human myelomonocytic cell line, THP-1 cells, in response to treatment with T. gondii lysate or lipopolysaccharide (LPS). Treatment of THP-1 cells with LPS induced production of IL-12, TNF-alpha, IL-8, and IL-10. Co-treatment of THP-1 cells with T. gondii lysate inhibited the LPS-induced IL-12, IL-8 and TNF-alpha expression, but increased the level of IL-10 synergistically. IL-12 and IL-10 production was down-regulated by anti-human toll-like receptor (TLR)-2 and TLR4 antibodies. T. gondii lysate triggered nuclear factor (NF)-kappaB-dependent IL-8 expression in HEK293 cells transfected with TLR2. It is suggested that immunosuppression induced by T. gondii lysate treatment might occur via TLR2-mediated NF-kappaB activation.     

Cytochrome c can be released into extracellular space and modulate functions of human astrocytes in a toll-like receptor 4-dependent manner

BackgroundChronic activation of glial cells contributes to neurodegenerative diseases. Cytochrome c (CytC) is a soluble mitochondrial protein that can act as a damage-associated molecular pattern (DAMP) when released into the extracellular space from damaged cells. CytC causes immune activation of microglia in a toll-like receptor (TLR) 4-dependent manner. The effects of extracellular CytC on astrocytes are unknown. Astrocytes, which are the most abundant glial cell type in the brain, express TLR 4 and secrete inflammatory mediators; therefore, we hypothesized that extracellular CytC can interact with the TLR 4 of astrocytes inducing their release of inflammatory molecules and cytotoxins.MethodExperiments were conducted using primary human astrocytes, U118 MG human astrocytic cells, BV-2 murine microglia, and SH-SY5Y human neuronal cells.ResultsExtracellularly applied CytC increased the secretion of interleukin (IL)-1β, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-12 p70 by cultured primary human astrocytes. Anti-TLR 4 antibodies blocked the CytC-induced secretion of IL-1β and GM-CSF by astrocytes. Supernatants from CytC-activated astrocytes were toxic to human SH-SY5Y neuronal cells. We also demonstrated CytC release from damaged glial cells by measuring CytC in the supernatants of BV-2 microglia after their exposure to cytotoxic concentrations of staurosporine, amyloid-β peptides (Aβ42) and tumor necrosis factor-α.ConclusionCytC can be released into the extracellular space from damaged glial cells causing immune activation of astrocytes in a TLR 4-dependent manner.General significanceAstrocyte activation by CytC may contribute to neuroinflammation and neuronal death in neurodegenerative diseases. Astrocyte TLR 4 could be a potential therapeutic target in these diseases.     

五倍子水煎剂对表皮葡萄球菌生物膜抑制的研究

通过五倍子水煎剂对表皮葡萄球菌MIC测定和生物膜形成干预的研究,为表皮葡萄球菌引起感染提供新的治疗途径。用微量肉汤稀释法分别测定五倍子水煎剂对表皮葡萄球菌的MIC;刚果红及刚果红红霉素、五倍子水煎剂琼脂平板测定表皮葡萄球菌PIA生成与抑制;五倍子水煎剂、红霉素干预表皮葡萄球菌生物膜形成,于光镜和电镜下观察其生物膜形态。134株表皮葡萄球菌五倍子水煎剂的MIC50为0.488 mg/mL,MIC90为0.977 mg/mL。134株表皮葡萄球菌中有50株为PIA阳性,PIA阳性的50株菌全部产生生物膜,红霉素对表皮葡萄球菌生物膜形成有抑制,而五倍子水煎剂则无。表皮葡萄球菌PIA的相互作用在其生物膜的生成中起主要作用;五倍子水煎剂对表皮葡萄球菌生长有明显的抑制但对生物膜形成无干预作用。     

Monokine modulation of human astroglial cell production of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. I. Effects of IL-1 alpha and IL-beta.

