of 14
1
L
ower respiratory tract
myeloid cells
harbor
SARS
-
CoV
-
2
and
display an inflammatory phenotype
William
Bain
1
,
Hernán F.
Pe
ñ
aloza
1
,
Mark
S.
Ladinsky
2
,
Rick van der Geest
1
,
Mara Sullivan
3
, Mark Ross
3
,
Georgios D.
Kitsios
1
,
Barbara Methe
1
, Bryan J.
McVerry
1
,
Alison
Morris
1
,
Alan
M.
Watson
3
,
Simon
C.
Watkins
3
,
Claudette
M.
St Croix
3
,
Donna B.
Stolz
3
,
Pamela
J.
Bjorkman
2
,
Janet S.
Lee
1
1
Acute Lung Injury Center of Excellence
,
Division of Pulmonary, Allergy, and Critical Care Medicine,
University of Pittsburgh
, Pittsburgh, Pennsylvania, United States
;
2
Division of Biology and Biological
Engineering, California Institute of Technology,
Pasadena,
California,
United States
;
3
Cente
r for Biologic
Imaging,
Department of Cell Biology,
University of Pittsburgh, Pittsburgh, Pennsylvania, United States
To whom correspondence should be addressed:
Janet S. Lee, MD, 3459 Fifth Avenue, Montefiore University
Hospital NW628, Pittsburgh, PA, 15213, U.S.A.; fax: 412.692
-
2260; email: leejs3@upmc.edu; tel:
412.692.2328
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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
2
Abstract:
SARS
-
CoV
-
2 pneumonia may induce an aberrant immune response with brisk recru
itment of myeloid
cells into the lower respiratory tract, which may contribute to morbidity and mortality. We describe
endotracheal aspirate samples from seven patients with SARS
-
CoV
-
2 pneumonia requiring mechanical
ventilation. We note SARS
-
CoV
-
2 virions
within lower respiratory tract myeloid cells shown by electron
tomography, immunofluorescence confocal imaging, and immuno
-
electron microscopy. Endotracheal aspirates
are primarily composed of mononuclear and polymorphonuclear leukocytes. These myeloid cel
ls that harbor
virus are frequently positive for CD14 and/or CD16 and most display an inflammatory phenotype marked by
expression of IL
-
6 and tissue factor mRNA transcript and protein expression.
Brief Report:
SARS
-
CoV
-
2 pneumonia may induce an aberrant i
mmune response with brisk recruitment of
myeloid
cells
into the airspaces.
1
3
Although the clinical implications are unclear, o
thers have
suggested that
infiltrating
myeloid cells may contribute to
morbidity and mortality during SARS
-
CoV
-
2 infectio
n.
1,4,5
We
examined endotracheal aspirates
(Supplemental methods)
from
seven
patients with
severe
COVI
D
-
19 pneumonia who required mechanical ventilation
(Table
1
)
:
t
hree patients required extra
-
corporeal membrane
oxygenation and two patients were deceased
by
60 days of follow
-
up from intensive care unit admission
.
Endotracheal aspirates were primarily composed of mononuclear
and polymorphonuclear
leukocytes
(Figure
1A
,
range 70.6
-
97.5% of nucleated cells
).
Electron tomography (ET)
of endotracheal aspirates re
vealed intra
-
cellular localization of presumptive SARS
-
CoV
-
2 virions in
mononuclear leukocytes
(
representative image,
Figure 1B)
and polymorphonuclear leukocytes (
representative image
,
Figure 1C)
.
The identification of SARS
-
CoV
-
2 virions by ET was
consistent with immune
-
electron microscopy using an antibody against
the
N
ucleocapsid
protein of SARS
-
CoV
-
2
that
confirmed the presence of virus in CD14
+
cells
in the lower airways
(
representative image
,
Figure 1D). Quantitative imaging
of ETA
cells
reveal
ed SARS
-
CoV
-
2
Nucleocapsid
protein
expression
(Figure 1E; n=6;
P
atient 7 did not have
sufficient
ETA available for imaging), many of
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3
which were also positive for CD14
or
CD16 immunostaining
(
Figure 1F; r
epresentative image
displayed
in
Figure 1
G)
.
Myeloid cells that
expressed
SARS
-
CoV
-
2 N
ucleocapsid
protein also frequently expressed IL
-
6
and
tissue factor
protein
(Figure 1H). Finally, we noted
that
endotracheal aspirate
myeloid cells showed
IL6
, F3
and
CD14
transcripts
(representative image,
Figure 1I).
