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Inflammation. 2012 Oct;35(5):1611-7. doi: 10.1007/s10753-012-9476-z.

Toll-like receptor-induced inflammatory cytokines are suppressed by gain of function or overexpression of Gα(i2) protein.

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Department of Neurosciences, Medical University of South Carolina, 173 Ashley Ave., BSB Room 403, Charleston, SC 29425, USA.


Previous studies have implicated a role of Gα(i) proteins as co-regulators of Toll-like receptor (TLR) activation. These studies largely derived from examining the effect of Gα(i) protein inhibitors or genetic deletion of Gα(i) proteins. However, the effect of increased Gα(i) protein function or Gα(i) protein expression on TLR activation has not been investigated. We hypothesized that gain of function or increased expression of Gα(i) 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 Gα(i2) protein; however, the Gα(i2) is insensitive to its negative regulator RGS thus rendering more sustained Gα(i2) protein activation following ligand/receptor binding. In subsequent studies, we generated Raw 264.7 cells that stably overexpress Gα(i2) protein (Raw Gα(i2)). 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 Gα(i2) cells also exhibit decreased TNF-α and IL-6 production in response to LPS and Pam3CSK4. These studies support our hypothesis that Gα(i2) proteins are novel negative regulators of TLR activation.

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