International Journal of

Molecular Sciences

Article
Nicotinic Receptor Subunits Atlas in the Adult
Human Lung
Zania Diabasana 1 , Jeanne-Marie Perotin 1,2 , Randa Belgacemi 1 , Julien Ancel 1,2 ,
Pauline Mulette 1,2 , Gonzague Delepine 1,3 , Philippe Gosset 4 , Uwe Maskos 5 ,
Myriam Polette 1,6 , Gaëtan Deslée 1,2 and Valérian Dormoy 1, *
1 Inserm UMR-S1250, P3Cell, University of Reims Champagne-Ardenne, SFR CAP-SANTE, 51092 Reims,
France; [email protected] (Z.D.); [email protected] (J.-M.P.);
[email protected] (R.B.); [email protected] (J.A.); [email protected] (P.M.);
[email protected] (G.D.); [email protected] (M.P.); [email protected] (G.D.)
2 Department of Respiratory Diseases, Centre Hospitalier Universitaire de Reims, Hôpital Maison Blanche,
51092 Reims, France
3 Department of Thoracic Surgery, Centre Hospitalier Universitaire de Reims, Hôpital Maison Blanche,
51092 Reims, France
4 CNRS UMR9017, Inserm U1019, University of Lille, Centre Hospitalier Régional Universitaire de Lille,
Institut Pasteur, CIIL—Center for Infection and Immunity of Lille, 59000 Lille, France;
[email protected]
5 Integrative Neurobiology of Cholinergic Systems, Institut Pasteur, CNRS UMR 3571, 75015 Paris, France;
[email protected]
6 Department of Biopathology, Centre Hospitalier Universitaire de Reims, Hôpital Maison Blanche,
51092 Reims, France
* Correspondence: [email protected]; Tel.: +33-(0)3-10-73-62-28; Fax: +33-(0)3-26-06-58-61




Received: 17 September 2020; Accepted: 6 October 2020; Published: 9 October 2020


Abstract: Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels
responsible for rapid neural and neuromuscular signal transmission. Although it is well documented
that 16 subunits are encoded by the human genome, their presence in airway epithelial cells (AECs)
remains poorly understood, and contribution to pathology is mainly discussed in the context of
cancer. We analysed nAChR subunit expression in the human lungs of smokers and non-smokers
using transcriptomic data for whole-lung tissues, isolated large AECs, and isolated small AECs.
We identified differential expressions of nAChRs in terms of detection and repartition in the three
modalities. Smoking-associated alterations were also unveiled. Then, we identified an nAChR
transcriptomic print at the single-cell level. Finally, we reported the localizations of detectable
nAChRs in bronchi and large bronchioles. Thus, we compiled the first complete atlas of pulmonary
nAChR subunits to open new avenues to further unravel the involvement of these receptors in lung
homeostasis and respiratory diseases.

Keywords: nicotinic receptors; airway epithelial cells; lung

1. Introduction
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated (cation-permeable) proteins expressed
in the brain and non-neuronal cells, including lung airway epithelial cells (AECs), macrophages,
neutrophils, and muscle cells [1]. These receptors are composed of five subunits organized as homo- or
hetero-pentamers forming a channel permeable to monovalent and divalent cations (predominantly
Na+ , K+ , and Ca2+ ) [1,2]. There are 16 mammalian subunits, namely α1–α7, α9–α10, β1–β4, δ, ε, and γ
(the corresponding gene names are, respectively, CHRN (cholinergic receptors nicotinic subunits)

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Int. J. Mol. Sci. 2020, 21, 7446 2 of 14

