Immune system dysregulation during COVID-19 illness remains an area of intense investigation. Excessive release of proinflammatory cytokines and multi-organ injury suggest aberrant control of the immune system. Given the established role of nicotinic acetylcholine receptors (nAChRs) in modulating inflammatory responses, we explored the possibility that the SARS-CoV-2 spike-protein may directly perturb this regulatory pathway. The spike protein contains a furin cleavage-site with sequence similarities to α-neurotoxins known to antagonize α7, α9, and α10 nAChR subtypes. To test whether this region of the spike protein can influence nAChR function, we synthesized short peptides corresponding to putative nAChR-interacting domains from several SARS-CoV-2 variants. Electrophysiological recordings from Xenopus laevis oocytes expressing human nAChRs revealed potent, virus-variant dependent inhibition of nAChR activity. In human monocytic THP-1 cells, these peptides reversed acetylcholine-mediated suppression of interleukin-1β release. Our findings identify a potential molecular mechanism by which SARS-CoV-2 may directly suppress nAChR signaling on immune cells thereby amplifying inflammatory cytokine release. This work supports the broader concept that structural motifs present in the SARS-CoV-2 spike-protein can disrupt cholinergic anti-inflammatory pathways and elucidates a mechanism that may contribute to the pathophysiology and immune system dysregulation that can occur in severe COVID-19.

IntroductionNicotinic acetylcholine receptors (nAChRs) on immune cells are promising therapeutic targets for the treatment of inflammatory diseases and pain. Both α7 and α9* nAChRs (*denotes the potential presence of other nAChR subunits) have been implicated as mediators of the cholinergic anti-inflammatory system (CAS). This study investigated the binding sites of α7-selective ligands on these receptors and their effects on ATP-dependent release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 by human mononuclear phagocytes.Materials and MethodsThe effects of classical ligands (e.g. ACh, nicotine), unconventional (phosphocholine), putative α7-specific ligands (S24795, PNU-282987 and methyllycaconitine), on the ATP-induced IL-1β release were studied in lipopolysaccharide-primed human monocytic THP-1 cells and THP-1-derived macrophages. Electrophysiological two-electrode voltage-clamp measurements were conducted on Xenopus laevis oocytes expressing human α7, α9 or α9α10 nAChRs. Molecular docking was performed using the crystal structure of the homomeric human α7 receptor (PDB ID: 7EKI) and a modeled pentameric assembly of the homomeric α9 extracellular domain (PDB ID: 6HY7). In addition, the homomeric α10 extracellular domain was generated by homology modeling using 6HY7 as the template.ResultsIn cytokine-release experiments, the nAChR agonists efficiently inhibited ATP-mediated IL-1β release. This inhibitory effect was reversed by specific antagonistic conopeptides [V11L;V16D]ArIB (α7 antagonist) and RgIA4 (α9 and α9α10 antagonist), indicating the involvement of nAChRs containing subunits α7, α9 and/or α10. Electrophysiological measurements suggested an interaction of putative α7-specific ligands with human α9* nAChRs. In molecular docking simulations, all tested ligands showed reasonable binding affinity to homomeric α7, α9 and α10 nAChR models near the C-loop region of the binding pocket.ConclusionOur findings provide a more nuanced framework for interpreting the roles of nAChR subtypes in non-neuronal immune modulation, highlighting the complexity and potential importance of α9* nAChRs in the context of inflammation and innate immunity. The results underscore the importance of considering the nAChR subunit α9 when developing α7-selective ligands for immunomodulation and provides novel insights into the role of α9* nAChRs as potential therapeutic targets for inflammatory diseases and pain.

