Toll-like receptor signaling in airborne Burkholderia thailandensis infection

Abstract

Melioidosis is a tropical disease endemic in southeast Asia and northern Australia caused by the gram-negative soil saprophyte Burkholderia pseudomallei. Although infection is often systemic, the lung is frequently involved. B. thailandensis is a closely related organism that at high doses causes lethal pneumonia in mice. We examined the role of Toll-like receptors (TLRs), essential components of innate immunity, in vitro and in vivo during murine B. thailandensis pneumonia. TLR2, TLR4, and TLR5 mediate NF-kappaB activation by B. thailandensis in transfected HEK293 or CHO cells. In macrophages, TLR4 and the adaptor molecule MyD88, but not TLR2 or TLR5, are required for tumor necrosis factor alpha production induced by B. thailandensis. In low-dose airborne infection, TLR4 is needed for early, but not late, bacterial containment, and MyD88 is essential for control of infection and host survival. TLR2 and TLR5 are not necessary to contain low-dose infection. In high-dose airborne infection, TLR2 deficiency confers a slight survival advantage. Lung and systemic inflammatory responses are induced by low-dose inhaled B. thailandensis independently of individual TLRs or MyD88. These findings suggest that redundancy in TLR signaling or other MyD88-dependent pathways may be important in pneumonic B. thailandensis infection but that MyD88-independent mechanisms of inflammation are also activated. TLR signaling in B. thailandensis infection is substantially comparable to signaling induced by virulent B. pseudomallei. These studies provide additional insights into the host-pathogen interaction in pneumonic Burkholderia infection.

FIG. 1.

TLR2, TLR4, and TLR5 mediate activation of NF-κB by B. thailandensis (Bt). (A) HEK293 cells were transiently transfected with murine TLR2, TLR2/1, TLR2/6, or TLR4 and coreceptors CD14 and MD-2, or with an empty vector and NF-κB-dependent firefly ELAM luciferase and control β-actin-dependent Renilla luciferase. Cells were stimulated with medium alone, 20 ng/ml IL-1β, 100 ng/ml Pam3CSK4, 10 ng/ml E. coli (Ec) O111:B4 LPS, or various concentrations of stationary-phase heat-killed B. thailandensis (CFU/ml). †† indicates a P value of <0.01, and ††† indicates a P value of <0.001, compared to TLR2-transfected cells stimulated with the same ligand. (B) CHO cells stably transfected with human TLR5, NF-κB-dependent firefly ELAM luciferase, and control thymidine kinase-driven Renilla luciferase were stimulated with medium alone, 20 ng/ml IL-1β, 10 ng/ml E. coli O111:B4 LPS, 100 ng/ml Salmonella serovar Typhimurium (St) FliC flagellin, or various concentrations of log- or stationary-phase heat-killed B. thailandensis (CFU/ml). NF-κB activation was measured by light emission (n-fold induction of relative light units over that of cells transfected with empty vector and stimulated with media). Data plotted are means ± standard deviations of duplicate conditions that represent one of two similar experiments performed independently (A) or quadruplicate conditions that represent one of six experiments performed independently (B) using either or both stationary- and/or log-phase heat-killed bacteria. Statistical testing was performed with ANOVA and Bonferroni's post-test (A) or Dunnett's post-test (B). *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared to empty vector-transfected cells stimulated by the same ligand (A) or medium (B).

FIG. 2.

B. thailandensis-induced TNF-α production by macrophages requires TLR4 and MyD88 but not TLR2 or TLR5. (A) Bone marrow-derived macrophages from WT, TLR2^−/−, TLR4^−/−, or MyD88^−/− mice were stimulated with medium or various bacterium/cell ratios of heat-killed B. thailandensis (Bt). Data plotted are means ± standard deviations of the results for triplicate samples (duplicate for MyD88^−/− cells) and represent one of three similar experiments performed independently comparing WT to TLR2^−/− and TLR4^−/− cells and one of two similar experiments performed independently comparing WT to MyD88^−/− cells. (B) Alveolar macrophages harvested from WT or TLR5^−/− mice were stimulated with medium alone, 100 ng/ml Salmonella serovar Typhimurium (St) FliC flagellin, or various bacterium/cell ratios of log- or stationary-phase heat-killed B. thailandensis. Data plotted are means ± standard deviations of triplicate samples and represent one of three independently performed experiments using either or both log- or stationary-phase bacteria. Supernatants were harvested after 16 h (A) or 24 h (B), and TNF-α production was measured by enzyme-linked immunosorbent assay. Statistical testing was performed with ANOVA and Bonferroni's post-test (A) or the t test (B). *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared to WT cells stimulated with the same ligand.

FIG. 3.

Survival after pneumonic infection with B. thailandensis is dependent on MyD88, whereas TLR2 deficiency extends survival. (A) Approximately 800 CFU/lung B. thailandensis was deposited by aerosol in the lungs of WT, TLR4^−/−, TLR5^−/−, and MyD88^−/− mice. (B) A total of 2 × 10⁴ CFU/lung B. thailandensis was deposited in the lungs of WT and TLR2^−/− mice. Survival of four (A) or eight (B) mice per strain was recorded. P values were calculated with the log rank test.