A1–A7, A9–A10, B1–B4, D, E, and G) [2]. Each subunit shares a common structure comprising a large
amino-terminal segment (about 200 residues), four transmembrane domains TM1, TM2, TM3, and TM4
(less than 30 residues each), a large cytoplasmic loop (90 to 270 residues) localized between TM3 and
TM4, and a variable carboxyl tail (10 to 30 residues) [2–4]. Muscle-type nAChRs are generally assembled
from 2(α1)/β1/δ/γ or 2(α1)/β1/δ/ε subtypes depending on muscle innervation [4–7]. Neuronal nAChRs
are assembled from α2–α7, α9–α10, and β2–β4 [8–10]. In the brain, homomeric α7 and heteromeric
α4/β2 subtypes are abundantly detected and are known to play an important role in memory and
learning due to their predominance in the hippocampal and cortical neurons [11,12]. Other subtypes
such as homomeric α9, heteromeric α2/α6, α7/β2, and α9/α10 have also been detected to a lesser
extent [1–3,12–18]. The diversity of nAChRs confers differential affinities to the ligands affecting several
parameters, including the channel opening and closing duration, the modulation of ionic conductance,
and cationic selectivity [1,2,12,14].
From a functional perspective, acetylcholine binding to nAChR at the extracellular interface
between two subunits leads to an allosteric conformational change permitting the channel opening,
followed by ion fluxes across the plasma membrane that participate in cell survival, differentiation,
and proliferation [3,9,19,20]. Nicotine, one of the components of cigarette smoke, acts as an agonist
implicated in the inhibition of apoptosis and oxidative stress responses, ultimately leading to lung
impairments due to long-term exposure [4,21,22]. Although nicotinic receptors are ionotropic complexes,
they may display metabotropic signalling properties via their association with trimeric GTP-binding
proteins to regulate downstream pathways and cytokine expression [23,24].
Previous studies have established that multiple single-nucleotide nAChR polymorphisms
are associated with risks of lung cancer and chronic obstructive pulmonary disease (COPD),
highlighting their potential implication in respiratory diseases [19,25,26]. In addition, it has been
hypothesized that the nAChRs may play a role in coronavirus disease 2019 (COVID-19) and might
represent a therapeutic target, particularly regarding their potential contribution in the regulation of
angiotensin-converting enzyme-2 (ACE-2), the main receptor for severe acute respiratory syndrome
coronavirus (SARS-CoV-2) [27–29]. Altogether, this underlines the requirement of deciphering the
atlas of pulmonary nAChR subunits.
Indeed, if the general expressions of muscle and neuronal nAChRs are well known,
little information is available regarding their expression in the lung and particularly in different
AECs [30]. Therefore, we conducted a transcriptomic and proteomic analysis of the localization and
expression of all human nAChR subunits in the adult lung.

2. Results

2.1. Smoking-Associated Pulmonary nAChR Subunit Transcript Expressions

Considering the whole lung contains all types of tissues, including epithelia, muscle, connective,
and nervous tissues (Figure 1a), the 16 nAChRs were detected among non-smoker subjects except for
CHRNA7, which was consistent in both datasets containing non-smokers (Figure 1b and Supplemental
Table S1). CHRNB1/E were very highly expressed; CHRNA6/A9/A10/B3/D were highly expressed
(see Section 4). There was a significant increase in CHRNA1/A2/A7/B3/B4 transcript levels in smokers
compared to non-smokers. Interestingly, CHRNA7 was only detected in smokers. On the contrary,
CHRNA3/A4/A9/B2/D/G transcript levels were significantly decreased in smokers. The global repartition
in non-smokers and smokers favoured CHRNA10/B1/E, representing almost half of all nAChR subunits
expressed in lung tissues (Figure 1c and Supplemental Table S2). The differential repartitions of
nAChRs between non-smokers and smokers matched their differential expressions.
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Figure1.1.Evaluation
Figure Evaluation of nicotinic acetylcholine
of nicotinic receptor
acetylcholine (nAChR)
receptor transcript
(nAChR) levels in
transcript human
levels in lung
humantissues.
lung
(a) Illustration depicting the origin of the samples. Whole-lung tissues contained all the tissues
tissues. (a) Illustration depicting the origin of the samples. Whole-lung tissues contained all the tissues present
inpresent
the lung ineither in parenchyma
the lung or in/aroundorbronchi
either in parenchyma and bronchioles.
in/around bronchi and (b) Histogram(b)
bronchioles. showing the
Histogram
detection of nAChRs in non-smokers (black) and smokers (red). * p < 0.05; *** p < 0.001
showing the detection of nAChRs in non-smokers (black) and smokers (red). * p < 0.05; *** p < 0.001 non-smokers
= 42) vs. smokers
(nnon-smokers (n = 42) = 111).
(nvs. (c) Pie
smokers (n charts
= 111).showing the repartition
(c) Pie charts showing theof nAChRs
repartitionin non-smokers
of nAChRs in(left)
non-
and smokers (right). Coloured subunits indicate upregulation (green) and
smokers (left) and smokers (right). Coloured subunits indicate upregulation (green) and downregulation (red) in
both groups when statistically significant.
downregulation (red) in both groups when statistically significant.

