Inhibition of mitophagy drives macrophage activation and antibacterial defense during sepsis

Abstract

Mitochondria have emerged as key actors of innate and adaptive immunity. Mitophagy has a pivotal role in cell homeostasis, but its contribution to macrophage functions and host defense remains to be delineated. Here, we showed that lipopolysaccharide (LPS) in combination with IFN-γ inhibited PINK1-dependent mitophagy in macrophages through a STAT1-dependent activation of the inflammatory caspases 1 and 11. In addition, we demonstrated that the inhibition of mitophagy triggered classical macrophage activation in a mitochondrial ROS-dependent manner. In a murine model of polymicrobial infection (cecal ligature and puncture), adoptive transfer of Pink1-deficient bone marrow or pharmacological inhibition of mitophagy promoted macrophage activation, which favored bactericidal clearance and led to a better survival rate. Reciprocally, mitochondrial uncouplers that promote mitophagy reversed LPS/IFN-γ-mediated activation of macrophages and led to immunoparalysis with impaired bacterial clearance and lowered survival. In critically ill patients, we showed that mitophagy was inhibited in blood monocytes of patients with sepsis as compared with nonseptic patients. Overall, this work demonstrates that the inhibition of mitophagy is a physiological mechanism that contributes to the activation of myeloid cells and improves the outcome of sepsis.

Keywords: Inflammation; Innate immunity; Macrophages; Metabolism; Mitochondria.

Figure 1. Macrophage activation is associated with the early inhibition of mitophagy.

(A–D) Flow cytometry assessment of mitochondrial ROS (upper) and mitochondrial density (lower) in raw 264.7 macrophages (A) exposed to TLR agonists, (B) mitophagy inhibitor (mdivi-1), or (C and D) to LPS and IFN-γ alone or in combination for 24 hours or a specified duration (n = 3 per condition). (E) Flow cytometry assessment of mitophagy in raw 264.7 macrophages with mt-mkeima (upper) or autophagic vacuole formation with CYTO-ID autophagy detection kit (lower) in raw 264.7 macrophages exposed to LPS/IFN-γ (n = 3 per condition). (F and G) Immunoblots of mitophagy and mitochondrial fission checkpoints on protein lysates from (F) stat1^+/+ or stat1^–/– BMDMs exposed to LPS/IFN-γ or (G) from raw 264.7 macrophages exposed for 24 hours to LPS/IFN-γ alone or in combination with a mitophagy inducer (2,4-DNP, 1 μM) (densitometry: ratio to β-actin is presented above the immunoblots). Bar graphs represent mean ± SEM with overlaid individual values; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 determined by ANOVA corrected for multiple comparisons; *P < 0.05, ***P < 0.001 determined by Student’s t test with Welch’s correction.

Figure 2. LPS/IFN-γ–mediated inhibition of mitophagy is associated with metabolic reprogramming of macrophages.

(A and B) Mitochondrial network of (A) mouse peritoneal macrophages or (B) raw 264.7 macrophages exposed to vehicle or LPS/IFN-γ for 6 hours (A) immunostained for Tom20 in combination or not with PINK1 or (B) stained with JC-1 (right panels represent the image binarization of JC-1 stained macrophages). Scale bars: 10 μm. (C) Flow cytometry assessment of mitochondrial density and Δψm in raw 264.7 macrophages exposed to LPS/IFN-γ and stained with JC-1 (n = 3 per time point). (D and E) (D) Oxygen consumption (OCR) and extracellular acidification (ECAR) profile measured with Seahorse XFe96 analyzer on BMDMs exposed to vehicle, LPS/IFN-γ for 6 hours, or mdivi-1 for 24 hours (n = 8 per condition). (F) Flow cytometry assessment of Δψm with TMRM in raw 264.7 macrophages exposed to LPS/IFN-γ for 6 hours (n = 3 per condition). (G) Cell death assessed by low cytometry (annexin V-PI) in raw 264.7 macrophages exposed to vehicle or LPS/IFN-γ and 2,4 DNP alone or in combination for 18 hours. Bar graphs represent mean ± SEM with overlaid individual values; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 determined by ANOVA corrected for multiple comparisons; **P < 0.01, ***P < 0.001 determined by Student’s t test with Welch’s correction. Veh, vehicle.