Providing transport routes for oxygen and nutrient supply in macroscopic tissue remains a major challenge in tissue engineering. Among fabrication methods for perfusable microtubular structures, two-photon stereolithography (TPS) stands out due to its superior resolution, achieving features at the capillary scale. This study uses TPS to fabricate hollow tubes and investigates their feasibility as microtubular supply structures in in vitro tissue models. With optimized parameters, porous hollow tubes of different sizes (8 μm to 2 mm in diameter) with pore diameters of 1 to 50 μm were printed with the two photoresins IP-S and IP-Visio. IP-S demonstrated superior printability and higher shape accuracy compared to IP-Visio. IP-Visio prints were compatible with fluorescence microscopy, whereas IP-S exhibited high autofluorescence, which interfered with fluorescence imaging. The prints displayed partial wettability with similar contact angles between 71° and 76° on both flat and porous curved surfaces, with none of the liquids passing through the pores. Printed hollow tubes with pores were integrated into a perfusion system, and the permeability of molecules of 4, 70 and 150 kDa into the surrounding hydrogel matrix was assessed under physiological flow conditions. Structures printed with both photoresins facilitated the attachment and survival of primary human endothelial cells on top of flat prints (viability ≥97 %) as well as inside printed tubes. Finally, flat prints were tested in cytokine release experiments and provoked neither pro- nor anti-inflammatory responses of human macrophages and monocytes.

ObjectiveThe clinical interest in mechanisms controlling the biosynthesis and release of the pro-inflammatory cytokine interleukin (IL)-1β is outstanding, as IL-1β is associated with life-threatening inflammatory diseases including hyperinflammation caused by extracellular ATP originating from damaged cells. Previously, we identified a cholinergic mechanism controlling ATP-dependent IL-1β release via metabotropic signaling of unconventional nicotinic acetylcholine receptors (nAChRs) containing subunits α7 and α9* (denoting homomeric or heteromeric α9) in monocytes. This study examines whether this mechanism is active in human macrophages (THP-1 cell-derived, peripheral blood mononuclear cell-derived, and peritoneal macrophages).MethodsExpression of nAChR subtypes (CHRNA7, CHRFAM7A, CHRNA9, CHRNA10) was analyzed using real-time RT-PCR. The efficiency of the differentiation protocols used was assessed by surface markers and metabolic conversion rate analysis. Cholinergic control of ATP-induced IL-1β, IL-18, and IL-1α release was tested using nAChR agonists and conopeptides antagonizing α7 and α9* nAChRs.ResultsAll nAChR subunits were expressed by all cells analyzed. Activation of nAChRs efficiently inhibited the ATP-mediated IL-1β release by macrophages, while ATP-independent release remained unaffected. Moreover, the nAChR agonists inhibited the release of IL-18 and IL-1α. The inhibitory effect was reversed by subunit-specific conopeptides, indicating the involvement of unconventional nAChRs containing subunits α7 and α9*.ConclusionWe conclude that the cholinergic control of ATP-mediated IL-1β release is active in human monocytes and in macrophages and that nAChR agonists can also regulate the release of IL-18 and IL-1α. This mechanism specifically regulates the ATP-induced cytokine release, without suppressing ATP-independent cytokine release. Thus, unconventional α9* nAChRs are promising therapeutic targets for ATP-induced inflammatory diseases, including sterile hyperinflammation.

2-(Cyclohexyldimethylammoniumethyl)ether of 4-stilbenol (2), and its styryl-modified analogues 21 and 22, were identified as lead compounds from a series targeting human α9α10, α9, and α7 nicotinic acetylcholine receptors (nAChRs). Compounds 2 and 21 exhibited potent, and subtype-selective modulation of α9-containing receptors, with low nanomolar IC50 values and dual agonist/antagonist activity in a concentration-dependent manner. In contrast, compound 22 acted as a selective, pure antagonist. Molecular dynamics (MD) simulations of compound 21 supported a concentration-dependent allosteric mechanism, with orthosteric binding at low concentrations and vestibular site interaction at higher levels. In a human monocytic cell line, all three compounds inhibited ATP-induced IL-1β release at nanomolar concentrations. These findings identify α9α10-selective ligands as promising scaffolds for the development of nonopioid analgesics and immunomodulators, with favorable selectivity over α7 nAChRs to minimize CNS-related side effects.