Granulocyte (G)-CSF and granulocyte-macrophage (GM)-CSF enhance phagocyte survival and function and are produced by fibroblasts and endothelial cells after induction by inflammatory mediators such as IL-1. Our ability to detect G-CSF and GM-CSF activity in the conditioned medium of the human astroglial tumor cell line, U87MG, and molecularly clone the cDNA for G-CSF from a U87MG cDNA library raised the possibility that astroglial cells are capable of G-CSF and GM-CSF production within the central nervous system; if so, the production of these CSF by astroglial cells may be inducible by IL-1. We examined the effects of IL-1 alpha and IL-1 beta on the production of G-CSF and GM-CSF by U87MG and U373MG, another astroglial tumor cell line that does not constitutively produce CSF. We demonstrate that both U87MG and U373MG can be induced to produce G-CSF and GM-CSF by exposure to IL-1 alpha and IL-1 beta. This response, measured by accumulation of increased CSF mRNA, is rapid, sensitive and due to the enhanced stability of CSF message following IL-1 exposure. The implications of these findings to the immunopathogenesis of central nervous system infections are discussed.     

Cyclic di-GMP signalling controls virulence properties of Salmonella enterica serovar Typhimurium at the mucosal lining

Cyclic di-GMP (c-di-GMP), a novel secondary signalling molecule present in most bacteria, controls transition between motility and sessility. In Salmonella enterica serovar Typhimurium ( S. typhimurium ) high c-di-GMP concentrations favour the expression of a biofilm state through expression of the master regulator CsgD. In this work, we investigate the effect of c-di-GMP signalling on virulence phenotypes of S. typhimurium. After saturation of the cell with c-di-GMP by overexpression of a di-guanylate cyclase, we studied invasion and induction of a pro-inflammatory cytokine in epithelial cells, basic phenotypes that are major determinants of S. typhimurium virulence. Elevated c-di-GMP had a profound effect on invasion into and IL-8 production by the gastrointestinal epithelial cell line HT-29. Invasion was mainly inhibited through CsgD and the extracellular matrix component cellulose, while inhibition of the pro-inflammatory response occurred through CsgD, which inhibited the secretion of monomeric flagellin. Our results suggest that transition between biofilm formation and virulence in S. typhimurium at the epithelial cell lining is mediated by c-di-GMP signalling through CsgD and cellulose expression.     

Cysteinyl Leukotriene Receptor Antagonists Inhibit Migration,Invasion, and Expression of MMP-2/9 in Human Glioblastoma

Glioblastoma is one of the most malignant and aggressive types of brain tumors. 5-lipoxygenase and cysteinyl leukotriene receptor 1 (CysLT1) play a role in human carcinogenesis. Leukotriene receptor antagonists (LTRAs), anti-asthmatic drugs with mild side effects, have anti-metastatic activity in epidermoid carcinoma, lung carcinoma, and colon cancers as well as neuroprotective effects. Herein, anti-migratory effects of two LTRAs, montelukast and zafirlukast, were investigated in glioblastoma cells. The level of CysLT1 in A172 cells was increased by 3.13 folds after IL-1β treatment. The median toxic concentration of LTRAs in A172, U373, and primary astrocytes ranged from 7.17 to 26.28 μM at 24-h post-exposure. Both LTRAs inhibited migration and invasion of glioma. Additionally, both drugs significantly inhibited the expression and activities of MMP-2 and MMP-9 in A172 and U373 glioblastoma cells and primary human astrocytes, suggesting that CysLT1 plays a role in migration and invasion of glioma, and LTRAs are potential drugs to reduce migration and invasion.     

