Neuroimmune nexus in the pathophysiology and therapy of inflammatory disorders: Role of α7 nicotinic acetylcholine receptors.

The α7-nicotinic acetylcholine receptor (α7nAChR) is a key protein in the cholinergic anti-inflammatory pathway (CAP) that links the nervous and immune systems. Initially, the pathway was discovered based on the observation that vagal nerve stimulation (VNS) reduced the systemic inflammatory response in septic animals. Subsequent studies form a foundation for the leading hypothesis about the central role of the spleen in CAP activation. VNS evokes noradrenergic stimulation of ACh release from T cells in the spleen, which in turn activates α7nAChRs on the surface of macrophages. α7nAChR-mediated signaling in macrophages reduces inflammatory cytokine secretion and modifies apoptosis, proliferation, and macrophage polarization, eventually reducing the systemic inflammatory response. A protective role of the CAP has been demonstrated in preclinical studies for multiple diseases including sepsis, metabolic disease, cardiovascular diseases, arthritis, Crohn's disease, ulcerative colitis, endometriosis, and potentially COVID-19, sparking interest in using bioelectronic and pharmacological approaches to target α7nAChRs for treating inflammatory conditions in patients. Despite a keen interest, many aspects of the cholinergic pathway are still unknown. α7nAChRs are expressed on many other subsets of immune cells that can affect the development of inflammation differently. There are also other sources of ACh that modify immune cell functions. How the interplay of ACh and α7nAChR on different cells and in various tissues contributes to the anti-inflammatory responses requires additional study. This review provides an update on basic and translational studies of the CAP in inflammatory diseases, the relevant pharmacology of α7nAChR-activated drugs and raises some questions that require further investigation.

Dual effects of supplemental oxygen on pulmonary infection, inflammatory lung injury, and neuromodulation in aging and COVID-19.

Clinical studies have shown a significant positive correlation between age and the likelihood of being infected with SARS-CoV-2. This increased susceptibility is positively correlated with chronic inflammation and compromised neurocognitive functions. Postmortem analyses suggest that acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), with systemic and lung hyperinflammation, can cause significant morbidity and mortality in COVID-19 patients. Supraphysiological supplemental oxygen, also known as hyperoxia, is commonly used to treat decreased blood oxygen saturation in COVID-19 patients. However, prolonged exposure to hyperoxia alone can cause oxygen toxicity, due to an excessive increase in the levels of reactive oxygen species (ROS), which can overwhelm the cellular antioxidant capacity. Subsequently, this causes oxidative cellular damage and increased levels of aging biomarkers, such as telomere shortening and inflammaging. The oxidative stress in the lungs and brain can compromise innate immunity, resulting in an increased susceptibility to secondary lung infections, impaired neurocognitive functions, and dysregulated hyperinflammation, which can lead to ALI/ARDS, and even death. Studies indicate that lung inflammation is regulated by the central nervous system, notably, the cholinergic anti-inflammatory pathway (CAIP), which is innervated by the vagus nerve and α7 nicotinic acetylcholine receptors (α7nAChRs) on lung cells, particularly lung macrophages. The activation of α7nAChRs attenuates oxygen toxicity in the lungs and improves clinical outcomes by restoring hyperoxia-compromised innate immunity. Mechanistically, α7nAChR agonist (e.g., GAT 107 and GTS-21) can regulate redox signaling by 1) activating Nrf2, a master regulator of the antioxidant response and a cytoprotective defense system, which can decrease cellular damage caused by ROS and 2) inhibiting the activation of the NF-κB-mediated inflammatory response. Notably, GTS-21 has been shown to be safe and it improves neurocognitive functions in humans. Therefore, targeting the α7nAChR may represent a viable therapeutic approach for attenuating dysregulated hyperinflammation-mediated ARDS and sepsis in COVID-19 patients receiving prolonged oxygen therapy.

Immunization with 674-685 fragment of SARS-Cov-2 spike protein induces neuroinflammation and impairs episodic memory of mice.