Introduction:Different innate immune cell types are known to release extracellular traps (ETs) in response to invasive pathogens, including parasites. These ETs function to trap, immobilize, and eventually kill pathogens. In line with this, monocytes and macrophages have been shown to release ETs, known as monocyte/macrophage extracellular traps (METs). Toxoplasma gondii (T. gondii) is an apicomplexan zoonotic parasite that infects humans and homeothermic animals. While most studies have focused on prolonged exposure of immune cells to T. gondii, this study characterized the early innate immune reaction of mononuclear phagocytes to vital T. gondii tachyzoites. Methods:Primary human and bovine monocytes, monocytic THP-1 cells, and THP-1 cell-derived macrophages (M0-, M1-, and M2-like) were exposed to T. gondii tachyzoites for 4 h. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), immunofluorescencemicroscopy, and confocal microscopy were used to visualize cell activation and the presence of METs. Additionally, the release of pro-inflammatory cytokines interleukin (IL)-1β and IL-6, and expression of Toll-like receptor (TLR) 2 and TLR4 were analyzed. Results and discussion:Microscopic analysis illustrated the activation of all cell types tested within 4 h of exposure to T. gondii tachyzoites. Numerous tachyzoites were found intracellularly in THP-1 cell-derived M1-like macrophages. Furthermore, the co-localization of extracellular DNA (extDNA) and histones in extracellular web-like fibers proved classical characteristics of extruded T. gondii-induced METs, although this was a rare event. In primary human monocytes, an increased release of IL-1β and IL-6 was observed following exposure to T. gondii tachyzoites. When co-stimulated with lipopolysaccharide (LPS), primary human monocytes showed an enhanced release of IL-1β and IL-6 in response to T. gondii. In contrast to monocytic THP-1 cells, THP-1 cell-derived M1-like macrophages released IL-1β in response to T. gondii tachyzoite exposure. When additionally stimulated by LPS, all THP-1 cell-derived macrophages showed an enhanced release of IL-1β, and monocytic THP-1 cells an increased release of IL-6 in response to T. gondii tachyzoites. This study provides insights into the early innate immune response of human and bovine mononuclear phagocytes to T. gondii. While cytokine secretion was prominent, MET formation was rare in the early response (i.e. < 4 h of exposure) to T. gondii tachyzoites.

Retro-muscular mesh augmentation is standard for repairing abdominal incisional or larger primary hernia. A wide variety of meshes with diverse properties are available. The knowledge on the immune-modulating effects of meshes is, however, insufficient. This study investigates the impact of two widely used lightweight meshes, ULTRAPRO® and ProGrip™, on macrophage activation (in vitro), systemic inflammation (in vivo), patient perioperative and long-term outcomes. Human THP-1 cell-derived macrophages were cultured in absence and presence of ULTRAPRO® or ProGrip™ meshes. The release of pro-inflammatory cytokines, interleukin (IL)-1β and IL-6, was measured following inflammasome activation. In a retrospective study, systemic inflammation and postoperative outcomes after retro-muscular hernia repair using ULTRAPRO® (321 patients) or ProGrip™ (161 patients) meshes were analyzed. In the presence of ULTRAPRO®, IL-1β and IL-6 release by macrophages was increased, whereas ProGrip™ tended to reduce cytokine levels (p ≤ 0.05; n = 7). Baseline characteristics were comparable between both groups; systemic C-reactive protein levels were likewise higher in patients receiving ULTRAPRO® compared to ProGrip™ (mean difference: 26.9 ± 7.5 mg/dl; p < 0.0001). No relevant differences were observed in perioperative morbidity or short-term outcomes, including complications and hospitalization after hernia repair, but hernia recurrence rates tended to be higher within three-year follow-up after ProGrip™ implantation compared to ULTRAPRO® (p = 0.0630). Meshes exhibit distinct immune-modulating effects on macrophages, leading to differential activation that may influence foreign-body reaction and systemic inflammation. These immune responses potentially impact clinical outcomes and recurrence after hernia repair. This study underscores the need for comparative prospective, randomized-controlled trials to further evaluate the clinical relevance of mesh-specific immunological effects.

We have characterized families of phenylpiperazine (PP) compounds, studying their relative activity with α7 and α9* nicotinic acetylcholine receptors (nAChRs) and focusing on the effects of side groups on the phenyl ring (R1) and the effects of different alkyl groups on the base nitrogen. In this study, we evaluated the impact of methyl substitution on the piperazine ring, which introduced a chiral center, enabling the generation and separation of stereoisomers. Methyl groups were added to either the C2 or C3 positions on the piperazine of the α9α10 agonist/α7 partial agonist PA-EMPP. Additions at the C3 position greatly reduced activity, while additions at the C2 position had selective effects on either α7 or α9/α10 activity. The 2-methyl S and R isomers of PA-EMPP contain a second chiral center at the nitrogen. Notably, replacing the terminal substitution with N,N-dimethyl abolished α9/α910 agonist activity, rendering the compound selective for α7. We also tested 2M isomers of the α9α10 agonist pCN-EMPP and obtained similar enantioselective activity as observed with the PA-EMPP isomers. Compounds were studied for their ability to reduce the ATP-dependent release of IL-1β from monocytes, one aspect of the cholinergic anti-inflammatory activity. Results were consistent with their apparent activation or antagonism of α9* receptors. These findings underscore the critical role of chirality and structural modifications in fine-tuning receptor selectivity, offering valuable insights for the rational design of selective nicotinic therapeutics.