In large AEC (LAEC) (Figure 2a) CHRNA1/A2/A4/B1/B3/B4/D were not detected in non-smokers
In large AEC (LAEC) (Figure 2a) CHRNA1/A2/A4/B1/B3/B4/D were not detected in non-
(Figure 2b and Supplemental Table S3). CHRNA5 was very highly expressed; CHRNA7/A10 were
smokers (Figure 2b and Supplemental Table S3). CHRNA5 was very highly expressed; CHRNA7/A10
highly expressed. Interestingly, CHRNB1/B4 were only detected in smokers. There was a significant
were highly expressed. Interestingly, CHRNB1/B4 were only detected in smokers. There was a
decrease in CHRNA5/A10 transcript levels in smokers when compared to non-smokers. The global
significant decrease in CHRNA5/A10 transcript levels in smokers when compared to non-smokers.
repartition in non-smokers and smokers favoured CHRNA5/A7/A9/A10, representing more than 75%
The global repartition in non-smokers and smokers favoured CHRNA5/A7/A9/A10, representing
of all nAChRs expressed in LAEC (Figure 2c and Supplemental Table S4). There were no significant
more than 75% of all nAChRs expressed in LAEC (Figure 2c and Supplemental Table S4). There were
differences in terms of nAChR repartitions between non-smokers and smokers.
no significant differences in terms of nAChR repartitions between non-smokers and smokers.
Int. J. Mol. Sci. 2020, 21, 7446 4 of 14
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Figure2.2. Evaluation
Figure of nAChR
Evaluation of nAChR transcript
transcript levels
levelsininhuman
humanlarge
largeairway
airwayepithelial
epithelial cells
cells (LAECs).
(LAECs). (a)
(a) Illustration depicting the origin of the samples. Isolated AECs were collected
Illustration depicting the origin of the samples. Isolated AECs were collected from bronchi. from bronchi.
Large
Large
airway airway epithelial
epithelial cellscells (LAECs)
(LAECs) areare depicted
depicted ininpurple.
purple.(b)(b)Histogram
Histogram showing
showing the
the detection
detectionofof
nAChRs in non-smokers (black) and smokers (red). * p < 0.05 non-smokers (n = 5) vs. smokers(n(n==5).
nAChRs in non-smokers (black) and smokers (red). * p < 0.05 non-smokers (n = 5) vs. smokers 5).
(c)
(c)Pie
Piecharts
chartsshowing
showingthetherepartition
repartitionofofnAChRs
nAChRsininnon-smokers
non-smokers(left)
(left)and
andsmokers
smokers(right).
(right).

