Developmental Biology / Research Article
Cells Tissues Organs 2020;209:155–164
DOI: 10.1159/000513040
Received: September 7, 2020
Accepted: November 12, 2020
Published online: January 22, 2021
Evidence of SARS-CoV2 Entry Protein
ACE2 in the Human Nose and Olfactory
Bulb
Moritz Klingenstein a Stefanie Klingenstein a Peter H. Neckel b
Andreas F. Mack b Andreas P. Wagner b Alexander Kleger c Stefan Liebau a
Alfio Milazzo a
aInstitute
of Neuroanatomy and Developmental Biology, Eberhard Karls University Tübingen, Tübingen, Germany;
of Clinical Anatomy and Cell Analysis, Eberhard Karls University Tübingen, Tübingen, Germany;
cDepartment of Internal Medicine I, University Medical Center Ulm, Ulm, Germany
bInstitute
Keywords
SARS-CoV2 · ACE2 · Human · Olfactory epithelium ·
Olfactory bulb
Abstract
Usually, pandemic COVID-19 disease, caused by SARS-CoV2,
presents with mild respiratory symptoms such as fever,
cough, but frequently also with anosmia and neurological
symptoms. Virus-cell fusion is mediated by angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) with their organ expression pattern determining viral tropism. Clinical presentation suggests rapid viral dissemination to the central nervous system leading
frequently to severe symptoms including viral meningitis.
Here, we provide a comprehensive expression landscape of
ACE2 and TMPRSS2 proteins across human postmortem nasal and olfactory tissue. Sagittal sections through the human
nose complemented with immunolabelling of respective
cell types represent different anatomically defined regions
including olfactory epithelium, respiratory epithelium of the
nasal conchae and the paranasal sinuses along with the
karger@karger.com
www.karger.com/cto
© 2021 S. Karger AG, Basel
hardly accessible human olfactory bulb. ACE2 can be detected in the olfactory epithelium as well as in the respiratory
epithelium of the nasal septum, the nasal conchae, and the
paranasal sinuses. ACE2 is located in the sustentacular cells
and in the glandular cells in the olfactory epithelium as well
as in the basal cells, glandular cells, and epithelial cells of the
respiratory epithelium. Intriguingly, ACE2 is not expressed in
mature or immature olfactory receptor neurons and basal
cells in the olfactory epithelium. Similarly, ACE2 is not localized in the olfactory receptor neurons albeit the olfactory
bulb is positive. Vice versa, TMPRSS2 can also be detected in
the sustentacular cells and the glandular cells of the olfactory epithelium. Our findings provide the basic anatomical
evidence for the expression of ACE2 and TMPRSS2 in the human nose, olfactory epithelium, and olfactory bulb. Thus,
they are substantial for future studies that aim to elucidate
the symptom of SARS-CoV2 induced anosmia via the olfactory pathway.
© 2021 S. Karger AG, Basel
M.K. and S.K. contributed equally to this work.
Stefanie Klingenstein
Institute of Neuroanatomy and Developmental Biology
Eberhard Karls University Tübingen
Oesterbergstr. 3, DE–72074 Tübingen (Germany)
stefanie.klingenstein @ uni-tuebingen.de
Introduction
The Coronavirus Disease 2019 (COVID-19) emerged
from East Asia and quickly spread all over the world
reaching a pandemic scale [Guan et al., 2020]. The infiltration of the virus SARS-CoV2 into different cell types
leads to different symptoms with varying severity. According to clinical studies worldwide, the most prevalent
symptoms are fever, cough, fatigue, headache, dyspnea,
sputum production, arthralgia, diarrhea, rhinorrhea, and
sore throat [Krajewska et al., 2020; Zhou et al., 2020].
Since many patients also report olfactory and gustatory
a
OB
b
CP
b
OE
IV
CE
SNC
I
III
NS
INC
II
a
b
a
a
IV
IV
I
I
III
III
II
c
Fig. 1. a Schematic illustration of a human head. Frontal section
highlights the nose specimen (a). Sagittal view of the olfactory bulb
section (b). Olfactory epithelium and olfactory bulb, both marked
in green, are located at the upper nasal cavity and above the cribriform plate. b Schematic representation of the nose specimen
from the frontal section through the head (a). The olfactory epithelium (OE) and the olfactory bulb (OB) are shown in green. Section plane for better comprehensibility of the olfactory bulb (b).