Several lines of evidence have indicated that nicotinic acetylcholine receptors (nAChR) that contain α9 subunits, probably in combination with α10 subunits, may be valuable targets for the management of pain associated with inflammatory diseases through a cholinergic anti-inflammatory system (CAS), which has also been associated with α7 nAChR. Both α7- and α9-containing neuronal nAChR can be pharmacologically distinguished from the high-affinity nicotinic receptors of the brain by their sensitivity to α-bungarotoxin, but in other ways, they have quite distinct pharmacological profiles. The early association of α7 with CAS led to the development of numerous new ligands, variously characterized as α7 agonists, partial agonists, or silent agonists that desensitized α7 receptors without activation. Subsequent reinvestigation of one such family of α7 ligands based on an N,N-diethyl-N′-phenylpiperazine scaffold led to the identification of potent agonists and antagonists for α9. In this paper, we characterize the α9/α10 activity of a series of compounds based on a 5-(quinuclidin-3-ylmethyl)-1,2,4-oxadiazole (QMO) scaffold and identify two new potent ligands of α9, QMO-28, an agonist, and QMO-17, an antagonist. We separated the stereoisomers of these compounds to identify the most potent agonist and discovered that only the 3R isomer of QMO-17 was an α9 antagonist, permitting an in silico model of α9 antagonism to be developed. The α9 activity of these compounds was confirmed to be potentially useful for CAS management of inflammatory pain in cell-based assays of cytokine release.

There is an urgent need for nonopioid treatments for chronic and neuropathic pain to provide effective alternatives amid the escalating opioid crisis. This study introduces novel compounds targeting the α9 nicotinic acetylcholine receptor (nAChR) subunit, which is crucial for pain regulation, inflammation, and inner ear functions. Specifically, it identifies novel substituted carbamoyl/amido/heteroaryl dialkylpiperazinium iodides as potent agonists selective for human α9 and α9α10 over α7 nAChRs, particularly compounds 3f, 3h, and 3j. Compound 3h (GAT2711) demonstrated a 230 nM potency as a full agonist at α9 nAChRs, being 340-fold selective over α7. Compound 3c was 10-fold selective for α9α10 over α9 nAChR. Compounds 2, 3f, and 3h inhibited ATP-induced interleukin-1β release in THP-1 cells. The analgesic activity of 3h was fully retained in α7 knockout mice, suggesting that analgesic effects were potentially mediated through α9* nAChRs. Our findings provide a blueprint for developing α9*-specific therapeutics for pain.

Host-derived succinate accumulates in the airways during bacterial infection. Here, we show that luminal succinate activates murine tracheal brush (tuft) cells through a signaling cascade involving the succinate receptor 1 (SUCNR1), phospholipase Cβ2, and the cation channel transient receptor potential channel subfamily M member 5 (TRPM5). Stimulated brush cells then trigger a long-range Ca 2+ wave spreading radially over the tracheal epithelium through a sequential signaling process. First, brush cells release acetylcholine, which excites nearby cells via muscarinic acetylcholine receptors. From there, the Ca 2+ wave propagates through gap junction signaling, reaching also distant ciliated and secretory cells. These effector cells translate activation into enhanced ciliary activity and Cl − secretion, which are synergistic in boosting mucociliary clearance, the major innate defense mechanism of the airways. Our data establish tracheal brush cells as a central hub in triggering a global epithelial defense program in response to a danger-associated metabolite.

