key: cord-1011395-aelha50x authors: Russo, Patrizia; Bonassi, Stefano; Giacconi, Robertina; Malavolta, Marco; Tomino, Carlo; Maggi, Fabrizio title: COVID-19 and smoking: is nicotine the hidden link? date: 2020-06-04 journal: Eur Respir J DOI: 10.1183/13993003.01116-2020 sha: 88f8346a3e1b6e61f81ee97593940073344f951c doc_id: 1011395 cord_uid: aelha50x Nicotine via alpha7-nicotinic receptor induces ACE-2 overexpression in human bronchial epithelial cells (HBEpC) https://bit.ly/3eJ5b35 treatment with nicotine (figure 1c), and is not able to induce phospho-S6 ribosomal protein (Ser235/236), Akt1, phospho-Akt (Ser473), phospho-Akt (Thr308) and phospho-p44/42 MAPK (Thr202/Tyr204) after nicotine treatment (figure 1d), was selected for further experiments. Nicotine did not induce ACE-2 in this clone (si-mRNA-α7-HBEpC) (figure 1a). This observation supports the hypothesis that ACE-2 increase is specifically mediated by α7-nAChR. Moreover, when HBEpC were incubated simultaneously with nicotine and α-BTX, an α7 nicotine antagonist [9] , no induction of ACE-2 was observed (figure 1d). Importantly, treatment with nicotine, α-BTX or with the combination is not cytotoxic (data not shown). On these bases, we suggest that smoking may promote cellular uptake mechanisms of SARS-CoV-2 through α7-nAChR signalling. A possible α7-nAChR down-stream mechanism may be the induction of phospho-Akt and phospho-p44/42 MAPK. This mechanism was hypothesised, partially, by OLDS and KABBANI [10] on their schematic model explaining how nicotine exposure increases the risk of SARS-CoV-2 entry into lung cells. α7-nAChR is present both in neuronal and non-neuronal cells (i.e. lung, endothelial, lymphocyte); consequently, smoking may impact COVID-19 pathophysiology and clinical outcome in several organ systems, including the brain. From the authors: We recently reported that current smokers and those with COPD had higher airway epithelial cell expression of the angiotensin-converting enzyme II (ACE-2) viral entry receptor [1] . We thus read with great interest the work of P. Russo and co-workers, which proposes a mechanism for this finding, namely that this upregulation is mediated by nicotine exposure specifically through the α7 subtype of nicotine acetylcholine receptors (α7-nAChR). While exposure to increasing concentrations of nicotine caused epithelial cells to increase ACE-2 levels, subsequent gene silencing of α7-nAChR appeared to significantly dampen this response. A secondary transcriptome sequencing analysis of our cohort (consisting of 42 subjects who underwent bronchoscopy for epithelial cell brushings [1] ) reveals evidence in support of this hypothesis. We found that airway epithelial cell expression of CHRNA7, encoding α7-nAChR, was significantly correlated with the expression of ACE2 (Pearson r=0.54, p=2.31×10 −8 ) ( figure 1 ). There was significantly higher CHRNA7 expression in those with COPD (2.75±0.73 versus 2.14±0.43 in those without COPD; p=1.47×10 −4 ), with a trend towards higher expression in current smokers compared to former and never smokers (2.86±0.92 in current smokers, 2.35±0.57 in former smokers, and 2.27±0.45 in never smokers; p=6.16×10 −2 ). CHRNA7 was also negatively correlated with forced expiratory volume in 1 s percent predicted (Pearson r=−0.37, p=2.83×10 −4 ). Interestingly, CHRNA7 was positively if weakly correlated with body mass index (Pearson r=0.14, p=6.31×10 −3 ), raising the intriguing possibility that nicotine receptor mediation of ACE-2 may also be related to why obese individuals have made up a considerable proportion of coronavirus disease 2019 (COVID-19) cases [2] . Together, these data further help to characterise the connections between airway epithelial ACE-2, and α7-nAChR, and the unique vulnerability of patients with COPD to severe COVID-19. α7-nAChR's widespread abundance in the human body, from neuronal tissue to immune cells to the lung and digestive tract, and its various roles in diseases such as schizophrenia [3] , Alzheimer's disease [4] and Parkinson's disease [5] has meant