In small AEC (SAEC) (Figure 3a), the 16 nAChRs were detected among non-smokers with
In small AEC (SAEC) (Figure 3a), the 16 nAChRs were detected among non-smokers with
moderate or low expressions (Figure 3b and Supplemental Table S5). There was a significant increase
moderate or low expressions (Figure 3b and Supplemental Table S5). There was a significant increase
in CHRNA5/A7/B2/B3 transcript levels in smokers compared to non-smokers. The global repartition in
in CHRNA5/A7/B2/B3 transcript levels in smokers compared to non-smokers. The global repartition
non-smokers and smokers favoured CHRNA7/A9/A10/B2, representing half of the nAChRs expressed in
in non-smokers and smokers favoured CHRNA7/A9/A10/B2, representing half of the nAChRs
SAEC (Figure 3c and Supplemental Table S6). There was a significant increase in CHRNA7/B2/B3 and a
expressed in SAEC (Figure 3c and Supplemental Table S6). There was a significant increase in
significant decrease in CHRNA2/A9 in the repartition of nAChRs in smokers compared to non-smokers.
CHRNA7/B2/B3 and a significant decrease in CHRNA2/A9 in the repartition of nAChRs in smokers
compared to non-smokers.
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Figure 3. Evaluation of nAChR transcript levels in human small airway epithelial cells (SAECs).
Figure 3. Evaluation of nAChR transcript levels in human small airway epithelial cells (SAECs). (a)
(a) Illustration depicting the origin of the samples. Isolated AECs were collected from bronchioles.
Illustration depicting the origin of the samples. Isolated AECs were collected from bronchioles. Small
Small airway epithelial cells (SAECs) are depicted in purple. (b) Histograms showing the detection of
airway epithelial cells (SAECs) are depicted in purple. (b) Histograms showing the detection of
nAChRs in non-smokers (black) and smokers (red). * p < 0.05; ** p < 0.01; *** p < 0.001 non-smokers
nAChRs in non-smokers (black) and smokers (red). * p < 0.05; ** p < 0.01; *** p < 0.001 non-smokers (n
(n = 63) vs. smokers (n = 72). (c) Pie charts showing the repartition of nAChRs in non-smokers (left)
= 63) vs. smokers (n = 72). (c) Pie charts showing the repartition of nAChRs in non-smokers (left) and
and smokers (right). Coloured subunits indicate upregulation (green) and downregulation (red) in
smokers (right). Coloured subunits indicate upregulation (green) and downregulation (red) in both
both groups when statistically significant.
groups when statistically significant.
2.2. Differential Pulmonary nAChRs Transcript Expressions at the Single-Cell Scale
2.2. Differential Pulmonary nAChRs Transcript Expressions at the Single-Cell Scale
At the level of single-cell transcriptomes (Figure 4), CHRNA5/A7/A9/A10/B1/E were highly
At the
expressed levelofofthe
in most single-cell transcriptomes
AEC populations (Figure
including 4), basal,
alveolar, CHRNA5/A7/A9/A10/B1/E
goblet, multiciliated, andwere
clubhighly
cells.
expressed in most of the AEC populations including alveolar, basal, goblet, multiciliated,
CHRNA1/A2/A4/A6/B2/B3/D/G showed low to no expression in AEC. Interestingly, functional AEC cell and club
cells. CHRNA1/A2/A4/A6/B2/B3/D/G showed low to no expression in AEC. Interestingly,
populations were distinguished with their nAChR signatures: pneumocytes expressed CHRNA5/A10/B1; functional
AECcells
basal cellexpressed
populations were distinguishedgoblet
CHRNA5/A7/A10/B1/E; withcells
their nAChRCHRNA7/A10/B1/E;
expressed signatures: pneumocytes expressed
multiciliated cells
CHRNA5/A10/B1; basal cells expressed CHRNA5/A7/A10/B1/E; goblet
expressed CHRNA9/10/B1/E; club cells expressed CHRNA7/A10/B1; ionocytes expressed CHRNA3/B4/E. cells expressed
CHRNA7/A10/B1/E; multiciliated cells expressed CHRNA9/10/B1/E; club cells expressed
CHRNA7/A10/B1; ionocytes expressed CHRNA3/B4/E.
Int. J. Mol. Sci. 2020, 21, 7446 6 of 14
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Considering
Considering non-epithelial cells, CHRNA1/A3/A5/A10/B1/E
non-epithelial cells, CHRNA1/A3/A5/A10/B1/E were were highly
highly expressed
expressed inin specific
specific
populations of immune cells including macrophages, B cells, dendritic cells, and mast cells.
populations of immune cells including macrophages, B cells, dendritic cells, and mast cells. CHRNB1CHRNB1
expression
expression was
was specific
specific to
to lymphatic
lymphatic cells. CHRNA5/B1/E were
cells. CHRNA5/B1/E were highly
highly expressed
expressed in
in fibroblasts;
fibroblasts;
CHRNA3/A10/B1 in smooth muscle cells; CHRNA5/B1/E in endothelial cells and macrophages.
CHRNA3/A10/B1 in smooth muscle cells; CHRNA5/B1/E in endothelial cells and macrophages. B cells
B
and dendritic cells mainly expressed CHRNB1/E.
cells and dendritic cells mainly expressed CHRNB1/E.

Figure 4. Evaluation of nAChR expressions in lung

4. Evaluation lung single-cell
single-cell populations.
populations. Dot plots of nAChRnAChR
expressions in the epithelial and non-epithelial compartments.
epithelial and non-epithelial compartments. The identities of cell populations are
shown on the y-axis, and the subunits on the x-axis.
the y-axis, and the subunits on the x-axis. The colour intensity represents the average
average
expression
expression level,
level, and
and the
the size
size of
of the
the dots
dots represents
represents the
the proportion
proportion of of the
the expressing
expressing cells
cells in
in each
each
population.
population. Raw expression values
Raw expression values were
were normalized,
normalized, log-transformed,
log-transformed, andand summarized
summarized by published
by published
cell
cell clustering.
clustering.

