Fig 1.
LTB4 synthesis is blunted during pneumonic plague.
(A) The LTB4 synthesis pathway. (B-I) C56BL/6J mice were infected with 10 x the LD50 Y. pestis KIM5+ and lungs were harvested at the indicated times (n = 5) to measure host lipids by LC-MS. UI = samples from uninfected animals. (B) LTB4 concentrations. (C) 20-hydroxy LTB4 concentrations. (D) LTB4 concentrations. (E) 20-hydroxy LTB4 concentrations. (F) 5-HETE concentrations. (G) PGE1 concentrations. (H) PGE2 concentrations. (I) PGA2 concentrations. Each symbol represents an individual mouse and the box plot represents the median of the group ± the range. Changes in lipid concentrations were compared to the UI sample using (B-C) One-way ANOVA with Dunnett’s post hoc test or (D-I) the LIMMA—Moderated t-test. ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, # = p ≤ 0.0001.
Fig 2.
BLT1-/- mice are not more susceptible to pneumonic plague than C57BL/6J mice.
(A) C57BL/6J (green circles) or BLT1-/- (purple squares) mice were infected intranasally with 10 x the LD50 of Y. pestis KIM5+ and lungs were harvested at 12 and 24 h post-infection. Bacterial proliferation within the lungs was determined by CFU enumeration. Each symbol represents an indivdual mouse and the box plot represents the median of the group ± the range. Combined data from two independent experiments. (B-C) C57BL/6J (green circles) or BLT1-/- (purple squares) mice were infected intranasally with 10x the LD50 of Y. pestis CO92 LUXpcysZK. (B) Bacterial proliferation in the lungs as a function of bioluminescence. Each symbol represents an indivdual mouse and the box plot represents the median of the group ± the range. Combined data from two independent experiments. (C) Survival curves of mice from B (n = 15). For A and B, T-test with Mann-Whitney’s post hoc test indicated no statistically significant (ns) differences between C57BL/6J and BLT1-/- groups. For C, Log-Rank anlysis revealed no statistically signficant (ns) differences in surival between the two groups.
Fig 3.
LTB4 treatment improves host killing of Y. pestis.
(A) C57BL/6J mice were administered 1 x DPBS (PBS) or 10 nmol LTB4 intraperitoneally and changes in neutrophil populations (Ly6G+CD11b+) were measured at 1 or 4 h post-treatment. (B) C57BL/6J (green circles) or BLT1-/- (purple squares) mice administered DPBS or 10 nmol LTB4 were infected 1 h later with 105 CFU of Y. pestis KIM5+ and bacterial numbers in the peritoneal cavities were enumerated 3 h post-inoculation. LOD = Limit of detection. (C) Neutrophil populations from a subset of animals from B. Each symbol represents an indivdual mouse and the box plot represents the median of the group ± the range. Combined data from three independent experiments. One-way ANOVA with Tukey’s post hoc test compared to each condition. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, # = p ≤ 0.0001.
Fig 4.
Neutrophils do not synthesize LTB4 in response to Y. pestis.
(A-B) Murine (BMNs) or (C-D) human (hPMNs) neutrophils were infected with E. coli DH5α (Ec), S. enterica Typhimurium LT2 (ST), or K. pneumoniae manC (ΔKp) at an MOI of 20, or with Y. pestis (Yp) at increasing MOIs. LTB4 was measured from supernatants 1 h post infection by ELISA. Each symbol represents an independent biological infection and the box plot represents the median of the group ± the range. UI or 0 = uninfected. One-way ANOVA with Dunnett’s post hoc test compared to uninfected. * = p ≤ 0.05, ** = p ≤ 0.01, # = p ≤ 0.0001.
Fig 5.
Y. pestis actively inhibits LTB4 synthesis.
(A) Murine (BMNs) or (B) human (hPMNs) neutrophils were infected with Y. pestis (Yp) or mutants that either lacked the Yop effectors (T3E) or the Yop effectors and the T3SS [T3(-)] at an MOI of 20 and LTB4 was measured at 30, 60, or 120 min. (C and D) Murine (BMNs) or (E and F) human (hPMNs) neutrophils were co-infected with the indicated bacteria at a combined MOI of 20 and LTB4 was measured at 60 min post-infection. (G) Murine neutrophils (BMNs) were infected with Yp, T3E, T3(-), or Y. pestis strains expressing only one Yop effector (+A = YpkA; +E = YopE; +H = YopH; +J = YopJ; +K = YopK; +M = YopM; or +T = YopT) at an MOI of 20 and LTB4 was measured at 60 min post-infection. Each symbol represents an independent biological infection and the box plot represents the median of the group ± the range. UI = uninfected. One-way ANOVA with Dunnett’s post hoc test compared to uninfected for A, B, and G, or Tukey’s post hoc test compared to each condition for C, D, E, and F. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, # = p ≤ 0.0001.
Fig 6.
Neutrophils synthesize LTB4 in response to the Y. pestis T3SS in the absence of the Yop effectors.
(A) Relative expression of LcrV based on western blots normalized to total protein loaded from bacteria cultured at 37 or 26°C. (B) Murine neutrophils (BMNs) were infected (MOI of 20) with Y. pestis (Yp) or mutants that either lacked the Yop effectors (T3E) or the Yop effectors and the T3SS [T3(-)] cultured at 37 or 26°C and LTB4 was measured at 60 min post-infection. (C) Murine neutrophils (BMNs) were infected (MOI of 20) with Yp, T3E, T3(-), a yopB mutant in the T3E background (ΔB), or ΔB complemented with yopB (ΔB::B) cultured at 37°C and LTB4 was measured at 60 min post-infection. Each symbol represents an independent biological infection and the box plot represents the median of the group ± the range. One-way ANOVA with Tukey’s post hoc test compared to each condition for A and B and Dunnett’s post hoc test compared to uninfected for C. ns = not significant, *** = p ≤ 0.001, # = p ≤ 0.0001.
Fig 7.
Lack of LTB4 response to Y. pestis is conserved in other leukocytes.
(A) Murine bone-marrow derived mast cells (BMMCs) were infected with Y. pestis (Yp) or with mutants that either lacked the Yop effectors (T3E) or the Yop effectors and the T3SS [T3(-)] at an MOI of 20, or treated with 100 mg/cm2 crystalline silica (Si) and LTB4 was measured at 2 h post-infection. (B and C) Murine bone-marrow derived macrophages (BMDMs) differentiated towards (B) M1 or (C) M2 phenotypes were infected with Yp, T3E, or T3(-) at an MOI of 20 and LTB4 was measured at 4 h post-infection. UI = uninfected. Each symbol represents an independent biological infection and the box plot represents the median of the group ± the range. One-way ANOVA with Dunnett’s post hoc test compared to uninfected. ** = p ≤ 0.01, # = p ≤ 0.0001, ns = not significant.
Fig 8.
Working model for inhibition of the inflammatory cascade during plague.
(A) Normal response by sentinel leukocytes results in rapid production of LTB4 that leads to autocrine signaling, neutrophil swarming, and induction of cytokine and chemokine release. (B) Y. pestis inhibits the production of LTB4 via the action of the Yop effectors, which delays resident neutrophil recruitment and subsequent production of cytokines and chemokines needed for inflammation.