Survey of innate immune responses to Burkholderia pseudomallei in human blood identifies a central role for lipopolysaccharide - PubMed (original) (raw)

. 2013 Nov 26;8(11):e81617.

doi: 10.1371/journal.pone.0081617. eCollection 2013.

Sarunporn Tandhavanant, Nicolle D Myers, Sudeshna Seal, Arkhom Arayawichanont, Aroonsri Kliangsa-Ad, Lauren E Hittle, Robert K Ernst, Mary J Emond, Mark M Wurfel, Nicholas P J Day, Sharon J Peacock, T Eoin West

Affiliations

Survey of innate immune responses to Burkholderia pseudomallei in human blood identifies a central role for lipopolysaccharide

Narisara Chantratita et al. PLoS One. 2013.

Abstract

B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-α production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B. pseudomallei lipopolysaccharide in blood is greater than the response to other lipopolysaccharide expressing isolates. Our findings suggest that B. pseudomallei lipopolysaccharide may play a central role in stimulating the host response in melioidosis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. WBC-normalized plasma cytokine concentrations induced by stimulation of whole blood from 300 healthy subjects at 37°C for six hours with medium alone, E. coli O111:B4 LPS 10 ng/ml (as a positive control), heat-killed B. pseudomallei 1026b 2.5 × 106 CFU/ml, or heat-killed B. pseudomallei K96243 2.5 × 106 CFU/ml.

Boxes show the median and interquartile range; whiskers show upper and lower adjacent values; outside values are not shown for clarity.

Figure 2

Figure 2. WBC-normalized plasma cytokine concentrations induced by stimulation of whole blood from 300 healthy subjects at 37°C for six hours with heat-killed B. pseudomallei 1026b 2.5 × 106 CFU/ml, stratified by age and sex.

Boxes show the median and interquartile range; whiskers show upper and lower adjacent values; outside values are not shown for clarity.

Figure 3

Figure 3. Heat map of correlation between cytokine concentration induced by stimulation of whole blood with a panel of purified innate immune ligands and heat-killed bacteria (as specified in the methods) with dendrogram of hierarchichal clustering results for the correlation matrix.

Lowest correlation is red and highest correlation is white.

Figure 4

Figure 4. Blockade of the LPS-TLR4 axis markedly impairs TNF-α production induced by B. pseudomallei in human monocytes.

Human monocytes (50,000/well) were stimulated with B. pseudomallei K96243 LPS 1 ng/ml (LPS) or heat-killed B. pseudomallei K96243 (bacteria:monocyte ratio of 1:1) (BP), with or without polymyxin B 100 μg/ml (PB) or a TLR4/MD2 neutralizing antibody 20 μg/ml (Ab) or isotype control 20 μg/ml (Iso). TNF-α was assayed in duplicate cell supernatants after 4 hours. Given substantial inter-individual variation in cytokine release, all TNF-α values for each individual were normalized to LPS-induced levels from the same subject. Relative TNF- α units for each individual (N=4) and mean values are displayed. Statistically significant differences were determined by ANOVA and a Bonferroni post-test. For clarity, only differences between each agonist alone (LPS or BP) and each agonist with polymyxin B or an antibody are shown. ***, p≤0.001. NS, p>0.05.

Figure 5

Figure 5. Plasma cytokine concentrations induced by stimulation of whole blood with B. pseudomallei K96243 LPS 10 ng/ml (BPlps), E. coli O111:B4 LPS 10 ng/ml (EClps), or S. minnesota Re595 LPS 10 ng/ml (SMlps).

Boxes show the median and interquartile range; whiskers show upper and lower adjacent values; outside values are not shown for clarity. Statistical comparisons are made by ANOVA with a Bonferroni post test on log10 transformed data. *, p≤0.05; **, p≤0.01; ***, p≤0.001.

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