Functional modulation of enterocytes by gram-positive and gram-negative microorganisms

Clinical studies have suggested that so-called probiotic bacteria may be effective as therapy in inflammatory bowel disease. However, the molecular mechanisms of their interaction with the intestinal surface remain undefined. The influence of whole probiotic bacteria [Escherichia coli Nissle 1917 (EcN); probiotic mixture VSL#3 (PM)], bacterial cell lysates, and conditioned media on transepithelial resistance (TER), IL-8 secretion, mucin gene expression, and tight junction proteins were determined in T84 and HT-29 intestinal epithelial cells (IEC). In addition, effects on pathogen (Salmonella dublin)-induced alterations were analyzed. EcN as well as debris and cell extracts induced IL-8 secretion from IEC, whereas no such effect was observed following incubation with the PM. The PM and soluble protein(s) released from the PM increased TER, prevented pathogen-induced decrease in TER, and were shown to stabilize tight junctions. The PM induced expression of mucins in IEC, and these organisms as well as EcN diminished S. dublin-induced cell death. Inhibition of MAPKs with PD-98059 or SB-203580 significantly decreased alterations in IL-8 synthesis and mucin expression and affected the regulation of TER. Probiotics and protein(s) released by these organisms may functionally modulate the intestinal epithelium of the host by different mechanisms, including the competition of whole organisms for contact with the epithelial surface as well as stabilization of the cytoskeleton and barrier function and the induction of mucin expression. Gram-negative and gram-positive organisms differ in the mechanisms activated, and a combination of organisms might be more effective than the application of a single strain.     

Decisive role of lipopolysaccharide in activating nitric oxide and cytokine production by the probiotic <Emphasis Type="Italic">Escherichia coli</Emphasis> strain Nissle 1917

Effects of Gram-negative probiotic E. coli strain Nissle 1917 (EcN) on the production of nitric oxide (NO) and cytokines were determined in cultures of resident peritoneal cells of rats. The cells (2 × 10⁶/mL) were cultured for 24 h in the presence of live EcN suspension (EcN-Susp), bacteria-free supernatant of this suspension (Sup-EcN), and LPS of EcN origin (LPS-EcN). The biosynthesis of NO was substantially enhanced using live bacteria counts as low as 10³/mL applied in the form of EcN-Susp. The same NO-enhancing effect was produced by the correspondingly diluted Sup-EcN. It was found that Sup-EcN contained relatively high amounts of LPS. Administration of the LPS-EcN mimicked the high NO-augmenting activities of both Sup-EcN and EcN-Susp. However, the activity of LPS-EcN was significantly less pronounced than were the activities of Sup-EcN and EcN-Susp containing identical amounts of LPS. The NOstimulatory effects of the EcN preparations were completely inhibited by polymyxin B. All LPS-EcN and correspondingly diluted Sup-EcN and EcN-Susp stimulated the secretion of cytokines TNF-α, IL-1β, IL-6, IL-10 and VEGF. Also these effects were abrogated by polymyxin B. In contrast to the effects on NO production, the cytokine-stimulatory effects were significantly less pronounced after the exposure of the cells to Sup-EcN and EcN-Susp than to the identical amounts of LPS-EcN. It may be concluded that the in vitro stimulatory effects of EcN on NO and cytokine production are mediated by LPS. It is suggested that the immunostimulatory activity of LPS is modulated by EcN-derived factor(s), the nature of which remains to be identified.     

Damage associated molecular pattern molecule-induced microRNAs (DAMPmiRs) in human peripheral blood mononuclear cells

Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10(-4) and p<3.7×10(-3)), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1(+/+)) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1(-/-) MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic "PAMPmiR". Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1β and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K(+) channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders.     