The dashed boxes show the section of the respiratory epithelium
of the nasal septum (NS) (l), the intermediate nasal conchae (INC)
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DOI: 10.1159/000513040
ACE2
d TUBB3
II
(II), the cellulae ethmoidales (CE) (III) and the olfactory epithelium (OE) (IV). CP, cribriform plate; SNC, superior nasal conchae.
c. Hematoxylin eosin staining of the right half of the nose specimen. Dashed boxes show the same areas found in the schematic
picture in b. d Immunofluorescence staining of the right half of
human nose specimen. TUBB3 is shown in green, ACE2 in red,
nuclei in blue. Dashed boxes show the same areas found in the
schematic picture in b. More details for area I, II, III of the respiratory epithelium can be found in Fig. 2. More details for area IV
olfactory epithelium can be found in Fig. 3. Scale bar, 2.5 mm.
Klingenstein/Klingenstein/Neckel/Mack/
Wagner/Kleger/Liebau/Milazzo
Ciliated Cell
Goblet Cell
Basal Cell
Gll. nasales
a
I
I
I
Respiratory Epithelium
Nasal Septum
ACE2
II
II
II
Respiratory Epithelium
Intermediate Nasal Conchae
ACE2
III
III
III
Respiratory Epithelium
Cellulae Ethmoidale
b
Fig. 2. a Schematic illustration of the pseudostratified respiratory
epithelium with ciliated cells, goblet cells, basal cells, and nasal
submucosal glands. b Hematoxylin eosin stainings of the respiratory epithelium in the area of the nasal septum (l), the intermediate
nasal conchae (II), and the cellulae ethmoidales (III). c Immunofluorescent stainings of the respiratory epithelium verified by absent TUBB3 expression in the epithelium from dashed boxes I, II
and III. TUBB3 is a highly specific marker for mature and immature ORN. ACE2 (red) positive basal cells of the respiratory epi-
Detection of ACE2 in Human Olfactory
Tissue
c
ACE2
thelium of the nasal septum as well as positive apical staining of
respiratory epithelial cells and nasal submucosal glands (l). ACE2
(red) protein expression is located in the ciliated epithelium cells
and the basal cells of the respiratory epithelium of intermediate
nasal conchae as well as in the underlying nasal submucosal glands
(II). ACE2 (red) expression in the cellulae ethmoidales can be
found in epithelial cells and basal cells (III). Nuclei are shown in
blue. Scale bar, 20 μm.
Cells Tissues Organs 2020;209:155–164
DOI: 10.1159/000513040
157
dysfunctions, these symptoms are considered typical of
the SARS-CoV2 infection [Lechien et al., 2020; Lee et al.,
2020]. There are many steps involved in the perception of
smell where the infection with SARS-CoV2 could potentially be the cause of anosmia, starting with the transport
of the odorants to the receptors in the olfactory neurons
extending to the signal transduction to different olfactory
cortex areas.
In the olfactory epithelium, a variety of histological
target structures, including the olfactory receptor neurons (ORN) with their ensheathed axons, sustentacular,
microvillar or glandular cells could serve as a viral target
and therefore influence olfactory function. Another area
of viral attack could be the olfactory bulb. Here, the fila
olfactoria or projection neurons in different layers of the
olfactory bulb could be targeted by the virus, causing disruption in olfactory perception.
Infection of host cells with SARS-CoV-2 is preceded
by a complex process of virus attaching, receptor recognition and proteolytic cleavage of the transmembrane spike
glycoprotein to promote virus-cell fusion mediated by
angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) [Hoffmann et
al., 2020].
Up to date, there is no clear evidence which cell types
of the olfactory and respiratory epithelium express ACE2
and TMPRSS2. Transcriptional but also data from murine and human olfactory tissue show as of now report
conflicting data [Bilinska et al., 2020; Brann et al., 2020;
Hou et al., 2020; Ueha et al., 2020]. In addition, the reports on localization of the viral entry proteins in different cell types from the epithelia vary [Bilinska et al., 2020;
Ueha et al., 2020]. Protein expression was found in sustentacular cells in all publications, but only one paper
claims a low ACE2 expression in ORN [Ueha et al., 2020].
