Targeting mitochondrial metabolism to suppress neutrophil overactivation in sepsis: Therapeutic potential of the mitochondrial uncoupler BAM15 Free

Background: Understanding the immunometabolic reprogramming that occurs during sepsis is crucial for designing future therapeutics that target specific aspects of this condition. While neutrophil overactivation significantly contributes to the severity of sepsis, the role of mitochondrial function in the neutrophil response has not yet been demonstrated. Thus, this study aimed to determine an alteration in the mitochondrial function in the neutrophils from sepsis and to point out the possibility of attenuating its overresponse by mitochondrial uncoupling mechanisms using mitochondrial uncoupler BAM15.

Method: Neutrophils were isolated by density gradient centrifugation from patients with sepsis admitted to the intensive care unit (ICU) at King Chulalonkorn Memorial Hospital (KCMH), as defined according to Sepsis-3 criteria, as well as from healthy controls. Metabolic parameters including mitochondrial respiration (oxidative phosphorylation) and glycolytic rate were assessed using extracellular flux (Seahorse) analysis with mito-stress and glycolysis-stress test kits. Mitochondrial membrane potential (MMP) and mitochondrial ROS (mtROS) were measured via flow cytometry using MitoTracker Red and MitoSOX, respectively. Neutrophil phenotypes were also analyzed by flow cytometry, while functional tests, including chemotaxis, phagocytosis, and neutrophil extracellular trap (NET) formation, were conducted using transwell migration assays, pHrodo Green–labeled E. coli, and immunofluorescence staining.

Result: Neutrophils from patients with sepsis exhibited an activated phenotype compared to healthy controls, marked by increased CD11b and reduced CD62L expression. Metabolic profiling using Seahorse analysis revealed a distinct energetic shift in sepsis neutrophils, characterized by elevated oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), in contrast to the quiescent metabolic profile (low OCR and ECAR) observed in neutrophils from healthy controls. Additionally, neutrophils from patients with sepsis demonstrated markedly increased levels of MMP, and mtROS were also identified in neutrophils, which paralleled their excessive functions. Treatment with the mitochondrial uncoupler BAM15 significantly attenuated basal OCR and ATP-linked OCR, due to enhanced mitochondrial uncoupling (proton leak). However, BAM15 had no significant effect on ECAR. These metabolic effects were consistent with the observed reductions in MMP and mtROS levels in sepsis neutrophils. Furthermore, in the context of excessive neutrophil activation in sepsis, BAM15 significantly suppressed NET formation and chemotaxis in dose-dependent manner, whereas phagocytic capacity remained preserved. Consistent with the ex vivo stimulation of sepsis neutrophils with lipopolysaccharide (LPS), the increased metabolic and functional alterations were observed. These LPS-induced changes were also attenuated by BAM15 treatment, further supporting the role of mitochondrial modulation in regulating neutrophil overactivations.

Conclusion: Mitochondria are key drivers of excessive neutrophil activation during sepsis. A proof of concept attenuation of excessive inflammation by mitochondrial uncoupler BAM15 through metabolic interference on neutrophils was demonstrated as a new strategy of anti-inflammation in sepsis.

Disclosures No relevant conflict of interest to declare.