2.3. Identification of nAChR Subunits in Bronchial and Large Bronchiolar Epithelia

2.3. Identification of nAChR Subunits in Bronchial and Large Bronchiolar Epithelia
To investigate nAChR subunit localization in the lung, we selected commercially available
To investigate nAChR subunit localization in the lung, we selected commercially available
validated primary antibodies displaying the antigenic sequences demonstrating the lowest percentage
validated primary antibodies displaying the antigenic sequences demonstrating the lowest
of identity with regard to cross-reactivity (Supplemental Tables S7 and S8; Supplemental Figures S1
percentage of identity with regard to cross-reactivity (Supplemental Tables S7 and S8; Supplemental
and S2). We focussed here on bronchi and large bronchioles as well as smooth muscle and blood
Figures S1 and S2). We focussed here on bronchi and large bronchioles as well as smooth muscle and
vessels on formalin-fixed paraffin-embedded (FFPE) tissues (Figure 5a,b). Subunits α1/α2/α4/β3/γ
blood vessels on formalin-fixed paraffin-embedded (FFPE) tissues (Figure 5a,b). Subunits
were not detected. Subunit α3 seemed restricted to the apical side of differentiated cells. Surprisingly,
α1/α2/α4/β3/γ were not detected. Subunit α3 seemed restricted to the apical side of differentiated
α5 was systematically found in AEC nuclei and the apical side of differentiated AECs, while its
cells. Surprisingly, α5 was systematically found in AEC nuclei and the apical side of differentiated
pattern was consistent with membrane-bound receptors on smooth muscle cells. Subunits α6 and α9
AECs, while its pattern was consistent with membrane-bound receptors on smooth muscle cells.
presented similar staining in differentiated AECs, such as α7/α10/β1/β2/δ/ε, which in addition were
Subunits α6 and α9 presented similar staining in differentiated AECs, such as α7/α10/β1/β2/δ/ε,
found in non-differentiated AECs. Finally, β4 appeared in multiciliated cells only. When available,
which in addition were found in non-differentiated AECs. Finally, β4 appeared in multiciliated cells
our observations were generally concordant with the data from the Human Protein Atlas (Figure 5b
only. When available, our observations were generally concordant with the data from the Human
and Supplemental Figure S3).
Protein Atlas (Figure 5b and Supplemental Figure S3).
 Int. J. Mol. Sci. 2020, 21, 7446 7 of 14
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Figure nAChRlocalizations
Figure 5. nAChR localizations in human
in human respiratory
respiratory epithelia.
epithelia. (a) Representative
(a) Representative micrographs
micrographs showing
showing the bronchial
the bronchial epitheliaepithelia on formalin-fixed
on formalin-fixed paraffin-embedded
paraffin-embedded (FFPE)(FFPE)
lung lung tissues
tissues stained
stained for for
the
the nAChRs
nAChRs (all (all
red),red), non-differentiated
non-differentiated cellscells
(p63(p63 or pan-cytokeratin,
or pan-cytokeratin, green),
green), andnuclei
and cell cell nuclei (DAPI,
(DAPI, blue).
blue). Magnification
Magnification corresponding
corresponding to thetoselected
the selected area
area is is shown.
shown. Drawings
Drawings depict
depict the localization
the localization of
of each
each
nAChRnAChR
subunit subunit (in(b)
(in red). red). (b)summarizing
Table Table summarizing nAChRcellular
nAChR subunit subunit cellular
and and sub-cellular
sub-cellular localization
and the available
localization andmicroscopic data frommicroscopic
the available the Human Protein
data Atlas
from(https://www.proteinatlas.org/).
the Human Protein Atlas NA,
not available; -, no detection. NA, not available; -, no detection.
(https://www.proteinatlas.org/).
Int. J. Mol. Sci. 2020, 21, 7446 8 of 14

