Cellular metabolic regulators: novel indicators of low-grade inflammation in humans.

OBJECTIVE: The Toll-like receptor 4 (TLR4) ligand endotoxin triggers robust systemic inflammatory responses in humans at doses equal to or greater than 1 ng/kg. In this study, we tested the hypothesis that evidence of TLR4-induced responses would be detectable in leukocytes challenged with endotoxin doses that are below the threshold needed to trigger a characteristic systemic inflammatory phenotype in humans. METHODS: Subjects were challenged with endotoxin at 1, 0.5, or 0.1 ng/kg (n = 5 per dose). Systemic responses were monitored for 24 hours. Blood samples, collected at designated intervals, were used to determine plasma cytokines levels, total and differential leukocyte counts, expression of leukocyte cell surface receptors, and changes in the leukocyte transcriptome. Western blotting was used to determine changes in leukocyte protein expression. RESULTS: We found that in vivo endotoxin at doses below 1.0 ng/kg triggers weak and variable responses in humans. In marked contrast, we show that endotoxin at a concentration as low as 0.1 ng/kg triggers a transient decline in cellular ATP levels in leukocytes. This is associated with the appearance of a unique protein expression signature in leukocytes. The protein expression signature includes 3 prominent features: (i) AMP-activated protein kinase subunit α (AMPKα) degradation, (ii) increased hypoxia inducible factor-1 (HIF-1) α expression, and (iii) autophagy, collectively indicative of a regulated metabolic response. An indistinguishable response phenotype was observed in human leukocytes treated with endotoxin in vitro. CONCLUSIONS: These data demonstrate for the first time in humans that a TLR4 ligand concentration that is below the threshold needed to trigger clinically evident systemic inflammatory manifestations initiates a transient decline in ATP levels, AMPKα degradation, HIF-1α expression, and autophagy in leukocytes. This establishes that low-grade TLR4 activation exerts control over leukocyte metabolism in the absence of systemic inflammatory indicators.

A novel model of common Toll-like receptor 4- and injury-induced transcriptional themes in human leukocytes.

INTRODUCTION: An endotoxin challenge, sepsis, and injury/trauma, trigger significant changes in human peripheral blood leukocytes (PBL) gene expression. In this study, we have sought to test the hypothesis that the Toll-like receptor 4 (TLR4) induced transcription patterns elicited in humans exposed to in vivo endotoxin would parallel gene expression patterns observed in trauma patients with initial non-infectious injury. In addition, we sought to identify functional modules that are commonly affected by these two insults of differing magnitude and duration. METHODS: PBL were obtained from seven adult human subject experimental groups. The groups included a group of healthy, hospitalized volunteers (n = 15), that comprised four study groups of subjects challenged with intravenous endotoxin, without or with cortisol, and two serial samplings of trauma patients (n = 5). The PBL were analyzed for gene expression using a 8,793 probe microarray platform (Gene Chip® Focus, Affymetrix). The expression of a subset of genes was determined using qPCR. RESULTS: We describe sequential selection criteria of gene expression data that identifies 445 genes that are significantly differentially expressed (both P ≤ 0.05 and > 1.2 fold-change) in PBL derived from human subjects during the peak of systemic inflammatory responses induced by in vivo endotoxin, as well as in PBL obtained from trauma patients at 1 to 12 days after admission. We identified two functional modules that are commonly represented by this analysis. The first module includes more than 50 suppressed genes that encode ribosomal proteins or translation regulators. The second module includes up-regulated genes encoding key enzymes associated with glycolysis. Finally, we show that several circadian clock genes are also suppressed in PBL of surgical ICU patients. CONCLUSIONS: We identified a group of > 400 genes that exhibit similar expression trends in PBL derived from either endotoxin-challenged subjects or trauma patients. The suppressed translational and circadian clock modules, and the upregulated glycolytic module, constitute a robust and long lasting PBL gene expression signature that may provide a tool for monitoring systemic inflammation and injury.

Polymorphisms of heat shock protein-70 (HSPA1B and HSPA1L loci) do not influence infection or outcome risk in critically ill surgical patients.