TMPRSS2 was found in murine and human respiratory
epithelium and murine olfactory epithelium [Bilinska et
al., 2020]. Similarly, experiments performed in the murine olfactory bulb showed partly contradictory results
for ACE2 staining. Until now, there are no protein verifications in human olfactory bulb due to its difficult accessibility.
Here, we employ a unique human postmortem tissue
resource to thoroughly study the ACE2 and TMPRSS2
protein expression patterns in the human olfactory system, including olfactory epithelium, respiratory epithelium of the nasal septum, the nasal conchae, and the paranasal sinuses as well as the olfactory bulb. These findings will help to explain the symptom of anosmia as well
as frequent dissemination to the central nervous system
in COVID-19 patients and give a starting point to further
investigations on how SARS-CoV2 can affect the olfactory system.
Fig. 3. a Schematic structure of human olfactory epithelium. The
mature olfactory receptor neurons are highlighted in green and are
surrounded by sustentacular cells. The sensory neurons arise via
an olfactory precursor step from basal cells. There are 2 types of
basal cells: horizontal and globose basal cells. Bowman glands are
secretory glands that are found exclusively in the olfactory epithelium. b Hematoxylin eosin staining of the olfactory epithelium in
the area of the nasal septum (IV). c Immunofluorescent staining
of the olfactory epithelium from dashed box (IV). Merged picture
shows ACE2 in red, OMP, a marker for mature ORN, in white and
TUBB3, a marker for mature and immature ORN, in green. Positive ACE2 staining is exclusively located to the region of the sustentacular cells, no appearance in basal cells or co-localization with
OMP or TUBB3. d Enlarged picture from the olfactory epithelium
and mucosa showing ACE2 positive sustentacular cells and positive Bowman glands. Upper dashed box shows an enlarged picture
of ACE2 positive sustentacular cell, marked with dashed white
lines, but negative for TUBB3. TUBB3 positive olfactory sensory
neuron marked with dashed yellow lines. Lower dashed box shows
ACE2 positive staining in Bowman glands in the olfactory submucosa. One ACE2 positive Bowman gland cell is marked with a
dashed white line. e Immunofluorescent picture with TMPRSS2
(green) and OMP (red) expression in the olfactory epithelium
(IV). TMPRSS2 is mainly located in the sustentacular cells and
shows a minor expression in Bowman glands, but no expression in
basal cell or OMP-positive mature ORN. Nuclei are shown in blue.
Scale bar, 20 μm.
(For figure see next page.)
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Cells Tissues Organs 2020;209:155–164
DOI: 10.1159/000513040
Materials and Methods
Tissue Processing
The tissue was obtained from human body donors. Surgery was
performed transcranial with particular attention to remove the
cribriform plate together with the olfactory bulb as well as the nasal septum and the nasal conchae. Fixation of the whole specimen
was performed for 4 days with daily changes of Roti Histofix (Carl
Roth, P087.1) fixation media, followed by 1-day washing step with
PBS. Decalcification was achieved with 10% EDTA for 70 days
with medium change twice a week. After a washing step for 1 day
with PBS, the specimen was incubated for another day in 30% sucrose, followed by embedding with Tissue-Tek (Thermo Fisher,
12351753) for frozen sections.
Immunohistological Staining
For hematoxylin-eosin staining, 12 μm frozen sections were
treated with filtered hematoxylin (Sigma, H9627) for 10 min, followed by a washing step with tap water for 2–5 min. Eosin (Sigma,
230251) counterstaining was performed for 7 min, followed by
careful rinses with distilled H2O and dehydration with increasing
Klingenstein/Klingenstein/Neckel/Mack/
Wagner/Kleger/Liebau/Milazzo
Sustentacular Cell
IV
Olfactory Receptor Neuron
Olfactory Precursor Cell
Globose Basal Cell
IV
Horizontal Basal Cell
Bowman‘s Gland
a
Olfactory Epithelium
Nasal Septum
b
IV
c
ACE2
TUBB3
OMP
IV
IV
Sustentacular Cell
Olfactory Receptor Neuron
Bowman‘s Gland Cell
d
ACE2
TUBB3
IV
e
TMPRSS2
OMP
3
Detection of ACE2 in Human Olfactory
Tissue
Cells Tissues Organs 2020;209:155–164
DOI: 10.1159/000513040
159
alcohol concentrations (70, 95, and 100%). After washing 2 times
in xylene, sections were mounted with DPX mounting media
(VWR, 13,514).
