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Drp1/Fis1-Dependent Pathologic Fission and Associated Damaged Extracellular Mitochondria Contribute to Macrophage
Riddhita Mukherjee1,2, Carly A Tompkins1,2, Nicolai P Ostberg2
1Division of Critical Care Medicine, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA.
Objectives:
Recent publications have shown that mitochondrial dynamics can govern the quality and quantity of extracellular mitochondria subsequently impacting immune phenotypes. This study aims to determine if pathologic mitochondrial fission mediated by Drp1/Fis1 interaction impacts extracellular mitochondrial content and macrophage function in sepsis-induced immunoparalysis.
Design:
Laboratory investigation.
Setting:
University laboratory.
Subjects:
C57BL/6 and BALB/C mice.
Interventions:
Using in vitro and murine models of endotoxin tolerance (ET), we evaluated changes in Drp1/Fis1-dependent pathologic fission and simultaneously measured the quantity and quality of extracellular mitochondria. Next, by priming mouse macrophages with isolated healthy mitochondria (MC) and damaged mitochondria, we determined if damaged extracellular mitochondria are capable of inducing tolerance to subsequent endotoxin challenge. Finally, we determined if inhibition of Drp1/Fis1-mediated pathologic fission abrogates release of damaged extracellular mitochondria and improves macrophage response to subsequent endotoxin challenge.
Measurements And Main Results:
When compared with naïve macrophages (NMs), endotoxin-tolerant macrophages (ETM) demonstrated Drp1/Fis1-dependent mitochondrial dysfunction and higher levels of damaged extracellular mitochondria (Mitotracker-Green + events/50 μL: ETM = 2.42 × 106 ± 4,391 vs NM = 5.69 × 105 ± 2,478; p < 0.001). Exposure of NMs to damaged extracellular mitochondria (MH) induced cross-tolerance to subsequent endotoxin challenge, whereas MC had minimal effect (tumor necrosis factor [TNF]-α [pg/mL]: NM = 668 ± 3, NM + MH = 221 ± 15, and NM + Mc = 881 ± 15; p < 0.0001). Inhibiting Drp1/Fis1-dependent mitochondrial fission using heptapeptide (P110), a selective inhibitor of Drp1/Fis1 interaction, improved extracellular mitochondrial function (extracellular mitochondrial membrane potential, JC-1 [R/G] ETM = 7 ± 0.5 vs ETM + P110 = 19 ± 2.0; p < 0.001) and subsequently improved immune response in ETMs (TNF-α [pg/mL]; ETM = 149 ± 1 vs ETM + P110 = 1,150 ± 4; p < 0.0001). Similarly, P110-treated endotoxin tolerant mice had lower amounts of damaged extracellular mitochondria in plasma (represented by higher extracellular mitochondrial membrane potential, TMRM/MT-G: endotoxin tolerant [ET] = 0.04 ± 0.02 vs ET + P110 = 0.21 ± 0.02; p = 0.03) and improved immune response to subsequent endotoxin treatment as well as cecal ligation and puncture.
Conclusions:
Inhibition of Drp1/Fis1-dependent mitochondrial fragmentation improved macrophage function and immune response in both in vitro and in vivo models of ET. This benefit is mediated, at least in part, by decreasing the release of damaged extracellular mitochondria, which contributes to endotoxin cross-tolerance. Altogether, these data suggest that alterations in mitochondrial dynamics may play an important role in sepsis-induced immunoparalysis.
Insights
Pathologic mitochondrial fission contributes to sepsis-induced immunoparalysis by increasing damaged extracellular mitochondria. Inhibiting this fission improves macrophage function and immune response, suggesting a therapeutic target for sepsis.
Area of Science:
- Immunology
- Cell Biology
- Mitochondrial Biology
Background:
- Mitochondrial dynamics influence extracellular mitochondrial content and immune responses.
- Sepsis can lead to immunoparalysis, a state of suppressed immune function.
Purpose of the Study:
- To investigate if pathologic mitochondrial fission, mediated by Drp1/Fis1 interaction, affects extracellular mitochondrial content and macrophage function in sepsis-induced immunoparalysis.
- To determine if inhibiting this fission can restore immune function.
Main Methods:
- Utilized in vitro and murine models of endotoxin tolerance (ET).
- Evaluated Drp1/Fis1-dependent mitochondrial fission and extracellular mitochondrial quantity/quality.
- Assessed macrophage response to healthy and damaged extracellular mitochondria.
- Administered a Drp1/Fis1 inhibitor (P110) in vitro and in vivo.
Main Results:
- Endotoxin-tolerant macrophages exhibited Drp1/Fis1-dependent mitochondrial dysfunction and increased damaged extracellular mitochondria.
- Exposure to damaged extracellular mitochondria induced cross-tolerance to endotoxin.
- Inhibition of Drp1/Fis1-mediated fission with P110 improved macrophage function and immune response in ET models.
- P110 treatment reduced damaged extracellular mitochondria in plasma and enhanced immune response in mice.
Conclusions:
- Inhibition of Drp1/Fis1-dependent mitochondrial fragmentation improves macrophage function and immune response in sepsis models.
- This improvement is partly due to reduced release of damaged extracellular mitochondria, which cause endotoxin cross-tolerance.
- Alterations in mitochondrial dynamics are implicated in sepsis-induced immunoparalysis.
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