Nicotinic acetylcholine receptors are not only expressed by the nervous system and at the neuro-muscular junction but also by mononuclear phagocytes, which belong to the innate immune system. Mononuclear phagocyte is an umbrella term for monocytes, macrophages, and dendritic cells. These cells play pivotal roles in host defense against infection but also in numerous often debilitating diseases that are characterized by exuberant inflammation. Nicotinic acetylcholine receptors of the neuronal type dominate in these cells, and their stimulation is mainly associated with anti-inflammatory effects. Although the cholinergic modulation of mononuclear phagocytes is of eminent clinical relevance for the prevention and treatment of inflammatory diseases and neuropathic pain, we are only beginning to understand the underlying mechanisms on the molecular level. The purpose of this review is to report and critically discuss the current knowledge on signal transduction mechanisms elicited by nicotinic acetylcholine receptors in mononuclear phagocytes.

Pain due to inflammation can be reduced by targeting the noncanonical nicotinic receptors (NCNR) in cells of the immune system that regulate the synthesis and release of pro- and anti-inflammatory cytokines. Although NCNR do not generate ion channel currents, the pharmacology of ion-channel forms of the receptors can predict drugs which may be effective regulators of the cholinergic anti-inflammatory system (CAS). Agonists of α7 type receptors have been definitively associated with CAS. Receptors containing α9 and α10 subunits have also been implicated. We have recently characterized two small molecules, pCN-diEPP and mCN-diEPP, as selective α9α10 agonists and antagonists, respectively. We used these drugs, along with nicotine, an α7 agonist and α9α10 antagonist, to probe the mixed populations of receptors that are formed when α7, α9, and α10 are all expressed together in Xenopus oocytes. We also evaluated the effects of the CN-diEPP compounds on regulating the ATP-induced release of interleukin-1β from monocytic THP-1 cells, which express NCNR. The compounds successfully identified separate populations of receptors when all three subunits were co-expressed, including a potential population of homomeric α10 receptors. The α9α10 agonist pCN-diEPP was the more effective regulator of interleukin-1β release in THP-1 cells. pCN-diEPP was also fully effective in a mouse model of inflammatory pain, while mCN-diEPP had only partial effects, requiring a higher dosage. The analgetic effects of pCN-diEPP and mCN-diEPP were retained in α7 knockout mice. Taken together, our results suggest that drugs that selectively activate α9α10 receptors may useful to reduce inflammatory pain through the CAS.

Mucociliary clearance is a pivotal physiological mechanism that protects the lung by cleaning the airways from pollution and colonization, thereby preventing infection. Ciliary function is influenced by various signal transduction cascades, and Ca2+ represents a key second messenger. A fixed 20:1 combination of cafedrine and theodrenaline has been widely used to treat perioperative hypotension and emergency hypotensive states since the 1960s; however, its effect on the intracellular Ca2+ concentration ([Ca2+]i) of respiratory epithelium remains unknown. Therefore, human tracheal epithelial cells were exposed to the clinically applied 20:1 mixture of cafedrine/theodrenaline and the individual substances separately. [Ca2+]i was assessed by FURA-2 340/380 fluorescence ratio. Pharmacological inhibitors were applied to elucidate relevant signal transduction cascades, and reverse transcription polymerase chain reaction (RT-PCR) was performed on murine tracheal epithelium to analyze ryanodine receptor (RyR) subtype expression. All three pharmacological preparations instantaneously induced a steep increase in [Ca2+]i that quickly returned to its baseline value despite the persistence of each substance. Peak [Ca2+]i following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone increased in a dose-dependent manner, with median effective concentrations of 0.35 mM (7.32 mM cafedrine and 0.35 mM theodrenaline), 3.14 mM, and 3.45 mM, respectively. When extracellular Ca2+ influx was inhibited using a Ca2+-free buffer solution, the peak [Ca2+]i following the administration of cafedrine alone and theodrenaline alone were reduced but not abolished. No alteration in [Ca2+]i compared with baseline [Ca2+]i was observed during β-adrenergic receptor inhibition. Depletion of caffeine-sensitive stores and inhibition of RyR, but not IP3 receptors, completely abolished any increase in [Ca2+]i. However, [Ca2+]i still increased following the depletion of mitochondrial Ca2+ stores using 2,4-dinitrophenol. RT-PCR revealed RyR-2 and RyR-3 expression on murine tracheal epithelium. Although our experiments showed that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone release Ca2+ from intracellular stores through mechanisms that are exclusively triggered by β-adrenergic receptor stimulation, which most probably lead to RyR activation, clinical plasma concentrations are considerably lower than those used in our experiments to elicit an increase in [Ca2+]i; therefore, further studies are needed to evaluate the ability of cafedrine/theodrenaline to alter mucociliary clearance in clinical practice.