Immunofluorescence Staining
For immunofluorescence staining, 12 μm frozen sections were
rehydrated for 5 min with PBS (Thermo Fisher, 10010056) followed by an ethanol gradient each 30 s (70, 95, 99, 95, and 70%).
The frozen sections were blocked in skimmed milk blocking buffer
(TBS + 10% NDS +1.25% BSA, 4% skimmed milk, 0.1% Triton X)
for 30 min at room temperature. The primary antibody was diluted in skimmed milk blocking solution and incubated over night
at 4°C. The following primary antibodies were used: ACE2 rb (Abcam, ab15348 1:100), glial fibrillary acidic protein (GFAP) ms
(Merck, MAB360, 1:100), TMPRSS2 ms (Santa Cruz, Sc515727,
1:50), ßIII-tubulin (TUBB3) ms (BioLegend, 802002, 1:1000), and
olfactory marker protein (OMP) gt (WAKO, 544-1001 1:500).
Synaptophysin rb (Abcam, 14,692-100, 1:100). All primary antibodies were verified in appropriate tissue (online suppl. Fig 1; see
www.karger.com/doi/10.1159/513040). After several washing
steps, secondary antibodies were diluted 1:100 in PBS together
with DAPI (Abcam, ab228549) and incubated for 45 min in room
temperature away from daylight. The following secondary antibodies were used: Dαrb 488 (Invitrogen, A32790), Dαms 488 (Invitrogen, A32766), Dαrb 546 (Invitrogen, A10040), Dαgt 546 (Invitrogen, A11056), and Dαms 647 (Abcam, ab150107). Sections
were embedded with Mowiol (Carl Roth, 0713). Immunofluorescence stainings were analyzed using the Axio Imager. M2 microscope with the AxioVision software (Zeiss).
Scans of immunolabelled nasal specimen were done by order
of Zeiss, pictures were analyzed with ZEN Blue software (Zeiss).
Results
Anatomical Regions Analyzed in Human Postmortem
Nasal and Olfactory Tissue
High ACE2 and TMPRSS2 expressions have been documented in the respiratory, gastrointestinal, and reproductive system [Fan et al., 2020; Ren et al., 2020]. Focusing on the upper respiratory tract, increased ACE2 and
Fig. 4. a Schematic illustration of the human olfactory bulb with
its layers: Under most is the outer nerve layer with the axons of the
olfactory receptor neurons followed by the glomerular layer with
olfactory glomerula connecting the axons with interneurons. Synaptic processing between the glomerular layer and the mitral cell
layer occurs in the external plexiform cell layer. In the mitral cell
layer, the mitral cells are located. They form synapses in the inner
plexiform layer with the granular cells of the granular cell layer.
b Hematoxylin eosin staining of a sagittal section of the human
olfactory bulb (b). Dashed box shows an enlarged picture in (c).
Scale bar, 2.5 mm. c Immunofluorescence staining of human olfactory bulb. Synaptophysin is shown in green. The specific layers of
the olfactory bulb are marked. ONL, outer nerve layer; GL, glo-
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TMPRSS2 are found in the respiratory and olfactory mucosa. First, a frontal section of human postmortem tissue
through the nose with distinct anatomical regions (shown
in Fig. 1a) is examined. The olfactory epithelium (IV) is
located at the roof of the nasal cavity between the nasal
septum and the superior nasal conchae. The paranasal
sinuses, in particular the cellulae ethmoidales (III), are
located next to the nasal turbinate. In addition, the nasal
septum (I), the superior nasal conchae, and the intermediate nasal conchae (II), all of which covered with respiratory epithelium, are present in this section (shown in
Fig. 1b). With hematoxylin-eosin staining, these different
regions of the nasal cavity were identified and visualized
(shown in Fig. 1c, 2b, 3b). Overview of ACE2 and TUBB3
immunolabelling in postmortem nasal tissue (shown in
Fig. 1d). TUBB3, a marker for mature and immature olfactory sensory neurons allows distinguishing the olfactory epithelium from the other non-neural areas within
the nose specimen (Fig. 1d).
