Serotype‐dependent expression patterns of stabilized lipopolysaccharide aggregates in Aggregatibacter actinomycetemcomitans strains

Above a critical concentration, amphiphilic lipopolysaccharide (LPS) molecules in an aqueous environment form aggregate structures, probably because of interactions involving hydrophobic bonds. Ionic bonds involving divalent cations stabilize these aggregate structures, making them resistant to breakdown by detergents. The aim of this study was to examine expression patterns of stabilized LPS aggregates in Aggregatibacter actinomycetemcomitans, a microorganism that causes periodontitis. A. actinomycetemcomitans strains of various serotypes and truncated LPS mutants were prepared for this study. Following treatment with a two‐phase separation system using the detergent Triton X‐114, crude LPS extracts of the study strains were separated into detergent‐phase LPS (DP‐LPS) and aqueous‐phase LPS (AP‐LPS). Repeated treatment of the aqueous phase with the two‐phase separation system produced only a slight decrease in AP‐LPS, suggesting that AP‐LPS was resistant to the detergent and thus distinguishable from DP‐LPS. The presence of divalent cations increased the yield of AP‐LPS. AP‐LPS expression patterns were serotype‐dependent; serotypes b and f showing early expression, and serotypes a and c late expression. In addition, highly truncated LPS from a waaD (rfaD) mutant were unable to generate AP‐LPS, suggesting involvement of the LPS structure in the generation of AP‐LPS. The two‐phase separation was able to distinguish two types of LPS with different physical states at the supramolecular structure level. Hence, AP‐LPS likely represents stabilized LPS aggregates, whereas DP‐LPS might be derived from non‐stabilized aggregates. Furthermore, time‐dependent expression of stabilized LPS aggregates was found to be serotype‐dependent in A. actinomycetemcomitans.     

Aggregatibacter actinomycetemcomitans biofilm killing by a targeted ciprofloxacin prodrug

A pH-sensitive ciprofloxacin prodrug was synthesized and targeted against biofilms of the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). The dose required to reduce the viability of a mature biofilm of Aa by ～80% was in the range of ng?cm^?2 of colonized area (mean biofilm density 2.33?×?10⁹?cells?cm^?2). A mathematical model was formulated that predicts the temporal change in the concentration of ciprofloxacin in the Aa biofilm as the drug is released and diffuses into the bulk medium. The predictions of the model were consistent with the extent of killing obtained. The results demonstrate the feasibility of the strategy to induce mortality, and together with the mathematical model, provide the basis for design of targeted antimicrobial prodrugs for the topical treatment of oral infections such as periodontitis. The targeted prodrug approach offers the possibility of optimizing the dose of available antimicrobials in order to kill a chosen pathogen while leaving the commensal microbiota relatively undisturbed.     

Characterization of a quinol peroxidase mutant in Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is an oral pathogen causing localized aggressive periodontitis (LAP). Recently, we characterized for the first time a quinol peroxidase (QPO) that catalyzes peroxidase activity using quinol in the respiratory chain of A. actinomycetemcomitans for the reduction of hydrogen peroxide. In the present study, we characterized the phenotype of a QPO null mutant. The QPO null mutant shows an oxidative stress phenotype, suggesting that QPO plays a certain role in scavenging endogenously generated reactive oxygen species. Notably, we discovered that the QPO null mutant exhibits a production defect of leukotoxin (LtxA), which is a secreted bacterial toxin and is known to target human leukocytes and erythrocytes. This result suggests that QPO would be considered as a potential drug target to inhibit the expression of LtxA from A. actinomycetemcomitans for the treatment and prevention of LAP.     

Nitric oxide production by a murine macrophage cell line (RAW264.7 cells) stimulated with Aggregatibacter actinomycetemcomitans surface-associated material

Nitric oxide (NO) may play a crucial role in the pathogenesis of periodontal disease and, hence, the aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans surface-associated material (SAM) stimulates inducible nitric oxide synthase (iNOS) activity and NO production by the murine macrophage cell line RAW264.7. Cells were stimulated with untreated or heat-treated A. actinomycetemcomitans SAM and with or without pre-treatment with l-N⁶-(1-Iminoethyl)-lysine (L-NIL) (an iNOS inhibitor), polymyxin B, interferon-gamma (IFN-γ) and Interleukin-4 (IL-4), IL-10, genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], bromophenacyl bromide (BPB) [a phospholipase A₂ (PLA2) inhibitor] or wortmannin [phosphatidylinositol 3-kinase (PI-3K) inhibitor]. The iNOS activity and nitrite production in the cell cultures were determined. Untreated but not heat-treated A. actinomycetemcomitans SAM-stimulated both iNOS activity and nitrite production in RAW264.7 cells. l-NIL, IL-4, IL-10, genistein, bisindolylmaleimide, or BPB, suppressed but IFN-γ enhanced both iNOS activity and nitrite production by A. actinomycetemcomitans SAM-stimulated cells. Wortmannin and polymyxin B failed to alter both iNOS activity or nitrite production by A. actinomycetemcomitans SAM treated cells. Therefore, the present study suggests that a heat-sensitive protein constituent(s) of A. actinomycetemcomitans SAM stimulates both iNOS activity and nitrite production by RAW264.7 cells in a cytokine, PTK, PKC, and PLA₂ but not PI-3K-dependent fashion.     

