Previous studies have implicated a role of heterotrimeric Gα(i)
proteins in
lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that
Toll-like receptor (TLR) signaling regulates Gα(i)
proteins, which are anti-inflammatory in
endotoxemia and polymicrobial
sepsis. RAW 264.7 cells were stimulated with LPS and the Gα(i)-
GTP protein complex was immunoprecipitated with a Gα(i)
protein activation assay. In subsequent in vivo studies, the Gα(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 Gα(i2) in
sepsis, Gα(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 Gα(i)
proteins are rapidly activated by LPS followed by rapid inactivation. These studies provide the first direct evidence that Gα(i)
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 Gα(i2) (-/-) 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 Gα(i2) (-/-) compared to WT mice. Gα(i2) (-/-) mice also exhibited increased Th1 and Th2 responses compared to WT mice. Taken together, Gα(i)
proteins are activated by LPS and negatively regulate
endotoxemia and
sepsis. Understanding the role of Gα(i2)
protein in regulation of the inflammatory response in
sepsis may provide novel targets for treatment of
sepsis.