Identification of ACE2 in Different Areas of the
Respiratory Epithelium
Schematic illustration and hematoxylin-eosin staining
show the anatomical stratification of the analyzed regions
of the nasal cavity (Fig. 1c, 2a, b). The different areas of
the respiratory epithelium, namely the nasal septum (I),
the intermediate nasal conchae (II), and the cellulae ethmoidales (III) show pseudostratified ciliated epithelial
cells arising from a basal cell layer. Mucus-producing
goblet cells can be found in the respiratory epithelium of
all 3 areas (Fig. 2b). In the respiratory epithelium of the
nasal septum, the intermediate nasal conchae and the paranasal sinus, ACE2 expression was located in epithelial
cells as well as in basal cells. High expression was found
in the nasal submucosal glands underneath the respiratory epithelium. The ACE2 expression can be found uni-
merular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GCL, glomerular cell layer.
d Overview picture with immunofluorescence stainings for ACE2
(green), TUBB3 (red), and OMP (white) illustrating the different
layers of the olfactory bulb. The white dashed box indicates the
magnified area illustrated in the next image, showing representative ACE2 positive cells, not co-localizing with TUBB3 or OMP.
High protein expression of TUBB3 and OMP can be found in the
outer nerve layer, whereas ACE2 is mainly located in the glomerular layer. e Picture of the olfactory bulb showing ACE2 (green) and
glial marker GFAP (red) positive cells. Some GFAP positive glia
cells co-localize with ACE2. Nuclei are shown in blue. Scale bar, 20
μm.
(For figure see next page.)
Klingenstein/Klingenstein/Neckel/Mack/
Wagner/Kleger/Liebau/Milazzo
show primary sensory neurons that are embedded between the supporting cells. These sustentacular cells provide mechanical strength to the epithelium, generate the
olfactory binding protein, and support the other cells
with nutrients [Choi and Goldstein, 2018]. Additionally,
sustentacular cells are responsible for the maintenance of
the ion and water balance within the olfactory epithelium
[Suzuki et al., 2000]. ORN arise from basal stem cells via
formly in the gland cells in the submucosa as well as in the
excretory part of the nasal glands, localized within the
epithelium (Fig. 2c).
Identification of ACE2 and TMPRSS2 in the Olfactory
Epithelium
Schematic illustration of the olfactory epithelium
(Fig. 3a) and histological staining of the same area (Fig. 3b)
Granular Cell Layer
Inner Plexiform Layer
Mitral Cell Layer
External Plexiform Layer
Glomerular Layer
Outer Nerve Layer
a
b
GCL
IPL
MCL
dorsal
ventral
SYNAPTOPHYSIN
occipital
frontal
EPL
GL
c
b
ONL
nuclei
EPL
GL
ACE2
d ONL
e
ACE2
TUBB3
OMP
GFAP
4
Detection of ACE2 in Human Olfactory
Tissue
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DOI: 10.1159/000513040
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immature and intermediate steps [Caggiano et al., 1994;
Fletcher et al., 2017]. Moreover, microvillar cells and excretory ducts from the specialized Bowman glands beneath the epithelium lie scattered in the epithelium
[Getchell and Getchell, 1992; Miller et al., 1995]. In addition, the data confirmed the expression of ACE2 in the
olfactory epithelium (Fig. 3c, d). The ACE2 staining was
mainly located in the supporting cells and could not be
found in basal cells, immature ORN, marked with ßIIItubulin (TUBB3) nor in mature ORN, marked with olfactory marker protein (OMP) (Fig. 3c). In the underlying
lamina propria, high concentrations of ACE2 were found
in the Bowman gland cells. ACE2 expression is mainly
limited to the sustentacular cells and shows no co-localization with adjacent TUBB3 positive sensory neurons
(Fig. 3d). TMPRSS2 was also expressed in the supporting
cells of the olfactory epithelium and in the glandular cells
of both epithelia, but no expression in the other cell types
(Fig. 3e).
