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TLR4-induced IFN-gamma production increases TLR2 sensitivity and drives Gram-negative sepsis in mice.

ABSTRACT: Gram-negative bacterial infection is a major cause of sepsis and septic shock. An important inducer of inflammation underlying both syndromes is the cellular recognition of bacterial products through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). We identified a novel antagonistic mAb (named 1A6) that recognizes the extracellular portion of the TLR4-MD-2 complex. If applied to mice before infection with clinical isolates of Salmonella enterica or Escherichia coli and subsequent antibiotic therapy, 1A6 prevented otherwise fatal shock, whereas application of 1A6 after infection was ineffective. In contrast, coapplication of 1A6 and an anti-TLR2 mAb up to 4 h after infection with Gram-negative bacteria, in combination with the start of antibiotic therapy (mimicking clinical conditions), provided robust protection. Consistent with our findings in mice, dual blockade of TLR2 and TLR4 inhibited TNF-alpha release from human peripheral blood mononuclear cells upon Gram-negative bacterial infection/antibiotic therapy. Both murine splenocytes and human PBMCs released IFN-gamma in a TLR4-dependent manner, leading to enhanced surface TLR2 expression and sensitivity for TLR2 ligands. Our results implicate TLR2 as an important, TLR4-driven sensor of Gram-negative bacterial infection and provide a rationale for blockade of both TLRs, in addition to antibiotic therapy for the treatment of Gram-negative bacterial infection.

SUBMITTER: Spiller S

PROVIDER: S-EPMC2525587 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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TLR4-induced IFN-gamma production increases TLR2 sensitivity and drives Gram-negative sepsis in mice.

Spiller Stephan S Elson Greg G Ferstl Ruth R Dreher Stefan S Mueller Thomas T Freudenberg Marina M Daubeuf Bruno B Wagner Hermann H Kirschning Carsten J CJ

The Journal of experimental medicine 20080721 8

Gram-negative bacterial infection is a major cause of sepsis and septic shock. An important inducer of inflammation underlying both syndromes is the cellular recognition of bacterial products through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). We identified a novel antagonistic mAb (named 1A6) that recognizes the extracellular portion of the TLR4-MD-2 complex. If applied to mice before infection with clinical isolates of Salmonella enterica or Escherichia coli and ...[more]

PMID: 18644971

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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.

Dataset Information

TLR4-induced IFN-gamma production increases TLR2 sensitivity and drives Gram-negative sepsis in mice.

Publications

TLR4-induced IFN-gamma production increases TLR2 sensitivity and drives Gram-negative sepsis in mice.

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