Molecular mechanisms underlying the probiotic effects of Escherichia coli Nissle 1917 involve ZO-2 and PKCzeta redistribution resulting in tight junction and epithelial barrier repair

The probiotic Escherichia coli strain Nissle 1917 (EcN) has been used for decades in human medicine in Central Europe for the treatment and prevention of intestinal disorders and diseases. However, the molecular mechanisms underlying its beneficial effects are only partially understood. To identify molecular responses induced by EcN that might contribute to its probiotic properties polarized T84 cells were investigated employing DNA microarrays, quantitative RT-PCR, Western blotting, immunofluorescence and specific protein kinase C (PKC) inhibitors. Polarized T84 epithelial cell monolayers were used as a model to monitor barrier disruption by infection with the enteropathogenic E. coli (EPEC) strain E2348/69. Co-incubation of EPEC with EcN or addition of EcN following EPEC infection abolished barrier disruption and, moreover, restored barrier integrity as monitored by transepithelial resistance. DNA-microarray analysis of T84 cells incubated with EcN identified 300+ genes exhibiting altered expression. EcN altered the expression, distribution of zonula occludens-2 (ZO-2) protein and of distinct PKC isotypes. ZO-2 expression was enhanced in parallel to its redistribution towards the cell boundaries. This study provides evidence that EcN induces an overriding signalling effect leading to restoration of a disrupted epithelial barrier. This is transmitted via silencing of PKCzeta and the redistribution of ZO-2. We suggest that these properties contribute to the reported efficacy in the treatment of inflammatory bowel diseases and in part rationalize the probiotic nature of EcN.     

The effect of fish oil supplementation on cytokine production in children

The ex vivo production of inflammatory cytokines during fish oil supplementation (n-3 polyunsaturated fatty acids, n-3 PUFA) is a matter of considerable controversy. Studies on human subjects have generally reported decreased lymphocyte proliferation and decreased production of IL-2, interferon-gamma, IL-1beta, IL-6 and TNF-alpha, but other studies showed no effect or even increased production. There are no published reports on ex vivo cytokine production in children on long-term, n-3 PUFA supplementation. The current double-blind study explored cytokine production by peripheral blood mononuclear cells (PBMCs), with and without lipopolysaccharide (LPS) stimulation in children on 12 weeks' supplementation with 300 mg/day of n-3 PUFA. Twenty-one children (aged 8-12 years) were randomized to receive 1 g canola oil (control) or 300 mg n-3 PUFA + 700 mg canola oil in a chocolate spread. Blood was then drawn and PBMCs were separated and cultured for 24 h in a culture medium with or without 10 microg/mL LPS for 5 x 10(6) PBMCs. The pro-inflammatory cytokines, IL-1beta, TNF-alpha and IL-6, and the anti-inflammatory cytokines, IL-10 and IL-1RA, were evaluated by ELISA. The levels of all the cytokines were higher in non-stimulated and LPS-stimulated cultures, from n-3 PUFA-treated subjects as compared to controls. There was no difference in the IL-1beta/IL-1RA ratio between the two groups, with and without LPS stimulation. Nevertheless, the ratio tended to be lower in the treated subjects on both occasions. In conclusion, our results indicate an increased production of both pro-inflammatory and anti-inflammatory cytokines, with and without LPS stimulation, in children on 12 weeks' n-3 PUFA supplementation.     

Lipopolysaccharide pretreatment of the udder protects against experimental Escherichia coli mastitis

Exposure to pathogen-associated molecular patterns such as LPS can cause an immune refractory state in mammals known as endotoxin tolerance (ET), resulting in a decreased inflammatory response after pathogen contact. This ET concept was used to reduce the severity of an experimentally-induced clinical mastitis. Cows were pretreated with 1?μg LPS per udder quarter and challenged 72?h (group L72EC) or 240?h (group L240EC) later with 500 CFU Escherichia coli. Pretreated animals showed no leukopenia after challenge, no (L72EC), or only slightly (L240EC), elevated body temperature and significantly reduced systemic and local clinical scores compared with cows that were not pretreated. Whereas an increase of milk somatic cell count after the E. coli challenge was abrogated in L72EC animals, it was significantly delayed in the L240EC group. In both pretreated groups the bacterial load in milk was markedly reduced. Based on the expression of inflammation-related genes in lobulo-alveolar mammary tissue, the tolerizing effect of LPS pretreatment is based on the inhibited up-regulation of inflammatory (TNF-α, IL-6, CXCL8, CCL20) and anti-inflammatory genes (IL-10, IRAK-M). These findings indicate that the concept of ET may be usefully applied as mastitis prophylaxis facilitating a rapid response to microbial infection and avoiding dysregulated inflammation.     