Identification of ACE2 in the Olfactory Bulb
To complete our view of ACE2 expression in the olfactory system, stainings of the human olfactory bulb were
performed. Axons from the ORN pervade through the
lamina cribrosa to the olfactory bulb. Here, the first synaptic interconnections to the downstream secondary
neurons appear [Nagayama et al., 2014]. These defined
interconnections are located in 6 distinct layers of the olfactory bulb (Fig. 4a, c). With hematoxylin-eosin staining, these different regions of the olfactory bulb were
identified and visualized (Fig. 4b). The processed information projects along the olfactory tract to different regions of the olfactory cortex [Scott et al., 1993]. The hallmark feature of the outer nerve layer is the ensheathed fila
olfactoria coming from the olfactory epithelium, penetrate the bone plate and enter the olfactory bulb. Different
cell types and their interconnections, like interneurons
and projection neurons are located in deeper layers of the
olfactory bulb and lead to processed and fine-tuned sensory information (Fig. 4a) [Au et al., 2002]. The olfactory
bulb has been analyzed on RNA and on protein level with
mouse tissue so far [Brann et al., 2020; Ueha et al., 2020].
In the human olfactory bulb, ACE2 could be found widely distributed, with high expression in the glomerular layer (Fig. 4d). Faint stainings of ACE2 in the mitral cell layer were also detected. However, no co-localization of
ACE2 and OMP or TUBB3 could be detected, suggesting
that ACE2 is not expressed in the axons of the ORN or
other neurons (Fig. 4d). While neurons in the olfactory
bulb lack the expression of ACE2, the protein could be
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DOI: 10.1159/000513040
localized in some glial fibrillary acidic protein (GFAP)positive glia cells (Fig. 4e), which is consistent with the
findings in other brain regions [Deffner et al., 2020].
Discussion and Conclusion
In our present work, we show that both virus entry proteins, ACE2 and TMPRSS2, are found in the sustentacular
cells and Bowman glands of the olfactory epithelium (IV),
but not in the primary sensory neurons. In the respiratory
epithelium from the nasal septum (I), the intermediate nasal conchae (II), and the cellulae ethmoidales (III), ACE2
expression was detected in the basal cell layer and in the
apical part of the respiratory epithelial cells as well as in
the nasal submucosal glands. We conclude, based on our
findings, that SARS-CoV2 may bind to ACE2 and
TMPRSS2 in epithelial cells of the respiratory, olfactory,
and paranasal sinus epithelium and can thus penetrate the
upper respiratory system. In the olfactory bulb, ACE2 was
widely distributed with the highest expression in the glomerular layer, but was not co-localized with OMP positive
neurons or other neuronal cell types.
Taken all findings together, this leads to the question,
how the symptoms of anosmia can be explained in COVID-19 patients. It could be hypothesized that anosmia
in COVID-19 is caused by viral infection of ORN, which
further leads to their damage. Corrupted ORN cannot
process and project odorant information to the brain via
the olfactory bulb and odorant information cannot be
processed and projected to the brain via the olfactory
bulb. However, in our work we can clearly demonstrate
that there is no expression of ACE2 in the primary neurons, which supports results from previous work [Brann
et al., 2020; Chen et al., 2020]. Probably, the sustentacular
cells are affected by the SARS-CoV2 virus, as these cells
express both proteins ACE2 and TMPRSS2. Sustentacular cells are essential for the olfactory system. They do not
only provide structural stability comparable to glia cells,
but also support all other cells of the epithelium in a nutritious and metabolic way as they are connected via tight
and adherens junctions with the other cell types [Suzuki
et al., 2000; Steinke et al., 2008; Bilinska et al., 2020].
Moreover, sustentacular cells perform phagocytosis and
are probably involved in protective mechanisms by expressing antiviral and antibacterial proteins [Suzuki et al.,
1996]. Sustentacular cells are in close contact to the ORN,
forming intercellular connections. Potentially, infected
sustentacular cells may also invade ORN across these
bridges, unassisted by the virus entry genes.