基于动脉管壁的脂联素球状域保护机理

脂肪细胞分泌产物脂联素(adiponectin,APN)的发现是脂肪内分泌学研究领域的重大进展。它主要通过与相应受体结合,发挥相应的生物学效应,且其心血管保护作用目前已成为研究热点。动脉管壁上也存在其受体,在此基础上,将APN活性区域的脂联素球状域(globular domain of adiponectin,gAd)设计为新靶点,研究其对动脉管壁的保护作用及其相关机理,将为动脉粥样硬化疾病的防治提供新方案。     

Access to gram scale amounts of functional globular adiponectin from E. coli inclusion bodies by alkaline-shock solubilization

The adipose tissue derived protein adiponectin exerts anti-diabetic, anti-inflammatory and anti-atherosclerotic effects. Adiponectin serum concentrations are in the microgram per milliliter range in healthy humans and inversely correlate with obesity and metabolic disorders. Accordingly, raising circulating adiponectin levels by direct administration may be an intriguing strategy in the treatment of obesity-related metabolic disorders. However production of large amounts of recombinant adiponectin protein is a primary obstacle so far.Here, we report a novel method for large amount production of globular adiponectin from E. coli inclusion bodies utilizing an alkaline-shock solubilization method without chaotropic agents followed by precipitation of the readily renaturing protein. Precipitation of the mildly solubilized protein capitalizes on advantages of inclusion body formation. This approach of inclusion body protein recovery provides access to gram scale amounts of globular adiponectin with standard laboratory equipment avoiding vast dilution or dialysis steps to neutralize the pH and renature the protein, thus saving chemicals and time. The precipitated protein is readily renaturing in buffer, is of adequate purity without a chromatography step and shows biological activity in cultured MCF7 cells and significantly lowered blood glucose levels in mice with streptozotocin induced type 1 diabetes.     

Purification of a fibrolast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

Abstract A factor showing inhibitory activity against human gingival fibrolasts was extracted from the cytosol fraction of Actinobacillus actinomycetemcomitans Y4. The activity markedly inhibited the proliferation of human gingival fibrolasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemcomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.     

Inhibitory effect of galectin-3 on the cytokine-inducing activity of periodontopathic Aggregatibacter actinomycetemcomitans endotoxin in splenocytes derived from mice

Galectins, a family of animal lectins, are involved not only in development and differentiation but also in immunoregulation and host–pathogen interactions. Galectin-3 interacts with lipopolysaccharides in gram-negative bacteria such as Escherichia coli, Salmonella minnesota and Pseudomonas aeruginosa . The present study investigated whether galectin-3 inhibited the cytokine-inducing activity of periodontopathic bacterial lipopolysaccharides using splenocytes derived from mice of different ages. Lipopolysaccharides were extracted from Aggregatibacter actinomycetemcomitans Y4 and Porphyromonas gingivalis ATCC 33277, and then purified. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that galectin-3 adhered to A. actinomycetemcomitans lipopolysaccharides, but not to the lipopolysaccharides of P. gingivalis . Splenocytes were prepared from 1- or 7-month-old C57BL/6 mice. Either A. actinomycetemcomitans lipopolysaccharides (200 ng mL⁻¹) alone or lipopolysaccharides and murine galectin-3 (10 μg mL⁻¹) were added to culture solutions, and the release of interleukin-6 (IL-6) and interferon-γ (IFNγ) from splenocytes was measured by ELISA after a 17-h incubation. In all mice tested, A. actinomycetemcomitans lipopolysaccharide stimulation significantly increased the production of IL-6 and IFNγ ( P <0.01). Murine galectin-3 suppressed lipopolysaccharide-induced cytokine production in the splenocytes of the 1-month-old mice ( P <0.02 for IL-6; P <0.05 for IFNγ), but not in the splenocytes of the 7-month-old mice. This suggests that responses change with age.     