Heat shock proteins (HSP) are induced in various stress conditions and have many cytoprotective effects, including formation of protein complexes for antigen presentation, stabilizing intracellular proteins, and facilitating protein folding. The HSP-70 gene exhibits polymorphisms at the HSPA1B and HSPA1L loci that reportedly influence cytokine levels and clinical outcomes in critically ill patients. These HSP variations also have been linked to TNF-beta polymorphisms associated with poor outcomes. This study further evaluated outcomes and risk of infection of HSP polymorphisms in critically ill patients. Seventy-six consecutive surgical intensive care unit uninfected patients with established systemic inflammatory response features were prospectively enrolled. Genomic DNA was isolated from whole blood samples and specific fragments, including the relevant polymorphic sites, were amplified by PCR, and restriction digestions were performed. Genotypes were determined by electrophoresis and all were confirmed by direct sequencing. Plasma cytokine levels for TNF-alpha were assayed in a subset of patients by enzyme-linked immunoabsorbent assay. None of the HSP alleles bore a significant relationship to nosocomial infection rates, organ specific dysfunctions, or mortality. No linkage of HSP genotype to common TNF-alpha or TNF-beta genotypes could be demonstrated, although the HSPA1L CT polymorphism was associated with higher levels of TNF-alpha compared with the TT genotype. These data suggest that polymorphisms of the HSPA1L or HSPA1B loci do not influence infection or other highly morbid outcomes in surgical intensive care unit patients.

Modulation of the lipopolysaccharide receptor complex (CD14, TLR4, MD-2) and toll-like receptor 2 in systemic inflammatory response syndrome-positive patients with and without infection: relationship to tolerance.

The lipopolysaccharide (LPS) receptor complex consists of two interacting receptors (CD14 and TLR4) and an associated protein (MD-2). When engaged by LPS, as in gram-negative infection, this complex transduces a signal detected by MyD88 and passed onward by a cascade of the IRAKs, TRAF6, and NIK, resulting in activation of NF-kappaB. A similar cascade, mediated by TLR2, occurs with ligands derived from gram-positive bacteria. In vitro studies of human monocytes have shown that TLR4 mRNA is paradoxically upregulated in response to "tolerizing" doses of LPS. This study evaluated changes in vivo of blood monocyte CD14, TLR4, TLR2, and MD-2 mRNA by reverse transcription followed by real-time polymerase chain reaction in surgical intensive care unit patients and in normal controls. In addition cell-surface receptor expression of TLR2, TLR4, and CD14 was assessed by flow cytometry in patients and normal controls. Inflammation-induced acute tolerance to LPS was evaluated by ex vivo whole blood tumor necrosis factor alpha production and was significantly reduced in patients compared with controls, confirming LPS hyporesponsiveness. Monocyte mRNA and cell-surface receptor expression of TLR4 were increased 2.4-fold (P < 0.05) and 1.7-fold (P <.002), respectively, in patients compared with normal controls. Monocyte TLR2 mRNA, MD-2 mRNA and CD14 and TLR2 cell-surface expression were not significantly changed compared with controls. The present study suggests that the acute inflammatory condition associated with peripheral cellular LPS hyporesponsiveness is neither specific to prior infectious challenge nor can be ascribed to significant alterations in expression of the cell-surface LPS binding complex proteins.

Insulin-like growth factor binding protein-3 is upregulated in LPS-treated THP-1 cells.

BACKGROUND: Lipopolysaccharide (LPS) is a potent activator of human monocytic cells. We have determined that LPS stimulation of the human monocytic cell line, THP-1, results in an increased apoptotic rate. We hypothesized that cDNA expression array analysis could be used to identify target genes involved in the regulation of this process. METHODS: THP-1 cells (1 x 10(6)/mL) were stimulated with LPS (1 microg/mL) or vehicle control. Apoptosis was measured at 0, 24, 48, 72 and 96 h using propidium iodide staining and flow cytometry to determine the percentage of cells with hypodiploid DNA. At 16 h, the Atlas Human cDNA expression array system, containing probes for 205 genes related to apoptosis, was used to survey and quantify transcript expression. The experiment was performed in duplicate and the membranes were normalized to cytoplasmic beta-actin. Standard Western blotting was performed on the conditioned medium to correlate secreted protein expression with RNA expression. Pretreatment with insulin-like growth factor I (IGF-I) was performed to determine whether the effects of insulin-like growth factor binding protein-3 (IGFBP-3) on apoptosis were IGF-dependent. RESULTS: LPS stimulation of THP-1 cells resulted in a greater than 2-fold increase in the rate of apoptosis when compared to vehicle control. When the cDNA expression arrays were compared, there was a 500-fold increase in the expression of the IGFBP-3 transcript in the LPS-stimulated cells. Western blotting of culture medium verified an approximately 2-fold increase in secreted IGFBP-3. Pretreatment with IGF-I did not prevent the increase in apoptosis seen with LPS stimulation. CONCLUSIONS: THP-1 cell apoptosis is increased in response to LPS stimulation and is associated with a significant induction of IGFBP-3 mRNA and protein. IGFBP-3, which reportedly promotes apoptosis and modulates the bioavailability of the pro-survival insulin-like growth factor 1, may serve to regulate apoptosis in monocytic cells in an IGF-independent manner. These data further support the investigation of the role of the IGF axis in programmed cell death of immune cells.

