Atg7 Deficiency Intensifies Inflammasome Activation and Pyroptosis in Pseudomonas Sepsis

Abstract

Sepsis is a severe and complicated syndrome that is characterized by dysregulation of host inflammatory responses and organ failure, with high morbidity and mortality. The literature implies that autophagy is a crucial regulator of inflammation in sepsis. In this article, we report that autophagy-related protein 7 (Atg7) is involved in inflammasome activation in Pseudomonas aeruginosa abdominal infection. Following i.p. challenge with P. aeruginosa, atg7^fl/fl mice showed impaired pathogen clearance, decreased survival, and widespread dissemination of bacteria into the blood and lung tissue compared with wild-type mice. The septic atg7^fl/fl mice also exhibited elevated neutrophil infiltration and severe lung injury. Loss of Atg7 resulted in increased production of IL-1β and pyroptosis, consistent with enhanced inflammasome activation. Furthermore, we demonstrated that P. aeruginosa flagellin is a chief trigger of inflammasome activation in the sepsis model. Collectively, our results provide insight into innate immunity and inflammasome activation in sepsis.

Figure 1

Infection progression of WT mice and atg7^fl/fl mice after P. aeruginosa challenge. (A) Whole body imaging of infected WT mice and atg7^fl/fl mice after i.p. injection of 1×10⁷ CFU PA-Xen41, respectively. Representative images of WT and atg7^fl/fl mice (n=8) at different time points using IVIS XRII imaging (arrows indicating spread of infection). Data are representative of three experiments. (B) WT mice and atg7^fl/fl mice were infected by i.p. injection of 5×10⁶ CFU of PAO1, survival was represented by Kaplan-Meier curves. (*P <0.05, n=8, log-rank test).

Figure 2

Disruption of Atg7 leads to severe bacterial dissemination, PMN penetration and oxidative lung injury in P. aeruginosa-induced sepsis. (A–C) Twenty-four hours after the onset of i.p. PAO1, blood, lung tissues and bronchoalveolar lavage fluid (BALF) of WT mice and atg7^fl/fl mice were collected to detect bacterial dissemination by CFU assay. (*P<0.05; n=5, T tests.) (D–E) At 0, 12 and 24 hours after i.p. injection of PAO1, PMN infiltration levels in the blood and BALF were measured by Hema staining. (*P<0.05; n=5, T tests.) (F) MPO activity in lungs following i.p. PAO1 challenge in WT mice and atg7^fl/fl mice. At 0, 12 and 24 hours after i.p. infection with PAO1, lung tissues were collected to determine MPO activity. (*P<0.05; n=5, T tests.) (G) Representative histological views of lungs of WT mice and atg7^fl/fl mice after 24 hour i.p. infection with PAO1 by H&E staining (Inset shows the enlarged view). Data are shown as mean ± SEM of three independent experiments (*P<0.05; n=3 mice; T tests.)

Figure 3

Hampered autophagosome formation and increased IL-1β in Atg7-deficient macrophages and septic atg7^fl/fl mice (A) AM cells were transfected with RFP-LC3 plasmid for 24 hours. Then the cells were infected with PAO1 at MOI of 10:1 for the indicated time periods. The number of LC3 puncta (arrows) within each cell was determined by confocal laser scanning microscopy. Arrows show RFP-LC3 punctation. WT mice and atg7^fl/fl mice (*P<0.05; n=5, T tests.) were infected with 5×10⁶ CFU of PAO1 for 24 hours. (B) IL-1β (eBioscience, Cat #, 88-7013) production in BALF of WT mice and atg7^fl/fl mice. (C) IL-1β levels in blood of WT mice and atg7^fl/fl mice. (D) TNF-α (88-7324) concentrations in BALF of WT mice and atg7^fl/fl mice. (E) IL-6 (88-7064) levels in BALF of WT mice and atg7^fl/fl mice. (*P<0.05; n=5, T tests.). (F) IL-1β (88-7013) concentrations in siRNA-transfected and WT MH-S cells at different times. (G) TNF-α (88-7324) concentrations in siRNA-transfected and WT MH-S cells at different times. Cytokines were measured by ELISA. Data are representative of three independent experiments (*P<0.05; n=5, T tests.)

FIGURE 4

Atg7 deficiency hyperactivates inflammasome in macrophages upon P. aeruginosa infection. (A–B) Bone marrow-derived macrophages (BMDMs) were isolated from atg7^fl/fl mice and C57BL/6J WT mice, then pre-stimulated with LPS for 5 hours. (A) Cells were infected with PAO1 at different MOIs. 4 hours later, cell lysates and cell culture media were harvested for western blot analysis. Atg7, pro-caspase-1, pro-IL-1, caspase-1 (P10), and IL-1β (P17) were evaluated by Western blotting. Densitometric analysis of caspase-1 (P10), and IL-1β (P17) by quantity one (B–D). Cell culture media were also assayed for IL-1β, TNF-α, IL-6 concentration by ELISA. (*P<0.05; n=3, T tests.)

FIGURE 5

Exacerbated P. aeruginosa-induced pyroptosis in AM cells from atg7^fl/fl mice and siRNA-transfected MH-S cell. (A) Alveolar macrophages of WT mice and atg7^fl/fl mice were infected with PAO1 at indicated MOI for 4 hours, cytotoxicity was determined by LDH release. (*P<0.05; n=3, T tests.) (B) MTT assay of MHS cells infected by PAO1. (*P<0.05; n=10, T tests.) (C) WT and Atg7 deficiency mouse lungs were tested by western blotting after infected for 5h, 10h and 20h, respectively. Caspase-1, IL-18, NLRC4, and ASC were evaluated. (D) WT and Atg7 siRNA-transfected MH-S cells were infected with PAO1 (MOI=10:1) for 2 h or stimulated for 5 h with or without LPS (500 ng/ml) and ATP (5 mmol). Cells were stained with FITC and Rhodamine secondary antibodies, caspase-1 speck and ASC cells were analyzed by immunofluorescence. Data are representative of three independent experiments. (*P<0.05; n=3, T tests.)

Figure 6

Flagellin mediates inflammasome hyperactivation in Atg7 deficient condition by P. aeruginosa infection. (A) MH-S and Atg7 siRNA-transfected MH-S cells were infected with PAO1 at different times (MOI=10:1). Western blotting of Atg7, caspase1P10, NLRC4, cleaved IL-1β, LC3 and β-actin was performed with no infection and 2 hour –starved controls. (B–C) Cells were transfected with PAO1 flagellin by Profect-P1 for indicated time points. Atg7, pro-caspase-1, pro-IL-1β, caspase-1 (P10), and IL-1β (P17) were evaluated by Western blotting. Densitometric analysis of caspase-1 (P10), and IL-1β (P17) by quantity one (B–D). Cell culture media were also assayed for IL-1β concentration by ELISA (bottom panel). (D–E) Cells were transfected with PAO1 flagellin by Profect P1 for the indicated time points. TNF-α and IL-6 levels in culture media were analyzed by ELISA. Data are shown as mean ± SEM of three independent experiments (*P<0.05; n=3, T tests.)