Mucociliary clearance is a pivotal physiological mechanism that protects the lung by cleaning the airways from pollution and colonization, thereby preventing infection. Ciliary function is influenced by various signal transduction cascades, and Ca 2+ represents a key second messenger. A fixed 20:1 combination of cafedrine and theodrenaline has been widely used to treat perioperative hypotension and emergency hypotensive states since the 1960s; however, its effect on the intracellular Ca 2+ concentration ([Ca 2+ ] i ) of respiratory epithelium remains unknown. Therefore, human tracheal epithelial cells were exposed to the clinically applied 20:1 mixture of cafedrine/theodrenaline and the individual substances separately. [Ca 2+ ] i was assessed by FURA-2 340/380 fluorescence ratio. Pharmacological inhibitors were applied to elucidate relevant signal transduction cascades, and reverse transcription polymerase chain reaction (RT-PCR) was performed on murine tracheal epithelium to analyze ryanodine receptor (RyR) subtype expression. All three pharmacological preparations instantaneously induced a steep increase in [Ca 2+ ] i that quickly returned to its baseline value despite the persistence of each substance. Peak [Ca 2+ ] i following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone increased in a dose-dependent manner, with median effective concentrations of 0.35 mM (7.32 mM cafedrine and 0.35 mM theodrenaline), 3.14 mM, and 3.45 mM, respectively. When extracellular Ca 2+ influx was inhibited using a Ca 2+ -free buffer solution, the peak [Ca 2+ ] i following the administration of cafedrine alone and theodrenaline alone were reduced but not abolished. No alteration in [Ca 2+ ] i compared with baseline [Ca 2+ ] i was observed during β-adrenergic receptor inhibition. Depletion of caffeine-sensitive stores and inhibition of RyR, but not IP 3 receptors, completely abolished any increase in [Ca 2+ ] i . However, [Ca 2+ ] i still increased following the depletion of mitochondrial Ca 2+ stores using 2,4-dinitrophenol. RT-PCR revealed RyR-2 and RyR-3 expression on murine tracheal epithelium. Although our experiments showed that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone release Ca 2+ from intracellular stores through mechanisms that are exclusively triggered by β-adrenergic receptor stimulation, which most probably lead to RyR activation, clinical plasma concentrations are considerably lower than those used in our experiments to elicit an increase in [Ca 2+ ] i ; therefore, further studies are needed to evaluate the ability of cafedrine/theodrenaline to alter mucociliary clearance in clinical practice.

Objective:The pro-inflammatory cytokine interleukin-1β (IL-1β) plays a central role in host defense against infections. High systemic IL-1β levels, however, promote the pathogenesis of inflammatory disorders. Therefore, mechanisms controlling IL-1β release are of substantial clinical interest. Recently, we identified a cholinergic mechanism inhibiting the ATP-mediated IL-1β release by human monocytes via nicotinic acetylcholine receptor (nAChR) subunits α7, α9 and/or α10. We also discovered novel nAChR agonists that trigger this inhibitory function in monocytic cells without eliciting ionotropic functions at conventional nAChRs. Here, we investigate the ion flux-independent signaling pathway that links nAChR activation to the inhibition of the ATP-sensitive P2X7 receptor (P2X7R).Methods:Different human and murine mononuclear phagocytes were primed with lipopolysaccharide and stimulated with the P2X7R agonist BzATP in the presence or absence of nAChR agonists, endothelial NO synthase (eNOS) inhibitors, and NO donors. IL-1β was measured in cell culture supernatants. Patch-clamp and intracellular Ca2+ imaging experiments were performed on HEK cells overexpressing human P2X7R or P2X7R with point mutations at cysteine residues in the cytoplasmic C-terminal domain.Results:The inhibitory effect of nAChR agonists on the BzATP-induced IL-1β release was reversed in the presence of eNOS inhibitors (L-NIO, L-NAME) as well as in U937 cells after silencing of eNOS expression. In peripheral blood mononuclear leukocytes from eNOS gene-deficient mice, the inhibitory effect of nAChR agonists was absent, suggesting that nAChRs signal via eNOS to inhibit the BzATP-induced IL-1β release. Moreover, NO donors (SNAP, S-nitroso-N-acetyl-DL-penicillamine; SIN-1) inhibited the BzATP-induced IL-1β release by mononuclear phagocytes. The BzATP-induced ionotropic activity of the P2X7R was abolished in the presence of SIN-1 in both, Xenopus laevis oocytes and HEK cells over-expressing the human P2X7R. This inhibitory effect of SIN-1 was absent in HEK cells expressing P2X7R, in which C377 was mutated to alanine, indicating the importance of C377 for the regulation of the P2X7R function by protein modification.Conclusion:We provide first evidence that ion flux-independent, metabotropic signaling of monocytic nAChRs involves eNOS activation and P2X7R modification, resulting in an inhibition of ATP signaling and ATP-mediated IL-1β release. This signaling pathway might be an interesting target for the treatment of inflammatory disorders.