Influence of the TNF-alpha and TNF-beta polymorphisms upon infectious risk and outcome in surgical intensive care patients.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a well-documented central inflammatory mediator in sepsis. Specific polymorphisms of the TNF-alpha and TNF-beta genes (TNF2 and LTA + 250, respectively) have been suggested to correlate with higher mortality in septic shock. This study sought to determine whether these polymorphisms of the TNF-alpha and -beta genes are associated with an increased risk of infection in an at-risk surgical intensive care population. MATERIALS AND METHODS: Forty-four consecutive patients with systemic inflammatory response syndrome were enrolled prospectively in the study. Genomic DNA was isolated from whole blood samples using standard phenol/chloroform extraction techniques. Specific fragments including the polymorphic sites of each gene were amplified by polymerase chain reaction, and restriction enzyme digestions were performed. Genotypes were determined by gel electrophoresis and confirmed by direct sequencing. RESULTS: Eighty-six percent of the patients were TNF1 homozygotes (G:G at -308 of the TNF-alpha promoter region), whereas 9% of the patients were homozygous for TNF2 (A:A). There was no difference in the incidence of sepsis, septic shock, or mortality between patients bearing the various alleles. Only 13.6% of the patients exhibited the G:G alleles for TNF-beta, whereas the homozygous A:A was present in 45.4% of the patients. CONCLUSION: The presence of the A allele at these polymorphic sites did not predispose critically ill surgical patients to either infection or septic shock.

A single nucleotide polymorphism in the Mdm2 promoter and risk of sepsis.

BACKGROUND: The Mdm2-SNP309(T/G) polymorphism has been shown to upregulate transcription of Mdm2 and subsequently attenuate the p53 pathway. Its role in regulating the human response to acute illness has not been reported. METHODS: Patients from the surgical intensive care unit were prospectively enrolled. SNP309 genotype was determined, and a genotype-based comparison of clinical outcomes was performed. RESULTS: Of the 85 enrolled patients, 41 had wild type (T/T) and 44 had mutant (32 T/G and 12 G/G) genotypes. The mutant-genotype group tended to have a longer LOS in both the surgical intensive care unit (P = .40) and the hospital (P = .08), but these trends did not reach significance. No observable genotype-based differences were noted in any other measured parameters. CONCLUSIONS: The Mdm2-SNP309(G) allele may be associated with longer LOS. However, it does not appear to influence any other clinical characteristics, nor can it be used to predict clinical outcome.

Response to systemic endotoxemia among humans bearing polymorphisms of the Toll-like receptor 4 (hTLR4).

Mutations (Asp299Gly and Thr399Ile) in the human Toll-like receptor 4 (hTLR4) gene are reportedly associated with hyporesponsiveness to inhaled LPS in humans. It was hypothesized that normal volunteers with these hTLR4 mutations would manifest altered physiological responses to intravenous LPS administration. Human subjects (n = 57) were administered LPS (2 ng/kg) intravenously and monitored for vital signs (temperature, heart rate, mean arterial pressure). Blood-derived genomic DNA samples were evaluated using PCR to detect hTLR4 and TLR2 mutations. Heterozygous hTLR4 mutations were identified in 8 of the 57 subjects studied. Subjects with hTLR4 mutations demonstrated similar responses to LPS administration. The moderate systemic inflammatory response produced by intravenous LPS administration in human subjects is not modulated by the presence of heterozygous mutations in the hTLR4 gene.

Human toll-like receptor 4 mutations but not CD14 polymorphisms are associated with an increased risk of gram-negative infections.

Human toll-like receptor 4 (hTLR4) and CD14 are known to be components of the lipopolysaccharide receptor complex. Our study investigated the association between TLR4 mutations (Asp299Gly and Thr399Ile) and CD14 polymorphism(s) with outcome in an intensive care unit (ICU) population at risk for sepsis. By use of a polymerase chain reaction-based restriction fragment-length polymorphism analysis technique, the hTLR4 gene was altered in 14 (18%) of 77 ICU patients (all positive for systemic inflammatory response syndrome) and in 5 (13%) of 39 volunteers. There was a significantly higher incidence of gram-negative infection among patients with the mutations (11 [79%] of 14), compared with that in the wild-type population (11 [17%] of 63; P=.004). No association between CD14 polymorphism(s) and the incidence of infection or outcome was observed. These findings indicate that hTLR4 mutations are associated with an increased incidence of gram-negative infections in critically ill patients in a surgical setting.