FIG. 4.

Containment of B. thailandensis infection is dependent on TLR4 and MyD88. Approximately 400 CFU/lung B. thailandensis was deposited by aerosol in the lungs of WT, TLR2^−/−, TLR4^−/−, and MyD88^−/− mice (A), and ∼550 CFU/lung B. thailandensis was deposited by aerosol in the lungs of WT and TLR5^−/− mice (B). Lung, liver, and spleen were harvested and quantitatively cultured at 24 and 72 h after infection. (A) Eight mice total per strain were sampled at each time point (seven for TLR2^−/− mice at 24 h), combining data from two comparable experiments performed independently. *** indicates a P value of <0.001 for MyD88^−/− versus WT. † indicates a P value of <0.05, and †† indicates a P value of <0.01 for TLR4^−/− versus WT. (B) Four mice per strain were sampled at each time point, and the data displayed represent one of two comparable experiments performed independently. For lung CFU, the lines connect mean values at each time point; otherwise, data are plotted as means ± standard deviations. Statistical testing was performed after log transformation of the data with ANOVA and Bonferroni's post-test (A) or the t test (B).

FIG. 5.

Bronchoalveolar inflammation induced by B. thailandensis is not dependent on individual TLRs or MyD88. BAL was performed in the mice infected as described in the legend for Fig. 4 after deposition of ∼400 CFU/lung (A) or ∼550 CFU/lung (B). At 24 and 72 h after infection, total cells, polymorphonuclear leukocytes (PMN), and mononuclear cells (MN) in the BAL fluid were enumerated. Data are displayed as means ± standard deviations. (A) Eight mice total per strain were sampled at each time point (seven for TLR2^−/− and MyD88^−/− mice at 24 h), and data from two comparable experiments performed independently are combined. (B) Four mice per strain were sampled at each time point, and the data displayed represent one of two comparable experiments performed independently. Statistical testing was performed with ANOVA and Bonferroni's post-test (A) or the t test (B). * indicates a P value of <0.05 for TLR5^−/− versus WT, and *** indicates a P value of <0.001 for MyD88^−/− versus WT. Mean values (± standard deviations) for BAL fluid cell counts in naïve C57BL/6 mice (n = 4) are 0 (± 0) × 10⁶ PMN/ml and 0.09 (± 0.03) × 10⁶ MN/ml.

FIG. 6.

Pulmonary cytokine responses to B. thailandensis are not dependent on individual TLRs or MyD88. Mice were infected as described in the legend for Fig. 4 after deposition of ∼400 CFU/lung (A) or ∼550 CFU/lung (B). Lung homogenate cytokines were measured in duplicate at 24 and 72 h after infection. (A) Eight mice total per strain were sampled at each time point (seven for TLR2^−/− mice at 24 h), and data from two comparable experiments performed independently are combined. (B) Four mice per strain were sampled at each time point, and the data displayed represent one of two comparable experiments performed independently. Data are plotted as means ± standard deviations. Statistical testing was performed after log transformation of data when displayed on a log scale with ANOVA and Bonferroni's post-test (A) or the t test (B). * indicates a P value of <0.05, ** P values of <0.01, and *** P values of <0.001 for MyD88^−/− versus WT. Mean values (± standard deviations) for lung homogenate cytokines in naïve C57BL/6 mice (n = 4) in pg/ml are as follows: TNF-α, 55 (± 0); MCP-1, 468 (± 0); IL-1β, 400 (± 0); IL-6, 84 (± 37); MIP-2, 50 (± 0); and KC, 137 (± 38).

FIG. 7.

Systemic cytokine responses to B. thailandensis are not dependent on individual TLRs or MyD88. Mice were infected as described in the legend for Fig. 4 after deposition of ∼400 CFU/lung (A) or ∼550 CFU/lung (B). Serum cytokines were measured in duplicate at 24 and 72 h after infection. (A) Seven mice total per strain were sampled at 24 h, and eight mice total per strain were sampled at 72 h; data from the two comparable experiments performed independently were combined. (B) Four mice per strain were sampled at each time point, and the data displayed represent one of two comparable experiments performed independently. Data are plotted as means ± standard deviations. Statistical testing was performed after log transformation of data when displayed on a log scale with ANOVA and Bonferroni's post-test (A) or the t test (B) after. * indicates a P value of <0.05, and *** indicates a P value of <0.001 for MyD88^−/− versus WT. Mean values (± standard deviations) for serum cytokines in naïve C57BL/6 mice (n = 4) in pg/ml are as follows: TNF-α, 55 (± 0); MCP-1, 468 (± 0); IL-1β, 400 (± 0); IL-6, 72 (± 0); MIP-2, 50 (± 0); and KC, 155 (± 43).