Modulation of peroxisome proliferator-activated receptor-alpha activity by N-acetyl cysteine attenuates inhibition of oligodendrocyte development in lipopolysaccharide stimulated mixed glial cultures

Glial cells secrete proinflammatory mediators in the brain in response to exogenous stimuli such as infection and injury. Previously, we documented that systemic maternal lipopolysaccharide (LPS)-exposure at embryonic gestation day 18 causes oligodendrocyte (OL)-injury/hypomyelination in the developing brain which can be attenuated by N-acetyl cysteine (NAC; precursor of glutathione). The present study delineates the underlying mechanism of NAC-mediated attenuation of inhibition of OL development in LPS-stimulated mixed glial cultures. Factors released by LPS-stimulated mixed glial cultures inhibited OL development as shown by decrease in both proliferation 3bromo-deoxyuridine+/chondroitin sulfate proteoglycan-NG2+, hereafter BrdU+/NG+ and differentiation (O4+ and myelin basic protein+) of OL-progenitors. Correspondingly, an impairment of peroxisomal proliferation was shown by a decrease in the level of peroxisomal proteins in the developing OLs following exposure to LPS-conditioned media (LCM). Both NAC and WY14643, a peroxisome proliferator-activated receptor (PPAR)-alpha agonist attenuated these LCM-induced effects in OL-progenitors. Similar to WY14643, NAC attenuated LCM-induced inhibition of PPAR-alpha activity in developing OLs. Studies conducted with cytokines and diamide (a thiol-depleting agent) confirmed that cytokines are active agents in LCM which may be responsible for inhibition of OL development via peroxisomal dysfunction and induction of oxidative stress. These findings were further corroborated by similar treatment of developing OLs generated from PPAR-alpha(-/-) and wild-type mice or B12 oligodendroglial cells co-transfected with PPAR-alpha small interfering RNAs/pTK-PPREx3-Luc plasmids. Collectively, these data provide evidence that the modulation of PPAR-alpha activity, thus peroxisomal function by NAC attenuates LPS-induced glial factors-mediated inhibition of OL development suggesting new therapeutic interventions to prevent the devastating effects of maternal infections.     

Interactions of adiponectin and lipopolysaccharide from Porphyromonas gingivalis on human oral epithelial cells

Background

Periodontitis is an inflammatory disease caused by pathogenic microorganisms, such as Porphyromonas gingivalis, and characterized by the destruction of the periodontium. Obese individuals have an increased risk for periodontitis and show decreased serum levels of adiponectin. This in-vitro study was established to examine whether adiponectin modulates critical effects of lipopolysaccharide (LPS) from P. gingivalis on oral epithelial cells (OECs).

Methodology/Principal Findings

The presence of adiponectin and its receptors in human gingival tissue samples and OECs was analyzed by immunohistochemistry and PCR. Furthermore, OECs were treated with LPS and/or adiponectin for up to 72 h, and the gene expression and protein synthesis of pro- and anti-inflammatory mediators, matrix metalloproteinases (MMPs) and growth factors were analyzed by real-time PCR and ELISA. Additionally, cell proliferation, differentiation and in-vitro wound healing were studied. The nuclear translocation of NFκB was investigated by immunofluorescence. Gingival tissue sections showed a strong synthesis of adiponectin and its receptors in the epithelial layer. In cell cultures, LPS induced a significant up-regulation of interleukin (IL) 1β, IL6, IL8, MMP1 and MMP3. Adiponectin abrogated significantly the stimulatory effects of LPS on these molecules. Similarly, adiponectin inhibited significantly the LPS-induced decrease in cell viability and increase in cell proliferation and differentiation. Adiponectin led to a time-dependent induction of the anti-inflammatory mediators IL10 and heme oxygenase 1, and blocked the LPS-stimulated NFκB nuclear translocation.

Conclusions/Significance

Adiponectin may counteract critical actions of P. gingivalis on oral epithelial cells. Low levels of adiponectin, as observed in obese individuals, may increase the risk for periodontal inflammation and destruction.     

