ArticleLiterature Review

Cellular reprogramming by gram-positive bacterial components: A review

November 2006
Journal of Leukocyte Biology 80(4):731-41

November 2006
80(4):731-41

Source
PubMed

Authors:

LPS tolerance has been the focus of extensive scientific and clinical research over the last several decades in an attempt to elucidate the sequence of changes that occur at a molecular level in tolerized cells. Tolerance to components of gram-positive bacterial cell walls such as bacterial lipoprotein and lipoteichoic acid is a much lesser studied, although equally important, phenomenon. This review will focus on cellular reprogramming by gram-positive bacterial components and examines the alterations in cell surface receptor expression, changes in intracellular signaling, gene expression and cytokine production, and the phenomenon of cross-tolerance.

Post-transcriptional regulatory feedback encodes JAK-STAT signal memory of interferon stimulation

Article

Full-text available

Sep 2022

Immune cells fine tune their responses to infection and inflammatory cues. Here, using live-cell confocal microscopy and mathematical modelling, we investigate interferon-induced JAK-STAT signalling in innate immune macrophages. We demonstrate that transient exposure to IFN-γ stimulation induces a long-term desensitisation of STAT1 signalling and gene expression responses, revealing a dose- and time-dependent regulatory feedback that controls JAK-STAT responses upon re-exposure to stimulus. We show that IFN-α/β1 elicit different level of desensitisation from IFN-γ, where cells refractory to IFN-α/β1 are sensitive to IFN-γ, but not vice versa . We experimentally demonstrate that the underlying feedback mechanism involves regulation of STAT1 phosphorylation but is independent of new mRNA synthesis and cognate receptor expression. A new feedback model of the protein tyrosine phosphatase activity recapitulates experimental data and demonstrates JAK-STAT network’s ability to decode relative changes of dose, timing, and type of temporal interferon stimulation. These findings reveal that STAT desensitisation renders cells with signalling memory of type I and II interferon stimulation, which in the future may improve administration of interferon therapy.

An Early Neutrophil Recruitment into the Infectious Site Is Critical for Bacterial Lipoprotein Tolerance–Afforded Protection against Microbial Sepsis

Article

Dec 2019

Bacterial lipoprotein (BLP)-induced tolerance represents an essential regulatory mechanism during bacterial infection and has been shown to protect against microbial sepsis. This protection is generally attributed to BLP-tolerized monocytes/macrophages characterized by hyporesponsiveness in producing inflammatory cytokines and, simultaneously, an augmented antimicrobial activity. However, the contribution of polymorphonuclear neutrophils (PMNs), another major player in innate immunity against bacterial infection, to BLP tolerance-afforded protection against microbial sepsis has not been identified. In this study, we report that induction of BLP tolerance protected mice against cecal ligation and puncture-induced polymicrobial sepsis, with significantly improved survival. Importantly, BLP tolerization via i.p. injection triggered an early PMN recruitment even before bacterial infection and promoted further PMN influx into the infectious site (i.e., the peritoneal cavity upon cecal ligation and puncture-associated septic challenge). Notably, this early PMN influx was mediated by BLP tolerization-induced PMN chemoattractant CXCL2-formed concentration gradient between the circulation and peritoneal cavity. Critically, blockage of PMN influx with the CXCR2 antagonist SB225002 abolished BLP tolerance-afforded protection and rendered BLP-tolerized mice more vulnerable to microbial infection with impaired bacterial clearance and increased overall mortality. Thus, our results highlight that an early recruitment of PMNs in the infectious site, as an important cellular mechanism, contributes to BLP tolerance-afforded protection against microbial sepsis.

NF-kappa B activation is critical for bacterial lipoprotein tolerance-enhanced bactericidal activity in macrophages during microbial infection

Article

Full-text available

Jan 2017

Tolerance to bacterial components represents an essential regulatory mechanism during bacterial infection. Bacterial lipoprotein (BLP)-induced tolerance confers protection against microbial sepsis by attenuating inflammatory responses and augmenting antimicrobial activity in innate phagocytes. It has been well-documented that BLP tolerance-attenuated proinflammatory cytokine production is associated with suppressed TLR2 signalling pathway; however, the underlying mechanism(s) involved in BLP tolerance-enhanced antimicrobial activity is unclear. Here we report that BLP-tolerised macrophages exhibited accelerated phagosome maturation and enhanced bactericidal activity upon bacterial infection, with upregulated expression of membrane-trafficking regulators and lysosomal enzymes. Notably, bacterial challenge resulted in a strong activation of NF-κB pathway in BLP-tolerised macrophages. Importantly, activation of NF-κB pathway is critical for BLP tolerance-enhanced antimicrobial activity, as deactivation of NF-κB in BLP-tolerised macrophages impaired phagosome maturation and intracellular killing of the ingested bacteria. Finally, activation of NF-κB pathway in BLP-tolerised macrophages was dependent on NOD1 and NOD2 signalling, as knocking-down NOD1 and NOD2 substantially inhibited bacteria-induced activation of NF-κB and overexpression of Rab10 and Acp5, two membrane-trafficking regulators and lysosomal enzymes contributed to BLP tolerance-enhanced bactericidal activity. These results indicate that activation of NF-κB pathway is essential for BLP tolerance-augmented antimicrobial activity in innate phagocytes and depends primarily on both NOD1 and NOD2.

Endotoxin tolerance and the immune system

Article

Elyce Marshfield

Endotoxin tolerance is a phenomenon known to cause innate immune cells, like macrophages, to produce a decreased pro-inflammatory response to a pathogen associated molecular pattern (PAMP), like LPS, after pre-stimulation. Innate immune cells involved have thought to be primarily monocytes/macrophages but evidence has been found for involvement of dendritic cells, neutrophils and T cells. The molecular mechanisms of endotoxin tolerance are vague. However, negative regulators such as SOCS1, IRAK-M and SHIP are believed to play a large role, along with the down-regulation of TLR4 on cell surface and gene re-programming. Clinically, sepsis is a major model of endotoxin tolerance due to the immunosuppression observed; however, new applications for endotoxin tolerance in pathology are becoming apparent, including ischemia-reperfusion injury. Little is known about cross-tolerance, but it does seem to have similarities and differences to homo-tolerance and also application into the clinical world. This review provides an overall picture of findings within endotoxin tolerance from the beginning to recent, including cellular and molecular mechanisms along with clinical applications.

Model of pathogenesis of psoriasis. Part 1. Systemic psoriatic process

Book

Full-text available

Oct 2011

Mikhail Peslyak

Analytical study of results of experimental and theoretical works on pathogenesis of psoriatic disease was conducted. Psoriasis is dermal implication of systemic psoriatic process (SPP). New SPP model explaining results of clinical and laboratory experiments was formulated. According to Y-model there are two main factors: hyperpermeability of small intestine for bacterial products and colonization of its walls by Gram+ bacteria (incl. psoriagenic bacteria PsB) and Gram(-) TLR4-active bacteria. Inside SPP there is a vicious cycle which is supported by disturbance of production and-or circulation of bile acids. SPP central subprocess is PAMP-nemia, namely chronic kPAMP-load on blood phagocytes (neutrophiles, monocytes and dendritic cells). The load results in increase of blood kPAMP level. The major key PAMP (kPAMP) are LPS and PG (incl. PG-Y - peptidoglycan of psoriagenic bacteria). Chronically increased kPAMP-load possibly provides tolerization of some neutrophils Neu, monocytes Mo and dendritic cells DC in blood flow. The chemostatus of tolerized blood Neu-T in process of their aging changes similarly to chemostatus nonactivated Neu and, hence, they carry endocytosed content from blood flow into the bone marrow. Chemostatuses of tolerized Mo-T and DC-T are similar to nonactivated ones. So they don't bring endocytosed content to lymph nodes or spleen and remain in blood. Tolerized phagocytes degrade endocytosed fragments of bacterial products containing kPAMP slowly and incompletely, Tolerized phagocytes appeared to be (PG-Y)-carriers are named by R-phagocytes and are designated as Neu-R, Mo-R and DC-R. SPP severity predetermines possibility of psoriasis initialization and maintenance because Mo-R and DC-R along with normal Mo and DC participate in homeostatic and inflammatory renewal of pool of dermal macrophages and DC of non-resident origin. Part 2 - arXiv:1201.2900

Staphylococcus aureus induces tolerance in human monocytes accompanied with expression changes of cell surface markers

Article

Full-text available

Mar 2023

Exposure of human monocytes to lipopolysaccharide (LPS) or other pathogen-associated molecular pattern (PAMPs) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance (ET), associated with the pathogenesis of sepsis. In this study, we aimed to characterize the cellular state of human monocytes from healthy donors stimulated with Staphylococcus aureus in comparison to TLR2-specific ligands. We analyzed S. aureus induced gene expression changes after 2 and 24 hours by amplicon sequencing (RNA-AmpliSeq) and compared the pro-inflammatory response after 2 hours with the response in re-stimulation experiments. In parallel, glycoprotein expression changes in human monocytes after 24 hours of S. aureus stimulation were analyzed by proteomics and compared to stimulation experiments with TLR2 ligands Malp-2 and Pam3Cys and TLR4 ligand LPS. Finally, we analyzed peripheral blood monocytes of patients with S. aureus bloodstream infection for their ex vivo inflammatory responses towards S. aureus stimulation and their glycoprotein expression profiles. Our results demonstrate that monocytes from healthy donors stimulated with S. aureus and TLR ligands of Gram-positive bacteria entered the tolerant cell state after activation similar to LPS treatment. In particular reduced gene expression of pro-inflammatory cytokines (TNF, IL1β) and chemokines (CCL20, CCL3, CCL4, CXCL2, CXCL3 and CXCL8) could be demonstrated. Glycoprotein expression changes in monocytes tolerized by the different TLR agonists were highly similar while S. aureus -stimulated monocytes shared some of the PAMP-induced changes but also exhibited a distinct expression profile. 11 glycoproteins (CD44, CD274, DSC2, ICAM1, LAMP3, LILRB1, PTGS2, SLC1A3, CR1, FGL2, and HP) were similarly up- or downregulated in all four comparisons in the tolerant cell state. Monocytes from patients with S. aureus bacteremia revealed preserved pro-inflammatory responsiveness to S. aureus stimulation ex vivo, expressed increased CD44 mRNA but no other glycoprotein of the tolerance signature was differentially expressed.

Post-transcriptional regulatory feedback encodes JAK-STAT signal memory of interferon stimulation

Preprint

Full-text available

May 2022

Immune cells fine tune their responses to infection and inflammatory cues. Here, using live-cell confocal microscopy and mathematical modelling, we investigate interferon induced JAK-STAT signalling in innate immune macrophages. We demonstrate that transient exposure to IFN-γ stimulation induces a long-term desensitisation of STAT1 signalling and gene expression responses, revealing a dose- and time-dependent regulatory feedback that controls JAK-STAT responses upon re-exposure to stimulus. We show that IFN-α/β1 elicit different level of desensitisation from IFN-γ, where cells refractory to IFN-α/β1 are sensitive to IFN-γ, but not vice versa . We experimentally demonstrate that the underlying feedback mechanism involves regulation of STAT1 phosphorylation but is independent of new mRNA synthesis and cognate receptor expression. A new feedback model of the protein tyrosine phosphatase activity recapitulates experimental data and demonstrates JAK-STAT network’s ability to decode relative changes of dose, timing, and type of temporal interferon stimulation. These findings reveal that STAT desensitisation renders cells with signalling memory of type I and II interferon stimulation, which in the future may improve administration of interferon therapy.

HMGB1 Binds to Lipoteichoic Acid and Enhances TNF-a and IL-6 Production through HMGB1-Mediated Transfer of Lipoteichoic Acid to CD14 and TLR2

Article

Full-text available

Feb 2015
J INNATE IMMUN

Lipoteichoic acid (LTA) is a component of the cell wall of Gram-positive bacteria and induces a toll-like receptor 2 (TLR2)-mediated inflammatory response upon initial binding to lipopolysaccharide-binding protein (LBP) and subsequent transfer to CD14. In this study, we identified a novel role for the nuclear protein high-mobility group box 1 (HMGB1) in LTA-mediated inflammation. Results of ELISA, surface plasmon resonance and native PAGE electrophoretic mobility shift analyses indicated that HMGB1 binds to LTA in a concentration-dependent manner and that this binding is inhibited by LBP. Native PAGE, fluorescence-based transfer and confocal imaging analyses indicated that HMGB1 catalytically disaggregates LTA and transfers LTA to CD14. NF-κB p65 nuclear transmigration, degradation of IκBα and reporter assay results demonstrated that NF-κB activity in HEK293-hTLR2/6 cells is significantly upregulated by a mixture of LTA and soluble CD14 in the presence of HMGB1. Furthermore, the production of TNF-α and IL-6 in J774A.1 and RAW264.7 cells increased significantly following treatment with a mixture of LTA and HMGB1 compared with treatment with LTA or HMGB1 alone. Thus, we propose that HMGB1 plays an important role in LTA-mediated inflammation by binding to and transferring LTA to CD14, which is subsequently transferred to TLR2 to induce an inflammatory response. © 2015 S. Karger AG, Basel.

An Endotoxin Tolerance Signature Predicts Sepsis and Organ Dysfunction at Initial Clinical Presentation

Article

Full-text available

Nov 2014

Sepsis involves aberrant immune responses to infection, but the exact nature of this immune dysfunction remains poorly defined. Bacterial endotoxins like lipopolysaccharide (LPS) are potent inducers of inflammation, which has been associated with the pathophysiology of sepsis, but repeated exposure can also induce a suppressive effect known as endotoxin tolerance or cellular reprogramming. It has been proposed that endotoxin tolerance might be associated with the immunosuppressive state that was primarily observed during late-stage sepsis. However, this relationship remains poorly characterised. Here we clarify the underlying mechanisms and timing of immune dysfunction in sepsis. We defined a gene expression signature characteristic of endotoxin tolerance. Gene-set test approaches were used to correlate this signature with early sepsis, both newly and retrospectively analysing microarrays from 593 patients in 11 cohorts. Then we recruited a unique cohort of possible sepsis patients at first clinical presentation in an independent blinded controlled observational study to determine whether this signature was associated with the development of confirmed sepsis and organ dysfunction. All sepsis patients presented an expression profile strongly associated with the endotoxin tolerance signature (p < 0.01; AUC 96.1%). Importantly, this signature further differentiated between suspected sepsis patients who did, or did not, go on to develop confirmed sepsis, and predicted the development of organ dysfunction. Our data support an updated model of sepsis pathogenesis in which endotoxin tolerance-mediated immune dysfunction (cellular reprogramming) is present throughout the clinical course of disease and related to disease severity. Thus endotoxin tolerance might offer new insights guiding the development of new therapies and diagnostics for early sepsis.

Gene expression profile of human peripheral blood mononuclear cells induced by Staphylococcus aureus lipoteichoic acid

Article

May 2012

Lipoteichoic acid (LTA) is a major virulence factor of Gram-positive bacteria including Staphylococcus aureus. Despite its pivotal role in causing sepsis, the systemic immune responses to LTA in human cells are poorly understood. Here, we produced highly-pure and structurally-intact LTA from S. aureus and examined the gene expression profile of LTA-stimulated human peripheral blood mononuclear cells (PBMCs). The LTA preparation did not contain any detectable biologically-active impurities and stimulated Toll-like receptor 2. Protein expression profiling using a cytokine array kit and ELISA revealed expression of MCP-1/CCL2, IL-6, and IL-1β. We performed transcriptional profiling of PBMCs in response to S. aureus LTA using an Affymetrix genechip microarray. A total of 208 genes were significantly (fold change>1.5 and P<0.05) altered, with 157 up-regulated and 51 down-regulated genes in response to S. aureus LTA treatment. The up-regulated genes were involved in recognition (30 genes), cellular adhesion (6 genes), signal transduction (42 genes), co-stimulation (4 genes), chemokines, cytokines and their receptors (51 genes), apoptosis (9 genes), and negative regulation (15 genes). The down-regulated genes were involved in recognition (12 genes), antigen processing and presentation (9 genes), signal transduction (27 genes), and chemotaxis (3 genes). The microarray results were validated using real-time RT-PCR with 21 up-regulated genes and 9 down-regulated genes. Our results provide a more comprehensive overview of the transcriptional changes in PBMCs in response to S. aureus LTA, and contribute to the understanding of the pathophysiological role of S. aureus LTA during the systemic inflammatory response.

