Impaired TLR5 functionality is associated with survival in melioidosis

Abstract

Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR5(1174C)>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney-293 cells transfected with TLR5 and found that B. pseudomallei induced TLR5(1174C)- but not TLR5(1174T)-dependent activation of NF-κB. We tested the association of TLR5(1174C)>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR5(1174C)>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08-0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR5(1174C)>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1β in carriers of TLR5(1174T) compared with carriers of TLR5(1174C). B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR5(1174T). We conclude that the hypofunctional genetic variant TLR5(1174C)>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis.

FIGURE 1.

B. pseudomallei induces TLR5-dependent NF-κB activation. CHO cells stably transfected with TLR5, NF-κB–dependent firefly ELAM luciferase, and control thymidine kinase–driven Renilla luciferase were stimulated with media alone, IL-1β 20 ng/ml, (B) E. coli 0111:B4 LPS 10 ng/ml, S. typhimurium flagellin 100 ng/ml, (A) stationary or (B) log phase heat-killed B. pseudomallei BP-1 at various concentrations in colony-forming units per milliliter. NF-κB activation was determined by the ratio of ELAM to Renilla light emission after stimulation overnight. Data plotted are means ± SDs of triplicate or quadruplicate conditions that represent one of seven experiments in total performed independently. **p ≤ 0.01, ***p ≤ 0.001 by t test for comparisons to cells in media alone.

FIGURE 2.

Survival from melioidosis is enhanced for carriers of TLR5_1174T. Kaplan–Meier in-hospital survival curves are plotted for melioidosis subjects, grouped by genotype. Curves are significantly different by the log rank test (p = 0.001).

FIGURE 3.

NF-κB activation induced by B. pseudomallei is abrogated by TLR5_1174T. HEK-293 cells transiently transfected with human TLR5_1174C or TLR5_1174T, NF-κB–dependent firefly ELAM luciferase, and control thymidine kinase–driven Renilla luciferase were stimulated with media alone, IL-1β 20 ng/ml, S. typhimurium flagellin 100 ng/ml, or log phase heat-killed B. pseudomallei 1026b at various concentrations in colony-forming units per milliliter. NF-κB activation was determined by the ratio of ELAM to Renilla light emission after 24 h. Data plotted are means ± SDs of triplicate or quadruplicate conditions that represent one of two similar experiments performed independently. ***p ≤ 0.001 by ANOVA with the Bonferroni posttest for comparisons between similarly stimulated cells transfected with empty vector TLR5_1174C or TLR5_1174T.

FIGURE 4.

B. pseudomallei induces lower IL-10 and IL-6 levels in white carriers of TLR5_1174T. Whole blood was stimulated with media or heat-killed B. pseudomallei 1026b 2.5 × 10⁶ CFU/ml for 6 h. Inflammatory mediators were measured in plasma by electrochemiluminescence assay, normalized to monocyte count, and log₁₀ transformed for statistical analysis by linear regression. n = 8 (TLR5_1174CC), n = 3 (TLR5_1174CT+TT). Boxes show the median and interquartile range; whiskers show upper and lower adjacent values; outside values are not shown for clarity. *p ≤ 0.05, **p ≤ 0.01.

FIGURE 5.

B. pseudomallei induces lower IL-10 levels in Thai carriers of TLR5_1174T. Whole blood was stimulated with media, E. coli 0111:B4 LPS 10 ng/ml, S. typhimurium flagellin 500 ng/ml, heat-killed B. pseudomallei 1026b 2.5 × 10⁶ CFU/ml, or heat-killed B. pseudomallei K96243 2.5 × 10⁶ CFU/ml (K9) for 6 h. Inflammatory mediators were measured in plasma by multiplex bead assay or ELISA (IL-1β for B. pseudomallei only), normalized to monocyte count, and log₁₀ transformed for statistical analysis by linear regression. Boxes show the median and interquartile range; whiskers show upper and lower adjacent values; outside values are not shown for clarity. n = 269 (TLR5_1174CC), n = 31 (TLR5_1174CT). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.