Activation of Toll-like receptors by Burkholderia pseudomallei

Abstract

Background: Melioidosis, a lethal tropical infection that is endemic in southeast Asia and northern Australia, is caused by the saprophytic Gram-negative bacterium Burkholderia pseudomallei. Overall mortality approaches 40% yet little is known about mechanisms of host defense. Toll-like receptors (TLRs) are host transmembrane receptors that recognize conserved pathogen molecular patterns and induce an inflammatory response. The lipopolysaccharide (LPS) of Gram-negative bacteria is a potent inducer of the host innate immune system. TLR4, in association with MD-2, is the archetype receptor for LPS although B. pseudomallei LPS has been previously identified as a TLR2 agonist. We examined TLR signaling induced by B. pseudomallei, B. pseudomallei LPS, and B. pseudomallei lipid A using gain-of-function transfection assays of NF-kappaB activation and studies of TLR-deficient macrophages.

Results: In HEK293 cells transfected with murine or human TLRs, CD14, and MD-2, heat-killed B. pseudomallei activated TLR2 (in combination with TLR1 or TLR6) and TLR4. B. pseudomallei LPS and lipid A activated TLR4 and this TLR4-mediated signaling required MD-2. In TLR2-/- macrophages, stimulation with heat-killed B. pseudomallei augmented TNF-alpha and MIP-2 production whereas in TLR4-/- cells, TNF-alpha, MIP-2, and IL-10 production was reduced. Cytokine production by macrophages stimulated with B. pseudomallei LPS or lipid A was entirely dependent on TLR4 but was increased in the absence of TLR2. TLR adaptor molecule MyD88 strongly regulated TNF-alpha production in response to heat-killed B. pseudomallei.

Conclusion: B. pseudomallei activates TLR2 and TLR4. In the presence of MD-2, B. pseudomallei LPS and lipid A are TLR4 ligands. Although the macrophage cytokine response to B. pseudomallei LPS or lipid A is completely dependent on TLR4, in TLR2-/- macrophages stimulated with B. pseudomallei, B. pseudomallei LPS or lipid A, cytokine production is augmented. Other MyD88-dependent signaling pathways may also be important in the host response to B. pseudomallei infection. These findings provide new insights into critical mechanisms of host defense in melioidosis.

Figure 1

B. pseudomallei activates TLR2 and TLR4. HEK293 cells were transiently transfected with (A) murine or (B) human TLR2, TLR2/1, TLR2/6, or TLR4; co-receptors CD14 and MD-2; and NF-κB-dependent firefly ELAM luciferase and control β-actin-dependent Renilla luciferase. In (C) HEK293 cells were transiently transfected with human TLR4 and co-receptor CD14 with or without MD-2; and firefly ELAM and Renilla luciferases. Cells were stimulated with media alone, (B) IL-1β 20 ng/mL, (A & B) Pam3CSK4 100 ng/mL, E. coli 0111:B4 LPS 10 ng/mL, or heat-killed BP-1 at various concentrations in CFU/mL. NF-κB activation was measured by light emission (relative light units). In (A&B) data plotted are means ± standard deviations of triplicate conditions. In (C) the means ± standard deviations of triplicate human TLR4-mediated relative light units (normalized to mean empty vector values) are plotted from parallel experiments with or without co-transfection of MD-2. For (A & B) * indicates p < 0.05 and § indicates p = 0.001 compared with empty vector stimulated with the same ligand, and † indicates p < 0.05 compared with TLR2 stimulated with the same ligand using analysis of variance followed by the Bonferroni post-test. Other comparisons are not shown for clarity. The data in (A) represent one of two independently performed experiments. The data in (B) represent one of three independently performed experiments, but the data displayed, in contrast to the two other experiments, show the response to a hyper-responding variant TLR1 plasmid. The data in (C) represent one of three independently performed experiments.

Figure 2

B. pseudomallei LPS signals via TLR4. HEK293 cells were transiently transfected with (A) murine or (B) human TLR2, TLR2/1, TLR2/6, or TLR4; co-receptors CD14 and MD-2; and NF-κB-dependent firefly ELAM luciferase and control β-actin-dependent Renilla luciferase. In (C) HEK293 cells were transiently transfected with human TLR4 and co-receptor CD14 with or without MD-2; and firefly ELAM and Renilla luciferases. Cells were stimulated with media alone, (A & B) Pam3CSK4 1000 ng/mL, E. coli 0111:B4 LPS 10 ng/mL, BP-1 LPS 10 ng/mL, K96243 LPS 10 ng/mL, or BP-1 lipid A 10 ng/mL. NF-κB activation was measured by light emission (relative light units). In (A&B) data plotted are means ± standard deviations of duplicate or triplicate conditions. In (C) the means ± standard deviations of triplicate human TLR4-mediated relative light units (normalized to mean empty vector values) are plotted from parallel experiments with or without co-transfection of MD-2. For (A & B) § indicates p < 0.001 compared with empty vector stimulated with the same ligand, and † indicates p < 0.001 compared with TLR2 stimulated with the same ligand using analysis of variance followed by the Bonferroni post-test. Other comparisons are not shown for clarity. Each graph represents one of two independently performed experiments.

Figure 3

Absence of TLR4, but not TLR2, impairs the macrophage cytokine response to B. pseudomallei. Bone marrow harvested from wild type, TLR2^-/-, TLR4^-/-, or TLR2/4^-/- mice was cultured in the presence of L929 cell conditioned media for 5–10 days to promote differentiation of macrophages before plating and stimulating with media alone, Pam3CSK4 1000 ng/mL, E. coli 0111:B4 LPS 100 ng/mL, heat-killed BP-1 at a bacteria to cell ratio of 100, BP-1 LPS 1000 ng/mL, K96243 LPS 1000 ng/mL, or BP-1 lipid A 1000 ng/mL. Supernatants were harvested after 24 hours and (A) TNF-α or (B) IL-10 production was measured by ELISA. Data plotted are means ± standard deviations of quadruplicate samples. * indicates p < 0.05 and § indicates p = 0.001 compared with wild type cells stimulated with the same ligand, using analysis of variance followed by the Bonferroni post-test. Other comparisons are not shown for clarity. The TNF-α data displayed are from one of six independently performed experiments stimulating various combinations of wild type, TLR2^-/-, TLR4^-/-, or TLR2/4^-/- macrophages with these ligands, and measuring TNF-α. In one of three experiments comparing cytokine responses from TLR2^-/- macrophages to wild type macrophages, production of TNF-α in response to heat-killed Bp was not significantly increased. The IL-10 data displayed are from one of five independently performed experiments stimulating various combinations of wild type, TLR2^-/-, TLR4^-/-, or TLR2/4^-/- macrophages with these ligands, and measuring IL-10. In two of three experiments comparing cytokine responses from TLR2^-/- macrophages to wild type macrophages, production of IL-10 in response to heat-killed Bp was not significantly increased.

Figure 4

MyD88 regulates TNF-α production by macrophages stimulated with B. pseudomallei. Bone marrow harvested from wild type or MyD88^-/- mice was cultured in the presence of L929 cell conditioned media for 5–10 days to promote differentiation of macrophages before plating and stimulating with media alone or heat-killed BP-1 at a bacteria to cell ratio of 100. Supernatants were harvested after 24 hours and TNF-α production was measured by ELISA. Data plotted are means ± standard deviations of quadruplicate samples. § indicates p < 0.001 compared with wild type cells, using the t test. The data displayed represent one of two independently performed experiments.