Lactate Inhibits Macrophage Phagocytosis and Bacterial Clearance During Polymicrobial Sepsis

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Introduction: Sepsis is a life-threatening disease that is characterized by organ dysfunction and dysregulated host innate and inflammatory responses to the infection. The defective eradiation of invading bacterial or other pathogens is a major cause of multiple organ dysfunction and death in sepsis. Elevated blood lactate levels are associated with severity and mortality of sepsis. Our previous studies have shown that lactate is a potent signaling molecule in regulating macrophage immune response. Macrophages are professional phagocytes and actively engulf/kill microorganisms. The present study investigated whether lactate impairs macrophage phagocytosis of bacterial during sepsis.

Methods: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Lactic acid (pH 6.8, 0.5g/kg body weight) was injected i.p. 6 hours after CLP or sham surgery. The Bronchoalveolar lavage fluid (BALF) and blood samples were collected 24 hours following surgery, serially diluted 10-fold with sterile PBS. Serial dilutions were spread onto typic soy agar plates and grown overnight at 37 ̊C, and bacterial colonies were counted. To investigate the role of YAP in macrophage bacterial clearance, GFP-labelled E.Coli bacterial (5x107 CFU/mouse, in PBS) were introduced to wildtype (WT) and macrophage-specific YAP deficient (mYAP-/-) mouse through intravitreal injection. Twenty minutes after bacterial injection, GFP-E.Coli bacterial burden was examined using flow cytometry, typic soy agar plate culture and confocal microscopy. For in vitro experiments, RAW 264.7 macrophages were stimulated with lactate (10 mM) for 24 hours followed by incubation with E.Coli BioParticles for 1 hour. In a separate experiment, peritoneal macrophages and bone marrow-derived macrophages (BMDMs) were isolated and prepared from WT and mYAP-/- mice. Phagocytosis of E.Coli BioParticles was compared between WT and mYAP-/- macrophages. Phagocytosis-related proteins were examined by western blotting.

Results: We observed that elevated serum lactate levels impair bacterial clearance in septic mice, as evidenced by increased bacterial burden, and increased mortality rate of septic mice by 74%. Lactate treatment also significantly suppressed macrophage phagocytosis of E.Coli Bioparticles. Mechanistically, we found that lactate induced GPR81/AMPK-mediated phosphorylation of YAP, suggesting YAP is involved in lactate-suppressed macrophage phagocytosis. Indeed, the clearance of GFP-E.Coli bacterial was dramatically impaired in mYAP-/- mice as compared to WT mice. Peritoneal macrophages and BMDMs from mYAP-/- mice had reduced phagocytosis of E.Coli Bio-particles. Notably, YAP deficiency in macrophages decreased the expression of phagocytosis-related proteins (Eea1, Rab5 and Eif5a), when compared with control groups.

Conclusion: Our results indicate a novel role of lactate in inhibiting macrophage phagocytosis and bacterial clearance during polymicrobial sepsis. Therefore, lactate/lactate-associated signaling may be promising target for sepsis treatment.


Big Island, HI

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