益生菌Escherichia coli Nissle1917功能研究进展

大肠埃希菌Nissle1917,简称EcN,是益生菌中为数不多的革兰氏阴性菌,在临床上主要用于克罗恩病、溃疡性结肠炎等胃肠功能障碍。其机制在于EcN能在人体肠道定殖,并阻止病原菌对肠道黏膜的侵袭,对肠道黏膜屏障具有保护和修护作用。EcN还参与机体的免疫调控,平衡免疫因子的分泌,增强宿主免疫能力,进而缓解和治疗炎症。最进研究发现,EcN具有肿瘤靶向作用,是良好的药物载体,且与化疗药物联用可增强药物抗肿瘤的疗效,为抗肿瘤治疗提供了新的思路。     

Probiotic Escherichia coli Nissle 1917 inhibits leaky gut by enhancing mucosal integrity

Background

Probiotics are proposed to positively modulate the intestinal epithelial barrier formed by intestinal epithelial cells (IECs) and intercellular junctions. Disruption of this border alters paracellular permeability and is a key mechanism for the development of enteric infections and inflammatory bowel diseases (IBDs).

Methodology and Principal Findings

To study the in vivo effect of probiotic Escherichia coli Nissle 1917 (EcN) on the stabilization of the intestinal barrier under healthy conditions, germfree mice were colonized with EcN or K12 E. coli strain MG1655. IECs were isolated and analyzed for gene and protein expression of the tight junction molecules ZO-1 and ZO-2. Then, in order to analyze beneficial effects of EcN under inflammatory conditions, the probiotic was orally administered to BALB/c mice with acute dextran sodium sulfate (DSS) induced colitis. Colonization of gnotobiotic mice with EcN resulted in an up-regulation of ZO-1 in IECs at both mRNA and protein levels. EcN administration to DSS-treated mice reduced the loss of body weight and colon shortening. In addition, infiltration of the colon with leukocytes was ameliorated in EcN inoculated mice. Acute DSS colitis did not result in an anion secretory defect, but abrogated the sodium absorptive function of the mucosa. Additionally, intestinal barrier function was severely affected as evidenced by a strong increase in the mucosal uptake of Evans blue in vivo. Concomitant administration of EcN to DSS treated animals resulted in a significant protection against intestinal barrier dysfunction and IECs isolated from these mice exhibited a more pronounced expression of ZO-1.

Conclusion and Significance

This study convincingly demonstrates that probiotic EcN is able to mediate up-regulation of ZO-1 expression in murine IECs and confer protection from the DSS colitis-associated increase in mucosal permeability to luminal substances.     

The fate of E. coli lipopolysaccharide after the uptake of E. coli by murine macrophages in vitro

The fate of bacterial lipopolysaccharide (LPS) after the uptake of Escherichia coli by macrophages in vitro was studied. The LPS of the galactose epimerase-deficient E. coli J5 mutant was specifically radiolabeled with [3H]galactose by growing the organism in a basic salts medium containing galactose. Control bacteria were uniformly radiolabeled by growth in [14C]glucose and unlabeled galactose-containing medium. Surface constituents of E. coli were also labeled with 125I. After in vitro phagocytosis of labeled E. coli by murine peritoneal exudate macrophages, the rate of exocytosis of LPS, as assessed by release of 3H over a 72-hr period, was considerably reduced in comparison with other bacterial constituents (14C and 125I release). The [3H]galactose-labeled material exocytosed from macrophages and that remaining intracellularly (obtained from macrophage lysates) were isolated by cesium chloride (CsCl) density gradients and were shown to have altered density profiles as compared with purified E. coli LPS. The macrophage-"processed" [3H] galactose-containing fractions from CsCl density gradients of culture supernatants or macrophage lysates were capable of clotting Limulus amebocyte lysate. The [3H]galactose material obtained from 48-hr macrophage lysates and culture supernatants could also induce a lethal response in actinomycin D-treated mice. These data suggest that bacterial LPS may be selectively retained by the macrophage and that the post-phagocytic events that result in bacterial degradation are not accompanied by the degradation of LPS. Furthermore, although the LPS may be modified by the macrophage, it retains its biologic activity.     

