Previous studies have implicated heterotrimeric Gi proteins in signaling leading to inflammatory mediator production induced by lipopolysaccharide (LPS). TLR4 has recently been shown to play a central role in response to LPS activation. We hypothesized that Gi proteins are coupled to TLR4 activation of signaling pathways. To inhibit Gi protein function, human embryonic kidney (HEK) 293 cells or RAW 264.7 cells were pretreated with pertussis toxin (PTx), an inhibitor of receptor-Galphai interaction, or transfected with dominant negative Galphai3 (Galphai3dn) or Galphai2 minigene (an inhibitory carboxyl terminus of Galphai2) plasmid. The cells were subsequently transfected with constitutively active TLR4 (TLR4ca) plasmid or TLR4ca together with an NFkappaB or AP-1 reporter construct. TLR4ca transfection induced ERK 1/2 activation (157 +/- 14%, P < 0.01), AP-1 activation (4.0 +/- 0.2-fold, P < 0.01), and NFkappaB activation (8.1 +/- 0.4-fold, P < 0.01) compared with empty vector controls. Pretreatment with PTx inhibited TLR4ca-induced ERK 1/2 phosphorylation (30 +/- 7%, P < 0.05) and AP-1 activation (36 +/- 3%, P < 0.05) but did not inhibit NFkappaB activation. Cotransfection of TLR4ca with Galphai3dn or Galphai2 minigene also reduced TLR4ca-induced ERK 1/2 phosphorylation (34 +/- 10% and 33 +/- 5%, respectively, P < 0.05). Constitutively active Galphai2 and Galphai3 plasmids potentiated TLR4ca-induced ERK 1/2 phosphorylation (27 +/- 3% and 41 +/- 6%, respectively, P < 0.05). betaARK-ct plasmid, which inhibits the function of betagamma subunit of G protein, has no effect on TLR4ca-induced ERK 1/2 phosphorylation. These data support our hypothesis and provide the first evidence that Galphai-coupled signaling pathways are activated by TLR4. The TLR4-activated Galphai signaling pathway activates ERK 1/2 phosphorylation and AP-1 activation independently of TLR4-mediated signaling to NFkappaB activation.

Heterotrimeric G(i) 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 G(i) proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of Galpha(i2) or Galpha(i1/3) protein in Galpha(i2)-knockout (Galpha(i2)-/-) or Galpha(i1/3)-knockout (Galpha(i1/3)-/-) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (MPhi) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF-alpha and IFN-gamma in splenocytes from Galpha(i2)-/- mice compared with wild-type (WT) mice. Also, LPS-induced TNF-alpha was increased in splenocytes from Galpha(i1/3)-/- mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-alpha, IL-10, and thromboxane B(2) (TxB(2)) production was decreased in MPhi harvested from Galpha(i2)-/- mice. Also, LPS-induced production of IL-10 and TxB(2) was decreased in MPhi from Galpha(i1/3)-/- mice. In subsequent in vivo studies, TNF-alpha levels after LPS challenge were significantly greater in Galpha(i2)-/- 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 Galpha(i2)-/- mice compared with WT mice. These data suggest that G(i) proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli.

Lipopolysaccharide (LPS), the gram-negative bacterial cell wall component, induces tolerance to a secondary challenge of LPS in macrophages (Mphi) as evidenced by reduced inflammatory mediator production. However, it is uncertain if heat-killed (HK) gram-positive bacteria Staphylococcus aureus (Sa) can induce a similar tolerance and alter responses to LPS. We hypothesized that HKSa induces homologous tolerance and cross tolerance to LPS stimulation in human promonocytic THP-1 cells. We measured TNF-alpha, TxB2, and IFN-gamma production and the phosphorylation of p38, JNK, and ERK-1/2 in human promonocytic THP-1 cells. HKSa (10 microg/mL) significantly stimulated naive (nonpretreated) cell TNF-alpha (P<0.05) and TxB2 production (P<0.05). However, HKSa-pretreated cells challenged secondarily with HKSa (10 microg/mL) exhibited a decrease in the production of TNF-alpha (89 +/- 5%, P<0.05) and TxB2 (85 +/- 3%, P<0.05) compared with HKSa-stimulated naive cells. By contrast, secondary LPS challenge of HKSa-pretreated cells augmented TNF-alpha (41 +/- 3%, P<0.05) and TxB2 (42 +/- 6%, P<0.05) compared with LPS-stimulated naive cells. In naive cells, HKSa and LPS stimulation also significantly phosphorylated the mitogen-activated kinases (MAPKs) p38, JNK, and ERK-1/2 (P<0.005) compared with basal levels. HKSa and LPS induced homologous tolerance as evidenced by the down-regulation of the three MAPK (P<0.05), thus paralleling data on mediator production. HKSa-pretreated cells' priming responses to LPS correlated with augmented phosphorylation of JNK and p38 (P<0.05), whereas ERK-1/2 phosphorylation remained down-regulated. In contrast to TNF-alpha and TxB2 production, HKSa-induced IFN-gamma was up-regulated (26 +/- 5%) in HKSa-pretreated cells compared with HKSa-stimulated naive cells. IFN-gamma antibody exhibited reversed priming in HKSa-pretreated cells as evidenced by a reduction in TNF-alpha. Exogenous human IFN-gamma- (1 microg/mL) and HKSa-pretreated cells secondarily stimulated with HKSa did not prevent the induction of tolerance. In contrast, exogenous IFN-gamma pretreatment prevented the induction of LPS homologous tolerance resulting in an increase in TNF-alpha production. The data demonstrate that HKSa induces homologous tolerance but causes priming to LPS.

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Our previous studies have demonstrated that although LPS and Staphylococcus aureus induce homologous tolerance, they induce priming to each other instead of cross-tolerance. The phosphatidylinositol 3 (PI3) kinase pathway has been implicated in microbial signaling and inflammatory gene expression regulation. We hypothesized that LPS or S. aureus induced tolerance and priming responses to each other are PI3 kinase pathway-dependent. CD1 mice received intraperitoneal injections of 1% Biogel and were treated intraperitoneally with vehicle, LPS, or S. aureus (5 mg/kg) 3 days later. Peritoneal macrophages (MØ) were harvested 24 h later and exposed to vehicle or the PI3 kinase inhibitors wortmannin (10 nmol/L) or LY294002 (10 nmol/L) 1 h before in vitro stimulation with LPS or S. aureus (10 microg/mL). Both LPS and S. aureus significantly induced tumor necrosis factor alpha and thromboxane B2 synthesis (P < 0.05, n = 3) in naive cells. LPS and S. aureus induced homologous tolerance were associated with suppressed tumor necrosis factor alpha and thromboxane B2 levels but augmented interleukin 10 production. However, LPS and S. aureus induced priming to each other, as shown by augmented mediator production. Wortmannin and LY294002 reversed LPS tolerance yet had no effect on S. aureus tolerance. PI3 kinase blockade attenuated the priming responses to both LPS and S. aureus. Mice pretreated with LPS and challenged with LPS were protected. In contrast, mice pretreated with LPS and wortmannin demonstrated LPS tolerance reversal. These data suggest that PI3 kinase is essential for LPS induced homologous tolerance and reciprocal LPS and S. aureus induced priming responses.