Heterotrimeric Gαi proteins are regulated by lipopolysaccharide and are anti-inflammatory in endotoxemia and polymicrobial sepsis

Previous studies have implicated a role of heterotrimeric Gαi proteins in lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that Toll-like receptor (TLR) signaling regulates Gαi proteins, which are anti-inflammatory in endotoxemia and polymicrobial sepsis. RAW 264.7 cells were stimulated with LPS and the Gαi–GTP protein complex was immunoprecipitated with a Gαi protein activation assay. In subsequent in vivo studies, the Gαi protein inhibitor pertussis toxin (PTx) or Gi protein agonist mastoparan (MP-7) were administrated prior to endotoxemia. LPS-induced pro-inflammatory cytokines and mortality were determined. To examine the role of Gαi2 in sepsis, Gαi2 (−/−) and wildtype (WT) mice were subjected to cecal ligation and puncture (CLP) and monitored every 24 h for 120 h. Other mice were sacrificed 24 h after CLP. Peritoneal fluid, blood, and tissue samples were collected. Plasma pro-inflammatory cytokine production, bacterial load in peritoneal fluid, blood and lung tissue, myeloperoxidase (MPO) activity in lung and liver and different immune cell populations in spleen were studied. We found that Gαi proteins are rapidly activated by LPS followed by rapid inactivation. These studies provide the first direct evidence that Gαi proteins are modulated by TLR signaling. In following studies, PTx augmented LPS-induced plasma TNFα, IL-6, whereas MP-7 suppressed LPS-induced TNFα and decreased LPS-induced mortality. In sepsis studies, the survival rate post-CLP was significantly decreased in the Gαi2 (−/−) mice compared to WT mice. CLP-induced plasma TNFα, IL-6, bacterial load in peritoneal fluid, blood and lung tissue and lung and liver MPO activity were significantly increased in Gαi2 (−/−) compared to WT mice. Gαi2 (−/−) mice also exhibited increased Th1 and Th2 responses compared to WT mice. Taken together, Gαi proteins are activated by LPS and negatively regulate endotoxemia and sepsis. Understanding the role of Gαi2 protein in regulation of the inflammatory response in sepsis may provide novel targets for treatment of sepsis.