Modulation of cytokine release from mononuclear cells by prostacyclin, IL-4 and IL-13

In a previous study, we reported that cicaprost, a stable prostacyclin analogue can inhibit the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from activated human peripheral mononuclear blood cells (PBMCs). Since interleukin (IL-4) and IL-13 have been shown to inhibit the release of cytokines from PBMCs we tested the hypothesis that prostacyclin in combination with IL-4 or IL-13 can act synergistically to modulate the release of IL-10, generally associated with anti-inflammatory properties, and the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha). For this purpose, PBMCs were isolated over Ficoll, stimulated with lipopolysaccharide (LPS) and incubated in the presence of cicaprost, IL-4 or IL-13. There was a significant reduction in TNF-alpha as well as IL-10 secretion from LPS-stimulated PBMCs following incubation with IL-4 or IL-13. In contrast, cicaprost reduced the secretion of TNF-alpha but led to a slight enhancement of IL-10 release from PBMCs. When LPS-activated PBMCs were incubated in the presence of cicaprost and IL-4 or IL-13 there was a selective, synergistic inhibition of the TNF-alpha release which was not observed for IL-10. Thus, our data suggest that prostacyclin can synergize with cytokines to selectively inhibit the release of pro-inflammatory cytokines from PBMCs.     

IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases

The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.     

Inhibitory action of telithromycin against Shiga toxin and endotoxin

Shiga toxin (Stx)-producing Escherichia coli (STEC) is associated with hemolytic uremic syndrome (HUS). High inflammatory cytokine [interleukin (IL)-6 and IL-8] levels and low anti-inflammatory cytokine (IL-10) levels are indicators of a high risk for developing HUS in STEC-infected children. In this study, we investigated inhibitory action of telithromycin, a ketolide, against STEC and against Stx and lipopolysaccharide (LPS). Telithromycin inhibited in vitro STEC growth without inducing Stx phage, in marked contrast to norfloxacin. Stx markedly induced inflammatory (but not anti-inflammatory) cytokine production in human peripheral blood monocytes, while LPS induced both inflammatory and anti-inflammatory cytokine production. Telithromycin selectively inhibited the IL-6 and IL-8 production from Stx-stimulated (but not LPS-stimulated) monocytes. The drug did not significantly inhibit IL-10 production. Our data suggest that Stx plays a crucial role in the stimulation of inflammatory cytokines and such inflammatory response is inhibited by telithromycin, an anti-bacterial agent.     

Divergent response to LPS and bacteria in CD14-deficient murine macrophages

Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock. The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-alpha and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-kappaB and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-alpha release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-alpha than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-alpha in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.     

Neutralization of IL-18 reduces neutrophil tissue accumulation and protects mice against lethal Escherichia coli and Salmonella typhimurium endotoxemia

In addition to stimulating IFN-gamma synthesis, IL-18 also possesses inflammatory effects by inducing synthesis of the proinflammatory cytokines TNF and IL-1beta and the chemokines IL-8 and macrophage inflammatory protein-1alpha. We hypothesized that neutralization of IL-18 would have a beneficial effect in lethal endotoxemia in mice. IL-1beta converting enzyme (ICE)-deficient mice, lacking the ability to process mature IL-18 and IL-1beta, were completely resistant to lethal endotoxemia induced by LPS derived from either Escherichia coli or Salmonella typhimurium. In contrast, both wild-type and IL-1beta-/- mice were equally susceptible to the lethal effects of LPS, implicating that absence of mature IL-18 or IFN-gamma but not IL-1beta in ICE-/- mice is responsible for this resistance. However, IFN-gamma-deficient mice were not resistant to S. typhimurium LPS, suggesting an IFN-gamma-independent role for IL-18. Anti-IL-18 Abs protected mice against a lethal injection of either LPS. Anti-IL-18 treatment also reduced neutrophil accumulation in liver and lungs. The increased survival was accompanied by decreased levels of IFN-gamma and macrophage inflammatory protein-2 in anti-IL-18-treated animals challenged with E. coli LPS, whereas IFN-gamma and TNF concentrations were decreased in treated mice challenged with S. typhimurium. In conclusion, neutralization of IL-18 during lethal endotoxemia protects mice against lethal effects of LPS. This protection is partly mediated through inhibition of IFN-gamma production, but mechanisms involving decreased neutrophil-mediated tissue damage due to the reduction of either chemokines (E. coli LPS) or TNF (S. typhimurium LPS) synthesis by anti-IL-18 treatment may also be involved.     