3. Discussion
This is the first study showing transcript levels and localizations of all nAChR subunits in the
human adult lung. Interestingly, we identified distinct variations in terms of nAChR transcript levels
between whole-lung tissues, LAEC, and SAEC, as well as important changes between non-smokers and
smokers. Since whole-lung transcriptomics encompasses all pulmonary tissues, isolated cell studies
represent the ideal strategy to unveil nAChR functions in airways. It has been successfully implemented
in the context of AEC differentiation analysis, asthma, and idiopathic pulmonary fibrosis [31–35].
If they summarize the transcriptomic profile of the organ, whole-lung microarray data require tissue
or single-cell analyses to distinguish the contribution of each cellular population to the specific gene
expression. Otherwise, it would be admitted that a gene is ubiquitously expressed in the lung, while it
is only found in one histological tissue. As such, our comparative analysis pointed mainly towards
CHRNA5/A7/A10/B2/B3 to tackle the association of nAChR expression and smoking. Furthermore,
the impact of smoking could be tied into the associated risks of respiratory diseases, provided complete
clinical data are available.
We included in the analysis of nAChR expression levels 298 subjects in three distinct modalities
(whole-lung tissues, LAEC, and SAEC) and performed a preliminary identification of single-cell
transcriptomic signature. Our immunostaining analyses provided important data regarding the
subcellular localization of nAChR subunits in bronchi and large bronchioles. Microscopic observations
and transcriptomic analysis were generally concordant. Because of their modalities of association
at the cell membrane and their high degree of amino acids identity, nAChR immunostainings were
generally sparse, rarely concordant, and performed on murine tissues in the literature [30,36–38].
A careful validation method including heterologous cells overexpressing the different human nAChR
subtypes is required to further validate all subunit nAChR antibodies in the human adult lung [39].
Nonetheless, since we selected all our antibodies based on thorough sequence alignments of the
antigenic sequences, we provided here a complete description of all nAChRs in bronchi and large
bronchioles. Only individual subunits were detected and not the receptors, which are assembled
of five subunits; in vitro experimental studies will be required to confirm the presence of various
pentamers at the cell surface of the lung tissues. Other caveats complicating the identification of
nAChRs include their dynamic of assembly/recycling at the cell surface [7,40] and their differential
requirement according to the cellular context (quiescence, proliferation, oxidative stress, etc.) [1].
Additional studies on larger cohorts are needed to complement and refine our analysis.
Deciphering the cellular and molecular impact of the observed differences in transcript expressions in
the context of smoking will be essential to understanding nAChR-associated pathogenesis. It will be
particularly insightful for at least three lung diseases where smoking may partly impact homeostasis:
lung cancers, COPD, and COVID-19. (i) nAChR single-nucleotide polymorphisms (SNPs) were
associated with lung cancer cells [41,42], and nAChRs were shown to be involved in cancer cell
proliferation and survival [43–46]. Interestingly, several subunits (including α5-7-10/β2-3) were
identified in cancer cell lines, and selective nAChR inhibitors induced anti-tumour effects [47,48].
In addition, acetylcholine-signalling proteins were involved in the progression of lung cancer [49].
Altogether, understanding the repartition and the possible assembly of nAChRs at the cancer cell
surface may pave the way towards the design of effective anti-cancer drugs. (ii) nAChR SNPs were
also associated with nicotine dependence and COPD [50–54]. CHRNA3/A5/B4 polymorphisms were
heavily discussed in the dissection of the genetic origins of COPD, but no functional studies have
been published so far [55]. In addition, α7 and its ligands received particular attention as potential
inflammatory players in COPD patients [54,56]. Exploring the involvement of nAChRs in COPD
pathogenesis and progression in light of their differential distribution in lung cell populations may
help improve health care for this pathology lacking treatments. (iii) Nicotine, an exogenous ligand
of nAChRs and, more generally, smoking, have been shown in vitro and in vivo to modulate the
expression of hACE2, the main receptor of the SARS-CoV-2 spike S protein [57–60]. In light of the
Int. J. Mol. Sci. 2020, 21, 7446 9 of 14

differential subunit expressions, it will be of interest to analyze the localizations of hACE2 and nAChRs
in COVID-19 tissues.
We provided the first atlas of nAChR subunits in the lung and invited cartographers to complete
the map in order to provide a fundamental understanding of these crucial actors of homeostasis that
may contribute to chronic and acute respiratory diseases. The identification of each potential subunit
that may assemble functional channels at the cell surface is a requisite for the optimal design of efficient
pharmacological modulations of nAChRs in the context of the pharmacology of the respiratory system.

4. Materials and Methods

4.1. Human Subjects

Patients scheduled for lung resection for cancer (University Hospital of Reims, France) were
prospectively recruited (n = 10) following standards established and approved by the institutional
review board of the University Hospital of Reims, France (IRB Reims-CHU, date of approval: 12 June
2011). In addition, 10 patients who underwent a routine large airway fiberoptic bronchoscopy with
bronchial brushings under local anaesthesia according to international guidelines were also recruited
(5 non-smokers, 5 smokers) [61]. Informed consent was obtained from all the patients. Subjects were
recruited from the Department of Pulmonary Medicine at the University Hospital of Reims (France)
and included in the cohort for Research and Innovation in Chronic Inflammatory Respiratory Diseases
(RINNOPARI, NCT02924818). The study was approved by the ethics committee for the protection of
human beings involved in biomedical research (CCP Dijon EST I, N◦ 2016-A00242-49, date of approval:
31 May 2016) and was conducted in accordance with the ethical guideline of the Declaration of
Helsinki. Patients with chronic obstructive pulmonary disease, asthma, cystic fibrosis, bronchiectasis,
or pulmonary fibrosis were excluded. At inclusion, age, sex, smoking history, and pulmonary function
test results were recorded to exclude patients with an alteration of lung functions. Ex-smokers were
considered for a withdrawal longer than 5 years.