Role of lipopolysaccharide on the structure and function of alpha-hemolysin from Escherichia coli

alpha-Hemolysin (HlyA) is a protein toxin (107 kDa) secreted by some pathogenic strains of E. coli. Several studies suggested the relationship between HlyA and lipopolysaccharide (LPS). We have studied experimentally the role of LPS on the stability and function of this toxin. The HlyA conformation in both, LPS-free and LPS-bound forms was investigated by tryptophan fluorescence. Studies about HlyA thermal and chemical denaturation indicated that its stability increased in the presence of LPS. On the other hand, the presence of negative and polar residues on the LPS reduced the tendency of HlyA to self-aggregation, and they may be the reservoir of calcium, cation essential for the lytic action of this toxin on red blood cells. These results suggest that HlyA and LPS are combined mainly via hydrophobic force to form an active toxin which stability is favored by the LPS.     

Synthesis and pharmacological activity of chalcones derived from 2,4,6-trimethoxyacetophenone in RAW 264.7 cells stimulated by LPS: quantitative structure-activity relationships

Inhibition of nitric oxide (NO) production by altering the expression of induced enzymes involved is potentially an important strategy for obtaining antiinflammatory agents. In the search for hits to obtain lead compounds for new drugs of this class, 14 synthetic chalcones derived from 2,4,6-trimethoxyacetophenone were evaluated in terms of their inhibitory action, in vitro, in relation to NO production in murine macrophages of the line RAW 264.7 induced by bacterial lipopolysaccharides (LPS). All the compounds were obtained by aldolic condensation between the acetophenone and corresponding aldehydes, under basic conditions. The mean IC(50) values, calculated through dose versus inhibitory effect curves, in four independent experiments, varied between 1.34 and 27.60microM, and were compared with the positive control, compound 1400W (IC(50)=3.78microM), a highly selective inhibitor of iNOS (induced nitric oxide synthase). Eight chalcones gave mean IC(50) values less than or equal to those obtained for 1400W, which suggests that these molecules may act as inhibitors of inflammatory process. The QSAR study reveals that electron-withdrawing groups in the B-ring seem to increase the inhibition of nitrite production, mainly when in position 2. A substitution in the ortho position of the A-ring seems to be necessary for the activity.     

Neurons reduce glial responses to lipopolysaccharide (LPS) and prevent injury of microglial cells from over-activation by LPS

The microenvironment of the CNS has been considered to tonically inhibit glial activities. It has been shown that glia become activated where neuronal death occurs in the aging brain. We have previously demonstrated that neurons tonically inhibit glial activities including their responses to the bacterial endotoxin lipopolysaccharide (LPS). It is not clear whether activation of glia, especially microglia in the aging brain, is the consequence of disinhibition due to neuronal death. This study was designed to determine if glia regain their responsiveness to LPS once the neurons have died in aged cultures. When cultured alone, glia from postnatal day one rat mesencephalons stimulated with LPS (0.1-1000 ng/mL) produced both nitric oxide (NO) and tumor necrosis factor alpha (TNFalpha), yielding a sigmoid and a bell-shaped curve, respectively. When neuron-containing cultures were prepared from embryonic day 14/15 mesencephalons, the shape of the dose-response curve for NO was monotonic and the bell-shaped curve for TNFalpha production was shifted to the right. After 1 month of culture under conditions where neurons die, the production curves for NO and TNFalpha in LPS-stimulated glia shifted back to the left compared to mixed neuron-glia cultures. Immunostaining of rat microglia for the marker CR3 (the receptor for complement component C3) demonstrated that high concentrations of LPS (1 microg/mL) reduced the number of microglia in mixed-glial cultures. In contrast, reduction of CR3 immunostaining was not observed in LPS-stimulated mixed neuron-glia cultures. Taken together, the results demonstrate that disinhibition of the glial response to LPS occurs after neurons die in aged cultures. Once neurons have died, the responsiveness of glia to LPS is restored. Neurons prevented injury to microglia by reducing their responsiveness to LPS. This study broadens our understanding of the ways in which the CNS microenvironment affects cerebral inflammation.     

连翘脂素对LPS诱导RAW264.7细胞炎症反应的影响

为研究连翘脂素的抗炎效应及其抗炎机制,以地塞米松作为阳性对照,建立脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7炎症模型,检测炎症因子的释放及相关蛋白和mRNA的表达,以期提高对连翘脂素抗炎作用的全面认识并为连翘脂素临床开发提供有力的科学依据。实验采用Griess法检测细胞上清液中NO含量,ELISA法检测TNF-α和IL-6的含量,Westernblot法检测iNOS、COX-2蛋白的表达,RT-qPCR法检测iNOS、COX-2mRNA的表达。与LPS组比较,连翘脂素组和地塞米松组可以明显降低LPS诱导的RAW264.7细胞释放NO、TNF-α和IL-6的量,并呈现浓度依赖关系。Westrenblot和RT-qPCR结果显示连翘脂素能抑制LPS诱导的iNOS、COX-2的蛋白表达以及mRNA的表达,并呈浓度依赖关系。实验研究表明连翘脂素能够明显抑制LPS诱导的RAW264.7细胞炎症因子的释放,iNOS、COX-2蛋白及mRNA的表达从而抑制炎症反应。     

