Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric Gαiproteins

Abstract

Heterotrimeric Gi proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of Gi proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of Gαi2 or Gαi1/3 protein in Gαi2-knockout (Gαi2−/−) or Gαi1/3-knockout (Gαi1/3−/−) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (MΦ) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF-α and IFN-γ in splenocytes from Gαi2−/− mice compared with wild-type (WT) mice. Also, LPS-induced TNF-α was increased in splenocytes from Gαi1/3−/− mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-α, IL-10, and thromboxane B2 (TxB2) production was decreased in MΦ harvested from Gαi2−/− mice. Also, LPS-induced production of IL-10 and TxB2 was decreased in MΦ from Gαi1/3−/− mice. In subsequent in vivo studies, TNF-α levels after LPS challenge were significantly greater in Gαi2−/− mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated Gαi2−/− mice compared with WT mice. These data suggest that Gi proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli.