, Volume 35, Issue 5, pp 1611-1617
Date: 12 May 2012

Toll-like Receptor-Induced Inflammatory Cytokines are Suppressed by Gain of Function or Overexpression of Gα i2 Protein

Rent the article at a discount

Rent now

* Final gross prices may vary according to local VAT.

Get Access

Abstract

Previous studies have implicated a role of Gαi proteins as co-regulators of Toll-like receptor (TLR) activation. These studies largely derived from examining the effect of Gαi protein inhibitors or genetic deletion of Gαi proteins. However, the effect of increased Gαi protein function or Gαi protein expression on TLR activation has not been investigated. We hypothesized that gain of function or increased expression of Gαi proteins suppresses TLR2- and TLR4-induced inflammatory cytokines. Novel transgenic mice with genomic “knock-in” of a regulator of G protein signaling (RGS)-insensitive Gnai2 allele (Gα i2 G184S/G184S ; GS/GS) were employed. These mice express essentially normal levels of Gαi2 protein; however, the Gαi2 is insensitive to its negative regulator RGS thus rendering more sustained Gαi2 protein activation following ligand/receptor binding. In subsequent studies, we generated Raw 264.7 cells that stably overexpress Gαi2 protein (Raw Gαi2). Peritoneal macrophages, splenocytes, and mouse embryonic fibroblasts (MEF) were isolated from WT and GS/GS mice and were stimulated with LPS, Pam3CSK4, or Poly (I:C). We also subjected WT and GS/GS mice to endotoxic shock (LPS, 25 mg/kg i.p.) and plasma tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 production were determined. We found that in vitro LPS and Pam3CSK4-induced TNF-α, and IL-6 production are decreased in macrophages from GS/GS mice compared with WT mice (p < 0.05). In vitro, LPS and Pam3CSK4 induced IL-6 production in splenocytes, and in vivo, LPS-induced IL-6 were suppressed in GS/GS mice. Poly (I:C)-induced TNF-α, and IL-6 in vitro demonstrated no difference between GS/GS mice and WT mice. LPS-induced IL-6 production was inhibited in MEFs from GS/GS mice similarly to macrophage and splenocytes. In parallel studies, Raw Gαi2 cells also exhibit decreased TNF-α and IL-6 production in response to LPS and Pam3CSK4. These studies support our hypothesis that Gαi2 proteins are novel negative regulators of TLR activation.