Variability of the innate immune response is globally constrained by transcriptional bursting

Article

Full-text available

Jun 2023

Transcription of almost all mammalian genes occurs in stochastic bursts, however the fundamental control mechanisms that allow appropriate single-cell responses remain unresolved. Here we utilise single cell genomics data and stochastic models of transcription to perform global analysis of the toll-like receptor (TLR)-induced gene expression variability. Based on analysis of more than 2000 TLR-response genes across multiple experimental conditions we demonstrate that the single-cell, gene-by-gene expression variability can be empirically described by a linear function of the population mean. We show that response heterogeneity of individual genes can be characterised by the slope of the mean-variance line, which captures how cells respond to stimulus and provides insight into evolutionary differences between species. We further demonstrate that linear relationships theoretically determine the underlying transcriptional bursting kinetics, revealing different regulatory modes of TLR response heterogeneity. Stochastic modelling of temporal scRNA-seq count distributions demonstrates that increased response variability is associated with larger and more frequent transcriptional bursts, which emerge via increased complexity of transcriptional regulatory networks between genes and different species. Overall, we provide a methodology relying on inference of empirical mean-variance relationships from single cell data and new insights into control of innate immune response variability.

Studies of the Impact of the Bifidobacterium Species on Inducible Nitric Oxide Synthase Expression and Nitric Oxide Production in Murine Macrophages of the BMDM Cell Line

Article

Full-text available

May 2023

Bifidobacterium species are one of the most important probiotic microorganisms which are present in both, infants and adults. Nowadays, growing data describing their healthy properties arise, indicating they could act at the cellular and molecular level. However, still little is known about the specific mechanisms promoting their beneficial effects. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is involved in the protective mechanisms in the gastrointestinal tract, where it can be provided by epithelial cells, macrophages, or bacteria. The present study explored whether induction of iNOS-dependent NO synthesis in macrophages stems from the cellular action of Bifidobacterium species. The ability of ten Bifidobacterium strains belonging to 3 different species (Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium animalis) to activate MAP kinases, NF-κB factor, and iNOS expression in a murine bone-marrow-derived macrophages cell line was determined by Western blotting. Changes in NO production were determined by the Griess reaction. It was performed that the Bifidobacterium strains were able to induce NF-қB-dependent iNOS expression and NO production; however, the efficacy depends on the strain. The highest stimulatory activity was observed for Bifidobacterium animalis subsp. animals CCDM 366, whereas the lowest was noted for strains Bifidobacterium adolescentis CCDM 371 and Bifidobacterium longum subsp. longum CCDM 372. Both TLR2 and TLR4 receptors are involved in Bifidobacterium-induced macrophage activation and NO production. We showed that the impact of Bifidobacterium on the regulation of iNOS expression is determined by MAPK kinase activity. Using pharmaceutical inhibitors of ERK 1/2 and JNK, we confirmed that Bifidobacterium strains can activate these kinases to control iNOS mRNA expression. Concluding, the induction of iNOS and NO production may be involved in the protective mechanism of action observed for Bifidobacterium in the intestine, and the efficacy is strain-dependent.

Induction of Trained Immunity Protects Neonatal Mice Against Microbial Sepsis by Boosting Both the Inflammatory Response and Antimicrobial Activity

Article

Full-text available

Jan 2022

Background: Neonates are susceptible to a wide range of microbial infection and at a high risk to develop severe sepsis and septic shock. Emerged evidence has shown that induction of trained immunity triggers a much stronger inflammatory response in adult monocytes/macrophages, thereby conferring protection against microbial infection. Methods: This study was carried out to examine whether trained immunity is inducible and exerts its protection against microbial sepsis in neonates. Results: Induction of trained immunity by Bacillus Calmette-Guerin (BCG) plus bacterial lipoprotein (BLP) protected neonatal mice against cecal slurry peritonitis-induced polymicrobial sepsis, and this protection is associated with elevated circulating inflammatory cytokines, increased neutrophil recruitment, and accelerated bacterial clearance. In vitro stimulation of neonatal murine macrophages with BCG+BLP augmented both inflammatory response and antimicrobial activity. Notably, BCG+BLP stimulation resulted in epigenetic remodeling characterized by histone modifications with enhanced H3K4me3, H3K27Ac, and suppressed H3K9me3 at the promoters of the targeted inflammatory and antimicrobial genes. Critically, BCG+BLP stimulation led to a shift in cellular metabolism with increased glycolysis, which is the prerequisite for subsequent BCG+BLP-triggered epigenetic reprogramming and augmented inflammatory response and antimicrobial capacity. Conclusion: These results illustrate that BCG+BLP induces trained immunity in neonates, thereby protecting against microbial infection by boosting both inflammatory and antimicrobial responses.

Anti-Inflammatory Mechanisms of Koreanaside A, a Lignan Isolated from the Flower of Forsythia koreana, against LPS-Induced Macrophage Activation and DSS-Induced Colitis Mice: The Crucial Role of AP-1, NF-κB, and JAK/STAT Signaling

Article

Full-text available

Sep 2019
CELL

The current treatment options for inflammatory bowel disease (IBD) are unsatisfactory. Therefore, novel and safer therapies are needed. We previously reported that koreanaside A (KA) showed high radical scavenging activity and suppressed vascular cell adhesion molecule 1 (VCAM-1) expression in vascular smooth muscle cells. However, the molecular mechanisms involved in its anti-inflammatory effect have not been reported. KA inhibited pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nitric oxide (NO), and prostaglandin E2 (PGE2). KA inhibited the production and mRNA expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) induced by LPS. KA downregulated the myeloid differentiation primary response 88 (MyD88)-dependent inflammatory gene expressions in the MyD88-overexpressed cells. KA suppressed the LPS-induced transcriptional and DNA-binding activities of activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB). KA was found to inhibit the phosphorylation of Janus kinase 1/2 (JAK1/2) and signal transducers and activators of transcription 1/3 (STAT1/3). In DSS-induced colitis mice, KA relieved the symptoms of colitis by suppressing inflammatory cell infiltration, restoring tight junction (TJ)-and epithelial-mesenchymal transition (EMT)-related protein expression, and inactivating AP-1, NF-κB, and STAT1/3. Therefore, KA reduced inflammatory responses by downregulating AP-1, NF-κB, and JAK/STAT signaling in LPS-induced macrophages and DSS-induced colitis mice.

Article

Full-text available

Sep 2019

The current treatment options for inflammatory bowel disease (IBD) are unsatisfactory. Therefore, novel and safer therapies are needed. We previously reported that koreanaside A (KA) showed high radical scavenging activity and suppressed vascular cell adhesion molecule 1 (VCAM-1) expression in vascular smooth muscle cells. However, the molecular mechanisms involved in its anti-inflammatory effect have not been reported. KA inhibited pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nitric oxide (NO), and prostaglandin E2 (PGE2). KA inhibited the production and mRNA expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) induced by LPS. KA downregulated the myeloid differentiation primary response 88 (MyD88)-dependent inflammatory gene expressions in the MyD88-overexpressed cells. KA suppressed the LPS-induced transcriptional and DNA-binding activities of activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB). KA was found to inhibit the phosphorylation of Janus kinase 1/2 (JAK1/2) and signal transducers and activators of transcription 1/3 (STAT1/3). In DSS-induced colitis mice, KA relieved the symptoms of colitis by suppressing inflammatory cell infiltration, restoring tight junction (TJ)- and epithelial–mesenchymal transition (EMT)-related protein expression, and inactivating AP-1, NF-κB, and STAT1/3. Therefore, KA reduced inflammatory responses by downregulating AP-1, NF-κB, and JAK/STAT signaling in LPS-induced macrophages and DSS-induced colitis mice.

Oral administration of Lipoteichoic acid from Lactobacillus rhamnosus GG overcomes UVB‐induced immunosuppression and impairs skin tumor growth in mice

Article

Full-text available

Jul 2019
EUR J IMMUNOL

There is increasing evidence of the relevant connection and regulation between the gut and skin immune axis. In fact, oral administration of lipoteichoic acid (LTA) from Lactobacillus rhamnosus GG (LGG) prevents the development of UV‐induced skin tumors in chronically exposed mice. Here we aim to evaluate whether this LTA is able to revert UV‐induced immunosuppression as a mechanism involved in its anti‐tumor effect and whether it has an immunotherapeutic effect against cutaneous squamous cell carcinoma. Using a mouse model of contact hypersensitivity, we demonstrate that LTA overcomes UV‐induced skin immunosuppression. This effect was in part achieved by modulating the phenotype of lymph node resident dendritic cells (DCs) and the homing of skin migratory DCs. Importantly, oral LTA reduced significantly the growth stablished skin tumors once UV radiation was discontinued, demonstrating that it has a therapeutic, besides the already demonstrated preventive antitumor effect. The data presented here strongly indicates that oral administration of LTA represents a promising immunotherapeutic approach for different conditions in which the skin immune system is compromised. This article is protected by copyright. All rights reserved

Innate Immune Memory: Time for Adopting a Correct Terminology

Article

Full-text available

Apr 2018

Staphylococcal LTA antagonizes the B cell-mitogenic potential of LPS

Article

Full-text available

Jan 2018

Lipoteichoic acid (LTA) of Gram-positive bacteria is regarded as the counterpart biomolecule of lipopolysaccharide (LPS) of Gram-negative bacteria because of their structural and immunological similarities. Although LPS induces a strong polyclonal expansion of B cells, little is known about the effect of LTA on B cell proliferation. In the present study, we prepared LTAs from Gram-positive bacteria and examined their effect on splenic B cell proliferation. Unlike LPS, LTA did not induce B cell proliferation. Instead, Staphylococcus aureus LTA (Sa.LTA) appeared to inhibit LPS-induced B cell proliferation in vitro, ex vivo, and in vivo models. Such effect was observed neither in splenocytes from Toll-like receptor 2 (TLR2)-deficient mice nor in the purified splenic B cells. Furthermore, decreased ERK phosphorylation appeared to be responsible for this phenomenon. Collectively, our results support that Sa.LTA inhibited LPS-induced B cell proliferation through the decrease of ERK phosphorylation via TLR2 signaling pathway.

Direct effects of fermented cow’s milk product with Lactobacillus paracasei CBA L74 on human enterocytes

Article

Full-text available

Oct 2017

Cow's milk fermented with Lactobacillus paracasei CBA L74 (FM-CBAL74) exerts a preventive effect against infectious diseases in children. We evaluated if this effect is at least in part related to a direct modulation of non-immune and immune defence mechanisms in human enterocytes. Human enterocytes (Caco-2) were stimulated for 48 h with FM-CBAL74 at different concentrations. Cell growth was assessed by colorimetric assay; cell differentiation (assessed by lactase expression), tight junction proteins (zonula occludens1 and occludin), mucin 2, and toll-like receptor (TRL) pathways were analysed by real-time PCR; innate immunity peptide synthesis, beta-defensin-2 (HBD-2) and cathelicidin (LL-37) were evaluated by ELISA. Mucus layer thickness was analysed by histochemistry. FMCBA L74 stimulated cell growth and differentiation, tight junction proteins and mucin 2 expression, and mucus layer thickness in a dose-dependent fashion. A significant stimulation of HBD-2 and LL-37 synthesis, associated with a modulation of TLR pathway, was also observed. FM-CBAL74 regulates non-immune and immune defence mechanisms through a direct interaction with the enterocytes. These effects could be involved in the preventive action against infectious diseases demonstrated by this fermented product in children.

Extremes of Interferon-Stimulated Gene Expression Associate with Worse Outcomes in the Acute Respiratory Distress Syndrome

Article

Full-text available

Sep 2016
PLOS ONE

Acute Respiratory Distress Syndrome (ARDS) severity may be influenced by heterogeneity of neutrophil activation. Interferon-stimulated genes (ISG) are a broad gene family induced by Type I interferons, often as a response to viral infections, which evokes extensive immunomodulation. We tested the hypothesis that over- or under-expression of immunomodulatory ISG by neutrophils is associated with worse clinical outcomes in patients with ARDS. Genome-wide transcriptional profiles of circulating neutrophils isolated from patients with sepsis-induced ARDS (n = 31) and healthy controls (n = 19) were used to characterize ISG expression. Hierarchical clustering of expression identified 3 distinct subject groups with Low, Mid and High ISG expression. ISG accounting for the greatest variability in expression were identified (MX1, IFIT1, and ISG15) and used to analyze a prospective cohort at the Colorado ARDS Network site. One hundred twenty ARDS patients from four urban hospitals were enrolled within 72 hours of initiation of mechanical ventilation. Circulating neutrophils were isolated from patients and expression of ISG determined by PCR. Samples were stratified by standard deviation from the mean into High (n = 21), Mid, (n = 82) or Low (n = 17) ISG expression. Clinical outcomes were compared between patients with High or Low ISG expression to those with Mid-range expression. At enrollment, there were no differences in age, gender, co-existing medical conditions, or type of physiologic injury between cohorts. After adjusting for age, race, gender and BMI, patients with either High or Low ISG expression had significantly worse clinical outcomes than those in the Mid for number of 28-day ventilator- and ICU-free days (P = 0.0006 and 0.0004), as well as 90-day mortality and 90-day home with unassisted breathing (P = 0.02 and 0.004). These findings suggest extremes of ISG expression by circulating neutrophils from ARDS patients recovered early in the syndrome are associated with poorer clinical outcomes.

Sodium Hypochlorite Inactivates Lipoteichoic Acid of Enterococcus faecalis by Deacylation

Article

Full-text available

Aug 2016

Introduction: Enterococcus faecalis is a pathogenic gram-positive bacterium closely associated with apical periodontitis. Although sodium hypochlorite (NaOCl) has been used as a common endodontic irrigant to eradicate bacteria in the root canal, it has not been elucidated whether NaOCl attenuates the inflammatory response induced by the E. faecalis virulence factor lipoteichoic acid (EfLTA). Methods: Structurally intact EfLTA purified from E. faecalis was treated with NaOCl at various concentrations and time periods. Murine macrophage cell line RAW 264.7 was treated with interferon gamma followed by treatment with intact or NaOCl-treated EfLTA to determine the inducibility of inflammatory mediators such as nitric oxide, interferon gamma-inducible protein 10, and macrophage inflammatory protein-1α. Reporter gene assays assessed by flow cytometry were used to examine the ability of intact or NaOCl-treated EfLTA to activate Toll-like receptor 2 (TLR2), which is known to recognize EfLTA on host cells. Structural damage of EfLTA by NaOCl was examined using silver staining and thin-layer chromatography. Results: NaOCl-treated EfLTA showed markedly less induction of nitric oxide, interferon gamma-inducible protein 10, and macrophage inflammatory protein-1α in RAW 264.7 cells compared with intact EfLTA. In contrast to intact EfLTA that potently stimulated TLR2 activation, NaOCl-treated EfLTA did not activate TLR2. Structural analysis showed that NaOCl damaged EfLTA structure by deacylation. Conclusions: NaOCl deacylates the glycolipid moiety of EfLTA, which fails to activate TLR2, leading to the reduced production of inflammatory mediators.

Receptor for the F4 fimbriae of enterotoxigenic Escherichia coli (ETEC)

Article

May 2015
APPL MICROBIOL BIOT

Infection with F4(+) enterotoxigenic Escherichia coli (ETEC) responsible for diarrhea in neonatal and post-weaned piglets leads to great economic losses in the swine industry. These pathogenic bacteria express either of three fimbrial variants F4ab, F4ac, and F4ad, which have long been known for their importance in host infection and initiating protective immune responses. The initial step in infection for the bacterium is to adhere to host enterocytes through fimbriae-mediated recognition of receptors on the host cell surface. A number of receptors for ETEC F4 have now been described and characterized, but their functions are still poorly understood. The current review summarizes the latest research addressing the characteristics of F4 fimbriae receptors and the interactions of F4 fimbriae and their receptors on host cells. These include observations that as follows: (1) FaeG mediates the binding activities of F4 and is an essential component of the F4 fimbriae, (2) the F4 fimbrial receptor gene is located in a region of chromosome 13, (3) the biochemical properties of F4 fimbrial receptors that form the binding site of the bacterium are now recognized, and (4) specific receptors confer susceptibility/resistance to ETEC F4 infection in pigs. Characterizing the host-pathogen interaction will be crucial to understand the pathogenicity of the bacteria, provide insights into receptor activation of the innate immune system, and develop therapeutic strategies to prevent this illness.