COVID-19 is accompanied by strong inflammatory reaction and is often followed by long-term cognitive disorders. The fragment 674-685 of SARS-Cov-2 spike protein was shown to interact with α7 nicotinic acetylcholine receptor involved in regulating both inflammatory reactions and cognitive functions. Here we show that mice immunized with the peptide corresponding to 674-685 fragment of SARS-Cov-2 spike protein conjugated to hemocyanin (KLH-674-685) demonstrate decreased level of α7 nicotinic acetylcholine receptors, increased levels of IL-1ß and TNFα in the brain and impairment of episodic memory. Choline injections prevented α7 nicotinic receptor decline and memory loss. Mice injected with immunoglobulins obtained from the blood of (KLH-674-685)-immunized mice also demonstrated episodic memory decline. These data allow suggesting that post-COVID memory impairment in humans is related to SARS-Cov-2 spike protein-specific immune reaction. The mechanisms of such effect are being discussed.

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.

SARS-Cov-2 spike protein fragment 674-685 protects mitochondria from releasing cytochrome c in response to apoptogenic influence.

In spite of numerous studies, many details of SARS-Cov-2 interaction with human cells are still poorly understood. The 674-685 fragment of SARS-Cov-2 spike protein is homologous to the fragment of α-cobratoxin underlying its interaction with α7 nicotinic acetylcholine receptors (nAChRs). The interaction of 674-685 peptide with α7 nAChR has been predicted in silico. In the present paper we confirm this prediction experimentally and investigate the effect of SARS-Cov-2 spike protein peptide on mitochondria, which express α7 nAChRs to regulate apoptosis-related events. We demonstrate that SARS-Cov-2 spike protein peptide 674-685 competes with the antibody against 179-190 fragment of α7 nAChR subunit for the binding to α7-expressing cells and mitochondria and prevents the release of cytochrome c from isolated mitochondria in response to 0.5 mM H2O2 but does not protect intact U373 cells against apoptogenic effect of H2O2. Our data suggest that the α7 nAChR-binding portion of SARS-Cov-2 spike protein prevents mitochondria-driven apoptosis when the virus is uncoated inside the cell and, therefore, supports the infected cell viability before the virus replication cycle is complete.

E-cigarette vape and lung ACE2 expression: Implications for coronavirus vulnerability.

Evidence in humans suggests a correlation between nicotine smoking and severe respiratory symptoms with COVID-19 infection. In lung tissue, angiotensin-converting enzyme 2 (ACE2) appears to mechanistically underlie viral entry. Here, we investigated whether e-cigarette vapor inhalation alters ACE2 and nicotinic acetylcholine receptor (nAChR) expression in male and female mice. In male lung, nicotine vapor inhalation induced a significant increase in ACE2 mRNA and protein, but surprisingly, these differences were not found in females. Further, both vehicle and nicotine vapor inhalation downregulated α5 nAChR subunits in both sexes, while differences were not found in α7 nAChR subunit expression. Finally, blood ACE2 levels did not differ with exposure, indicating that blood sampling is not a sufficient indicator of lung ACE2 changes. Together, these data indicate a direct link between e-cigarette vaping and increased ACE2 expression in male lung tissue, which thereby reveals an underlying mechanism of increased vulnerability to coronavirus infection in individuals vaping nicotine.

Nicotinic cholinergic system and COVID-19: In silico identification of interactions between α7 nicotinic acetylcholine receptor and the cryptic epitopes of SARS-Co-V and SARS-CoV-2 Spike glycoproteins.

