Research Article: GTS-21 has cell-specific anti-inflammatory effects independent of α7 nicotinic acetylcholine receptors

Date Published: April 4, 2019

Publisher: Public Library of Science

Author(s): Brijesh K. Garg, Ralph H. Loring, Alain Haziot.


α7 Nicotinic acetylcholine receptors (nAChRs) reportedly reduce inflammation by blocking effects of the important pro-inflammatory transcription factor, nuclear factor kappa-light chain-enhancer of B cells (NFκB). The α7 nAChR partial agonist GTS-21 reduces secretion of pro-inflammatory cytokines including interleukin-6 (IL6) and tumor-necrosis factor (TNF) in models of endotoxemia and sepsis, and its anti-inflammatory effects are widely ascribed to α7 nAChR activation. However, mechanistic details of α7 nAChR involvement in GTS-21 effects on inflammatory pathways remain unclear. Here, we investigate how GTS-21 acts in two cell systems including the non-immune rat pituitary cell line GH4C1 expressing an NFκB-driven reporter gene and cytokine secretion by ex vivo cultures of primary mouse macrophages activated by lipopolysaccharide (LPS). GTS-21 does not change TNF-stimulated NFκB signaling in GH4C1 cells expressing rat α7 nAChRs, suggesting that GTS-21 requires additional unidentified factors besides α7 nAChR expression to allow anti-inflammatory effects in these cells. In contrast, GTS-21 dose-dependently suppresses LPS-induced IL6 and TNF secretion in primary mouse macrophages endogenously expressing α7 nAChRs. GTS-21 also blocks TNF-induced phosphorylation of NFκB inhibitor alpha (IκBα), an important intermediary in NFκB signaling. However, α7 antagonists methyllycaconitine and α-bungarotoxin only partially reverse GTS-21 blockade of IL6 and TNF secretion. Further, GTS-21 significantly inhibited LPS-induced IL6 and TNF secretion in macrophages isolated from knockout mice lacking α7 nAChRs. These data indicate that even though a discrete component of the anti-inflammatory effects of GTS-21 requires expression of α7 nAChRs in macrophages, GTS-21 also has anti-inflammatory effects independent of these receptors depending on the cellular context.

Partial Text

Chronic inflammation may lead to several inflammatory disorders including sepsis, rheumatoid arthritis, asthma, diabetes and Crohn’s disease [1, 2] and involves production and secretion of various pro-inflammatory cytokines [3] including IL6, TNF and high mobility group box-1 (HMGB1). Koopman et al. [4] recently proposed vagus nerve stimulation as therapy against the inflammation found in rheumatoid arthritis, based in part on previous work [5] that vagus nerve stimulation requires cholinergic activation to prevent TNF secretion in response to endotoxins, such as lipopolysaccharide (LPS). LPS is a gram-negative bacteria cell wall component that activates NFκB-mediated inflammatory signaling in cells expressing Toll-like 4 receptors (TLR4) [6]. Later, Wang et al. [7] reported that activated α7 nicotinic acetylcholine receptors (nAChRs) inhibit LPS-induced pro-inflammatory cytokine secretions without affecting anti-inflammatory mediators such as IL-10. Wang et al. [8] found that α7 nAChRs mediate anti-inflammatory signaling in part by blocking NFκB activation in a mouse sepsis model. Follow-up studies have since established that during macrophage activation, the afferent vagus nerve senses pro-inflammatory mediators in the periphery to relay the message to the brain which releases acetylcholine via efferent vagus activity to inhibit inflammation locally through α7 nAChRs activation [9]. This pathway is known as the ‘cholinergic anti-inflammatory pathway’ [9, 10] and has increased interest in α7 nAChR agonists, such as GTS-21, as potential anti-inflammatory drugs.

Chronic inflammation often occurs due to either unresolved injuries or pathogenic infections that cause over-production of multiple pro-inflammatory cytokines such as TNF, IL1, IL6, IL17, HMGB1 and other mediators such as nitric oxide. Monoclonal antibodies are used clinically to prevent inflammation associated with various disease states by binding to specific pro-inflammatory cytokines. An example is etanercept, a TNF antibody used to treat rheumatoid arthritis [38]. However, a major challenge has been to find orally bioavailable small molecules that simultaneously suppress multiple pro-inflammatory cytokines. Wang et al. [7] reported that vagus nerve stimulation activates cholinergic signaling via α7 nAChRs to inhibit chronic inflammation. Since then, many reports (based on in vivo and in vitro experiments) have shown that like vagus nerve stimulation, α7 nAChR agonists inhibit multiple pro-inflammatory cytokines [10, 17]. GTS-21 is an α7 nAChR partial agonist commonly used as an anti-inflammatory agent [24–27]. In this report, we investigated whether α7 nAChRs are required for GTS-21’s proposed anti-inflammatory effects, both in immune-derived cells and in a heterologous expression model in a non-immune cell system.




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