Assessing Cellular Responses in Sepsis

Article

Full-text available

Nov 2014

Jean-Louis Vincent

Antibacterial studies on in vivo plant parts of medicinally important Eurycoma longifolia (Tongkat Ali)

Data

Full-text available

Apr 2013

The various crude extracts of the In vivo plant parts of medicinally important Eurycoma longifolia were observed to demonstrate antibacterial activity to all the tested pathogenic bacteria. The most effective antibacterial agent was the extracted compound from the roots of Eurycoma longifolia on Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis (CDR), Staphylococcus aureus ATCC 25923 and Shigella flexneri ATCC 12022. In addition, there are also several other parts of this plants extract that produced good antibacterial activity against some tested pathogenic bacteria. Thus, the results obtained can be used to facilitate the development of antibacterial medicines for specific pathogenic bacteria to treat diseases caused by these bacteria.

Exposure-Dependent Control of Malaria-Induced Inflammation in Children

Article

Full-text available

Apr 2014
PLOS PATHOG

In malaria-naïve individuals, Plasmodium falciparum infection results in high levels of parasite-infected red blood cells (iRBCs) that trigger systemic inflammation and fever. Conversely, individuals in endemic areas who are repeatedly infected are often asymptomatic and have low levels of iRBCs, even young children. We hypothesized that febrile malaria alters the immune system such that P. falciparum re-exposure results in reduced production of pro-inflammatory cytokines/chemokines and enhanced anti-parasite effector responses compared to responses induced before malaria. To test this hypothesis we used a systems biology approach to analyze PBMCs sampled from healthy children before the six-month malaria season and the same children seven days after treatment of their first febrile malaria episode of the ensuing season. PBMCs were stimulated with iRBC in vitro and various immune parameters were measured. Before the malaria season, children's immune cells responded to iRBCs by producing pro-inflammatory mediators such as IL-1β, IL-6 and IL-8. Following malaria there was a marked shift in the response to iRBCs with the same children's immune cells producing lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines (IL-10, TGF-β). In addition, molecules involved in phagocytosis and activation of adaptive immunity were upregulated after malaria as compared to before. This shift was accompanied by an increase in P. falciparum-specific CD4+Foxp3- T cells that co-produce IL-10, IFN-γ and TNF; however, after the subsequent six-month dry season, a period of markedly reduced malaria transmission, P. falciparum-inducible IL-10 production remained partially upregulated only in children with persistent asymptomatic infections. These findings suggest that in the face of P. falciparum re-exposure, children acquire exposure-dependent P. falciparum-specific immunoregulatory responses that dampen pathogenic inflammation while enhancing anti-parasite effector mechanisms. These data provide mechanistic insight into the observation that P. falciparum-infected children in endemic areas are often afebrile and tend to control parasite replication.

Muramyl dipeptide potentiates staphylococcal lipoteichoic acid induction of cyclooxygenase-2 expression in macrophages

Article

Nov 2013

Gram-positive bacteria contains lipoteichoic acid (LTA) and peptidoglycan (PGN) layers, both of which are considered as major virulence factors associated with inflammation. Cyclooxygenase-2 (COX-2) plays an important role in the inflammation by generating prostaglandins at infections. Since LTA and PGN are thought to cooperate in the establishment of inflammation, we examined the ability of staphylococcal LTA (Sa.LTA) to induce COX-2 expression in the presence of muramyl dipeptide (MDP), which is the minimal structural unit of PGN required for inflammation, in macrophages. While MDP failed to induce COX-2 expression, Sa.LTA alone was sufficient to induce COX-2 production. Treatment with MDP enhanced Sa.LTA-induced COX-2 and prostaglandin E2 production. The cooperative effect between Sa.LTA and MDP was not observed in COX-2 expression by macrophages derived from Toll-like receptor 2 (TLR2)- or nucleotide-binding oligomerization domain 2 (NOD2)-deficient mice. In addition, MDP enhanced Sa.LTA-induced activation of the transcription factors NF-κB and CRE, which are known to modulate COX-2 gene transcription. Conclusively, these results suggest that MDP and Sa.LTA cooperatively induce inflammatory response by overproducing COX-2 through NOD2 and TLR2.

Exploring the immunomodulatory potential of microbial-associated molecular patterns derived from the enteric bacterial microbiota

Article

Full-text available

Jul 2013
MICROBIOL-SGM

The human intestinal lumen represents one of the most densely populated microbial niches in the biological world and, as a result, the intestinal innate immune system exists in a constant state of stimulation. A key component in the innate defence system is the intestinal epithelial layer, which not only acts as a physical barrier, but also as an immune sensor. The expression of pattern recognition receptors, such as Toll-like receptors, in epithelial cells allows innate recognition of a wide range of highly conserved bacterial moieties, termed microbial-associated molecular patterns (MAMPs), from both pathogenic and non-pathogenic bacteria. To date, studies of epithelial immunity have largely concentrated on inflammatory pathogenic antigens; however, this review discusses the major types of MAMPs likely to be produced by the enteric bacterial microbiota and, using data from in vitro studies, animal model systems and clinical observations, speculates on their immunomodulatory potential.

Role of Macrophage Receptor with Collagenous Structure in Innate Immune Tolerance

Article

Full-text available

May 2013
J IMMUNOL

Macrophages play a key role in host defense against microbes, in part, through phagocytosis. Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor on the cell surface of macrophages that mediates opsonin-independent phagocytosis. The goal of our study is to investigate the role of MARCO in LPS or lipotechoic acid-induced macrophage tolerance. Although it has been established that expression of MARCO and phagocytosis is increased in tolerant macrophages, the transcriptional regulation and biological role of MARCO in tolerant macrophages have not been investigated. In this study, we confirm that tolerized mouse bone marrow-derived macrophages (BMDM) selectively increase expression of MARCO (both transcript and cell surface receptor) and increase phagocytosis. We found that H3K4me3 dynamic modification of a promoter site of MARCO was increased in tolerized BMDM. Blocking methylation by treatment with 5-aza-2'-deoxycytidine resulted in reduced H3K4me3 binding in the promoter of MARCO, decreased expression of MARCO, and impaired phagocytosis in tolerized BMDM. However, 5-aza-2'-deoxycytidine had no effect on the inflammatory component of innate immune tolerance. In aggregate, we found that histone methylation was critical to MARCO expression and phagocytosis in tolerized macrophages, but did not affect the inflammatory component of innate immune tolerance.

MicroRNA-146a Is Upregulated by and Negatively Regulates TLR2 Signaling

Article

Full-text available

Apr 2013
PLOS ONE

TLR signaling is a crucial component of the innate immune response to infection. MicroRNAs (miRNAs) have been shown to be upregulated during TLR signaling. Specifically, microRNA-146a (miR-146a) plays a key role in endotoxin tolerance by downregulating interleukin-1 receptor-associated kinase 1 (IRAK-1). The aim of this study was to assess the role of miR-146a in the TLR2 signaling and development of bacterial lipoprotein (BLP) self-tolerance and cross-tolerance to bacteria. Expression of miR-146a increased in a dose- and time-dependent manner in BLP-stimulated human THP-1 promonocytic cells. In BLP-tolerised cells miR-146a was even further upregulated in response to BLP re-stimulation (p<0.001). Re-stimulation of BLP-tolerised cells with heat-killed gram-negative Salmonella typhimurium (S. typhimurium), but not gram-positive Staphylococcus aureus (S. aureus), led to significant overexpression of miR-146a (p<0.05). Transfection of naive cells with a miR-146a mimic substantially suppressed TNF-α production (p<0.05). Furthermore, overexpression of miR-146a resulted in strong reduction in IRAK-1 and phosphorylated IκBα expression in naive and S. typhimurium-stimulated THP-1 cells. Collectively, miR-146a is upregulated in response to BLP and bacterial stimulation in both naive and BLP-tolerised cells. Overexpression of miR-146a induces a state analogous to tolerance in BLP-stimulated cells and therefore may represent a future target for exogenous modulation of tolerance during microbial infection and sepsis.

Nonpathogenic Lactobacillus rhamnosus activates the inflammasome and antiviral responses in human macrophages

Article

Full-text available

Nov 2012

In this study, we have utilized global gene expression profiling to compare the responses of human primary macrophages to two closely related, well-characterized Lactobacillus rhamnosus strains GG and LC705, since our understanding of the responses elicited by nonpathogenic bacteria in human innate immune system is limited. Macrophages are phagocytic cells of the innate immune system that perform sentinel functions to initiate appropriate responses to surrounding stimuli. Macrophages that reside on gut mucosa encounter ingested and intestinal bacteria. Bacteria of Lactobacillus genus are nonpathogenic and used in food and as supplements with health-promoting probiotic potential. Our results demonstrate that live GG and LC705 induced quantitatively different gene expression profiles in macrophages. A gene ontology analysis revealed functional similarities and differences in responses to GG and LC705 that were reflected in host defense responses. Both GG and LC705 induced interleukin-1β production in macrophages that required caspase-1 activity. LC705, but not GG, induced type I interferon -dependent gene activation that correlated with its ability to prevent influenza A virus replication and production of viral proteins in macrophages. Our results indicate that nonpathogenic bacteria are able to activate the inflammasome. In addition, our results suggest that L. rhamnosus may prime the antiviral potential of human macrophages.

Nodakenin Suppresses Lipopolysaccharide-Induced Inflammatory Responses in Macrophage Cells by Inhibiting Tumor Necrosis Factor Receptor-Associated Factor 6 and Nuclear Factor- B Pathways and Protects Mice from Lethal Endotoxin Shock

Article

May 2012
J PHARMACOL EXP THER

Nodakenin, a coumarin isolated from the roots of Angelicae gigas, has been reported to possess neuroprotective, antiaggregatory, antibacterial, and memory-enhancing effects. In the present study, we investigated the anti-inflammatory effects of nodakenin by examining its in vitro inhibitory effects on inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and mouse peritoneal macrophages and its in vivo effects on LPS-induced septic shock in mice. Our results indicate that nodakenin concentration-dependently inhibits iNOS and COX-2 at the protein, mRNA, and promoter binding levels, and these inhibitions cause attendant decreases in the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). Furthermore, we found that nodakenin inhibits the production and mRNA expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β induced by LPS. Molecular data revealed that nodakenin suppressed the transcriptional activity and translocation of nuclear factor-κB (NF-κB) by inhibiting inhibitory κB-α degradation and IκB kinase-α/β phosphorylation. In addition, nodakenin was found to significantly inhibit the LPS-induced binding of transforming growth factor-β-activated kinase 1 to tumor necrosis factor receptor-associated factor 6 (TRAF6) by reducing TRAF6 ubiquitination. Pretreatment with nodakenin reduced the serum levels of NO, PGE₂, and proinflammatory cytokines and increased the survival rate of mice with LPS-induced endotoxemia. Taken together, our data suggest that nodakenin down-regulates the expression of the proinflammatory iNOS, COX-2, TNF-α, IL-6, and IL-1β genes in macrophages by interfering with the activation of TRAF6, thus preventing NF-κB activation.

Lipoteichoic acid from Lactobacillus plantarum induces nitric oxide production in the presence of interferon-?? in murine macrophages

Article

Aug 2011

Lipoteichoic acid (LTA) is a major immuno-stimulating component of Gram-positive bacteria. LTA from the beneficial bacterium Lactobacillus plantarum induces weak nitric oxide (NO) production in murine macrophages. Currently, it is not clear if LTA from L. plantarum is able to stimulate the innate immune response, even in the presence of inflammation. In the present study, we prepared highly pure and structurally intact LTA from L. plantarum and investigated its ability to induce NO in the presence of interferon (IFN)-γ in the RAW 264.7 murine macrophage cell line and bone marrow-derived macrophages (BMMs) from mice. L. plantarum LTA alone was unable to induce NO production, even at 30μg/ml. However, LTA in the presence of IFN-γ significantly induced NO production in RAW 264.7 cells. The observed NO production was inhibited by a NO synthase (NOS) inhibitor l-NAME and an inducible NOS (iNOS) inhibitor l-NIL, suggesting that iNOS is specifically required for this action. Western blot analysis and reverse transcription and polymerase chain reaction further confirmed that L. plantarum LTA increased protein and mRNA levels of iNOS, respectively. However, such induction was substantially inhibited in BMMs from Toll-like receptor 2 (TLR2)-deficient mice and the macrophages treated with an inhibitor blocking platelet-activating factor receptor. In addition, L. plantarum LTA plus IFN-γ induced IFN-β expression and STAT1 phosphorylation, which are key pathways for inducing iNOS expression. Electrophoretic mobility shift assay demonstrated that L. plantarum LTA in the presence of IFN-γ remarkably increased the DNA-binding activity of NF-κB transcription factor, which is known to be involved in the iNOS gene expression. Collectively, these results suggest that LTA from L. plantarum alone has no inflammatory potential but does induce NO production under conditions of inflammation, such as the presence of IFN-γ.

Lipoteichoic Acid Inhibits Lipopolysaccharide-Induced TLR4 Signaling by Forming an Inactive TLR4/MD-2 Complex Dimer

Article

Mar 2023
J IMMUNOL

LPS interacts with TLR4, which play important roles in host-against-pathogen immune responses, by binding to MD-2 and inducing an inflammatory response. In this study, to our knowledge, we found a novel function of lipoteichoic acid (LTA), a TLR2 ligand, that involves suppression of TLR4-mediated signaling independently of TLR2 under serum-free conditions. LTA inhibited NF-κB activation induced by LPS or a synthetic lipid A in a noncompetitive manner in human embryonic kidney 293 cells expressing CD14, TLR4, and MD-2. This inhibition was abrogated by addition of serum or albumin. LTAs from different bacterial sources also inhibited NF-κB activation, although LTA from Enterococcus hirae had essentially no TLR2-mediated NF-κB activation. The TLR2 ligands tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4) and macrophage-activating lipopeptide-2 (MALP-2) did not affect the TLR4-mediated NF-κB activation. In bone marrow-derived macrophages from TLR2-/- mice, LTA inhibited LPS-induced IκB-α phosphorylation and production of TNF, CXCL1/KC, RANTES, and IFN-β without affecting cell surface expression of TLR4. LTA did not suppress IL-1β-induced NF-κB activation mediated through signaling pathways shared with TLRs. LTAs including E. hirae LTA, but not LPS, induced association of TLR4/MD-2 complexes, which was suppressed by serum. LTA also increased association of MD-2, but not TLR4 molecules. These results demonstrate that, under serum-free conditions, LTA induces association of MD-2 molecules to promote formation of an inactive TLR4/MD-2 complex dimer that in turn prevents TLR4-mediated signaling. The presence of LTA that poorly induces TLR2-mediated activation but inhibits TLR4 signaling provides insight into the role of Gram-positive bacteria in suppressing inflammation induced by Gram-negative bacteria in organs such as the intestines where serum is absent.

Induction of Innate Memory in Human Monocytes Exposed to Mixtures of Bacterial Agents and Nanoparticles

Article

Full-text available

Nov 2022
INT J MOL SCI

We assessed whether concomitant exposure of human monocytes to bacterial agents and different engineered nanoparticles can affect the induction of protective innate memory, an immune mechanism that affords better resistance to diverse threatening challenges. Monocytes were exposed in vitro to nanoparticles of different chemical nature, shape and size either alone or admixed with LPS, and cell activation was assessed in terms of production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra). After return to baseline conditions, cells were re-challenged with LPS and their secondary “memory” response measured. Results show that nanoparticles alone are essentially unable to generate memory, while LPS induced a tolerance memory response (less inflammatory cytokines, equal or increased anti-inflammatory cytokines). LPS-induced tolerance was not significantly affected by the presence of nanoparticles during the memory generation phase, although with substantial donor-to-donor variability. This suggests that, despite the overall lack of significant effects on LPS-induced innate memory, nanoparticles may have donor-specific effects. Thus, future nanosafety assessment and nanotherapeutic strategies will need a personalized approach in order to ensure both the safety and efficacy of nano medical compounds for individual patients.