Figure 3. Polymicrobial infection triggers the early inhibition of mitophagy in myeloid cells.

(A and B) Flow cytometry assessment of the mitochondrial density in (A) total monocytes (CD45⁺ CD115⁺ CD11b^hi) and in (B) inflammatory monocyte subpopulation (CD45⁺ CD115⁺ CD11b^hi Ly6C^hi) in the blood of C57BL6/J mice after sham or CLP surgery (n = 3–4 sham; n = 5 CLP). (C) Flow cytometry assessment of the mitochondrial density of peritoneal macrophages (CD45⁺ F4/80^hi CD11b^hi) in peritoneal fluid of mice treated as in A and B. (D) Flow cytometry assessment of mitochondrial density in CD64^hi Mitotracker^hi subpopulation of peritoneal macrophages of mice treated as in A and B. Graphs with plots represent mean plus individual values; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 determined by ANOVA corrected for multiple comparisons.

Figure 4. Polymicrobial infection and endotoxemia trigger the early inhibition of mitophagy in myeloid cells in a STAT1-dependent manner.

(A and B) Flow cytometry assessment of CD64^hi Mitotracker^hi peritoneal macrophage subpopulation and mitochondrial density in blood monocytes of stat1^+/+ or stat1^–/– mice after (A) sham or CLP surgery (n = 6–7 sham; n = 5–7 CLP) or (B) i.p. injection of saline (sal.) or LPS (0.5 mg/kg, 24 hours) (n = 5–7 sal.; n = 5–7 LPS). (C and D) Flow cytometry assessment of mitochondrial density in (C) stat1^+/+ or stat1^–/– BMDMs or in (D) raw 264.7 macrophages targeted with control (CTL) or stat1 siRNA and then exposed to vehicle or LPS/IFN-γ for 24 hours. (E and F) Flow cytometry assessment of (E) mitochondrial ROS production and (F) Δψm in raw 264.7 macrophages targeted with CTL or stat1 siRNA and then exposed to vehicle or LPS/IFN-γ for 24 hours. (G) Oxygen consumption profile measured with Seahorse XFe96 analyzer on stat1^+/+ or stat1^–/– BMDMs exposed to vehicle or LPS/IFN-γ for 6 hours (n = 6–8 per condition). Graphs with plots represent mean plus individual values; bar graphs represent mean ± SEM with overlaid individual values; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 determined by ANOVA corrected for multiple comparisons. Veh, vehicle.

Figure 5. LPS/IFN-γ inhibits mitophagy in macrophages through the STAT1-dependent regulation of caspase-1.

(A and B) Caspase-1 expression in stat1^+/+ or stat1^–/– BMDMs exposed to vehicle or LPS/IFN-γ for 18 hours or indicated duration assessed by (A) qPCR or (B) immunoblotting (n = 3 per condition). (C and D) Assessment by (C) immunoblotting or (D) flow cytometry of the impact of zVAD-FMK–dependent inhibition of caspases on LPS/IFN-γ–dependent inhibition of mitophagy in raw 264.7 macrophages exposed to vehicle or LPS/IFN-γ for 18 hours (n = 3 per condition) (densitometry: ratio to β-actin is presented above the immunoblots). (E) Proteolytic activity of recombinant human caspase-1 (hCASP1) against recombinant Tribolium castaneum PINK1 (T. cast. PINK1) in the presence of zVAD-FMK and LPS (100 ng/mL) alone or in combination. Protein levels were assessed with 2,2,2-TCE gels after UV transillumination (the table below C presents densitometry data). Bar graphs represent mean ± SEM with overlaid individual values; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 determined by ANOVA corrected for multiple comparison.

Figure 6. The inhibition of mitophagy triggers classical activation of macrophages through mitochondrial ROS.

