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Toll-like receptor 4 decoy, TOY, attenuates gram-negative bacterial sepsis.

ABSTRACT: Lipopolysaccharide (LPS), the Gram-negative bacterial outer membrane glycolipid, induces sepsis through its interaction with myeloid differentiation protein-2 (MD-2) and Toll-like receptor 4 (TLR4). To block interaction between LPS/MD-2 complex and TLR4, we designed and generated soluble fusion proteins capable of binding MD-2, dubbed TLR4 decoy receptor (TOY) using 'the Hybrid leucine-rich repeats (LRR) technique'. TOY contains the MD-2 binding ectodomain of TLR4, the LRR motif of hagfish variable lymphocyte receptor (VLR), and the Fc domain of IgG1 to make it soluble, productive, and functional. TOY exhibited strong binding to MD-2, but not to the extracellular matrix (ECM), resulting in a favorable pharmacokinetic profile in vivo. TOY significantly extended the lifespan, when administered in either preventive or therapeutic manners, in both the LPS- and cecal ligation/puncture-induced sepsis models in mice. TOY markedly attenuated LPS-triggered NF-kappaB activation, secretion of proinflammatory cytokines, and thrombus formation in multiple organs. Taken together, the targeting strategy for sequestration of LPS/MD-2 complex using the decoy receptor TOY is effective in treating LPS- and bacteria-induced sepsis; furthermore, the strategy used in TOY development can be applied to the generation of other novel decoy receptor proteins.

SUBMITTER: Jung K

PROVIDER: S-EPMC2754608 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Toll-like receptor 4 decoy, TOY, attenuates gram-negative bacterial sepsis.

Jung Keehoon K Lee Jung-Eun JE Kim Hak-Zoo HZ Kim Ho Min HM Park Beom Seok BS Hwang Seong-Ik SI Lee Jie-Oh JO Kim Sun Chang SC Koh Gou Young GY

PloS one 20091009 10

Lipopolysaccharide (LPS), the Gram-negative bacterial outer membrane glycolipid, induces sepsis through its interaction with myeloid differentiation protein-2 (MD-2) and Toll-like receptor 4 (TLR4). To block interaction between LPS/MD-2 complex and TLR4, we designed and generated soluble fusion proteins capable of binding MD-2, dubbed TLR4 decoy receptor (TOY) using 'the Hybrid leucine-rich repeats (LRR) technique'. TOY contains the MD-2 binding ectodomain of TLR4, the LRR motif of hagfish varia ...[more]

PMID: 19816595

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Project description:BackgroundToll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis.Methods and findingsPatient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury.ConclusionsPatients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.

Project description:Objective: It is unclear whether the host response of gram-positive sepsis differs from gram-negative sepsis at a transcriptome level. Using microarray technology, we compared the gene-expression profiles of gram-positive sepsis and gram-negative sepsis in critically ill patients. Design: A prospective cross-sectional study. Setting: A 20-bed general intensive care unit of a tertiary referral hospital. Patients: Seventy-two patients admitted to the intensive care unit. Interventions: Intravenous blood was collected for leukocyte separation and RNA extraction. Microarray experiements were then performed examing the expression level of 19,232 genes in each sample. Measurements and Main Results: There was no difference in the expression profile between gram-positive and gram-negative sepsis. The finding remained unchanged even when genes with lower expression level were included or after statistical stringency was lowered. There were, however, ninety-four genes differentially expressed between sepsis and control patients. These genes included those involved in immune regulation, inflammation and mitochondrial function. Hierarchical cluster analysis confirmed that the difference in gene expression profile existed between sepsis and control patients, but not between gram-positive and gram-negative patients. Conclusion: Gram-positive and gram-negative sepsis share a common host response at a transcriptome level. These findings support the hypothesis that the septic response is non-specific and is designed to provide a more general response that can be elicited by a wide range of different micro-organisms. The study included seventy-two critically ill patients admitted to the intensive care unit (ICU) of Nepean Hospital, Sydney, Australia. Of these, fifty-five patients were diagnosed to have sepsis, as confirmed by microbiological culture. The remaining seventeen patients did not have sepsis and were therefore used as controls. The study was approved by the hospital ethics committee and informed consent was obtained from all patients or their relatives. Patient Samples. Whole blood was taken from each patient on admission to ICU. Neutrophils were separated from whole blood using density-gradient separation with Ficoll-PaqueP P(Amersham). Subsequent neutrophil RNA extraction was performed using guanidinium thiocyanate (Ambion). Microarray Experiment. The neutrophil RNA was converted to cDNA, fluorescently labeled and hybridized to its complimentary sequences on the microarray (Invitrogen). The fluorescent signals on each micrroarray were captured using the GenePix 4000B laser scanner (Axon Instruments). Expression level of each gene was represented by the intensity of its fluorescent signal. Data Extraction. All signal intensity values were processed using background-subtraction method. Prior to analysis, all values were log-transformed and normalized by fitting a print-tip group Lowess curve. Normalization minimizes bias due to dye chemistry, signal intensity or location of a gene on the array. It ensures the detection of genes that are truly differentially expressed, instead of those caused by experimental artifacts or variation in the hybridization process. After normalization, genes that had more than 50% of data missing were removed. We then selected genes that had at least 80% of the data showing two-fold changes from the geneâs median values. After filtering, 1617 genes were available for further analysis.

Project description:Sepsis is an inflammatory response to pathogens (such as Gram?positive and Gram?negative bacteria), which has high morbidity and mortality in critically ill patients. The present study aimed to identify the key genes in Gram?positive and Gram?negative sepsis. GSE6535 was downloaded from Gene Expression Omnibus, containing 17 control samples, 18 Gram?positive samples and 25 Gram?negative samples. Subsequently, the limma package in R was used to screen the differentially expressed genes (DEGs). Hierarchical clustering was conducted for the specific DEGs in Gram?negative and Gram?negative samples using cluster software and the TreeView software. To analyze the correlation of samples at the gene level, a similarity network was constructed using Cytoscape software. Functional and pathway enrichment analyses were conducted for the DEGs using DAVID. Finally, stochastic perturbation was used to determine the significantly differential functions between Gram?positive and Gram?negative samples. A total of 340 and 485 DEGs were obtained in Gram?positive and Gram?negative samples, respectively. Hierarchical clustering revealed that there were significant differences between control and sepsis samples. In Gram?positive and Gram?negative samples, myeloid cell leukemia sequence 1 was associated with apoptosis and programmed cell death. Additionally, NADH:ubiquinone oxidoreductase subunit S4 was associated with mitochondrial respiratory chain complex I assembly. Stochastic perturbation analysis revealed that NADH:ubiquinone oxidoreductase subunit B2 (NDUFB2), NDUFB8 and ubiquinol?cytochrome c reductase hinge protein (UQCRH) were associated with cellular respiration in Gram?negative samples, whereas large tumor suppressor kinase 2 (LATS2) was associated with G1/S transition of the mitotic cell cycle in Gram?positive samples. NDUFB2, NDUFB8 and UQCRH may be biomarkers for Gram?negative sepsis, whereas LATS2 may be a biomarker for Gram?positive sepsis. These findings may promote the therapies of sepsis caused by Gram?positive and Gram?negative bacteria.

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Toll-like receptor 4 decoy, TOY, attenuates gram-negative bacterial sepsis.

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Toll-like receptor 4 decoy, TOY, attenuates gram-negative bacterial sepsis.

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