Molecular characterization of the acute inflammatory response to infections with gram-negative versus gram-positive bacteria

Abstract

Sepsis caused by gram-negative bacteria and that caused by gram-positive bacteria often manifest similar clinical features. We investigated plasma proinflammatory cytokine profiles in patients with sepsis due to gram-positive and gram-negative bacteria and studied the cytokine production and differential gene regulation of leukocytes stimulated ex vivo with Escherichia coli lipopolysaccharide or heat-killed Staphylococcus aureus. Concentrations of tumor necrosis factor alpha, interleukin 1 receptor antagonist (IL-1Ra), IL-8, IL-10, IL-18 binding protein, procalcitonin, and protein C in plasma did not differ between patients with sepsis due to gram-negative and gram-positive bacteria. However, plasma IL-1beta, IL-6, and IL-18 concentrations were significantly higher in patients with sepsis due to gram-positive bacteria. Ex vivo stimulation of whole blood with heat-killed S. aureus markedly increased IL-1beta and IL-18 levels more than E. coli lipopolysaccharide stimulation. Microarray analysis revealed at least 359 cross-validated probe sets (genes) significant at the P < 0.001 level whose expression discriminated among gram-negative-organism-stimulated, gram-positive-organism-stimulated, and unstimulated whole-blood leukocytes. The host inflammatory responses to gram-negative and gram-positive stimuli share some common response elements but also exhibit distinct patterns of cytokine appearance and leukocyte gene expression.

Figures

FIG. 1.

Plasma cytokine concentrations in patients with sepsis due to gram-negative (n = 25) and gram-positive (n = 27) bacteria. Plasma was obtained at admission to a phase II clinical trial of PAFase prior to administration of test drug or placebo. Measurements of protein C were obtained from 34 of the 52 patients. Values are means ± standard errors of the means. *, P < 0.05 by Student's t test.

FIG. 2.

Different patterns of gene expression in human leukocytes after stimulation with LPS and S. aureus. Hierarchical clustering of variance-normalized gene expression data from unstimulated human leukocytes and from leukocytes that had been exposed to either LPS or heat-killed S. aureus for 2 h prior to RNA harvest. Samples were obtained from three subjects. Expression and variation filters were applied to the data set prior to clustering. Probe sets whose hybridization signal intensity was at or below background levels (absent) on all arrays tested were eliminated from further analysis. The resulting data set was culled by ranking on the coefficient of variation and eliminating the bottom half of the data set to remove probe sets whose expression did not vary between the treatment regimens. The gene expression observations were variance normalized to a mean of 0 and a standard deviation of 1, and this normalized data set was subjected to hierarchical cluster analysis with average linkage clustering of the nodes. The variation in gene expression for a given gene is expressed as distance from the mean observation for that gene (S.D., standard deviations). The scale adjacent to the dendrogram is for Pearson's correlationcoefficient. CON, control unstimulated leukocytes; LPS, leukocytes exposed to LPS for 2 h prior to RNA harvest; SAC, leukocytes exposed to S. aureus Cowan for 2 h prior to RNA harvest; r, replicate number.

FIG. 3.

Hierarchical clustering of hybridization signal intensity (variance normalized gene expression) of probe sets significant at the P ≤ 0.001 level in unstimulated leukocytes and leukocytes that had been exposed to either LPS or heat-killed S. aureus for 2 h prior to RNA harvest. Samples were obtained from three subjects. A total of 359 genes were identified by using BRB Array Tools at the P < 0.001 level. Hierarchical clustering reveals that the LPS-stimulated and unstimulated samples were more similar than the S. aureus-stimulated samples. Also see Table 3.

FIG. 4.

k-means clustering of hybridization signal intensity (gene expression) differences of 758 probe sets significant at the P ≤ 0.001 level in unstimulated leukocytes and leukocytes that had been exposed to either LPS or heat-killed S. aureus for 2 h prior to RNA harvest. Samples were obtained from a single subject with three replicates. The gene expression observations were variance normalized and subjected to k-means clustering to six bins, labeled A through F. The variation in gene expression for a given gene is expressed as distance from the mean observation for that gene (S.D., standard deviations). CON,control unstimulated leukocytes; LPS, leukocytes exposed to LPS for 2 h prior to RNA harvest; SAC, leukocytes exposed to S. aureus Cowan for 2 h prior to RNA harvest; r, replicate number.

FIG. 5.

IL-1β and IL-18 production from whole blood stimulated overnight with increasing concentrations of LPS or heat-killed S. aureus. Heparin-anticoagulated whole blood was obtained from three healthy volunteers and incubated with 100 ng, 1 μg, or 10 μg of E. coli LPS/ml or 0.01, 0.1, or 1% (wt/vol) heat-killed S. aureus Cowan (SAC) overnight at 37°C. The following morning, plasma was separated from the cells, and plasma cytokine concentrations were determined. The concentrations of IL-1β and IL-18 were markedly higher with S. aureus stimulation than with LPS (P < 0.05 by ANOVA and the Student-Newman-Keuls multiple-range test). In contrast, TNF-α, IL-6, and IL-18BP production was unaffected (data not shown). *, P < 0.05 versus control (unstimulated); ‡, P < 0.05 versus low-dose SAC or LPS (0.001% and 1 ng, respectively); †, P < 0.05 versus 0.01% SAC.

FIG. 6.

Hierarchical clustering of hybridization signal intensity (gene expression) differences for pro- and anti-inflammatory cytokine probe sets. The gene expression observations from the single subject with three replicates were variance normalized and subjected to hierarchical clustering. The variation in gene expression for a given gene is expressed as the distance from the mean observation for that gene (S.D., standard deviations). Several genes were represented by more than one probe set on the Affymetrix U95aVer2 chip, and the different probe sets are identified by their Affymetrix identification numbers. CON, control unstimulated leukocytes; LPS, leukocytes exposed to LPS for 2 h prior to RNA harvest; SAC, leukocytes exposed to S. aureus Cowan for 2 h prior to RNA harvest; r, replicate number.