(A) Flow cytometry assessment of classical macrophage activation in raw 264.7 cells exposed to antimycin A for 6 hours. (B and C) Flow cytometry assessment of classical macrophage activation in raw 264.7 cells exposed to LPS/IFN-γ for 18 hours alone or in combination with (B) the mitochondrial ROS scavenger MitoTEMPOL or with (C) the HIF-1α inhibitor echinomycin (n = 3 per condition). (D–I) Assessment of macrophage activation profile by (D and G) flow cytometry, (E and H) gene expression, and (F and I) bactericidal activity in raw 264.7 cells (D, E, G, and H) incubated (24 hours) or (F and I) preincubated (24 hours) with (D–F) the mitophagy inhibitor mdivi-1 or (G–I) the mitophagy inducer CCCP (n = 3 per condition). Bar graphs represent mean ± SEM with overlaid individual values; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 determined by ANOVA corrected for multiple comparisons; *P < 0.05, ***P < 0.001 determined by Student’s t test with Welch’s correction.

Figure 7. Inhibition of mitophagy in myeloid cells protects against bacterial infection and improves survival during sepsis.

(A) Survival curve of C57BL6/J mice treated with saline (sal.) or 2,4-DNP (10 mg/kg) 24 hours before sham or CLP surgery (sham, n = 5 per group; CLP, n = 13 per group). P value was determined by Gehan-Breslow-Wilcoxon test. (B) Percentage of classically activated macrophages (percentage of CD11b^hi F4/80^hi macrophages) in C57BL6/J mice treated as in A (n = 7 per condition). (C) Bacterial load in the peritoneal cavity of C57BL6/J mice treated as in A (n = 8 per condition). (D) Correlation of the mitochondrial density in Ly6C^hi blood monocytes 2 hours after CLP surgery versus the survival (in hours) after CLP surgery (n = 26). P and r values were determined by Spearman’s rank correlation. (E) Survival curve of C57BL6/J mice treated with vehicle or mdivi-1 for 24 hours before CLP surgery (CLP + vehicle, n = 15; CLP + mdivi-1, n = 15). P value was determined by Gehan-Breslow-Wilcoxon test. (F and G) Percentage of classically activated macrophages (F) (percentage of CD11b^hi F4/80^hi macrophages) and bacterial load (G) in the peritoneal cavity of C57BL6/J mice treated as in E (n = 6–7 per condition) (H) Survival curve of C57BL6/J mice transplanted with Pink1^+/+ (BMT Pink1^+/+) or Pink1^–/– bone marrow (BMT Pink1^–/–) 5 weeks before CLP surgery (BMT Pink1^+/+, n = 13; BMT Pink1^–/–, n = 15). P value was determined by Gehan-Breslow-Wilcoxon test. (I) Flow cytometry assessment of the percentage of classically activated macrophages (percentage of CD11b^hi F4/80^hi macrophages) in the peritoneal cavity of mice that underwent transplantation and surgery as in H (n = 11–12 per group). (J) Bacterial load in the cavity of mice that underwent transplantation and surgery as in H (n = 11–12 per group). Graphs with plots represent mean plus individual values; *P < 0.05 determined by Student’s t test with Welch’s correction.

Figure 8. Increased mitochondrial density in blood monocytes is a biomarker of sepsis in critically ill patients.

(A) Flow cytometry assessment of mitochondrial density in blood monocytes of critically ill patients (ICU patients without [n = 16] or with sepsis [n = 16] according to sepsis-3 task force criteria). (B) Levels of inflammatory and tissue perfusion biomarkers in the blood of critically ill patients as described in A. Data were collected from patient medical records and were not available for all patients (n indicated above graphs). The graphs represent median plus individual values. The median value of each group is presented at the bottom of each graph. The normal range of healthy patients is presented in bold italics enclosed in square brackets. *P < 0.05, **P < 0.01, ***P < 0.001 determined by Student’s t test with Welch’s correction. (C) Correlation matrix of blood biomarkers in critically ill patients as described in A (n = 15–32). *P < 0.05, **P < 0.01, ***P < 0.001 were determined by Spearman’s rank correlation.