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

Previous studies have implicated a role of heterotrimeric G alpha(i) proteins in lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that Toll-like receptor (TLR) signaling regulates G alpha(i) proteins, which are anti-inflammatory in endotoxemia and polymicrobial sepsis. RAW 264.7 cells were stimulated with LPS and the G alpha(i)-GTP protein complex was immunoprecipitated with a G alpha(i) protein activation assay. In subsequent in vivo studies, the G alpha(i) protein inhibitor pertussis toxin (PTx) or G(i) protein agonist mastoparan (MP-7) were administrated prior to endotoxemia. LPS-induced pro-inflammatory cytokines and mortality were determined. To examine the role of G alpha(i2) in sepsis, G alpha(i2) (-/-) 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 G alpha(i) proteins are rapidly activated by LPS followed by rapid inactivation. These studies provide the first direct evidence that G alpha(i) proteins are modulated by TLR signaling. In following studies, PTx augmented LPS-induced plasma TNF alpha, IL-6, whereas MP-7 suppressed LPS-induced TNF alpha and decreased LPS-induced mortality. In sepsis studies, the survival rate post-CLP was significantly decreased in the G alpha(i2) (-/-) mice compared to WT mice. CLP-induced plasma TNF alpha, IL-6, bacterial load in peritoneal fluid, blood and lung tissue and lung and liver MPO activity were significantly increased in G alpha(i2) (-/-) compared to WT mice. G alpha(i2) (-/-) mice also exhibited increased Th1 and Th2 responses compared to WT mice. Taken together, G alpha(i) proteins are activated by LPS and negatively regulate endotoxemia and sepsis. Understanding the role of G alpha(i2) protein in regulation of the inflammatory response in sepsis may provide novel targets for treatment of sepsis. (c) 2011 Elsevier B.V. All rights reserved.