Anugraha Gandhirajan, PhD
Post Doctoral Fellow
Cleveland Clinic Lerner Research Institute
Cleveland, Ohio, United States
Disclosure information not submitted.
Sanjoy Roychowdhury, PhD
Project Scientist
Cleveland Clinic Lerner Research Institute
Cleveland, Ohio, United States
Disclosure information not submitted.
Christopher Kibler, B. S.
Laboratory Technician
Cleveland Clinic Lerner Research Institute
Cleveland, Ohio, United States
Disclosure information not submitted.
Vidula Vachharajani, MD, FCCP, FCCM
Professor of Medicine
Cleveland Clinic Lerner College of Medicine
Westlake, Ohio
Disclosure information not submitted.
Title: Sirtuin 2 dysregulates glycolysis and impairs phagocytosis in ethanol exposed macrophages
Introduction: Alcohol use disorder, reported by 1 in 8 critically ill patients, is an independent risk factor for sepsis-mortality. Immune dysfunction and impaired pathogen clearance are reported with alcohol use disorder but the molecular mechanisms and specific therapeutic targets remain unclear. Our recent publication indicates that increased in sirtuin 2 (SIRT2) expression is associated with immune dysfunction and decreased bacterial clearance in ethanol with sepsis-mice. Glycolysis is critical for immune cell-phagocytosis and pathogen killing. We hypothesized that with ethanol exposure, SIRT2 dysregulates macrophage phagocytosis via impaired glycolysis. Using ethanol/vehicle-exposed mouse bone marrow derived macrophages (BMDM), we studied the role of SIRT2 in impaired pro-inflammatory cytokine response, glycolysis and phagocytosis.
Methods: We exposed BMDM from C57Bl/6 (WT) and global SIRT2 knock out (SIRT2KO) mice, to ethanol (25mM) or vehicle (phosphate-buffered saline: PBS) and stimulated with lipopolysaccharide (LPS; 100 ng/ml)/control (normal saline) for 4h. We studied TNF-α protein expression (ELISA), phagocytosis (pHrodo E.coli bioparticles), glycolysis (Seahorse XF24) and glycolytic enzyme-expression (Western blot analysis). Results and discussion: Consistent with our previous work, we found muted TNF-α expression in ethanol-exposed WT-BMDM with a trend towards higher SIRT2 expression with LPS stimulation vs vehicle-exposure. The phagocytosis in ethanol-exposed cells was significantly lower ± LPS stimulation vs. vehicle. The basal glycolysis, glycolytic capacity and a key glycolysis-regulator enzyme, phosphofructokinase P (PFKP) expressions were significantly lower in ethanol exposed WT-BMDM ± LPS vs. vehicle-exposed cells. Ethanol-exposed SIRT2KO-BMDM showed significantly higher TNF-α expression, phagocytosis, basal glycolysis, glycolytic capacity and increased PFKP expression vs. WT-BMDM with LPS stimulation. With co-immunoprecipitation, we found that SIRT2 directly interacts with PFKP.
Conclusion: SIRT2 mutes immunometabolic response to LPS stimulation in ethanol-exposed macrophages and directly interacts with a key glycolytic enzyme, PFKP. Glycolysis is critical for phagocytosis. SIRT2 inhibition is a potential therapeutic strategy to treat immunometabolic dysregulation in ethanol with sepsis.