| Summary: | Alpha7 nicotinic acetylcholine receptors (α7 nAChRs) are ligand-gated ion-channels located in brain, retina, autonomic ganglia and some immune cells such as macrophages. α7 nAChRs have emerged as a novel target to inhibit peripheral inflammation by blocking the transcription factor, nuclear factor kappa-light chain-enhancer of B cells (NFΚB). α7 nAChR agonists including nicotine and GTS-21 have shown great promise in reducing levels of the pro-inflammatory cytokines such as interleukin-6 (IL6) and tumor-necrosis factor (TNF) in murine endotoxemia and severe sepsis models in vivo. However, mechanistic aspects of GTS-21effects on inflammatory pathways are largely unexplored. Here we used GH4C1 cells overexpressing α7 nAChR to evaluate the effects of nicotine and GTS-21 on TNF-induced NFΚB signaling. We also compared cell-type dependent responses of both α7 nAChR agonists in GH4C1 cells (non-immune cells), RAW264.7 cells (macrophage-like immune cells) and in ex vivo cultures of primary mouse macrophages. Nicotine and GTS-21 do not block TNF-stimulated NFΚB signaling in GH4C1 cells overexpressing α7 nAChRs, suggesting that they require additional unidentified factors, other than α7 nAChRs, for this anti-inflammatory effect. GTS-21 dose-dependently suppressed lipopolysaccharide (LPS)-induced TNF secretion in RAW264.7 cells lacking α7 nAChRs and primary mouse macrophages expressing them. In contrast, GTS-21 blocked IL6 secretion in primary mouse macrophages but had no effect in RAW264.7 cells. Similarly, nicotine inhibited LPS-activated IL6 and TNF secretion in primary mouse macrophages; however it had no effect on endotoxin-activated RAW264.7 cells. Moreover, α7 nAChR antagonism, using either methyllycaconitine (MLA) or α-bungarotoxin (αBGT), partially reversed nicotine or GTS-21 blockade of IL6 and TNF secretion in primary mouse macrophages. Since anti-inflammatory effects of nicotine were smaller than those of GTS-21 in mouse macrophages and because α7 nAChR involvement in nicotine's effects have been well characterized in macrophages, we used α7 nAChR-knockout (Chrna7-/-) mouse models to measure the importance of α7 nAChR in GTS-21 mediated anti-inflammatory effects. GTS-21 significantly inhibited LPS-induced IL6 and TNF production in Chrna7-/- mouse macrophages. These data indicate that even though a component of the GTS-21 anti-inflammatory effects requires an α7 nAChR presence, GTS-21 also has anti-inflammatory effects independent of α7 nAChR receptors, and these effects depend on the cell type.
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