Klingenstein/Klingenstein/Neckel/Mack/
Wagner/Kleger/Liebau/Milazzo
How does the localization of ACE2 and TMPRSS2 in
Bowman gland cells and nasal submucosal glands of the
respiratory epithelium fit to our hypothesis? Glandular
cells in the nasal cavity, together with goblet cells, which
are found in respiratory epithelium, are necessary for
the production of mucus. Mucus is a viscous solution,
which contains mainly water, ions, proteins and mucins
and covers the apical side of the nasal epithelium
[Escada et al., 2009]. It helps to maintain the physiological barrier of the epithelium against foreign substances
from the air. In particular, the Bowman glands are supposed to produce enzymes providing xenobiotic-metabolizing functions [Renne et al., 2007]. Due to the
virus entry protein expression in the Bowman gland
and nasal submucosal glands, SARS-CoV2 could destroy many glandular cells which would probably result
in a reduced or incorrect mucus production, leading to
dysfunction or even damage of ORN. In addition, sustentacular cells and Bowman glands are supposed to
produce odorant-binding proteins (OBPs), which are
indispensable for the perception of odorants [Vogt et
al., 2002; Nagnan-Le Meillour et al., 2019]. Without
OBPs in the mucus of the olfactory epithelium, the
binding of odorants to the olfactory receptors is hampered.
That in mind, we hypothesize that a disruption of high
numbers of sustentacular cells as well as mucus producing glandular cells could lead to a decreased perception of
smell and leave the olfactory epithelium less protected
against other viral or bacterial threats. The stem cells of
the olfactory epithelium, the basal cells, are most probably not affected by COVID-19 infections, maintaining the
potential of reproducing ORN, sustentacular cells as well
as Bowman glands [Maddux et al., 1993]. This could explain the relatively fast recovery in most patients suffering
from COVID-19 triggered anosmia [Hopkins et al., 2020;
Lee et al., 2020]. However, some cases have been reported
with very slow or nearly no recovery of anosmia [Kosugi
et al., 2020]. Depending on the dimension of destruction
of sustentacular and glandular cells, reproduction from
basal cells followed by recovery of the nasal mucus may
be prolonged or, as in severe cases, the destruction of sustentacular cells may also affect the basal cells leading to
extended symptoms. In this context, it is noteworthy that
we also found ACE2 expression in the basal cells of the
respiratory epithelium.
Based on our findings, we presume that SARS-CoV2
can enter the cells from the upper respiratory system via
the viral entry proteins ACE2 and TMPRSS2. The infection of the sustentacular cells of the olfactory epithelium
together with the underling Bowman gland cells may lead
to altered mucus production, metabolism and structural
instability in the olfactory epithelium. In addition, infection may result in the inability of the ORNs to connect to
odorants via OBPs. Nevertheless, most patients regain
their ability of smell perception, due to the fact that the
basal cells are presumably not affected by the virus and
can therefore replace destroyed cells of the olfactory epithelium.
Detection of ACE2 in Human Olfactory
Tissue
Cells Tissues Organs 2020;209:155–164
DOI: 10.1159/000513040
Statement of Ethics
Sampling of human material from body donors and all following experiments were made in accordance to local laws and regulations approved by the responsible ethical committee at the Medical
Department of the University of Tübingen (Project Nr.
284/2020BO2). The body donors gave their informed consent in
concert with the declaration of Helsinki to use the cadaver for research purposes. The procedure was approved by the ethics commission at the Medical Department of the University of Tübingen
(Project Nr. 237/2007 BO1).
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
No specific funding was received for this study.
Author Contributions
M.K., S.K., A.M. invented and designed the project. A.M.,
P.H.N., and A.W. performed the surgeries and extraction of human tissues. A.M. and M.K. performed the tissue processing procedures and generated the frozen sections. A.M. performed the
histological stainings. S.K. optimized and performed the immunofluorescence stainings. M.K. took and edited the microscope pictures and created the Figures and schemata. S.K. and M.K. wrote
the manuscript. A.W., A.F.M., P.H.N. helped in discussion of the
project and performed proofreading of the manuscript. M.K., A.K.
and S.L. were proofreading of the manuscript. Financial support
was provided by S.L.
Acknowledgment
This study has been published on pre-print server [Klingenstein et al., 2020].
163
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