Monocyte-mediated inhibition of TLR9-dependent IFN-α induction in plasmacytoid dendritic cells questions bacterial DNA as the active ingredient of bacterial lysates

Bacterial DNA contains unmethylated CpG dinucleotides and is a potent ligand for TLR9. Bacterial DNA has been claimed the active ingredient in bacterial lysates used for immunotherapy. Whereas the detection of viral DNA by TLR9 expressed in plasmacytoid dendritic cells (PDCs) with subsequent IFN-α production is well defined, the role of bacterial DNA during microbial infection is less clear. In fact, IFN-α is not a hallmark of antibacterial immune responses. Unlike in mice, TLR9 expression in humans is restricted to PDCs and B cells; thus, conclusions from murine models of infection have limitations. In this study, we demonstrate that lysates of heat-killed Escherichia coli containing bacterial DNA induced IFN-α in isolated PDCs but not in the mixed cell populations of human PBMCs. Depletion of monocytes restored IFN-α secretion by PDCs within PBMCs. We found that monocyte-derived IL-10 and PGs contribute to monocyte-mediated inhibition of IFN-α release in PDCs. We conclude that human PDCs can be stimulated by bacterial DNA via TLR9; however, in the physiological context of mixed-cell populations, PDC activation is blocked by factors released from monocytes stimulated in parallel by other components of bacterial lysates such as LPS. This functional repression of PDCs by concomitantly stimulated monocytes avoids production of antiviral IFN-α during bacterial infection and thus explains how the innate immune system is enabled to distinguish bacterial from viral CpG DNA and thus to elicit the appropriate responses despite the presence of CpG DNA in both types of infection.     

Autoregulation of CCL26 synthesis and secretion in A549 cells: a possible mechanism by which alveolar epithelial cells modulate airway inflammation

Eotaxins (CCL11, CCL24, CCL26) originating from airway epithelial cells and leukocytes have been detected in bronchoalveolar lavage of asthmatics. Although the alveolar epithelium is the destination of uncleared allergens and other inflammatory products, scanty information exists on their contribution to the generation and regulation of the eotaxins. We envisioned a state whereby alveolar type II cells, a known source of other inflammatory proteins, could be involved in both the production and regulation of CCL24 and CCL26. Herein, we demonstrated that all three eotaxins are constitutively expressed in A549 cells. IL-4 and IL-13 stimulated a concentration-dependent secretion of CCL24 and CCL26. The cytokines did not act synergistically. Cycloheximide and actinomycin D abrogated IL-4- and IL-13-dependent CCL26 but not CCL24 secretion. Both IL-13 and IL-4 stimulated CCL26 synthesis that was inhibited in a concentration-dependent manner by CCL26 but not CCL24. Only CCL26 reduced expression of CCR3 receptors by 30-40%. On the other hand, anti-CCR3 pretreatment reduced IL-4+IL-13-dependent CCL26 secretion, implying autoregulation. A CCR3-specific antagonist (SB-328437) significantly decreased IL-4-dependent synthesis and release of CCL26. Eosinophils treated with medium from IL-4-stimulated A549 cells preincubated with anti-CCL26 showed a marked decrease of superoxide anion production compared with anti-CCL24 treated. These results suggest that CCL26 is a major eotaxin synthesized and released by alveolar epithelial cells and is involved in autoregulation of CCR3 receptors and other eotaxins. This CCL26-CCR3 ligand-receptor system may be an attractive target for development of therapeutics that limits progress of inflammation in airway disease.     

