Fig 1.
Caspase-1 and caspase-11 are protective in melioidosis.
(A) Mice were infected intranasaly with B. thailandensis (105 CFU) and their survival and body weight were monitored. (B, C) Mice infected with B. thailandensis (5x105 CFU) were sacrificed 48 hours p.i. and the bacterial burden in organs and cytokines in BALF and serum were measured. (D, E) Mice infected intranasaly with B. thailandensis (105 CFU) were sacrificed at the shown time points and cytokine levels and cellular infiltrates in BALF were measured. One representative experiment of three (A-C) or two (D, E) is shown. Data are expressed as mean ± S.D. *p<0.05, **p<0.01, ***p<0.001. (A) log rank Kaplan-Meier test, (B) Mann-Whitney U test, (C) Unpaired t-test, (D, E) One-way ANOVA.
Fig 2.
Caspase-1, not caspase-11, controls IL-1β/IL-18 secretion and pyroptosis in BMM infected with B. thailandensis.
BMM were primed O/N with IFNγ (100 ng/ml) and infected with B. thailandensis (MOI 50). Cytokine secretion (A) and LDH release (B) were measured 4 hours p.i.. Intracellular bacteria replication was measured 7 hours post infection. One representative experiment of four is shown. Data are expressed as mean ± S.D. *p<0.05, **p<0.01, ***p<0.001. (B) One-way ANOVA.
Fig 3.
Caspase-11 protective role in hematopoietic and radio-resistant cell types.
(A) Mice that underwent adoptive bone marrow transplant were infected with B. thailandensis (5x105 CFU) and sacrificed 48 hours p.i. to measure the organ bacterial burdens. (B) The total numbers of CD45-positive cells in bone marrow, spleen, and BALF of mice from A were calculated. One representative experiment of two is shown. Data are expressed as mean ± S.D. *p<0.05, **p<0.01, ***p<0.001 (A) Mann-Whitney U test, (B) One-way ANOVA.
Fig 4.
Caspase-11 controls pyroptosis of lung epithelial cells.
(A) TC-1 cells were infected with B. thailandensis or the bsaZ mutant in presence or absence of IFNγ. Active caspase-11 was pulled-down from cell lysates using Biotin-VAD-FMK and detected by immunoblot. (B) Caspase-11 expression in control (WT) and knocked-out (C11KO) TC-1 cells. (C) WT and C11KO TC-1 cells were stimulated with IFNγ (100 ng/ml) and infected with B. thailandensis (MOI 100). LDH release and intracellular bacteria replication were measured 8 hours p.i.. (D, E) Mice infected for 48 hours with B. thailandensis (5x105 CFU) were treated intranasaly with Image-iT DEAD to stain necrotic cells and euthanized. Lung histological sections were stained with anti-EpCAM antibody to visualize epithelial cells. White arrow heads indicate necrotic epithelial cells, white dots indicate live epithelial cells. One representative experiment of four (A-C) or two (D, E) is shown. *p<0.05, **p<0.01, ***p<0.001 Unpaired t-test.
Fig 5.
IFNγ is necessary and sufficient for the protective effect of IL-18 in melioidosis.
Mice of shown genotype were infected intranasaly with B. thailandensis (5x105 CFU) and treated with IFNγ (A) or IL-18 (B). Mice were sacrificed 48 hours p.i. and the organ bacterial burdens were measured. (C) IFNγ was measured in the BALF and serum of mice from B. One representative experiment of two is shown. Data are expressed as mean ± S.D. *p<0.05, **p<0.01. (A, B) Mann-Whitney U test (C) Unpaired t-test.
Fig 6.
IFNγ protection in melioidosis depends on ROS generation.
(A) Mice were infected intranasaly with B. thailandensis (105 CFU) and treated with IFNγ. Mice were sacrificed 48 hours p.i. and the organ bacterial burdens were measured. (B) BMM were infected with B. thailandensis (MOI 50) in presence or absence of IFNγ (100 ng/ml)). Cells were lysed 4 hours later and intracellular bacteria growth was measured (left panel). Induction of pyroptosis was measured as LDH release in conditioned supernatants (right panel). One experiment representative of three is shown. Data are expressed as mean ± S.D. *p<0.05, **p<0.01, ***p<0.001 (A) Mann-Whitney U test (B) One-way ANOVA.
Fig 7.
IFNγ stimulates ROS production that protects from B. thailandensis infection.
Mice were infected intranasaly with B. thailandensis (105 CFU). (A) ROS generation was measured ex vivo in neutrophils or macrophages obtained from BALF of mice of shown genotype 14 or 48 hours p.i., respectively. (B) Mice were sacrificed 48 hours p.i. and the organ bacterial burdens were measured. (C, D) Wild type mice were infected intranasaly with B. thailandensis (105 CFU) and treated daily with IFNγ or NAC. Mice were sacrificed 48 hours p.i. and the organ bacterial burdens (C) or cytokine levels in BALF (D) were measured. One representative experiment of two is shown. Data are expressed as mean ± S.D. *p<0.05, **p<0.01, ***p<0.001 (A, D) Unpaired t-test, (B, C) Mann-Whitney U test.