BACKGROUND AND PURPOSE:ATP plays an important role as an extracellular messenger acting via different types of purinoceptors. Whereas most of the actions of ATP at intestinal epithelia are thought to be mediated by metabotropic P2Y receptors, the role of ionotropic P2X receptors remains unclear. Consequently, we investigated the role of P2X4 and P2X7 receptors on ion transport across rat colonic epithelia by using BzATP, a potent agonist at P2X7 (and weak agonist at P2X4).EXPERIMENTAL APPROACH:Ussing chamber and Ca2+ imaging experiments were performed on rat colonic epithelia, combined with P2X receptor expression studies.KEY RESULTS:Ussing chamber experiments revealed that serosal BzATP induced a neuronally mediated increase in short-circuit current caused by Cl- secretion. In contrast, the effect of mucosal BzATP was smaller, insensitive to tetrodotoxin and Cl- -independent. When epithelia were basolaterally depolarized to measure currents across the apical membrane, BzATP stimulated a cation current consistent with the activation of apical nonselective cation channels. Experiments with isolated colonic crypts revealed a BzATP-induced increase in the cytosolic Ca2+ concentration. Sensitivity to antagonists indicates stimulation of P2X4 and P2X7 receptors by serosal BzATP and of P2X7 receptors by mucosal BzATP. A similar pattern was observed with native ATP, which induced larger transepithelial currents in comparison to BzATP. RT-PCR and immunohistochemistry experiments confirmed the expression of P2X4 and P2X7 receptors in the colon localized in the epithelium and in submucosal ganglia.CONCLUSIONS AND IMPLICATIONS:Epithelial and neuronal ionotropic P2X receptors are involved in the regulation of intestinal ion transport.

The expression of the acute-phase reactants C-reactive protein (CRP), α1-antitrypsin (AAT), and secretory leukocyte protease inhibitor (SLPI), is induced in response to inflammation by pro-inflammatory mediators, including interleukin-1β. It is conceivable that acute-phase proteins exert protective functions, when the integrity of an organism is challenged by pathogens or trauma, which result in uncontrolled release of endogenous damage-associated molecular patterns like Toll-like receptor agonists and ATP. Acute-phase proteins can enhance or down-modulate immunity against infections or protect the host against damage caused by over-shooting effector functions of the immune system. CRP is mainly regarded as a pro-inflammatory opsonizing agent that binds to bacteria and damaged host cells thereby contributing to their inactivation and elimination. AAT and SLPI are well known for their anti-protease activity, which protects the lung extracellular matrix against degradation by proteases that are released by activated neutrophil granulocytes. In addition, there is growing evidence, that CRP, AAT, and SLPI can control the biosynthesis, maturation, and secretion of pro-inflammatory cytokines. The purpose of this narrative mini review is to summarize these anti-inflammatory functions with a focus on the negative control of the ATP-induced, inflammasome-dependent secretion of interleukin-1β by monocytes. CRP-, AAT- and SLPI-mediated control of interleukin-1β release involves the activation of unconventional nicotinic acetylcholine receptors that inhibits the ionotropic function of the ATP receptor P2X7. Apart from other functions, CRP, AAT, and SLPI seem to be central elements of systemic negative feedback loops that protect the host against systemic hyperinflammation, barrier dysfunction, and death by multiple organ damage.