SARS-CoV-2 is the coronavirus that originated in Wuhan in December 2019 and has spread globally. Studies have shown that smokers are less likely to be diagnosed with or be hospitalized for COVID-19 but, once hospitalized, have higher odds for an adverse outcome. We have previously presented the potential interaction between SARS-CoV-2 Spike glycoprotein and nicotinic acetylcholine receptors (nAChRs), due to a "toxin-like" epitope on the Spike glycoprotein, with homology to a sequence of a snake venom toxin. This epitope coincides with the well-described cryptic epitope for the human anti-SARS-CoV antibody CR3022. In this study, we present the molecular complexes of both SARS-CoV and SARS-CoV-2 Spike glycoproteins, at their open or closed conformations, with the model of the human α7 nAChR. We found that all studied protein complexes' interface involves a large part of the "toxin-like" sequences of SARS-CoV and SARS-CoV-2 Spike glycoproteins and toxin binding site of human α7 nAChR. Our findings provide further support to the hypothesis about the protective role of nicotine and other cholinergic agonists. The potential therapeutic role of CR3022 and other similar monoclonal antibodies with increased affinity for SARS-CoV-2 Spike glycoprotein against the clinical effects originating from the dysregulated cholinergic pathway should be further explored.

Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity.

(1) Background: Nicotine is implicated in the SARS-COV-2 infection through activation of the α7-nAChR and over-expression of ACE2. Our objective was to clarify the role of nicotine in SARS-CoV-2 infection exploring its molecular and cellular activity. (2) Methods: HBEpC or si-mRNA-α7-HBEpC were treated for 1 h, 48 h or continuously with 10-7 M nicotine, a concentration mimicking human exposure to a cigarette. Cell viability and proliferation were evaluated by trypan blue dye exclusion and cell counting, migration by cell migration assay, senescence by SA-ß-Gal activity, and anchorage-independent growth by cloning in soft agar. Expression of Ki67, p53/phospho-p53, VEGF, EGFR/pEGFR, phospho-p38, intracellular Ca2+, ATP and EMT were evaluated by ELISA and/or Western blotting. (3) Results: nicotine induced through α7-nAChR (i) increase in cell viability, (ii) cell proliferation, (iii) Ki67 over-expression, (iv) phospho-p38 up-regulation, (v) EGFR/pEGFR over-expression, (vi) increase in basal Ca2+ concentration, (vii) reduction of ATP production, (viii) decreased level of p53/phospho-p53, (ix) delayed senescence, (x) VEGF increase, (xi) EMT and consequent (xii) enhanced migration, and (xiii) ability to grow independently of the substrate. (4) Conclusions: Based on our results and on evidence showing that nicotine potentiates viral infection, it is likely that nicotine is involved in SARS-CoV-2 infection and severity.

Nicotinic acetylcholine receptors regulate clustering, fusion and acidification of the rat brain synaptic vesicles.

The brain nicotinic acetylcholine receptors (nAChRs) expressed in pre-synaptic nerve terminals regulate neurotransmitter release. However, there is no evidence for the expression of nAChRs in synaptic vesicles, which deliver neurotransmitter to synaptic cleft. The aim of this paper was to investigate the presence of nAChRs in synaptic vesicles purified from the rat brain and to study their possible involvement in vesicles life cycle. According to dynamic light scattering analysis, the antibody against extracellular domain (1-208) of α7 nAChR subunit inhibited synaptic vesicles clustering. Sandwich ELISA with nAChR subunit-specific antibodies demonstrated the presence of α4ß2, α7 and α7ß2nAChR subtypes in synaptic vesicles and showed that α7 and ß2 nAChR subunits are co-localized with synaptic vesicle glycoprotein 2A (SV2A). Pre-incubation with either α7-selective agonist PNU282987 or nicotine did not affect synaptic vesicles clustering but delayed their Ca2+-dependent fusion with the plasma membranes. In contrast, nicotine but not PNU282987 stimulated acidification of isolated synaptic vesicles, indicating that α4ß2 but not α7-containing nAChRs are involved in regulation of proton influx and neurotransmitter refilling. Treatment of rats with levetiracetam, a specific modulator of SV2A, increased the content of α7 nAChRs in synaptic vesicles accompanied by increased clustering but decreased Ca2+-dependent fusion. These data for the first time demonstrate the presence of nAChRs in synaptic vesicles and suggest an active involvement of cholinergic regulation in neurotransmitter release. Synaptic vesicles may be an additional target of nicotine inhaled upon smoking and of α7-specific drugs widely discussed as anti-inflammatory and pro-cognitive tools.