Functional Transcriptomic Studies of Immune Responses and Endotoxin Tolerance In early Human Sepsis

Article

Jan 2022

Background: Limited studies have functionally evaluated the heterogeneity in early ex vivo immune responses during sepsis. Our aim was to characterize early sepsis ex vivo functional immune response heterogeneity by studying whole blood endotoxin responses and derive a transcriptional metric of ex vivo endotoxin response. Methods: Blood collected within 24 h of hospital presentation from 40 septic patients was divided into two fractions and incubated with media (unstimulated) or endotoxin. Supernatants and cells were isolated, and responses measured using: supernatant cytokines, lung endothelial permeability after supernatant exposure, and RNA expression. A transcriptomic signature was derived in unstimulated cells to predict the ex vivo endotoxin response. The signature was tested in a separate cohort of 191 septic patients to evaluate for association with clinical outcome. Plasma biomarkers were quantified to measure in vivo host inflammation. Results: Ex vivo response to endotoxin varied and was unrelated to immunosuppression, white blood cell count, or the causative pathogen. Thirty-five percent of patients demonstrated a minimal response to endotoxin, suggesting early immunosuppression. High ex vivo cytokine production by stimulated blood cells correlated with increased in vitro pulmonary endothelial cell permeability and was associated with attenuated in vivo host inflammation. A four-gene signature of endotoxin response detectable without the need for a functional assay was identified. When tested in a separate cohort of septic patients, its expression was inversely associated with hospital mortality. Conclusions: An attenuated ex vivo endotoxin response in early sepsis is associated with greater host in vivo inflammation and a worse clinical outcome.

Investigation of innate immune function in adult and geriatric horses

Article

Feb 2021
VET IMMUNOL IMMUNOP

In order to better understand the influence of age on innate immune function in horses, blood was collected from twelve adult horses (aged 10 – 16 years; mean: 13 years) and ten geriatric horses (aged 18 – 26 years; mean: 21.7 years) for analysis of plasma myeloperoxidase, complete blood counts, and cytokine and receptor expression in response to in vitro stimulation with heat-inactivated Rhodococcus equi, heat-inactivated Escherichia coli, and PMA/ionomycin. Gene expression was measured using RT-PCR for IFNγ, IL-1β, IL-6, IL-8, IL-10, IL-12α, IL-13, IL-17α, TLR2, TLR4, and TNFα. Endocrine function and body weight were measured to assess any potential impacts of ACTH, insulin, or body weight on immune function; none of the horses had pituitary pars intermedia dysfunction. The geriatric horse group had lower concentrations of plasma myeloperoxidase (P = 0.0459) and lower absolute monocyte counts (P = 0.0477); however, the difference in monocyte counts was no longer significant after outliers were removed. Additionally, only two significant differences in cytokine/receptor expression in whole blood were observed. Compared with adult horses, the geriatric horses had increased TNFα expression after in vitro stimulation with heat-inactivated R. equi (P = 0.0224) and had decreased IL-17α expression after PMA/ionomycin stimulation when one outlier was excluded (P = 0.0334). These changes may represent a compensatory mechanism by which geriatric horses could ensure adequate immune responses despite potentially dysfunctional neutrophil activity and/or decreased monocyte counts. Aging may influence equine innate immune function, and additional research is warranted to confirm and further explore these findings.

Immunologische Mechanismen der Pathogenese und Prävention von Asthma bronchiale

Thesis

Oct 2018

Marcus Peters

Kumulative Habilitationsschrift zur Erlangung der Venia legendi für das Fach "Immunologie"

K ATP channel block inhibits the Toll-like receptor 2-mediated stimulation of NF-κB by suppressing the activation of Akt, mTOR, JNK and p38-MAPK

Article

Sep 2017
EUR J PHARMACOL

Changes in the KATP channel activity have been shown to regulate inflammation and immune responses. Using human keratinocytes, we investigated the effect of KATP channel inhibition on inflammatory mediator production in relation to the Toll like receptor-2-mediated-Akt, mTOR and NF-κB pathways, as well as JNK and p38-MAPK, which regulate the transcription genes involved in immune and inflammatory responses. 5-Hydroxydecanoate (a selective KATP channel blocker), glibenclamide (a cell surface and mitochondrial KATP channel inhibitor), the Akt inhibitor, rapamycin, Bay 11-7085 and N-acetylcysteine reduced the lipoteichoic acid- or peptidoglycan-induced production of cytokines and chemokines, and production of reactive oxygen species and increased the levels and activities of Kir 6.2, NF-κB, phosphorylated-Akt and mTOR, and the activation of JNK and p38-MAPK in keratinocytes. Inhibitors of c-JNK (SP600125) and p38-MAPK (SB203580) attenuated the lipoteichoic acid- or peptidoglycan-induced production of inflammatory mediators, the activation of the JNK and p38-MAPK, and the production of reactive oxygen species in keratinocytes. The results show that KATP channel blockers may reduce the bacterial component-stimulated production of inflammatory mediators in keratinocytes by suppressing the Toll-like receptor-2-mediated activation of the Akt, mTOR and NF-κB pathways, as well as JNK and p38-MAPK. The suppressive effect of KATP channel blockers appears to be achieved by the inhibition of reactive oxygen species production.

Immunostimulatory Activity of Opuntia ficus-indica var. Saboten Cladodes Fermented by Lactobacillus plantarum and Bacillus subtilis in RAW 264.7 Macrophages

Article

Feb 2017

To increase the functionality of Opuntia ficus-indica var. saboten cladodes, it was fermented by Lactobacillus plantarum and Bacillus subtilis. Eighty percent methanol extracts were investigated for their effects on nitric oxide (NO) production, cytokine secretion, nuclear factor-κB (NF-κB) activity, and mitogen-activated protein kinase (MAPK) phosphorylation in RAW 264.7 cells. Methanol extracts of L. plantarum culture medium (LPCME) and B. subtilis culture medium (BSCME) did not affect lipopolysaccharide (LPS)-induced NO production but, at 500 μg/mL, increased interferon (IFN)-γ-induced NO production by 55.2 and 66.5 μM, respectively, in RAW 264.7 cells. In RAW 264.7 cells not treated with LPS and IFN-γ, LPCME did not affect NO production, but BSCME increased NO production significantly in a dose-dependent manner. In addition, BSCME induced the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells in a dose-dependent manner. BSCME at 500 μg/mL increased TNF-α and IL-1β mRNA levels by 83.8% and 82.2%, respectively. BSCME increased NF-κB-dependent luciferase activity in a dose-dependent manner; 500 μg/mL BSCME increased activity 9.1-fold compared with the control. BSCME induced the phosphorylation of p38, c-JUN NH2-terminal protein kinase (JNK), and extracellular signal-regulated kinase (ERK) in a dose-dependent manner, but did not affect total ERK levels. In conclusion, BSCME exerted immunostimulatory effects, which were mediated by MAPK phosphorylation and NF-κB activation, resulting in increased TNF-α and IL-1β gene expression in RAW 264.7 macrophages. Therefore, BSCM shows promise for use as an immunostimulatory therapeutic. © Copyright 2017, Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition.

Bioactive Bacterial Components: Could they Change the Probiotic World?

Article

Dec 2011

Probiotic agents are defined as live microorganisms, which confer a health benefit to the host when administered in adequate amounts. However, also molecules produced by both probiotic and non-probiotic bacteria and administered alone seem to provide beneficial effects similar to those obtained with live cells. These molecules can stimulate different signalling pathways by activating pattern recognition receptors and modulate different immune responses in the host. This review focuses on bacterial components, such as surface molecules and DNA, with beneficial immunomodula-tory properties. Lipopolysaccharides from Gram-negative bacteria, lipoteichoic acid from Gram-positive bacteria and DNA of specific strains are the most effective molecules in modulating the host immune response, as they can modify the pattern of released cytokines. Bacterial components with beneficial properties could be an interesting substitute to traditional probiotics, as they could eliminate some of the risks associated with the consumption of live bacteria. Data presented in this review show that the concept of probiotic agent should change, as a large portion of the beneficial effects of probiotic bacteria does not depend on their viability and integrity. A more clear understanding of molecular mechanisms resulting in the health benefits provided by probiotics could be the basis for a more rational application of probiotics in functional foods or as supporting therapy for specific disorders.

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

Article

May 2014
INT IMMUNOPHARMACOL

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 in MAC-T cells. 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.

Bacteria and Toll-like receptor and cytokine mRNA expression profiles associated with canine arthritis

Article

Aug 2014

The major forms of inflammatory canine arthritis are immune-mediated arthritis (IMA) and septic arthritis (SA), although some cases of cruciate disease (CD) are associated with significant levels of synovitis. In this study, the bacteria associated with canine arthritis were identified and mRNA expression levels of Toll-like receptors (TLRs) and pro-inflammatory cytokines determined. Of the 40 synovial fluid samples analysed, bacteria were isolated from 12 samples by culture (two CD, 10 SA) and detected in four samples (three CD, one SA) using culture-independent methods. Statistically significant increases in TLR2, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-12 mRNA expression were seen in all disease groups compared to normal controls. All disease groups had decreased mRNA expression of other TLRs compared to normal controls, but this did not reach statistical significance. Synovial fluid cell counts revealed that the highest number and proportion of mononuclear cells and neutrophils were found in the IMA and SA samples, respectively. Age had an effect on the TLR and cytokine mRNA expression profiles: TNF-α (p = 0.043) and IL-12 (p = 0.025) mRNA expression was increased and TLR4 mRNA expression was reduced (p = 0.033) in dogs up to fours years of age compared to older animals. In the 10 SA samples from which bacteria were isolated, statistically significant increases in TLR2, TLR7, TNF-α and IL-6 mRNA expression were observed. It is concluded that canine arthritis is associated with increased mRNA levels of pro-inflammatory cytokines, which could in some cases be mediated by bacteria through activation of TLR2.

Rotundarpene inhibits Toll-like receptor 2 activation-induced production of inflammatory mediators in keratinocytes by suppressing the Akt and NF-κB pathways

Article

Dec 2013

Microbial components have been shown to be involved in the pathogenesis of inflammatory skin diseases. The extract of from the barks of Ilex rotunda Thunb has demonstrated anti-inflammatory and anti-oxidant effects. However, the effect of hemiterpene rotundarpene (4-caffeoyl-3-methyl-but-2-ene-1,4-diol) on the Toll-like receptor (TLR)-2 activation-induced production of inflammatory mediators in keratinocytes has not been studied. Using human keratinocytes, we investigated the effect of rotundarpene on the inflammatory mediator production in relation to the TLR-2-mediated-Akt and NF-κB pathways, which regulates the transcription genes involved in immune and inflammatory responses. Rotundarpene, Akt inhibitor, Bay 11-7085 and N-acetylcysteine each attenuated the lipoteichoic acid- or peptidoglycan-induced production of cytokines and chemokines, expression of TLR-2, activation of NF-κB and Akt, and formation of reactive oxygen species in keratinocytes. Cyclosporine A attenuated the bacterial component-induced production of inflammatory mediators but did not reduce the formation of reactive oxygen species. The results show that rotundarpene may attenuate the bacterial component-stimulated production of inflammatory mediators in keratinocytes by suppressing the TLR-2-mediated activation of the Akt and NF-κB pathways. The effect of rotundarpene may be attributed to its inhibitory effect on the formation of reactive oxygen species. Rotundarpene may exert a preventive effect against the bacterial component-mediated inflammatory skin diseases.

The compromised inflammatory response to bacterial components after pediatric cardiac surgery is associated with cardiopulmonary bypass-suppressed Toll-like receptor signal transduction pathways

Article

Oct 2013

Cardiopulmonary bypass (CPB) during pediatric cardiac surgery often elicits a systemic inflammatory response followed by a compromised immune response, which has been attributed to the morbidity of postoperative infection; however, the underlying mechanism(s) has not yet been fully elucidated. We hypothesized that CPB inhibits the activation of Toll-like receptor (TLR) signal transduction pathways, thereby causing an immunosuppressive state after pediatric cardiac surgery. We examined 20 children with congenital heart disease undergoing pediatric cardiac surgery. Cardiopulmonary bypass differentially affected lipopolysaccharide (LPS)- or bacterial lipoprotein (BLP)-stimulated ex vivo production of proinflammatory and anti-inflammatory cytokines, with significantly diminished tumor necrosis factor α, interleukin (IL) 1β, IL-6, and IL-8, but substantially enhanced IL-10 production. Consistent with the reduced inflammatory response, CPB strongly inhibited LPS- or BLP-activated TLR signal transduction pathways in monocytes with down-regulated expression of CD14, TLR4, and TLR2 and with suppressed phosphorylation of nuclear factor κB p65, p38, and extracellular signal-regulated kinase 1/2. These results indicate that CPB during pediatric cardiac surgery causes substantially reduced production of inflammatory cytokines in response to bacterial component LPS or BLP stimulation, which is associated with CPB-induced suppression of TLR-mediated signal transduction pathways. This reduced inflammatory response after CPB in children with congenital heart disease may predispose them to an increased risk of postoperative infection.

A novel synthetic lipopeptide is allergy-protective by the induction of LPS-tolerance

Article

Jul 2013
CLIN EXP ALLERGY

Exposure to the environment of traditional farms can protect children from some allergic disease. Due to this exposure, TLR2 expression in these children is increased. TLR2 ligands derived from gram-positive bacteria are found in the dust of these farms. We proved whether a synthetic lipopeptide binding to the TLR1/2 heterodimer is able to protect from allergic disease in two different murine models of allergy. We also investigated the immunological mechanisms underlying the protective properties of the lipopeptide. We synthesized a lipopeptide derived from a germination lipoprotein of Bacillus cereus (LPGerD). We evaluated the immunomodulatory activity of LPGerD in a murine model of systemic sensitization (OVA/Alum) and in a model in which mice were sensitized with OVA pulsed bone-marrow-derived dendritic cells (BMDCs) via the airways. Furthermore, the induction of LPS tolerance was studied. Treatment of mice with LPGerD in a mouse model of asthma led to protection against sensitization and airway inflammation. Similarly, bone-marrow-derived dendritic cells (BMDCs) pre-treated with LPGerD were not able to prime mice for allergic immune response. We observed that pre-treatment with LPGerD led to the induction of a LPS-tolerant state in BMDCs. These cells secreted markedly lower amounts of pro-inflammatory cytokines upon LPS stimulation. Furthermore, we observed an up-regulation of IRAK-M mRNA in BMDCs pre-treated with LPGerD. Our results suggest that induction of a LPS-tolerant state in antigen-presenting cells (APCs) may contribute to the protective effect of a farming environment. TLR2 agonists similar to those appearing in cowshed dust extracts, such as our synthetic LPGerD, lead to the ignorance of the LPS stimulus, which is important for the activation of APCs to mount a Th2 immune response. This substance might be a promising candidate for allergy-preventive treatments as LPGerD had only low pro-inflammatory characteristics.

Lipoteichoic acid from Lactobacillus rhamnosus GG as an oral photoprotective agent against UV-induced carcinogenesis

Article

Full-text available

Jul 2012
BRIT J NUTR

Probiotics are live micro-organisms that when administered in adequate amounts confer a health benefit on the host. Cell surface molecules of these micro-organisms are being studied in relation to their ability to interact with the host. The cell wall of lactobacilli possesses lipoteichoic acids (LTA) which are molecules with immunomodulatory properties. UV radiation (UVR) has been proposed as the main cause of skin cancer because of its mutagenic and immunosuppressive effects. Photoprotection with some nutrition interventions including probiotics has recently been shown. The aim of the present study was to investigate whether the oral administration of purified LTA from Lactobacillus rhamnosus GG can modulate the immune-suppressive effect of UVR and skin tumour development in female Crl:SKH-1-hrBR mice. For this purpose, two irradiation models were studied: (1) a chronic irradiation scheme consisting of daily irradiations during twenty consecutive days and (2) a long-term irradiation schedule, irradiating the animals three times per week, during 34 weeks for tumour development. The results showed that T-cells in the inguinal lymph node of LTA-treated mice produced higher levels of (1) interferon-γ and (2) a number of total, helper and cytotoxic T-cells compared with non-treated mice. Moreover, a significant delay in tumour appearance was found in LTA-treated mice. An increased IgA+ cell number was found in the small intestine together with a higher number of activated dendritic cells in the mesenteric lymph nodes. The latter results might be indicative of a direct effect of LTA in the gut, affecting the cutaneous immune system and restoring homeostasis through the gut-skin axis.

