Figure 1.
Deleterious role of IL-1β during infection with B. thailandensis.
Mice were infected intranasaly with B. thailandensis (A, 5×104 CFU or B, 5×105 CFU) and their survival was monitored. (C, D) Mice infected with B. thailandensis (5×105 CFU) were sacrificed 72 h post-infection and the organs bacterial burden in the indicated tissues was measured. (E) Flow cytometric analysis of BAL cells and MPO and elastase activity levels in the lung homogenates obtained from the indicated mouse strains at 72 h p.i. (n = 5 for all except Il-1a−/− n = 3). (F) The total area of the inflammatory nodules of lung sections stained with H&E was measured and expressed as percentage of the total lung lobe area (n = 5). (G) IgM and collagen were measured in the BALF collected 72 h p.i. from mice (n = 5) infected with B. thailandensis (5×105 CFU). Data are expressed as mean + S.D. *p<0.05, **p<0.01. (A, B) log rank Kaplan-Meier test, (C–G) Mann-Whitney U test. DKO is Il-1r1−/−/Il-18−/−double deficient mouse. One representative experiment of three (A–C, F, G) or one (D–E) is shown.
Figure 2.
Neutrophil elastase reduces host tolerance to infection with B. thailandensis.
(A) Mice were intranasaly infected with 5×105 CFU B. thailandensis and their survival was monitored. (B) Elastase inhibitor sivelestat (125 µg/mouse, i.n.) and serine protease inhibitor BBI (3 mg/mouse, oral gavage) were administered on day 1, 2, 3 p.i. to C57BL/6 intranasaly infected with 1×105 CFU B. thailandensis. (C) Flow cytometric analysis of BAL cells, levels of MPO activity in lung homogenates, and total area of the inflammatory nodules for mice infected with 5×105 CFU B. thailandensis (n = 5). (D, E) Organs bacterial burden in the indicated tissues of mice infected with 5×105 CFU (D) or 106 CFU (E) B. thailandensis. (F) Wild type and Cxcr2−/− mice (n = 7, pool of two experiments) were intranasaly infected with 2×105 CFU B. thailandensis and survival and BALF myeloid cells composition (G) (n = 4 C57BL/6J, n = 5 Cxcr2−/−) were measured. (H) Cxcr2−/− mice (n = 4) were intranasaly infected with 2×105 CFU B. thailandensis and their survival was monitored. C57BL/6J or Elane−/− neutrophils (1.5×106) were adoptively transferred by intravenous injection 24 and 96 hours p.i. Data are expressed as mean +S.D.*p<0.05, **p<0.01. # p<0.05 Elane−/− respect to Nos2−/−.(A, B, F, H) log rank Kaplan-Meier test, (C, D, E, G) Mann-Whitney U test. One representative experiment of three (A, C, D, E, G) or two (B, F, H) is shown.
Figure 3.
Neutrophil elastase reduces host tolerance to infection with B. pseudomallei.
Mice (n = 5) were intranasaly infected with 400 CFU B. pseudomallei and their survival and weight were monitored (A). (B) Flow cytometric analysis of BAL cells and levels of MPO in BALF collected 72 h p.i. from mice (n = 5) infected as in A. (C) Representative lung sections stained with H&E and the total area of the inflammatory nodules was measured and expressed as percentage of the total lung lobe area (n = 5). (D) Tissue burdens of B. pseudomallei at 72 h p.i. Data are expressed as mean + S.D. *p<0.05. (A) log rank Kaplan-Meier test, (B–D) Mann-Whitney U test.
Figure 4.
Role of elastase in IL-1β and IL-18 processing and pyroptosis.
(A) Neutrophils were purified from bone marrow and infected in vitro with B. thailandensis 1∶100 m.o.i., and release of IL-1β was measured. (B, C) IL-1β and IL-18 were measured in BALF of mice infected with 5×105 CFU B. thailandensis (B) or 400 CFU B. pseudomallei (C). (D) Pyropoptosis and intracellular bacteria replication in neutrophils infected in vitro as in (A). Data are expressed as mean + S.D. *p<0.05. (A, D) One way ANOVA Tukey Post-test, (B, C) Mann-Whitney U test. One representative experiment of three (A, B, D) or one (C) is shown.
Figure 5.
Inflammatory cytokines and acute phase proteins in BALF and sera of infected mice.
Cytokines were measured in BALF of mice infected with 400 CFU B. pseudomallei (A) or in BALF and serum of mice infected with 5×105 CFU B. thailandensis (B) 72 hours p.i. (C) Levels of C Reactive Protein in BALF of mice infected with 5×105 CFU B. thailandensis. Data are expressed as mean + S.D. **p<0.01. Mann-Whitney U test. One representative experiment of two is shown.
Figure 6.
Neutrophil elastase causes lung tissue damage during infection with Burkholderia species.
Total protein, IgM, and collagen were measured in the BALF collected at 72 h p.i. from mice infected with 5×105 CFU B. thailandensis (n = 5) (A) or 400 CFU B. pseudomallei (n = 5) (B). (C) Extravascular leakage of intravenously administered Evans blue in mice infected with B. thailandensis as in (A) was measured 72 h p.i in the lung tissue (µg/g tissue). (D) Representative photographs of histological sections of lung tissues stained with H&E. Alveolar septa are significantly thicker in C57BL/6J than in Elane−/− or Il-1r1−/− mice (arrow heads). (E) Area of alveolar septa was measured by Image J software. (F) Verhoeff-van Gieson stain for elastic fibers. Reduced elastic fiber staining was observed in C57BL/6J mice but not in Elane−/− or Il-1r1−/− mice (arrow heads). Bar is 50 µm. Data are expressed as mean + S.D. *p<0.05, **p<0.01, ***p<0.001. Mann-Whitney U test. One representative experiment of three (A, C–F) or one (B) is shown.
Figure 7.
Bradykinin contributes to NE-mediated lung damage and decreased host tolerance.
(A) Levels of bradykinin were measured 72 h p.i. in the BALF of mice (n = 5) infected with 5×105 CFU B. thailandensis. (B) Mice (n = 12) infected with 5×105 CFU B. thailandensis were administered bradykinin antagonist HOE-140 (10 µg i.n., daily) and their survival monitored. Results pooled from three experiments are shown. (C) Extravascular leakage of intravenously administered Evans blue (µg/g tissue) and bacteria burdens (D) were measured at 72 h p.i. Data are expressed as mean + S.D. *p<0.05, **p<0.01, ***p<0.001. (A, C, D) Mann-Whitney U test. (B) log rank Kaplan-Meier test. One representative experiment of three (A) or two (C, D) is shown.