Cell and agonist-specific regulation of genes for matrix metalloproteinases and their tissue inhibitors by primary glial cells

An imbalance in the matrix metalloproteinase (MMP) : tissue inhibitor of MMP (TIMP) ratio may be associated with tissue injury. Here, we studied the regulation of TIMP and MMP gene expression in primary glial cultures to ascertain the factors involved in the regulation of these genes in conditions of inflammatory neuropathology. Astrocytes were found to basally express TIMP-1 and TIMP-3 mRNA while microglia expressed only TIMP-2 mRNA. TIMP-4 mRNA was not detectable in either cell type. Treatment with interferon-alpha (IFN-alpha), IFN-gamma, interleukin-3 (IL-3), IL-6 or tumor necrosis factor-alpha (TNF-alpha) did not alter expression of the TIMP genes. However, in astrocytes, but not in microglia, serum, IL-1beta or lipopolysaccharide (LPS) evoked a dose- and time-dependent increase in TIMP-1 mRNA and a coincident down-regulation of the TIMP-3 gene. Astrocytes were found to express mRNA constitutively for MMPs -3, -11 and -14. In contrast, microglia expressed only MMP-12 mRNA under basal conditions. IL-1beta enhanced MMP-3 mRNA levels while LPS increased the MMP-3, -9, -12, -13 and -14 mRNAs. Our findings reveal that regulatory control of TIMP and MMP gene expression by glial cells is agonist- and cell-type specific, and suggest that innate immune signals govern the temporal and spatial expression patterns of TIMP and MMP genes in neuroinflammatory conditions of the CNS.     

Effect of Escherichia coli lipopolysaccharide on phosphatidylcholine biosynthesis by rat lung and alveolar type II cells

Summary Alterations in pulmonary surfactant are partly responsible for the respiratory insufficiency seen under septic shock process. We have used an experimental model of LPS-induced shock in rats to examine the cells responsible for the pulmonary surfactant synthesis and its relationship to lung injury. (¹⁴C)Choline incorporation into phosphatidylcholine was significantly reduced in lung homogenates or type II cells obtained from LPS-treated animals. Addition of LPS in vitro fails to increase (¹⁴C)choline incorporation in type II cells obtained from LPS-treated animals. We suggest that this depression of pulmonary phosphatidylcholine synthesis may partly explain the occurrence of respiratory failure with septic shock.     

Modulation of barrier function of small intestinal epithelial cells by lamina propria fibroblasts in response to lipopolysaccharide: possible role in TNFalpha in inducing barrier dysfunction.

Recent evidence suggests an interaction between immune, enteric neural and fibroblasts in the regulation of intestinal function. Earlier, we have reported that lipopolysaccharide (LPS) induced cell proliferation, collagen synthesis and production of proinflammatory mediators in lamina propria fibroblasts. In this report, we investigated the change in transepithelial resistance (TER) as a marker of epithelial barrier function by lipopolysaccharide (LPS) and its modulation by human small intestinal lamina propria fibroblasts (HSILPF). Epithelial cells incubated with LPS alone did not show any change in the TER at any concentration or prolonged exposure. However, co-cultivation of epithelial cells with lamina propria fibroblasts which had been exposed to LPS resulted in a rapid decrease in TER by 2 hr. The decrease in the TER was continued till 8 hr followed by returning to the basal level by 24 hr. The supernatant of LPS-treated HSILPF was less effective in causing a fall in the TER than HSILPF itself. The fall in TER was accompanied by loosening of tight junctions as depicted by increased penetration of horse radish peroxidase (HRP) across the epithelial cells from the apical to the basal side. Increased incorporation of 3[H]thymidine (tritiated thymidine) in epithelial cells was observed at 48 hr in the presence of LPS-treated HSILPF. The decrease in TER during the early time period in epithelial cells was abrogated to 70% by incubating the LPS-treated HSILPF and the conditioned medium of LPS-treated HSILPF with anti-TNFalpha antibody, and not with antibody to other cytokines like IL1alpha, IL1beta, IL6 and IL8. Overall, these results suggest that TNFalpha produced by HSILPF in response to LPS as a soluble form cause a decrease in the TER and loosening of tight junctions, and such early changes in the epithelial barrier may contribute to local inflammation in the gut.