Stem cells of intestinal epithelium. The mechanisms of survival and the role of microbiota

Article

Sep 2009

The thanatogenetic mechanisms of stem cells (sc) of rapidly renewing system of intestinal epithelium still remain unclear. on the one hand they are definitely involved into basic mechanisms of carcinogenesis in the gastrointestinal tract, because dysregulation of programs responsible for elimination of “unwanted” mutant cells (which are normally under immune and own intrinsic control) is one of the reasons of neoplastic expansion. on the other hand elucidation and characterization of the regulatory machinery controlling sc survival are interrelated with problems of clinical medicine, including the increase of therapeutic efficiency of treatment of inflammatory and ulcer lesions of the gut, traumatic and surgical wounds, as well as restriction of side effects in normal tissues induced by application of intensive methods chemo- and radiotherapy of cancer. the latter is especially important for treatment of blood diseases and tumors of peritoneal cavity organs mainly due to bone marrow and intestinal epithelium damage. (These tissues are the most sensitive to these treatments.) The review considers data on exogenous and genetic modifiers of sc survival, and also the basic principles of mechanisms involved into renewal and regeneration of sc and the effects of microbiota on these processes.

Модель патогенеза псориаза. Часть 2. Локальные процессы.

Book

Full-text available

Apr 2012

Mikhail Peslyak

Выполнено обзорно-аналитическое исследование результатов экспериментальных и теоретических работ по изучению этиологии и патогенеза псориатической болезни. Сформулирована новая модель патогенеза псориаза – кожной реакции на системный псориатический процесс SPP. Из-за PAMP-немии в кровотоке образуются фракции толеризованных моноцитов Mo-T и дендритных клеток DC-T. Толеризованные Mo-T и DC-T также являются kPAMP носителями. В рамках PAMP-немии также имеет место (PG-Y)-немия. Поэтому внутри толеризованных фракций образуются подфракции моноцитов Mo-R и дендритных клеток DC-R. Эти Mo-R и DC-R также являются (PG-Y)-носителями. Толеризованные Mo-T и DC-T (в т.ч. Mo-R и DC-R) имеют хемостатусы подобные неактивированным. Поэтому эти клетки участвуют в гомеостатическом и/или воспалительном обновлении пула дермальных макрофагов и дендритных клеток нерезидентного происхождения. Псориатическое воспаление рассматривается как реакция кожной иммунной системы на действия привлеченных в дерму из кровотока Mo-R и DC-R. Они содержат Y антиген внутри себя и, попадая в дерму, могут трансформироваться в зрелые maDC-Y и осуществить презентацию этого антигена Y специфическим T лимфоцитам и активировать их. Y-антиген является частью межпептидного мостика IB Y. Поэтому кожная иммунная система может ошибочно интерпретировать презентацию Y-антигена как признак внешней PsB-инфекции и включить один из механизмов защиты от бактериальной инфекции - эпидермальную гиперпролиферацию. Псориатическое пятно может инициироваться только во время действия в дерме локального воспалительного процесса LP2, вызывающего не только врожденный, но и приобретенный ответ против себя. В частности это возможно при LP2(IN) – открытой травме дермы или при LP2(HPV) – HPV-носительстве кератиноцитов. Уровень Y примирования (наличие и концентрация Y-специфических T-лимфоцитов в препсориатической дерме и лимфоузлах) также определяет возможность инициации псориатического пятна. Существование и тяжесть пятна определяется интенсивностью поступления Y антигена в дерму (внутри Mo-R и DC-R). Тяжесть пятна усугубляется интенсивностью поступления kPAMP в дерму (внутри Mo-T и DC-T). Интенсивность обоих поступлений зависит от тяжести SPP. Тяжесть пятна усугубляется LP2 воспалением, если оно сохраняется после инициации этого пятна. В пятна из кровотока постоянно привлекаются новые Mo T, DC-T (в т.ч. Mo R, DC-R) и Y-специфические T-лимфоциты, что поддерживает действие порочных циклов. Только при снижении тяжести SPP порочные циклы слабеют и происходит естественная ремиссия пятен, вплоть до полного их исчезновения. Проведен детальный сравнительный анализ новой модели патогенеза с пятью другими моделями, опубликованными в последние годы.

Модель патогенеза псориаза. Часть 1. Системный псориатический процесс. Издание r4.0

Book

Full-text available

Apr 2012

Mikhail Peslyak

Выполнено обзорно-аналитическое исследование результатов экспериментальных и теоретических работ по изучению этиологии и патогенеза псориатической болезни. Сформулирована новая модель патогенеза псориаза – кожного проявления системного псориатического процесса (SPP = systemic psoriatic process), объясняющая результаты клинических и лабораторных экспериментов. Эта модель (далее Y-модель) предполагает решающую роль повышенной проницаемости тонкого кишечника для бактериальных продуктов и колонизации его стенок грамположительными бактериями (в т.ч. псорагенными PsB) и грамотрицательными TLR4 активными бактериями. Внутри SPP есть порочный цикл, который поддерживается нарушением производства и/или циркуляции желчных кислот. Центральным подпроцессом SPP является PAMP-немия, а именно хроническая kPAMP-нагрузка на фагоциты крови (нейтрофилы, моноциты и дендритные клетки), приводящая также к повышенному уровню kPAMP в кровотоке. Главными ключевыми PAMP (kPAMP) являются LPS и PG (в т.ч. PG-Y – пептидогликан псорагенных бактерий). Хронически повышенная kPAMP-нагрузка вероятно обеспечивает толеризацию части нейтрофилов Neu, моноцитов Mo и дендритных клеток DC, находящихся в кровотоке. Хемостатус толеризованных Neu-T по мере их старения меняется аналогично хемостатусу неактивированных Neu и, следовательно, они несут эндоцитированный в кровотоке контент в костный мозг. Хемостатусы толеризованных Mo-T и DC-T подобны хемостатусам неактивированных и, как следствие, они не уносят эндоцитированный контент в лимфоузлы или селезенку, а остаются в кровотоке. Толеризованные фагоциты медленно и не полностью деградируют эндоцитированные фрагменты бактериальных продуктов содержащие kPAMP. Толеризованные фагоциты оказавшиеся (PG Y) носителями названы R-фагоцитами и обозначаются как Neu-R, Mo-R и DC-R. Тяжесть SPP пропорциональна суммарному (PG-Y)-носительству Mo-R и DC-R в кровотоке. Тяжесть SPP предопределяет возможность возникновения и поддержки псориаза, поскольку Mo-R и DC-R наряду с нормальными Mo и DC участвуют в гомеостатическом и воспалительном обновлении пула дермальных макрофагов и DC нерезидентного происхождения. Mo-R и DC-R попадая в дерму могут превратиться в зрелые maDC-Y, осуществляющие презентацию Y-антигена специфическим TL-Y. Локальные процессы, происходящие при этом в дерме и эпидермисе, подробно описаны во второй части монографии.

Model of pathogenesis of psoriasis. Part 2. Local processes

Book

Full-text available

Jan 2012

Mikhail Peslyak

Observational and analytical research of results of experimental and theoretical studies on etiology and pathogenesis of psoriatic disease is carried out. The new model of pathogenesis of psoriasis –skin reaction to systemic psoriatic process SPP is formulated. Because of PAMP-nemia in blood flow fractions of tolerized monocytes Mo-T and dendritic cells DC-T are formed. Tolerized Mo-T and DC-T also are kPAMP-carriers. Within the limits of PAMP-nemia also (PG-Y)-nemia takes place. Therefore in tolerized fractions subfractions of monocytes Mo-R and dendritic cells DC-R are formed. These Mo-R and DC-R also are (PG-Y)-carriers. Tolerized Mo-T and DC-T (incl. Mo-R and DC-R) have chemostatuses similar to nonactivated ones. Therefore these cells participate in homeostatic and-or inflammatory renewal of pool of dermalmacrophages and dendritic cells of non-resident origin. Psoriatic inflammation is regarded as a reaction of the skin immune system to activity of Mo-R and DC-R involved in derma from blood flow. They contain Y-antigen and, getting to derma, can be transformed in mature maDC-Y and present this antigen to Y-specific T-lymphocytes as well as activate them. Y-antigen is a part of the interpeptide bridge IB-Y. Therefore, the skin immune system can incorrectly interpret Y-antigen presentation as a sign of external PsB-infection and switch one of mechanisms of protection against bacterial infection –epidermal hyperproliferation. Psoriatic plaque can be initiated only during action of local inflammatory process LP2 in derma causing not only innate, but also adaptive response. In particular, it is possible at LP2(IN) -open trauma of derma or at LP2(HPV) -HPV-carriage of keratinocytes. The level of Y-priming (presence and concentration of Y-specific T-lymphocytes in prepsoriatic derma and in lymph nodes) also determines possibility of psoriatic plaque initiation. Existence and severity of psoriatic plaque is determined by intensity of Y-antigen income into derma (inside Mo-R and DC-R). Severity of plaque is aggavatedby intensity of kPAMP income into derma (insideMo-Tand DC-T). Intensity of both incomes depends on SPP severity. Severity of plaque is aggravated by LP2-inflammation if it persists after this plaque initiation. New Mo-T, DC-T (incl. Mo-R, DC-R)and Y-specific T-lymphocytes are constantly attracted into plaquesfrom blood flow, and sosupport vicious cycles. Only at decrease of SPP severity, these vicious cycles weaken and natural remission of plaques takes place, up to their complete disappearance. The detailed analysis comparing the new model of pathogenesis with five other previously published models is carried out. Part 1. arXiv:1110.0584

Cytokine-inducible expression in endothelial cells of an I kappa B alpha-like gene is regulated by NF kappa B

Article

Full-text available

Jul 1993

The transient expression of many different genes is mediated by the inducible transcription factor p50‐p65 NF kappa B, which in turn is regulated by complex formation with its inhibitor I kappa B alpha. We describe here that in porcine aortic endothelial cells, either IL‐1 alpha, TNF alpha or LPS upregulates an inhibitor of NF kappa B which we refer to as ECI‐6. ECI‐6 is by structural and functional criteria an I kappa B alpha protein, the porcine homologue of MAD‐3, pp40 and RL/IF‐1. We have studied the promoter of the ECI‐6/I kappa B alpha gene and provide three lines of evidence that its expression is directly regulated by NF kappa B. First, the 5′ regulatory region of ECI‐6/I kappa B alpha contains two sites that bind NF kappa B in electrophoretic mobility shift assays. Second, expression following transfection of an ECI‐6/I kappa B alpha promoter‐luciferase reporter construct is dependent on a co‐transfected NF kappa B‐p65 subunit. Third, pretreatment of endothelial cells with antioxidants, agents that inhibit activation of NF kappa B, inhibit the expression of ECI‐6/I kappa B alpha. We conclude that the regulated expression of ECI‐6/I kappa B alpha could represent a novel feedback mechanism by which NF kappa B downregulates its own activity after transient activation of target genes has been achieved.

Synergy and Cross-Tolerance Between Toll-Like Receptor (TLR) 2- and TLR4-Mediated Signaling Pathways

Article

Full-text available

Dec 2000

A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-α production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-α in response to LPS. LPS-induced activation of both NF-κB and c-Jun NH2-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.

MyD88 Is an Adaptor Protein in the hToll/IL1 Receptor Family Signaling Pathways

Article

Full-text available

Aug 1998
MOL CELL

The Toll-mediated signaling cascade using the NF-κB pathway has been shown to be essential for immune responses in adult Drosophila, and we recently reported that a human homolog of the Drosophila Toll protein induces various immune response genes via this pathway. We now demonstrate that signaling by the human Toll receptor employs an adaptor protein, MyD88, and induces activation of NF-κB via the Pelle-like kinase IRAK and the TRAF6 protein, similar to IL-1R-mediated NF-κB activation. However, we find that Toll and IL-1R signaling pathways are not identical with respect to AP-1 activation. Finally, our findings implicate MyD88 as a general adaptor/regulator molecule for the Toll/IL-1R family of receptors for innate immunity.

Mammalian MAP kinase signaling cascades

Article

Full-text available

Jan 2001
NATURE

IRAK-M Is a Novel Member of the Pelle/Interleukin1 Receptor-associated Kinase (IRAK) Family

Article

Full-text available

Jul 1999

The interleukin-1 receptor-associated kinase (IRAK) was first described as a signal transducer for interleukin-1 (IL-1) and has later been implicated in signal transduction of other members of the Toll/IL-1 receptor family. We now report the identification and characterization of a novel IRAK-like molecule. In contrast to the ubiquitously expressed IRAK and IRAK-2, this new IRAK-like molecule is found mainly in cells of monomyeloic origin and is, therefore, designated IRAK-M. Although IRAK-M and IRAK-2 exhibit only a negligible autophosphorylation activity, they can reconstitute the IL-1 response in a 293 mutant cell line lacking IRAK. In addition, we show for the first time that members of the IRAK family are indispensable elements of lipopolysaccharide signal transduction. The discovery of IRAK-M adds another level of complexity to our understanding of signaling by members of the Toll/IL-1 receptor family.

Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades

Article

Full-text available

Jan 2000

MyD88 is an adaptor molecule essential for signaling via the Toll-like receptor (TLR)/IL-1 receptor family. TLR4 is a member of the TLR family and a point mutation in the Tlr4 gene causes hyporesponsiveness to lipopolysaccharide (LPS) in C3H/HeJ mice. We have previously shown that both TLR4- and MyD88-deficient mice are hyporesponsive to LPS. In this study we examined the responsiveness of these two knockout mice to various bacterial cell wall components. Cells from TLR4-deficient mice responded to several kinds of LPS, peptidoglycan and crude cell wall preparation from Gram-positive bacteria and mycobacterial lysates. In contrast, macrophages and splenocytes from MyD88-deficient mice did not respond to any of the bacterial components we tested. These results show that MyD88 is essential for the cellular response to bacterial cell wall components.

Soluble CD14 participates in the response of cells to lipopolysaccharide

Article

Full-text available

Jan 1993

CD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononuclear phagocytes and as a soluble protein in the blood. CD14 on the cell membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothelial-leukocyte adhesion molecule 1 expression by human umbilical vein endothelial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial cells. None of these cell types express mCD14, yet all respond to LPS in a serum-dependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

RelB, a new Rel family transcription activator that can interact with p50-NF-??B

Article

Full-text available

Mar 1992

We have identified a serum-inducible gene, relB, which encodes a protein of 558 amino acids containing a region with high similarity to c-Rel and other members of the Rel family. Transcriptional activation analysis of GAL4-RelB fusion proteins in yeast cells reveals that RelB contains in its C-terminal 180 amino acids a transcriptional activation domain. The N-terminal part including the region of similarity with the Rel family shows no detectable transcriptional activity. RelB does not bind with high affinity to NF-kappa B sites, but heterodimers between RelB and p50-NF-kappa B do bind to different NF-kappa B-binding sites with a similar affinity to that shown by p50-NF-kappa B homodimers. However, RelB/p50-NF-kappa B heterodimers, in contrast to p50-NF-kappa B homodimers, transactivate transcription of a promoter containing a kappa B-binding site.