that considerable work has already been done to target α7-nAChR as a therapeutic modality. As an example, α7-nAChR antagonists for the purpose of smoking cessation have long been proposed [6] and the idea of potentially repurposing these compounds for a pandemic with few therapeutic options currently available is certainly appealing. Whether α7-nAChR-selective antagonists, such as methyllycaconitine [7] and α-conotoxin [8] , can meaningfully alter ACE-2 expression to prevent severe acute respiratory syndrome coronavirus 2 entry into the airway epithelium seems the next logical investigation in our furious pursuit for better therapeutics. Janice M. Leung ACE-2 expression in the small airway epithelia of smokers and COPD patients: implications for COVID-19 COVID-19 and smoking: a systematic review of the evidence Sex difference and smoking predisposition in patients with COVID-19 Nicotine and the renin-angiotensin system Nicotine: specific role in angiogenesis, proliferation and apoptosis A pneumonia outbreak associated with a new coronavirus of probable bat origin Tobacco smoking: risk to develop addiction, chronic obstructive pulmonary disease, and lung cancer The expression and pharmacological characterization of nicotinic acetylcholine receptor subunits in HBE16 airway epithelial cells Nicotine promotes cell proliferation and induces resistance to cisplatin by α7 nicotinic acetylcholine receptor-mediated activation in Raw264.7 and El4 cells Is nicotine exposure linked to cardiopulmonary vulnerability to COVID-19 in the general population? Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesothelioma cells: role of mitogen-activated protein kinase pathway Data were obtained using PathScan® cell growth Multi-target Sandwich ELISA kit n.7239 (Cell Signaling) PathScan® Cell Growth Multi-Target Sandwich ELISA Kit is a solid phase sandwich ELISA that combines the reagents necessary to detect endogenous levels of S6 ribosomal protein, phospho-S6 ribosomal protein (Ser235/236), Akt1, phospho-Akt (Ser473), phospho-Akt (Thr308) and phospho-p44/42 MAPK (Thr202/Tyr204). Data are mean±SEM; p-value was evaluated using t-test. Experiments were performed twice in duplicate. c) α7-nAChR protein detection. Western blotting was performed as described previously [11]. Human α7-nAchR antibody NBP1-49348 was purchased from Novus Biologicals (www.novusbio.com). 1-2 si-mRNA-α7-HBEpC treated with zero HBEpC treated with zero (lane 3) or 1.0×10 −7 M nicotine (lane 4) for 1 h. Experiments were performed twice. d) as in panel b ACE-2 expression in the small airway epithelia of smokers and COPD patients: implications for COVID-19 Hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019-COVID-NET, 14 States Linkage disequilibrium for schizophrenia at the chromosome 15q13-14 locus of the alpha7-nicotinic acetylcholine receptor subunit gene (CHRNA7) Decreased protein levels of nicotinic receptor subunits in the hippocampus and temporal cortex of patients with Alzheimer's disease Cellular expression of alpha7 nicotinic acetylcholine receptor protein in the temporal cortex in Alzheimer's and Parkinson's disease-a stereological approach Targeting the alpha4beta2-and alpha7-subtypes of nicotinic acetylcholine receptors for smoking cessation medication development Effects of methyllycaconitine (MLA), an alpha 7 nicotinic receptor antagonist, on nicotine-and cocaine-induced potentiation of brain stimulation reward Dimerization of alpha-conotoxins as a strategy to enhance the inhibition of the human alpha7 and alpha9alpha10 nicotinic acetylcholine receptors This version is distributed under the terms of the Creative Commons Attribution Non-Commercial Licence 4 Conflict of interest: J.M. Leung has nothing to disclose. C.X. Yang has nothing to disclose. D.D. Sin reports grants from Merck, personal fees for advisory board work from Sanofi-Aventis and Regeneron, grants and personal fees for lectures from Boehringer Ingelheim and AstraZeneca, personal fees for lectures and advisory board worm from Novartis, outside the submitted work.