4.2. Sample Processing

Fresh airway epithelial cells (AECs) obtained from bronchial brushings (right lower lobe, 5th to
8th divisions) were suspended for 30 min in Roswell Park Memorial Institute Medium (RPMI) (1%
penicillin/streptomycin + 10% Bovine Serum Albumin (BSA)) before centrifugation (13,500g ×2 times).
The cell pellet was dissociated in 1 mL of Trypsin Versene (Lonza), centrifuged (13,500g ×2 times),
and kept at −20 ◦ C until further steps.

4.3. RT-qPCR Analyses

Total RNA from AEC bronchial brushings was isolated by a High Pure RNA isolation kit (Roche
Diagnostics), and 250 ng was reverse-transcribed into cDNA by a Transcriptor First Stand cDNA
Synthesis kit (Roche Diagnostics, Meylan, France). Quantitative PCR reactions were performed with a
Fast Start Universal Probe Master kit and UPL-probe system in a LightCycler 480 Instrument (Roche
Diagnostics) as recommended by the manufacturer. Primers listed in Supplementary Table S9 were
designed via the Universal Probe Library Assay Design Center (Roche, Manheim, Germany). Results for
all expression data regarding transcripts were normalized to the expression of the house-keeping
gene GAPDH amplified with the following primers: forward 50 -ACCAGGTGGTCTCCTCTGAC-30 ,
reverse 50 -TGCTGTAGCCAAATTCGTTG-30 . We verified that GAPDH transcript detection levels
were highly similar between non-smokers and smokers to validate the housekeeping gene (average
Ct = 25.54 ± 0.17 in non-smokers vs. 25.35 ± 0.34 in smokers; p = 0.64). Relative gene expression was
assessed by the ∆∆Ct method [62] and expressed as 2−∆∆Ct . To compare data generated via PCR with
RNAseq analysis, we transformed the transcript expressions to a percentage scale considering the
highest and lowest values per subunit for the detection, or across all the subunits for the repartition.
Int. J. Mol. Sci. 2020, 21, 7446 10 of 14

4.4. Immunofluorescent Staining and Analyses

Immunohistochemistry was performed on formalin-fixed paraffin-embedded (FFPE) lung tissues
distant from the tumour as previously described [63]. Only patients having no respiratory diseases
were included (smokers and ex-smokers). Five micrometer sections were processed for hematoxylin
and eosin staining and observed on a microscope (×20) to confirm the presence of bronchi and large
bronchioles (pseudostratified epithelia). The bronchial epithelium was analysed on the entire slide
including 2 to 7 units per patient. FFPE lung tissue section slides were deparaffinised and blocked with
10% BSA in phosphate-buffered saline (PBS) for 30 min at room temperature. Tissue sections were then
incubated with the primary antibodies as listed in Supplementary Table S7 for one night at 4 ◦ C in 3%
BSA in PBS. After the PBS wash, a second primary antibody was used to highlight non-differentiated
cells on epithelia for 2h at room temperature: p63 (AF1916, 1:200, R&D Systems, Noyal Châtillon
sur Seiche, France) or pan-cytokeratin (CK, 1:1000, E-AB-33599, Elabscience, Clinisciences, Nanterre,
France). Sections were washed with PBS and incubated with the appropriate secondary antibodies in
3% BSA in PBS for 30 min at room temperature: Alexa Fluor® (Invitrogen, Fisherscientific, Illkirch,
France) donkey anti-rabbit IgG 594 (A21207), donkey anti-goat IgG 488 (A11055), goat anti-mouse IgG
594 (A11005), and goat anti-rabbit IgG 488 (A11008). DNA was stained with DAPI during incubation
with the secondary antibodies. Micrographs were acquired on a Zeiss AxioImageur (20× Ph) with
ZEN software (8.1, 2012) and processed with ImageJ (National Institutes of Health) for analysis.
For each patient, five random fields per section containing bronchi and large bronchioles were taken to
evaluate the localization of nicotinic receptors on epithelial and stromal cells. We selected the most
suitable primary antibodies directed against each subunit, considering external validations, identity,
and staining optimization, to highlight the localization of nAChRs on bronchi and large bronchioles.