The p65 subunit is responsible for the strong transcription activating potential of NF-kappa B

Article

Full-text available

Jan 1992

The nuclear form of the NF-kappa B transcription factor binds to DNA as a heterodimer of a 50 kDa (p50) and 65 kDa (p65) polypeptide. The two polypeptides are encoded by different genes but share a long region of homology, the NRD motif, encompassing domains required for DNA binding and dimerization. In this study we have analysed the contribution of the two subunits to the strong transactivating potential of NF-kappa B. Transient expression of the p65 subunit alone resulted in a potent transactivation of a CAT reporter construct under the control of two NF-kappa B binding sites in monkey COS and mouse L cells. The strongly DNA binding p50 subunit showed only very weak, if any, induction of gene expression. Co-expression of p50 suppressed the transactivation by p65 presumably by competitive DNA binding of transcriptionally inactive p50 dimers (KBF1). Fusion of p65 sequences to DNA binding domain of the yeast GAL4 transcription factor allowed detection of the principal transactivation domain of p65 (TA1) in the C-terminal 30 amino acid sequence. TA1 is likely to adopt an amphipathic alpha-helical structure which clusters serine residues on the hydrophilic surface, a structural feature conserved between human, mouse and Xenopus p65. The unique C-terminal third of p65 contained at least one more activation domain, TA2, within a 90 amino acid sequence directly adjacent to TA1. In two mammalian cell lines, TA1 and TA2 acted separately, while in an insect cell line, the two domains were inactive after their separation. Our study suggests that the p50 subunit in NF-kappa B might only serve a helper function in DNA binding whereas the p65 subunit is responsible for initiating transcription. Homodimers of p50 seem to have the potential of down-regulating kappa B-specific gene expression.

Tumor necrosis factor alpha mediates lethal activity of killed gram-negative and gram-positive bacteria in D-galactosamine-treated mice

Article

Full-text available

Jul 1991

Treatment with D-galactosamine increases sensitivity of lipopolysaccharide (LPS)-responder mice to the lethal effects of LPS, while nonresponder mice remain resistant (M.A. Freudenberg, D. Keppler, and C. Galanos, Infect. Immun. 51:891-895, 1986). In the present study it is shown that, in contrast to LPS, killed gram-negative bacteria (Salmonella abortus equi and S. typhimurium) were highly toxic for D-galactosamine-treated LPS-responder (C57BL/10 ScSN and C3H/HeN) and -nonresponder (C57BL/10 ScCR and C3H/HeJ) mice, although to a higher extent in the former strains. Also, killed gram-positive bacteria (Staphylococcus aureus, Propionibacterium acnes, and Mycobacterium phlei) exhibited toxicity for D-galactosamine-treated mice, LPS-responder and -nonresponder mice being equally susceptible. Evidently, bacterial components other than LPS may exhibit lethal effects in sensitized animals. In all cases, the lethality of LPS and of bacteria was inhibited by anti-tumor necrosis factor alpha (TNF-alpha) serum. While LPS induced TNF-alpha in vitro only in macrophages from LPS-responder mice, gram-negative and gram-positive bacteria induced TNF-alpha also in macrophages from LPS-nonresponder mice. The data show that TNF-alpha is a common endogenous mediator of the lethal activity of gram-negative and gram-positive bacteria.

Lipopolysaccharide Induces Hyporesponsiveness to Its Own Action in RAW 264.7 Cells

Article

Full-text available

Jan 1990

Bacterial endotoxin (lipopolysaccharide, LPS) has the property of inducing hyporesponsiveness or tolerance to its own effects. This phenomenon has been demonstrated in man and experimental animals. The cellular changes that contribute to LPS tolerance are not understood. One mechanism of tolerance could involve a diminished response to LPS by key effector cells such as macrophages. Here we describe experiments designed to determine the mechanism whereby LPS produces a hyporesponsive state to its own effects. Because of the importance of the monokine known as tumor necrosis factor-alpha (TNF-alpha) in mediating many of the diverse effects of LPS, we have studied induction of TNF-alpha at the mRNA and activity level in the murine macrophage-like cell line RAW 264.7. Hyporesponsiveness can be induced by exposure of RAW 264.7 cells to low doses of LPS for more than 6 h prior to challenge with a second, normally stimulatory dose of LPS. This hyporesponsiveness is characterized by a diminished ability of LPS to increase steady state levels of TNF-alpha mRNA, is not due to an increased rate of TNF-alpha mRNA degradation, and is specific for LPS since LPS-pretreated and control cells produce similar amounts of TNF-alpha in response to challenge with heat-killed Staphylococcal aureus. The presence of indomethacin during the primary and/or challenge LPS treatment has no effect on the induction of acquired hyporesponsiveness. Thus, cyclooxygenase products are probably not involved in the development of LPS-induced hyporesponsiveness. These studies provide the basis for a better understanding of the cellular mechanisms that contribute to LPS tolerance.

Enhanced Phagocytosis and Bactericidal Activity of Hepatic Reticuloendothelial System During Endotoxin Tolerance

Article

Full-text available

Apr 1980

The effects of tolerance to Escherichia coli endotoxin on the phagocytic and bactericidal activity of the hepatic reticuloendothelial system against viable E. coli were examined using ex vivo perfused rat livers. Livers were isolated from control and endotoxin-tolerant rats and perfused with a medium containing 5% homologous serum from either control or tolerant rats. After the addition of the E. coli (2 x 10(7) cells per ml) to the perfusate, the hepatic clearance of the bacteria was followed for 30 min. The highest activation of the hepatic reticuloendothelial system was observed when serum from tolerant animals was added to the perfusate. Under these conditions phagocytosis was 47% (12% in controls), and 37 to 38% of the bacteria were killed (5% in controls). This activation was less when livers obtained from tolerant rats were perfused with serum from controls or with saline only. The data suggests that, during endotoxin tolerance, humoral factors play an important role in the activation of the hepatic reticulendothelial system, although a direct stimulation of Kupffer cells also occurs. The enhancement of phagocytosis by tolerant serum did not require the presence of homologous antibodies and involved the activation of the alternative complement pathway, since it was lost after removal of factor B activity. On the other hand, stimulation of intracellular killing required both complement and specific antibodies. The data suggest a role of endotoxin in the activation of humoral and cellular mechanisms involved in the host resistance to gram-negative bacterial infection.

Tolerance to lipopolysaccharide involves mobilization of nuclear factor kappa-B with predominance of p50 homodimers

Article

Full-text available

Jul 1994

Stimulation of the human monocytic cell line Mono Mac 6 with lipopolysaccharide (LPS) leads to rapid and transient expression of cytokines like tumor necrosis factor (TNF). When such cells are precultured for 2 days with a low dose of LPS (20 ng/ml) followed by stimulation with a high dose of LPS (1 microgram/ml), expression of the TNF gene is minimal, i.e. the cells are tolerant. In nuclear run-on analysis, such tolerant cells show only a low degree of transcription, indicating that tolerance operates at or upstream of the transcription level. The CD14 LPS receptor is, however, up-regulated (not down-regulated) in tolerant cells, and LPS can, in fact, still lead to activation of tolerant cells as evidenced by mobilization of the transcription factor nuclear factor kappa B (NF-kappa B). Resolution of the NF-kappa B complex in gel shift analysis shows that the binding protein, mobilized in naive Mono Mac 6 cells, consists mainly of p50-p65 heterodimers, while in tolerant cells, the p50 homodimer is predominant. This increase in p50 homodimers coincides with an increase in p105 mRNA, suggestive of a transcriptional up-regulation of p50. Reporter gene analysis reveals that the NF-kappa B complex mobilized in tolerant cells is functionally inactive in that NF-kappa B-dependent luciferase constructs containing the human immunodeficiency virus long terminal repeat or the TNF 5'-region show only minimal transactivation after LPS stimulation. Similar to Mono Mac 6 cells, primary blood monocytes, when precultured with a low dose of LPS, also become tolerant and produce little TNF after LPS stimulation. The tolerant blood monocytes also up-regulate CD14, and they mobilize NF-kappa B with a predominance of p50 homodimers. Taken together, these results demonstrate that tolerance to LPS is determined by post-receptor mechanisms that involve an altered composition of the NF-kappa B complex.

de Martin R, Vanhove B, Cheng Q, Hofer E, Csizmadia V, Winkler H, Bach FHCytokine-inducible expression in endothelial cells of an IB-like gene is regulated by NF-B. EMBO J 12: 2773-2779

Article

Full-text available

Aug 1993

The transient expression of many different genes is mediated by the inducible transcription factor p50-p65 NF kappa B, which in turn is regulated by complex formation with its inhibitor I kappa B alpha. We describe here that in porcine aortic endothelial cells, either IL-1 alpha, TNF alpha or LPS upregulates an inhibitor of NF kappa B which we refer to as ECI-6. ECI-6 is by structural and functional criteria an I kappa B alpha protein, the porcine homologue of MAD-3, pp40 and RL/IF-1. We have studied the promoter of the ECI-6/I kappa B alpha gene and provide three lines of evidence that its expression is directly regulated by NF kappa B. First, the 5' regulatory region of ECI-6/I kappa B alpha contains two sites that bind NF kappa B in electrophoretic mobility shift assays. Second, expression following transfection of an ECI-6/I kappa B alpha promoter-luciferase reporter construct is dependent on a co-transfected NF kappa B-p65 subunit. Third, pretreatment of endothelial cells with antioxidants, agents that inhibit activation of NF kappa B, inhibit the expression of ECI-6/I kappa B alpha. We conclude that the regulated expression of ECI-6/I kappa B alpha could represent a novel feedback mechanism by which NF kappa B downregulates its own activity after transient activation of target genes has been achieved.

Cleveland MG, Gorham JD, Murphy TL, Tuomanen E, Murphy KMLipoteichoic acid preparations of Gram-positive bacteria induce interleukin-12 through a CD14-dependent pathway. Infect Immun 64:1906-1912

Article

Full-text available

Jul 1996

Interleukin 12 (IL-12) strongly augments gamma interferon production by natural killer (NK) and T cells. IL-12 also promotes effective cell-mediated immune responses, which are particularly important against intracellular bacteria such as Listeria monocytogenes. While the lipopolysaccharide (LPS) of gram-negative bacteria induces monocyte production of IL-12, the relevant gram-positive components which induce IL-12 production are uncharacterized. We used the human monocytic cell line THP-1 to study IL-12 induction by gram-positive bacteria. Muramyl dipeptides as well as the major muramyl tetrapeptide component of Streptococcus pneumoniae were inactive for inducing IL-12. In contrast, lipoteichoic acid (LTA), a predominant surface glycolipid of gram-positive bacteria, potently induced IL-12 p40 gene expression. A competitive LPS antagonist, Rhodobacter sphaeroides LPS, inhibited LTA-induced IL-12 production, suggesting a common pathway for LPS and LTA in IL-12 activation. Pretreatment of cells with anti-CD14 monoclonal antibody blocked both LPS and LTA induction of IL-12 p40 expression. LTA also induced Thl development in naive CD4 T cells by an IL-12-dependent mechanism, indicating direct induction of physiologic levels of IL-12. Together, these results show that LTA is a potent surface structure of gram-positive bacteria which induces IL-12 in monocytes through a CD14-mediated pathway.

Innate Immunity: The Virtues of a Nonclonal System of Recognition

Article

Full-text available

Nov 1997
CELL

One of the predictions of the clonal selection theory was the ability of mature peripheral lymphocytes to discriminate between self and nonself. All self-reactive lymphocytes were thought to be deleted during maturation, and any molecule presented to the mature lymphocytes would be regarded as nonself and should therefore induce an adaptive immune response. Indeed, early experiments with model antigens, such as simple chemicals conjugated to self serum proteins and injected with adjuvants showed that responses to such antigens were the norm rather than the exception. However, if unmodified self antigens, and even nonself proteins, were administered alone in the absence of adjuvant, they gave rise to tolerance rather than an immune response. Immune responses specific even for self antigens could be induced, however, if the self antigen was mixed with adjuvants.Although adjuvants have a long history, so far they have only been defined operationally as any substance that increases the immunogenicity of admixed antigens. The reason for the failure of pure antigens to induce immune response is now known—the antigens used in these studies failed to induce the costimulatory signal necessary for the activation of lymphocytes. Accordingly, the mechanism of adjuvant activity has been proposed to be due to the induction of costimulatory signals by microbial constituents carrying PAMPs that are routinely mixed in adjuvants (Janeway 1989xCold Spring Harbor Symp. Janeway, C.A. Jr. Quant. Biol. 1989; 54: 1–13Crossref | PubMedSee all ReferencesJaneway 1989). Recognition of these PAMPs by PRRs is suggested to induce the signals necessary for lymphocyte activation (such as B7) and differentiation (effector cytokines; see above). In other words, adjuvants induce the innate immune system to produce the signals that are required for activation of an adaptive immune response. While adjuvants are potent immunostimulators, most of them cannot be used in the clinic because of unwanted side effects, and much adjuvant research has been directed toward identification of the active components of the adjuvants and their subsequent modification to minimize side effects (for review, seeAudibert and Lise 1993xAudibert, F.M. and Lise, L.D. Immunol. Today. 1993; 14: 281–284Abstract | Full Text PDF | PubMedSee all ReferencesAudibert and Lise 1993). Characterization of the nonclonal receptors of the innate immune system responsible for the adjuvant activity, and, evidently, for the associated side effects, would provide a powerful alternative approach, which would ultimately allow one to target these receptors directly. In one example of such a rational approach, fusion of an antigen with C3dg, a product of complement activation, resulted in dramatic potentiation of a specific immune response to the fused antigen (Dempsey et al 1996xDempsey, P.W., Allison, M.E., Akkaraju, S., Goodnow, C.C., and Fearon, D.T. Science. 1996; 271: 348–350Crossref | PubMedSee all ReferencesDempsey et al 1996).Rational approaches to vaccine design based on the detailed understanding of the molecular mechanisms of innate immunity should ultimately allow one not only to induce a specific adaptive immune response but also the desired effector mechanism without the accompanying damage to the host tissues.

The Role of CD14 in Signaling Mediated by Outer Membrane Lipoproteins of Borrelia burgdorferi

Article

Full-text available

Jul 1998

Borrelia burgdorferi possesses membrane lipoproteins that exhibit stimulatory properties and, consequently, have been implicated in the pathology related to Lyme disease. As CD14 has been shown to mediate signaling by a number of lipid-modified bacterial products, the involvement of CD14 in signaling mediated by two B. burgdorferi lipoproteins, outer surface protein A (OspA) and OspC, was determined. Lipoprotein-mediated induction of nuclear factor-kappaB nuclear translocation and production of IL-8 and IL-6 in HUVEC was enhanced in the presence of serum or soluble rCD14. CD14-specific Abs that block LPS-mediated signaling also inhibited lipoprotein-dependent signaling in HUVEC and neutrophils. The formation of stable complexes between OspA and CD14 was demonstrated by native gel electrophoresis. LPS was found to compete with OspA for binding with CD14, suggesting that LPS and OspA bind similar regions on CD14. The similarity in binding was further supported by the finding that a mutant soluble CD14, lacking the LPS binding site, did not facilitate lipoprotein signaling, nor did it form a complex with OspA. Binding of OspA to CD14 was dependent on the lipid modification, as unlipidated OspA did not form a complex with CD14 or stimulate cells. In contrast, the lipopeptide remaining after proteinase K digestion both formed a complex with CD14 and retained stimulatory properties. These findings indicate that CD14 facilitates bacterial lipoprotein signaling in mammalian cells.

Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene

Article

Full-text available

Jan 1999

Mutations of the gene Lps selectively impede lipopolysaccharide (LPS) signal transduction in C3H/HeJ and C57BL/10ScCr mice, rendering them resistant to endotoxin yet highly susceptible to Gram-negative infection. The codominantLps d allele of C3H/HeJ mice was shown to correspond to a missense mutation in the third exon of the Toll-like receptor-4 gene (Tlr4), predicted to replace proline with histidine at position 712 of the polypeptide chain. C57BL/10ScCr mice are homozygous for a null mutation of Tlr4. Thus, the mammalian Tlr4 protein has been adapted primarily to subserve the recognition of LPS and presumably transduces the LPS signal across the plasma membrane. Destructive mutations of Tlr4 predispose to the development of Gram-negative sepsis, leaving most aspects of immune function intact.

Navarre, W. W. & Schneewind, O. Surface proteins of Gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol. Mol. Biol. Rev. 63, 174-229

Article

Full-text available

Apr 1999

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.