Assessment of lipopolysaccharide-binding activity of Bifidobacterium and its relationship with cell surface hydrophobicity, autoaggregation, and inhibition of interleukin-8 production

This study was performed to screen probiotic bifidobacteria for their ability to bind and neutralize lipopolysaccharides (LPS) from Escherichia coli and to verify the relationship between LPS-binding ability, cell surface hydrophobicity (CSH), and inhibition of LPS-induced interleukin-8 (IL-8) secretion by HT-29 cells of the various bifidobacterial strains. Ninety bifidobacteria isolates from human feces were assessed for their ability to bind fluorescein isothiocyanate (FITC)-labeled LPS from E. coli. Isolates showing 30-60% binding were designated LPS-high binding (LPS-H) and those with less than 15% binding were designated LPS-low binding (LPS-L). The CSH, autoaggregation (AA), and inhibition of LPS-induced IL-8 release from HT-29 cells of the LPS-H and LPS-L groups were evaluated. Five bifidobacteria strains showed high levels of LPS binding, CSH, AA, and inhibition of IL-8 release. However, statistically significant correlations between LPS binding, CSH, AA, and reduction of IL-8 release were not found. Although we could isolate bifidobacteria with high LPS-binding ability, CSH, AA, and inhibition of IL-8 release, each characteristic should be considered as strain dependent. Bifidobacteria with high LPS binding and inhibition of IL-8 release may be good agents for preventing inflammation by neutralizing Gram-negative endotoxins and improving intestinal health.     

Effect of erythropoietin on primed leucocyte expression profile

Resistance to erythropoietin (EPO) affects a significant number of anaemic patients with end-stage renal disease. Previous reports suggest that inflammation is one of the major independent predictors of EPO resistance, and the effects of EPO treatment on inflammatory mediators are not well established. The aim of this study was to investigate EPO-induced modification to gene expression in primary cultured leucocytes. Microarray experiments were performed on primed ex vivo peripheral blood mononuclear cells (PBMCs) and treated with human EPO-α. Data suggested that EPO-α modulated genes involved in cell movement and interaction in primed PBMCs. Of note, EPO-α exerts anti-inflammatory effects inhibiting the expression of pro-inflammatory cytokine IL-8 and its receptor CXCR2; by contrast, EPO-α increases expression of genes relating to promotion of inflammation encoding for IL-1β and CCL8, and induces de novo synthesis of IL-1α, CXCL1 and CXCL5 in primed cells. The reduction in MAPK p38-α activity is involved in modulating both IL-1β and IL-8 expression. Unlike the induction of MAPK, Erk1/2 activity leads to upregulation of IL-1β, but does not affect IL-8 expression and release. Furthermore, EPO-α treatment of primed cells induces the activation of caspase-1 upstream higher secretion of IL-1β, and this process is not dependent on caspase-8 activation. In conclusion, our findings highlight new potential molecules involved in EPO resistance and confirm the anti-inflammatory role for EPO, but also suggest a plausible in vivo scenario in which the positive correlation found between EPO resistance and elevated levels of some pro-inflammatory mediators is due to treatment with EPO itself.     

Macrophage inflammatory protein-3 beta enhances IL-10 production by activated human peripheral blood monocytes and T cells.

We report that the addition of human macrophage inflammatory protein-3 beta (MIP-3 beta) to cultures of human PBMCs that have been activated with LPS or PHA results in a significant enhancement of IL-10 production. This effect was concentration-dependent, with optimal MIP-3 beta concentrations inducing more than a 5-fold induction of IL-10 from LPS-stimulated PBMCs and a 2- to 3-fold induction of IL-10 from PHA-stimulated PBMCs. In contrast, no significant effect on IL-10 production was observed when 6Ckine, the other reported ligand for human CCR7, or other CC chemokines such as monocyte chemoattractant protein-1, RANTES, MIP-1 alpha, and MIP-1 beta were added to LPS- or PHA-stimulated PBMCs. Similar results were observed using activated purified human peripheral blood monocytes or T cells. Addition of MIP-3 beta to nonactivated PBMCs had no effect on cytokine production. Enhancement of IL-10 production by MIP-3beta correlated with the inhibition of IL-12 p40 and TNF-alpha production by monocytes and with the impairment of IFN-gamma production by T cells, which was reversed by addition of anti-IL-10 Abs to the cultures. The ability of MIP-3 beta to augment IL-10 production correlated with CCR7 mRNA expression and stimulation of intracellular calcium mobilization in both monocytes and T cells. These data indicate that MIP-3 beta acts directly on human monocytes and T cells and suggest that this chemokine is unique among ligands binding to CC receptors due to its ability to modulate inflammatory activity via the enhanced production of the anti-inflammatory cytokine IL-10.