Research Project

Control of the ATP-mediated release of the pro-inflammatory cytokines by mononuclear phagocytes by unconventional nicotinic receptors

Monocytes, macrophages, and epithelial cells, initiate the innate immune response, the first line of defense against infections and cell damage. The pro-inflammatory cytokine interleukin (IL)-1β released by innate immune cells plays an essential role in this host defense against infections. The danger signal extracellular ATP originating from dead or injured cells is sensed by the ionotropic P2X7 receptor (P2RX7) resulting in NLRP3 inflammasome activation and, thus, the of IL-1β. High levels of IL-1β seem to be involved in tissue damage and can contribute to life-threatening inflammatory diseases. Therefore, mechanisms controlling IL 1β release are of substantial clinical interest but so far have remained largely unexplored.

We identified a novel cholinergic mechanism that inhibits the ATP-mediated release of IL-1β from mononuclear phagocytes via activation of nicotinic acetylcholine receptors (nAChRs) containing the evolutionary highly conserved subunits α7 (CHRNA7), α9 (CHRNA9) and/or α10 (CHRNA10). Activation of nAChRs by classical agonists like ACh, nicotine and choline results in an inhibition of the ionotropic function of the ATP-sensitive P2X7R and thus, the release of IL-1β. This mechanism specifically targets the P2X7R-mediated IL-1β release, while sparing other pathways of inflammasome activation induced by pathogens. Moreover, we found that metabolites of phosphatidylcholine (lecithin), an important bio membrane phospholipid of eukaryotic cells, function as novel endogenous agonists of these nAChRs. At the same time, a completely new biological function of the acute-phase reactants C-reactive protein (CRP), α1-antitrypsin (AAT), and secretory leukocyte protease inhibitor (SLPI), was identified: We demonstrated that CRP, AAT and SLPI control the ATP-mediated release of IL-1β release by mononuclear phagocytes by  directly (CRP) or indirectly (AAT and SLPI) activating the unconventional nAChRs. CRP, AAT, and SLPI seem to be central elements of systemic negative feedback loops that against hyperinflammation. Later, we provided evidence that amyloid-β peptide (Aβ1-42), mainly known from research on Alzheimer´s disease, is an opponent of the cholinergic control mechanism, allowing  ATP-induced IL-1β release in the presence of cholinergic agonists.

From the traditional view, nAChR are known as ligand-gated ion channels from the neuronal system that mediate electrical transmission at neurons. By doing electrophysiological patch clamp measurements I showed that stimulation of monocytic nAChRs does not cause ion-currents, but results in a clear-cut inhibition of the ion-channel function of the P2RX7 [3,4, 6]. Metabotropic functions of nAChRs are a new emerging field of nAChR research that attracts tremendous interest in the scientific community. We provided evidence that the cholinergic control of the ATP-induced IL-1β release mechanism involves activation of endothelial NO synthase and P2RX7 modification to inhibit its ionotropic function.

Compounds that function as potent agonists eliciting metabotropic functions in innate immune cells but no ionotropic functions at nAChR are currently understated as nAChR silent agonists. Nevertheless, they are promising therapeutics for the prevention and treatment of excessive inflammation involving IL-1β without entailing the risk of adverse effects in the nervous system. We compared the endogenous agonists phosphocholine with synthetic nAChR silent agonists and α9-nAChR specific ligands. In addition to the efficient anti-inflammatory effects, we identified that α9-nAChR specific ligands also have potent analgesic effects, and thus, have great therapeutic potential against hyperinflammation and inflammatory pain.

Research Methods

• Cytokine release measurements (ELISA), flow cytometry
• Immunocytochemistry and microscopy
• Cell culture techniques, isolation of primary immune cells, spheroids and organoids
• Molecular biology, protein biochemistry
• Electrophysiological characterization of ion channels and receptors (Two-Electrode Voltage-Clamp, Patch-Clamp, Calcium-imaging)
• Heterologous expression of ion channels and receptors in Xenopus oocytes and HEK293-cells