NF-kappaB1 (p50) Is Upregulated in Lipopolysaccharide Tolerance and Can Block Tumor Necrosis Factor Gene Expression

Article

Full-text available

May 1999

Monocytes respond to lipopolysaccharide (LPS) stimulation with a rapid expression of the tumor necrosis factor (TNF) gene. Upon repeated LPS stimulation there is, however, little production of TNF mRNA and protein; i.e., the cells are tolerant to LPS. Analysis of NF-kappaB proteins in gel shift assays demonstrated that the DNA binding activity that is induced by LPS stimulation in tolerant cells consists mainly of p50-p50 homodimers. Since p50 can bind to DNA but lacks a transactivation domain, this may explain the blockade of TNF gene expression. We now show that in the monocytic cell line Mono Mac 6, this inability to respond can be largely ascribed to NF-kappaB, since a reporter construct directed by a trimeric NF-kappaB motif is strongly transactivated by LPS stimulation of naive cells whereas LPS-tolerant cells exhibit only low activity. Also, Western blot analyses of proteins extracted from purified nuclei showed mobilization of threefold-higher levels of p50 protein in tolerant compared to naive cells, while mobilization of p65 was unaltered. Overexpression of p50 in HEK 293 cells resulted in a strong reduction of p65-driven TNF promoter activity at the levels of both luciferase mRNA and protein. These data support the concept that an upregulation of p50 is instrumental in LPS tolerance in human monocytes.

TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling

Article

Full-text available

May 1999
GENE DEV

Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.

Peptidoglycan- and Lipoteichoic Acid-induced Cell Activation Is Mediated by Toll-like Receptor 2

Article

Full-text available

Jul 1999

The life-threatening complications of sepsis in humans are elicited by infection with Gram-negative as well as Gram-positive bacteria. Recently, lipopolysaccharide (LPS), a major biologically active agent of Gram-negative bacteria, was shown to mediate cellular activation by a member of the human Toll-like receptor family, Toll-like receptor (TLR) 2. Here we investigate the mechanism of cellular activation by soluble peptidoglycan (sPGN) and lipoteichoic acid (LTA), main stimulatory components of Gram-positive bacteria. Like LPS, sPGN and LTA bind to the glycosylphosphatidylinositol-anchored membrane protein CD14 and induce activation of the transcription factor NF-κB in host cells like macrophages. We show that whole Gram-positive bacteria, sPGN and LTA induce the activation of NF-κB in HEK293 cells expressing TLR2 but not in cells expressing TLR1 or TLR4. The sPGN- and LTA-induced NF-κB activation was not inhibited by polymyxin B, an antibiotic that binds and neutralizes LPS. Coexpression together with membrane CD14 enhances sPGN signal transmission through TLR2. In contrast to LPS signaling, activation of TLR2 by sPGN and LTA does not require serum. These findings identify TLR2 as a signal transducer for sPGN and LTA in addition to LPS.

Cutting Edge: Recognition of Gram-Positive Bacterial Cell Wall Components by the Innate Immune System Occurs Via Toll-Like Receptor 21

Article

Full-text available

Aug 1999

Invasive infection with Gram-positive and Gram-negative bacteria often results in septic shock and death. The basis for the earliest steps in innate immune response to Gram-positive bacterial infection is poorly understood. The LPS component of the Gram-negative bacterial cell wall appears to activate cells via CD14 and Toll-like receptor (TLR) 2 and TLR4. We hypothesized that Gram-positive bacteria might also be recognized by TLRs. Heterologous expression of human TLR2, but not TLR4, in fibroblasts conferred responsiveness to Staphylococcus aureus and Streptococcus pneumoniae as evidenced by inducible translocation of NF-kappaB. CD14 coexpression synergistically enhanced TLR2-mediated activation. To determine which components of Gram-positive cell walls activate Toll proteins, we tested a soluble preparation of peptidoglycan prepared from S. aureus. Soluble peptidoglycan substituted for whole organisms. These data suggest that the similarity of clinical response to invasive infection by Gram-positive and Gram-negative bacteria is due to bacterial recognition via similar TLRs.

Cutting Edge: Inflammatory Signaling by Borrelia burgdorferi Lipoproteins Is Mediated by Toll-Like Receptor 2

Article

Full-text available

Oct 1999

The agent of Lyme disease, Borrelia burgdorferi, produces membrane lipoproteins possessing potent inflammatory properties linked to disease pathology. The recent association of toll-like receptors (TLR) 2 and 4 with LPS responses prompted the examination of TLR involvement in lipoprotein signaling. The ability of human cell lines to respond to lipoproteins was correlated with the expression of TLR2. Transfection of TLR2 into cell lines conferred responsiveness to lipoproteins, lipopeptides, and sonicated B. burgdorferi, as measured by nuclear translocation of NF-kappaB and cytokine production. The physiological importance of this interaction was demonstrated by the 10-fold greater sensitivity of TLR2-transfected cells to lipoproteins than LPS. Futhermore, TLR2-dependent signaling by lipoproteins was facilitated by CD14. These data indicate that TLR2 facilitates the inflammatory events associated with Lyme arthritis. In addition, the widespread expression of lipoproteins by other bacterial species suggests that this interaction may have broad implications in microbial inflammation and pathogenesis.

Cutting Edge: Preferentially the R-Stereoisomer of the Mycoplasmal Lipopeptide Macrophage-Activating Lipopeptide-2 Activates Immune Cells Through a Toll-Like Receptor 2- and MyD88-Dependent Signaling Pathway

Article

Full-text available

Jan 2000

Mycoplasmas and their membranes are potent activators of macrophages, the active principle being lipoproteins and lipopeptides. Two stereoisomers of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 (MALP-2) differing in the configuration of the lipid moiety were synthesized and compared in their macrophage-activating potential, the R-MALP being >100 times more active than the S-MALP in stimulating the release of cytokines, chemokines, and NO. To assess the role of the Toll-like receptor (TLR) family in mycoplasmal lipopeptide signaling, the MALP-2-mediated responses were analyzed using macrophages from wild-type, TLR2-, TLR4-, and MyD88-deficient mice. TLR2- and MyD88-deficient cells showed severely impaired cytokine productions in response to R- and S-MALP. The MALP-induced activation of intracellular signaling molecules was fully dependent on both TLR2 and MyD88. There was a strong preference for the R-MALP in the recognition by its functional receptor, TLR2.

Cutting Edge: Cell Surface Expression and Lipopolysaccharide Signaling Via the Toll-Like Receptor 4-MD-2 Complex on Mouse Peritoneal Macrophages

Article

Full-text available

May 2000

The human MD-2 molecule is associated with the extracellular domain of human Toll-like receptor 4 (TLR4) and greatly enhances its LPS signaling. The human TLR4-MD-2 complex thus signals the presence of LPS. Little is known, however, about cell surface expression and LPS signaling of the TLR4-MD-2 complex in vivo. We cloned mouse MD-2 molecularly and established a unique mAb MTS510, which reacted selectively with mouse TLR4-MD-2 but not with TLR4 alone in flow cytometry. Mouse MD-2 expression in TLR4-expressing cells enhanced LPS-induced NF-kappaB activation, which was clearly inhibited by MTS510. Thioglycolate-elicited peritoneal macrophages expressed TLR4-MD-2, which was rapidly down-regulated in the presence of LPS. Moreover, LPS-induced TNF-alpha production by peritoneal macrophages was inhibited by MTS510. Collectively, the TLR4-MD-2 complex is expressed on macrophages in vivo and senses and signals the presence of LPS.

Interleukin-10 and interleukin-10 receptor

Article

Feb 2001
ANNU REV IMMUNOL

Interleukin-10 (IL-10), first recognized for its ability to inhibit activation and effector function of T cells, monocytes, and macrophages, is a multifunctional cytokine with diverse effects on most hemopoietic cell types. The principal routine function of IL-10 appears to be to limit and ultimately terminate inflammatory responses. In addition to these activities, IL-10 regulates growth and/or differentiation of B cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and endothelial cells. IL-10 plays a key role in differentiation and function of a newly appreciated type of T cell, the T regulatory cell, which may figure prominently in control of immune responses and tolerance in vivo. Uniquely among hemopoietic cytokines, IL-10 has closely related homologs in several virus genomes, which testify to its crucial role in regulating immune and inflammatory responses. This review highlights findings that have advanced our understanding of IL-10 and its receptor, as well as its in vivo function in health and disease.

Gene Expressions of Toll-Like Receptor 2, But Not Toll-Like Receptor 4, Is Induced by LPS and Inflammatory Cytokines in Mouse Macrophages1

Article

Dec 2000

Toll-like receptors (TLRs) are a family of mammalian homologues of Drosophila Toll and play important roles in host defense. Two of the TLRs, TLR2 and TLR4, mediate the responsiveness to LPS. Here the gene expression of TLR2 and TLR4 was analyzed in mouse macrophages. Mouse splenic macrophages responded to an intraperitoneal injection or in vitro treatment of LPS by increased gene expression of TLR2, but not TLR4. Treatment of a mouse macrophage cell line with LPS, synthetic lipid A, IL-2, IL-15, IL-1beta, IFN-gamma, or TNF-alpha significantly increased TLR2 mRNA expression, whereas TLR4 mRNA expression remained constant. TLR2 mRNA increase in response to synthetic lipid A was severely impaired in splenic macrophages isolated from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role in the process. Specific inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase and p38 kinase did not significantly inhibit TLR2 mRNA up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a high concentration of curcumin, suggesting that NF-kappaB activation may be essential for the process. Taken together, our results indicate that TLR2, in contrast to TLR4, can be induced in macrophages in response to bacterial infections and may accelerate the innate immunity against pathogens.

Inhibitory kappa B alpha control of nuclear factor-kappa B is dysregulated in endotoxin tolerant macrophages

Article

Apr 1999

The transcription factor nuclear factor (NF)-KB is thought to be required for endotoxin-stimulated tumor necrosis factor (TNF) and interleukin (IL)-1 gene transcription. Nuclear translocation of NF-KB is regulated by the cytoplasmic inhibitory factor IKB[alpha]. Low-dose lipopolysaccharide (LPS) pretreatment modulates cytokine release by altering subsequent LPS-activated signal transduction pathways. In this study, we examined the effect of LPS pretreatment exposure on IKB[alpha] and NF-KB following activation with LPS. Murine macrophages (Mo) were exposed to a range of LPS concentrations +/- 24 h PreRx with 10 ng/mL LPS pretreatment. Cytoplasmic IKB[alpha] (Western immunoblot) and NF-KB (gel-shift assay) were assayed 30 min after LPS activation. Gene transcription for TNF was measured 6 h after LPS activation using RT-PCR. In the absence of LPS pretreatment, IKB[alpha] disappeared from the cytoplasm coincident with nuclear translocation of NF-KB. Tolerant Mo had markedly enhanced levels of IKB[alpha] and normal to increased levels of NF-KB translocation with a different electrophoretic shift. LPS activation enhanced cytokine gene transcription in a dose-dependent manner, and this was unaltered by LPS pretreatment. Endotoxin-tolerant Mo also had increased cytoplasmic levels of the p65 subunit of NF-KB. LPS tolerance is associated with increases of cytoplasmic IKB[alpha] p65, as well as enhanced NF-KB. We conclude that control of NF-KB translocation by IKB[alpha] is dysregulated in endotoxin-tolerant Mo. (C)1999The Shock Society

Development of tolerance to typhoid bacterial pyrogen and its abolition by reticulo-endothelial blockade

Article

Mar 1946

P B BEESON

CD14, a Receptor for Complexes of Lipopolysaccharide (LPS) and LPS Binding Protein

Article

Oct 1990

Leukocytes respond to lipopolysaccharide (LPS) at nanogram per milliliter concentrations with secretion of cytokines such as tumor necrosis factor-alpha (TNF-alpha). Excess secretion of TNF-alpha causes endotoxic shock, an often fatal complication of infection. LPS in the bloodstream rapidly binds to the serum protein, lipopolysaccharide binding protein (LBP), and cellular responses to physiological levels of LPS are dependent on LBP. CD14, a differentiation antigen of monocytes, was found to bind complexes of LPS and LBP, and blockade of CD14 with monoclonal antibodies prevented synthesis of TNF-alpha by whole blood incubated with LPS. Thus, LPS may induce responses by interacting with a soluble binding protein in serum that then binds the cell surface protein CD14.

The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein

Article

Feb 1988
CELL

The Toll gene of Drosophila, a maternal effect gene that plays a central role in the establishment of the embryonic dorsal-ventral pattern, has been cloned using P element tagging. A 5.3 kb poly(A)+ ovarian transcript from the cloned region was purified by hybrid selection with the cloned DNA. This purified transcript complements the Toll mutant phenotype when injected into Toll- embryos, which proves that it is the Toll transcript. The sequence of cDNAs suggests that the Toll protein is an integral membrane protein with a cytoplasmic domain and a large extracytoplasmic domain. The putative extracytoplasmic domain contains at least 15 repeats of a 24 amino acid, leucine-rich sequence found in both human and yeast membrane proteins.

Muramyl dipeptide increases tolerance to shock and bacterial challenge

Article

Feb 1984

We measured the phagocytic capacity of the reticulo-endothelial system by assay of the clearance of colloidal carbon from the blood stream in both normal mice and mice in which shock had been induced by intraperitoneal injection of hypertonic glucose. The group of shocked mice was further subdivided into those pretreated with the immunoadjuvant, muramyl dipeptide (MDP), and those given placebo. Shock reduced reticulo-endothelial phagocytosis (P less than 0.01), whereas pretreatment of shocked mice with MDP led to a hyperphagocytic state (P less than 0.01). In a second series of experiments we subjected shocked mice, pretreated with MDP or placebo, to a virulent Klebsiella pneumoniae infection. MDP significantly improved survival (P less than 0.01).

Interleukin10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance

Article

Dec 1995

Previous studies in vivo have shown that IL-10 infusion can prevent lethal endotoxic shock. Mice deficient in the production of IL-10 (IL10T) were used to investigate the regulatory role of IL-10 in the responses to LPS in three experimental systems. In a model of acute endotoxic shock, it was found that the lethal dose of LPS for IL10T mice was 20-fold lower than that for wild type (wt) mice suggesting that endogenous IL-10 determines the amount of LPS which can be tolerated without death. The high mortality rate of IL10T mice challenged with modest doses of LPS was correlated to the uncontrolled production of TNF as treatment with anti-TNF antibody (Ab) resulted in 70% survival. Additional studies suggested that IL-10 mediates protection by controlling the early effectors of endotoxic shock (e.g., TNF alpha) and that it is incapable of directly antagonizing the production and/or actions of late appearing effector molecules (e.g., nitric oxide). We also found that IL10T mice were extremely vulnerable to a generalized Shwartzman reaction where prior exposure to a small amount of LPS primes the host for a lethal response to a subsequent sublethal dose. The priming LPS dose for IL10T mice was 100-fold lower than that required to prime wt mice implying that IL-10 is important for suppressing sensitization. In agreement with this assumption, IL-10 infusion was found to block the sensitization step. Interestingly, IL-10 was not the main effector of endotoxin tolerance as IL10T mice could be tolerized to LPS. Furthermore, IL-10 infusion could not substitute for the desensitizing dose of LPS. These results show that IL-10 is a critical component of the host's natural defense against the development of pathologic responses to LPS although it is not responsible for LPS-induced tolerance.

Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide

Article

Sep 1993

Recent studies have suggested that soluble CD14 found in serum is involved in the LPS-induced activation of endothelial cells (EC). To more fully investigate the relevance of sCD14 to LPS-induced activation of EC, we have used recombinant soluble CD14 (rsCD14) and have examined, under serum-free conditions, its role in the LPS-induced EC response in the presence of LPS alone as well as in the presence of LPS-binding protein. Our studies show that EC can be activated by high concentrations of LPS in the presence of rsCD14 alone. However, at low concentrations of LPS (5 and 10 ng/ml), the rsCD14-stimulated activation is strongly enhanced by LPS-binding protein. In addition, we show that LPS binds to rsCD14 directly; in the presence of low concentrations of LPS this binding is enhanced by the presence of LPS-binding protein. These results show that while the membrane form of CD14 can function as a receptor, its soluble form can function as a co-ligand with LPS in the EC-LPS response.

Lipopolysaccharide structure-function relationship in activation versus reprogramming of mouse peritoneal macrophages

Article

Dec 1993

Lipopolysaccharide (LPS) is one of the most potent stimuli for macrophages. The activities of LPS have been attributed to the lipid A region of the molecule. We have previously shown that pretreatment of macrophages with very low doses of LPS can selectively "reprogram" these cells to respond differentially to subsequent activation, as assessed by tumor necrosis factor-alpha and nitric oxide (NO) production. Here we demonstrate that the relative capacity of various LPS preparations for induction of down-regulation of subsequent LPS-activated NO production correlates well with their relative potency for initiation of NO formation. Although LPS-dependent activation can be regulated by pertussis toxin (PT)-sensitive factor, the LPS pretreatment-induced reprogramming is shown here to be refractory to regulation by PT. These results suggest that, although the structural components of LPS dictating the relative activities of the molecule for activation versus reprogramming are similar, there may exist different pathways in initiation of LPS-induced activation versus reprogramming.