4.5. Transcriptome Profiling Microarray Analysis

Gene expressions of non-smoking and smoking subjects with no chronic respiratory diseases were
collected from datasets available online (GEO database; http://www.ncbi.nlm.nih.gov/geo) including
whole-lung tissue samples in 153 subjects (42 non-smokers, 111 smokers; GSE103174, 76925, and 47460)
or small airway bronchoscopic samples (10th to 14th divisions) in 135 subjects (63 non-smokers,
72 smokers; GSE11784).
In order to compare transcriptomic data extracted from various datasets or PCR reactions,
we formatted the absolute values to a percentage scale. Concerning the detection of genes, we first
identified for each gene the highest and lowest expression values in both non-smokers and smokers to
set the maximal value at 100%. After proportionally expressing each of the single expression values for
all the subunits, the average was calculated and plotted on a graph. To discuss the relative level of
expressions, we arbitrarily categorized 4 groups: (1) very high expressions, the average percentage of
expression is over 75% of the maximum; (2) high expressions, the average percentage of expression is
between 50 and 75% of the maximum; (3) moderate expressions, the average percentage of expression
is between 25 and 50% of the maximum; (4) and low expression, the average percentage of expression
is below 25% of the maximum. Concerning the repartition, the total expressions of absolute values for
all nAChR were summed for each patient of the considered dataset to express the proportion of each
subunit. The comparative average percentage of expression of each subunit for all patients was plotted
in a pie chart.

4.6. Single-Cell Sequencing

The published dataset can be found at lungcellatlas.org and https://www.covid19cellatlas.org.
We retained cell clustering based on the original studies and considered only lung samples (brushing
and parenchyma from resected tissues) from subjects with no respiratory disease [33]. An Illumina
Hiseq 4000 per 10× Genomics chip position was used (n = 6; 2000–5000 cells/sample). Additional
sequencing was performed to obtain coverage, or at least mean coverage, of 100,000 reads per cell.
Int. J. Mol. Sci. 2020, 21, 7446 11 of 14

4.7. Statistics
The data are expressed as mean values and percentages. Differences between the two groups
(non-smokers and smokers) for each gene were determined using the Student t test. A p-value < 0.05
was considered significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/20/7446/s1.

Author Contributions: Conceptualization, V.D.; methodology, V.D.; formal analysis, Z.D., J.-M.P., M.P., P.G., U.M.,
G.D. (Gaëtan Deslée) and V.D.; investigation, Z.D., R.B. and V.D.; resources, J.-M.P., J.A., P.M., G.D. (Gonzague
Delepine) and G.D. (Gaëtan Deslée); data curation, J.-M.P., J.A., P.M. and G.D. (Gaëtan Deslée); writing—original
draft preparation, Z.D. and V.D.; writing—review and editing, Z.D., J.-M.P., M.P., P.G., U.M., G.D. (Gaëtan Deslée)
and V.D.; supervision, G.D. (Gaëtan Deslée) and V.D.; project administration, V.D.; funding acquisition, P.G., U.M.,
G.D. (Gaëtan Deslée) and V.D. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by funding from the University of Reims Champagne-Ardenne (URCA),
the French National Institute of Health and Medical Research (Inserm) and a grant from the Research Institute
in Public Health (IReSP) in association with the National Institute of Cancer (INCa). It was carried out in the
framework of the Federative Research Structure CAP-Santé and benefited from the Project Research and Innovation
in Inflammatory Respiratory Diseases (RINNOPARI).
Acknowledgments: We thank the members of the Inserm UMR-S 1250 unit and our collaborators for their
helpful comments and insights. We thank the Platform of Cell and Tissue Imaging (PICT) for technical assistance.
The drawings were created with BioRender.com.
Conflicts of Interest: G.D. (Gaëtan Deslée) reports personal fees from Nuvaira, personal fees from BTG/PneumRx,
personal fees from Chiesi, personal fees from Boehringer, personal fees from Astra Zeneca, outside the submitted
work. V.D. reports personal fees from Chiesi outside the submitted work. The funders had no role in the design of
the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision
to publish the results.

Abbreviations
nAChRs Nicotinic acetylcholine receptors
AEC Airway epithelial cells
COPD Chronic obstructive pulmonary disease
COVID-19 Coronavirus disease 2019
ACE-2 Angiotensin-converting enzyme-2
SARS-CoV-2 Severe acute respiratory syndrome coronavirus
FFPE Formalin-fixed paraffin-embedded
LAEC Large airway epithelial cell
SAEC Small airway epithelial cell
SNP Single-nucleotide polymorphisms
RAS Renin–angiotensin system

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