Interleukin-6-Mediated Autocrine Growth Promotion in Human Glioblastoma Multiforme Cell Line U87MG

Abstract: Human glioblastoma multiforme cell lines, brain tumor biopsy tissue, and normal human fetal brain synthesize interleukin (IL)-6 and IL-6 receptor (IL-6R). Neither of these is expressed in human neurons or neuroblastoma cell lines in culture. Astrocytes from fetal brain grown in culture retain the ability to synthesize IL-6 but do not express IL-6R as inferred from RT-PCR and Southern blot studies. Coexpression of IL-6 and IL-6R in the glioblastoma cell line U87MG is confirmed by immunofluorescence staining. Both specific monoclonal antibodies against IL-6 and IL-6R and antisense oligonucleotide to IL-6 mRNA inhibit the growth of U87MG cells in culture, suggesting the existence of a functional autocrine growth loop. Anti-IL-6 antibodies also inhibit the growth of glioblastoma cell lines U373 and U118. The expression of IL-6 by human fetal astrocytes in culture is highly suggestive of its role as an oncofetal protein responsible for rapid proliferation of fetal and tumor cells but not cells of adult brain.     

Requirement of Rab21 in LPS-induced TLR4 signaling and pro-inflammatory responses in macrophages and monocytes

Lipopolysaccharide (LPS) induces macrophage/monocyte activation and pro-inflammatory cytokines production by activating Toll-like receptor 4 (TLR-4) signaling. Rab GTPase 21 (Rab21) is a member of the Rab GTPase subfamily. In the present study, we show that LPS induced TLR4 and Rab21 association and endosomal translocation in murine bone marrow–derived macrophages (BMDMs) and primary human peripheral blood mononuclear cells (PBMCs). In BMDMs, shRNA-mediated stable knockdown of Rab21 inhibited LPS-induced expression and production of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α). Conversely, forced overexpression of Rab21 by an adenovirus construct potentiated LPS-induced IL-1β, IL-6 and TNF-α production in BMDMs. Further studies show that LPS-induced TLR4 endosomal traffic and downstream c-Jun and NFκB (nuclear factor-kappa B) activation were significantly inhibited by Rab21 shRNA, but intensified with Rab21 overexpression in BMDMs. Finally, in the primary human PBMCs, siRNA-induced knockdown of Rab21 significantly inhibited LPS-induced IL-1β, IL-6 and TNF-α production. Taken together, we suggest that Rab21 regulates LPS-induced pro-inflammatory responses by promoting TLR4 endosomal traffic and downstream signaling activation.     

ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus

Recent progress in elucidating the role of the icaADBC-encoded polysaccharide intercellular adhesin (PIA) or polymeric N-acetyl-glucosamine (PNAG) in staphylococcal biofilm development has in turn contributed significantly to our understanding of the pathogenesis of device-related infections. Nevertheless, our understanding of how the ica locus and PIA/PNAG biosynthesis are regulated is far from complete and many questions remain. Moreover, beyond ica, evidence is now emerging for the existence of ica-independent biofilm mechanisms in both Staphylococcus aureus and Staphylococcus epidermidis. Teichoic acids, which are a major carbohydrate component of the S. epidermidis biofilm matrix and the major cell wall autolysin, play an important role in the primary attachment phase of biofilm development, whereas the cell surface biofilm-associated protein and accumulation-associated protein are capable of mediating intercellular accumulation. These findings raise the exciting prospect that other surface proteins, which typically function as antigenic determinants or in binding to extracellular matrix proteins, may also act as biofilm adhesins. Given the impressive array of surface proteins expressed by S. aureus and S. epidermidis, future research into their potential role in biofilm development either independent of PIA/PNAG or in cooperation with PIA/PNAG will be of particular interest.     

Interferon gamma stimulates beta-secretase expression and sAPPbeta production in astrocytes

Neurons, but not astrocytes, are known as the major source of Abeta, because astrocytes express low levels of putative beta-secretase (BACE). Astrocytes near senile plaque cores show enhanced levels of BACE protein expression, however, suggesting that astrocytes can contribute to Abeta production under pathological conditions. To investigate factors that stimulate BACE protein expression in astrocytes, we tested the effects of interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) on BACE protein expression in U373MG astrocytoma cells and primary astrocyte cultures from Tg2576 mouse brains. BACE protein expression and sAPPbeta production were dramatically increased, without changes in holo APP levels, following IFN-gamma treatment in both cell types. AG490, which is a blocker of IFN-gamma-induced STAT signaling, decreased IFN-gamma-induced BACE protein expression and sAPPbeta production in a dose-dependent manner. These results show that astrocytes are capable of expressing BACE and producing sAPPbeta in response to certain stimulating factors, and IFN-gamma is one such factor.     

Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system

The skin commensal and opportunistic pathogen Staphylococcus epidermidis is the leading cause of nosocomial and biofilm-associated infections. Little is known about the mechanisms by which S. epidermidis protects itself against the innate human immune system during colonization and infection. We used scanning electron microscopy to demonstrate that the exopolysaccharide intercellular adhesin (PIA) resides in fibrous strands on the bacterial cell surface, and that lack of PIA production results in complete loss of the extracellular matrix material that has been suggested to mediate immune evasion. Phagocytosis and killing by human polymorphonuclear leucocytes was significantly increased in a mutant strain lacking PIA production compared with the wild-type strain. The mutant strain was also significantly more susceptible to killing by major antibacterial peptides of human skin, cationic human beta-defensin 3 and LL-37, and anionic dermcidin. PIA represents the first defined factor of the staphylococcal biofilm matrix that protects against major components of human innate host defence.     

Serotonin via 5-HT7 receptors activates p38 mitogen-activated protein kinase and protein kinase C epsilon resulting in interleukin-6 synthesis in human U373 MG astrocytoma cells

Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neurotransmitter which is involved in neuroimmunomodulatory processes. Previously, it has been demonstrated that 5-HT may induce interleukin (IL)-6 expression in primary rat hippocampal astrocytes. The present study was undertaken to investigate the molecular pathways underlying this induction of IL-6 synthesis. As a model system, we used the human astrocytoma cell line U373 MG, which synthesizes IL-6 upon stimulation with various inducers. 5-HT dose- and time-dependently induced IL-6 protein synthesis. We identified several 5-HT receptors to be expressed on U373 MG cells, including the 5-HT1D, 5-HT2A, 5-HT3 and 5-HT7 receptors. In this report, we show that the 5-HT-induced IL-6 release is mediated by the 5-HT7 receptor based on several agonist/antagonists that were used. 5-HT-induced IL-6 synthesis is inhibited by the partially selective 5-HT7 receptor antagonist, pimozide, and the selective antagonist SB269970. Furthermore, IL-6 synthesis was induced by the 5-HT7 receptor agonist carboxamidotryptamin. In addition, we found p38 MAPKs and protein kinase C (PKC) epsilon to be involved in 5-HT-induced IL-6 synthesis as specific inhibitors of these enzymes (SB202190 and RO-31-8425, respectively) blocked 5-HT-induced IL-6 synthesis. Furthermore, 5-HT mediated the phosphorylation of both p38 MAPK as well as the PKC epsilon isoform. The p42/44 MAPKs, however, were not involved in 5-HT-induced IL-6 synthesis. This study shows, for the first time, a central role of 5-HT7 receptor linked to p38 MAPK and PKC epsilon for the induction of cytokine synthesis in astrocytic cells.     

Herbal melanin activates TLR4/NF-κB signaling pathway

Expression of many pro-inflammatory cytokines is controlled by the NF-κB signaling pathway. NF-κB is induced by LPS through activation of TLR4. Melanins extracted from fungal, plant and human sources modulate cytokine production and activate NF-κB pathway. We showed that a herbal melanin (HM) from Nigella sativa L. modulates cytokine production and suggested it as a ligand for TLR4. In this study we investigated the possibility that the HM-induced cytokine production is via an NF-κB signaling pathway. We found that HM induced the degradation of IκBα, a key step in the activation of NF-κB. Moreover, addition of IκB kinase (IKK) specific inhibitors effectively inhibited the observed HM-induced production of IL-8 and IL-6 by TLR4-transfected HEK293 cells and THP-1 cells. Our results have also shown that HM induced cleavage of caspase 8, and that this cleavage was partially abrogated by IKK inhibitors. We suggest that HM can modulate the inflammatory response by inducing IL-8 and IL-6 production via TLR4-dependent activation of the NF-κB signaling pathway.     

Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells

The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.