A human homologue of the Drosophila Toll protein signals activation of adaptive immunity

Article

Aug 1997

Induction of the adaptive immune response depends on the expression of co-stimulatory molecules and cytokines by antigen-presenting cells. The mechanisms that control the initial induction of these signals upon infection are poorly understood. It has been proposed that their expression is controlled by the non-clonal, or innate, component of immunity that preceded in evolution the development of an adaptive immune system in vertebrates. We report here the cloning and characterization of a human homologue of the Drosophila toll protein (Toll) which has been shown to induce the innate immune response in adult Drosophila. Like Drosophila Toll, human Toll is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin (IL)-1 receptor. Both Drosophila Toll and the IL-1 receptor are known to signal through the NF-kappaB pathway. We show that a constitutively active mutant of human Toll transfected into human cell lines can induce the activation of NF-kappaB and the expression of NF-kappaB-controlled genes for the inflammatory cytokines IL-1, IL-6 and IL-8, as well as the expression of the co-stimulatory molecule B7.1, which is required for the activation of naive T cells.

A comparative analysis of cytokine production and tolerance induction by bacterial lipopeptides, lipopolysaccharides and Staphyloccous aureus in human monocytes

Article

Dec 1997

Monocytes (MO) and macrophages (MAC) are important producers of cytokines involved in the pathophysiology of bacterial sepsis. Most studies concentrate on the effects of bacterial lipopolysaccharides (LPS) regarding the induction of cytokine gene expression and secretion in MO/MAC. Here we report that besides LPS, the synthetic lipoprotein analogue lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyl)-(2RS)-propyl)-(R)-cysteinyl-alanyl- glycine (Pam3-Cys-Ala-Gly), another component of the outer membrane of Gram-negative bacteria, as well as heat-killed Staphyloccocus aureus (S. aureus/SAC) are potent stimuli for cytokines in human MO. For all three investigated stimuli we found an individual pattern of cytokine induction: LPS was most potent in inducing interleukin-6 (IL-6) synthesis, whereas for tumour necrosis factor-alpha (TNF-alpha) secretion SAC was the best stimulus. Comparable amounts of IL-8 were induced by either LPS or Pam3-Cys-Ala-Gly, with SAC being less effective even at higher concentrations. The addition of serum led to an increase in LPS-, SAC- and Pam3-Cys-Ala-Gly-stimulated TNF-alpha secretion, indicating that the presence of serum is critical not just for LPS stimulation. Furthermore, as is known for LPS, Pam3-Cys-Ala-Gly and SAC rendered MO refractory to a second bacterial stimulus. Pam3-Cys-Ala-Gly and SAC induced tolerance for itself, but LPS could partially overcome this effect. As the CD14 molecule is discussed as a common receptor for different bacterial components, we investigated whether the TNF-alpha response of MO could be blocked by anti-CD14 antibodies. MY4, a CD14 antibody, selectively blocked the TNF-alpha secretion induced by LPS but not by Pam3-Cys-Ala-Gly or SAC. In summary, we conclude that besides LPS, lipopeptide Pam3-Cys-Ala-Gly and SAC are potent stimuli for human MO, while the mechanisms of activation seem to be partially different from LPS.

Hyporesponsiveness to lipopolysaccharide alters the composition of NF-??B binding to the regulatory regions of inducible nitric oxide synthase gene

Article

Sep 1998

Repeated exposure to bacterial endotoxin causes a diminished response by the host to further exposure. One important feature of this hyporesponsiveness is a reduced macrophage production of nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) pathway. Using a murine macrophage model, we observed that hyporesponsiveness was accompanied by a decrease in the levels of NO release (measured as nitrite), iNOS protein and iNOS gene transcription. The expression of the putative lipopolysaccharide (LPS) receptor, CD14, was not altered. In vivo genomic footprinting showed that the same binding sites are occupied in the iNOS promoter and enhancer of desensitized macrophages and of LPS-responsive macrophages, yet the composition of NF-kappaB in the nuclei of these cells was found to be altered. The transcriptionally inactive homodimer p50-p50 represented the predominant binding activity in nuclei from LPS-pretreated cells before and after stimulation. Nuclei from cells which had not been pretreated but were stimulated contained more of the transcriptionally active p50-p65 heterodimer than their pretreated counterparts. Consistent with this, the cytosolic steady-state level of an inhibitor of NF-kappaB activity, I-kappaBalpha, was decreased in normal cells but not in pretreated cells. We propose that the presence of an overwhelming excess of transcriptionally inactive p50 homodimers on their kappaB sites in the iNOS control region in pretreated cells may block kappaB site binding by p50-p65, thereby reducing the activity of the protein complex governing iNOS transcription.

Lipoprotein Release by Bacteria: Potential Factor in Bacterial Pathogenesis

Article

Dec 1998

Lipoprotein (LP) is a major component of the outer membrane of bacteria in the family Enterobacteriaceae. LP induces proinflammatory cytokine production in macrophages and lethal shock in LPS-responsive and -nonresponsive mice. In this study, the release of LP from growing bacteria was investigated by immuno-dot blot analysis. An immuno-dot blot assay that could detect LP at levels as low as 100 ng/ml was developed. By using this assay, significant levels of LP were detected in culture supernatants of growing Escherichia coli cells. During mid-logarithmic growth, approximately 1 to 1.5 microgram of LP per ml was detected in culture supernatants from E. coli. In contrast, these culture supernatants contained 5 to 6 microgram/ml of lipopolysaccharide (LPS). LP release was not unique to E. coli. Salmonella typhimurium, Yersinia enterocolitica, and two pathogenic E. coli strains also released LP during in vitro growth. Treatment of bacteria with the antibiotic ceftazidime significantly enhanced LP release. Culture supernatants from 5-h cultures of E. coli were shown to induce in vitro production of interleukin-6 (IL-6) by macrophages obtained from LPS-nonresponsive C3H/HeJ mice. In contrast, culture supernatants from an E. coli LP-deletion mutant were significantly less efficient at inducing IL-6 production in C3H/HeJ macrophages. These results suggest, for the first time, that LP is released from growing bacteria and that this released LP may play an important role in the induction of cytokine production and pathologic changes associated with gram-negative bacterial infections.

Tolerance to Pyrogens

Article

Oct 1998

In humans or experimental animals, the repeated confrontation with lipopolysaccharides (LPS) from gram-negative bacteria, but not with muramyl dipeptide (MDP) from gram-positive bacteria, leads to attenuation of almost all pathophysiologic effects mediated by proinflammatory cytokines. Our experiments in guinea pigs and rats demonstrate that attenuation of the febrile response during the development of LPS tolerance is associated with a reduced production of cytokines rather than a decrease in responsiveness to cytokines. Cross-tolerance experiments demonstrate that different stimuli influencing LPS-induced tumor necrosis factor (TNF) release and nitric oxide (NO) synthesis can modify the development of tolerance. On the other hand, the lack of cross-tolerance between LPS and MDP indicates that MDP can activate the cytokine cascade and induce the febrile response in animals tolerant to LPS. This may indicate distinct receptors and signal pathways for LPS and MDP, leading to activation of the cytokine cascade. LPS tolerance has also been demonstrated in ex vivo and in vitro studies. In cultures of monocytes, diminished synthesis of TNF and NO reported after LPS restimulation could be prevented and reversed by interferon and granulocyte-macrophage colony-stimulating factor. These findings add an additional hypothesis in tolerance development.

In vitro macrophage endotoxin tolerance: Defective in vitro macrophage MAP kinase signal transduction after LPS pretreatment is not present in macrophages from C3H/HeJ endotoxin resistant mice

Article

Feb 1999

Altered endotoxin (LPS) signal transduction in macrophages (Mphi) may mediate development of organ dysfunction in sepsis. C3H/HeJ Mphi have a specific genetic defect that renders them "tolerant" to in vitro LPS activation. LPS tolerance can be induced in normal C3H/HeN Mphi following in vitro LPS pretreatment. In these experiments, in vitro LPS-stimulated activation of Mphi mitogen-activated protein (MAP) kinases were compared in C3H/HeJ and C3H/HeN mice. C3H/HeJ and C3H/HeN Mphi were cultured+/-10 ng/mL LPS pretreatment for 24 h, then stimulated with 0-1,000 ng/mL LPS for 6 h. Western blots were performed on lysates with monoclonal antibody to active ERK1,2 (p42/44), stress-activated protein kinase (SAPK, p54/46), and p38 kinase. Supernatant TNF or IL-1 was determined by bioassay. High dose LPS stimulation activated ERK, SAPK, and p38 kinases in both C3H/HeN and C3H/HeJ Mphi. ERK activation, p46 SAPK, and p38 activation were inhibited in C3H/HeN Mphi after LPS pretreatment, whereas they were unchanged or increased in HeJ Mphi. TNF secretion was significantly decreased in C3H/HeN Mphi following LPS pretreatment, but absent in C3H/HeJ Mphi at all times. Mphi from normal C3H/HeN mice rendered endotoxin tolerant by in vitro, low dose LPS pretreatment have specific signal transduction defects that are not present in genetically LPS hyporesponsive C3H/HeJ mice.

Inhibitory κBα control of nuclear factor-κB is dysregulated in endotoxin tolerant macrophages

Article

May 1999

The transcription factor nuclear factor (NF)-kappaB is thought to be required for endotoxin-stimulated tumor necrosis factor (TNF) and interleukin (IL)-1 gene transcription. Nuclear translocation of NF-kappaB is regulated by the cytoplasmic inhibitory factor IkappaBalpha. Low-dose lipopolysaccharide (LPS) pretreatment modulates cytokine release by altering subsequent LPS-activated signal transduction pathways. In this study, we examined the effect of LPS pretreatment exposure on IkappaBalpha and NF-kappaB following activation with LPS. Murine macrophages (Mphi were exposed to a range of LPS concentrations +/-24 h PreRx with 10 ng/mL LPS pretreatment. Cytoplasmic IkappaBalpha (Western immunoblot) and NF-kappaB (gel-shift assay) were assayed 30 min after LPS activation. Gene transcription for TNF was measured 6 h after LPS activation using RT-PCR. In the absence of LPS pretreatment, IkappaBalpha disappeared from the cytoplasm coincident with nuclear translocation of NF-kappaB. Tolerant Mphi had markedly enhanced levels of IkappaBalpha and normal to increased levels of NF-kappaB translocation with a different electrophoretic shift. LPS activation enhanced cytokine gene transcription in a dose-dependent manner, and this was unaltered by LPS pretreatment. Endotoxin-tolerant Mphi also had increased cytoplasmic levels of the p65 subunit of NF-kappaB. LPS tolerance is associated with increases of cytoplasmic IkappaBalpha p65, as well as enhanced NF-kappaB. We conclude that control of NF-kappaB translocation by IkappaBalpha is dysregulated in endotoxin-tolerant Mphi.

Lipopolysaccharide tolerance in murine peritoneal macrophages induces downregulation of the lipopolysaccharide signal transduction pathway through mitogen-activated protein kinase and nuclear factor-κB cascades, but not lipopolysaccharide-incorporation steps

Article

Jul 1999
Biochim Biophys Acta

Endotoxin/lipopolysaccharide (LPS) tolerance, a hyporesponsive state to endotoxin or LPS stimulation, was induced in murine peritoneal macrophages by previous exposure of macrophages to LPS. Expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNA in response to LPS stimulation was suppressed in LPS-tolerant macrophages. Tyrosine phosphorylations in response to LPS of 40-45-kDa proteins in non-tolerant macrophages were also suppressed in LPS-tolerant macrophages. These proteins corresponded to two members of the mitogen-activated protein kinase (MAPK) family, ERK and p38. In addition to these proteins, another MAPK family protein, JNK, was also suppressed in LPS-tolerant macrophages. Activation of Raf-1, located in the upstream portion of ERK cascades, was also suppressed by LPS-tolerance induction. These suppressions in LPS-tolerant macrophages were exhibited against stimulation by an LPS agonist like taxol, but not towards stimulation by an unrelated activator like phorbol ester (PMA). Activation of the transcription factor NF-kappaB, which is supposed to be one of the components of another important pathway for transduction of LPS-stimulated cytokine producing signals, was strongly suppressed and degradation of IkappaB, an inhibitor of NF-kappaB, was also severely diminished in LPS-tolerant macrophages. Although a monosaccharide lipid A analog, GLA-58, was able to stimulate macrophages to activate ERK proteins without cytokine production, pretreatment of macrophages with this compound suppressed both LPS-stimulated activation of ERK and cytokine production. Furthermore, downregulation of LPS-uptake in LPS-tolerant macrophages was not observed. Based on all these findings, LPS tolerance might be caused by the previous activation of some components on LPS-signaling pathways. This may then induce a refractory state in key LPS-signal transducer molecules located downstream of the cell membrane LPS receptor and upstream of the branching point in intracellular cascades for activation of MAPK and NF-kappaB, probably in some initial steps of intracellular signaling.

IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase (IRAK) family

Article

Aug 1999

Cell Activation and Apoptosis by Bacterial Lipoproteins Through Toll-like Receptor-2

Article

Aug 1999

Apoptosis is implicated in the generation and resolution of inflammation in response to bacterial pathogens. All bacterial pathogens produce lipoproteins (BLPs), which trigger the innate immune response. BLPs were found to induce apoptosis in THP-1 monocytic cells through human Toll-like receptor–2 (hTLR2). BLPs also initiated apoptosis in an epithelial cell line transfected with hTLR2. In addition, BLPs stimulated nuclear factor–κB, a transcriptional activator of multiple host defense genes, and activated the respiratory burst through hTLR2. Thus, hTLR2 is a molecular link between microbial products, apoptosis, and host defense mechanisms.

Host Defense Mechanisms Triggered by Microbial Lipoproteins Through Toll-Like Receptors

Article

Aug 1999

The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (IL-12), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL-12 production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of IL-12 production by human macrophages, and that induction is mediated by Toll-like receptors (TLRs). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.

Unresponsiveness of MyD88-Deficient Mice to Endotoxin

Article

Aug 1999
IMMUNITY

MyD88 is a general adaptor protein that plays an important role in the Toll/IL-1 receptor family signalings. Recently, Toll-like receptors 2 and 4 (TLR2 and TLR4) have been suggested to be the signaling receptors for lipopolysaccharide (LPS). In this study, we demonstrate that MyD88 knockout mice lack the ability to respond to LPS as measured by shock response, B cell proliferative response, and secretion of cytokines by macrophages and embryonic fibroblasts. However, activation of neither NF-kappaB nor the mitogen-activated protein (MAP) kinase family is abolished in MyD88 knockout mice. These findings demonstrate that signaling via MyD88 is essential for LPS response, but the inability of MyD88 knockout mice to induce LPS-dependent gene expression cannot simply be attributed to lack of the activation of MAP kinases and NF-kappaB.

Structures of Gram-Negative Cell Walls and Their Derived Membrane Vesicles

Article

Sep 1999

T J Beveridge

How NF-κB is activated: the role of the IκB kinase (IKK) complex

Article

Dec 1999
ONCOGENE

Michael Karin

Rel/NF-kappaB transcription factors are primarily regulated by association with inhibitor IkappaB proteins. Thus, in most cells NF-kappaB exists in the cytoplasm in an inactive complex bound to IkappaB. Most agents that activate NF-kappaB do so through a common pathway based on phosphorylation-induced, proteasome-mediated degradation of IkappaB. The key regulatory step in this pathway involves activation of a high molecular weight IkappaB kinase (IKK) complex, whose catalysis is generally carried out by a heterodimeric kinase consisting of IKKalpha and IKKbeta subunits. This review describes the identification of proteins in the IKK complex, and the regulation and physiological functions of IKK.

Cellular reprogramming by gram-positive bacterial components: A review

Abstract

No full-text available

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