Taken together, our findings suggest that myeloid cells
found in
endotracheal aspirate
samples
harbor
SARS
-
CoV
-
2 and display an inflammatory phenotype marked by expression of IL
-
6 and tissue factor.
The
mechanisms by which virions enter
lower r
espiratory tract myeloid cells
and survive phagocytic degradation
are
unclear
. However,
others have noted
human monocyte
-
derived macrophages to harbor
SARS
-
CoV
-
1
virus
without productive replication
in vitro
and
prior reports have
shown
survival of HIV virions in bone marrow
macrophages in a humanized mouse model
.
6,7
The clinical implications of our findings are unknown, but i
t is
intriguing to consider that the purported benefits of dexamethasone in patients
with SARS
-
CoV
-
2 pneumonia
req
uiring mechanical ventilation
8
may potentially result from modulation
of inflammatory
myeloid cells
recruited to lung airspaces
, which are deleterious
in mouse models of SARS
-
CoV
-
1 pneumonia.
9
Further work
remains to determine the clinical implications of myeloid cells harboring SARS
-
CoV
-
2 virus during severe
SARS
-
CoV
-
2 pneumonia.
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4
Figure 1
(A)
Representative images of
cytospins prepared from
endotracheal aspirate
(ETA) samples
P
atient 3 is on
the
left and
P
atient 6 is on
the
right. Black arrowheads denote mononuclear cells and red arrowheads
denote polymorphonuclear cells. Black scale bar in lower right
portion
of each image indicates 20
microns.
(B)
Electron
microscopy (EM) overview of lower respiratory tract mononuclear leukocyte (presumptive
macrophage) from Patient 4 with upper inset demonstrating the region indicated by the square that
shows tomographic slice with presumptive SARS
-
CoV
-
2 virion in a smooth
-
walled compartment or
surface invagination. Lower inset shows higher magnification tomographic view of presumptive virion
with apparent spike proteins indicated by red arrowheads.
(C)
Polymorphonuclear leukocyte (presumptive neutrophil) from Patient 7 with ins
et showing the region
indicated by the square in the overview containing presumptive SARS
-
CoV
-
2 virions (red arrowheads).
(D)
ImmunoTEM of lower respiratory tract mononuclear leukocyte from Patient 6 with CD14 (18 nm gold
colloid, open arrowhead) surface immu
nostaining and internal immunostaining of SARS
-
CoV
-
2
Nucleocapsid protein (6 nm gold colloid, black arrowheads at clusters of staining)
(E)
Quantitative immunofluorescence with median percentage (n=3 slides per patient) of total ETA cells
that expressed SARS
-
CoV
-
2 Nucleocapsid protein (n=6 patients, Patient 7 did not have sufficient
endotracheal aspirate for immunofluorescence staining).
(F)
Percentage of endotracheal aspirate cells that co
-
expressed Nucleocapsid protein and CD14 or CD16
(n=6 patients).
(G)
Representa
tive montage from a single polymorphonuclear cell
illustrating co
-
localization by
immunofluorescence. P
anels from left to right
show:
a) merge
,
b)
CD14 (green),
c)
IL
-
6 (red),
d)
SARS
-
CoV
-
2 Nucleocapsid protein (white), and
e)
Imaris (Bitplane) surface ren
dered image
of the
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5
overlapping areas of labeling
. The blue nuclear stain
in all panels
is DAPI. White scale bar
is
10
microns.
(H)
Quantitative immunofluorescence of Nucleocapsid protein positive cells that co
-
expressed IL
-
6 or tissue
factor (TF) (n=6 patients
).
(I)
Representative in situ localization of
CD14
(green),
IL6
(white), and tissue factor or
F3
(red) transcript
as well as DAPI nuclear staining (blue) in ETA myeloid cell.
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6
Table 1
: Clinical characteristics of seven patients with severe
SARS
-
CoV
-
2 pneumonia
Patient
Age
Range
Gender
Days from symptom onset to MV
Days from MV to ETA sample
1
60
-
69
M
3
2
2
50
-
59
M
11
5
3
50
-
59
M
7
1
4
70
-
79
F
3
14
5
70
-
79
F
7
2
6
50
-
59
M
5
10
7
50
-
59
M
9
6
Abbreviations: ETA =
endotracheal aspirate; F = female; M = male; MV = mechanical ventilation
.
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7
Supplemental Methods
Patients
Samples were prospectively collected from
patients
hospitalized in intensive care units (ICU) at the
University of Pittsburgh Medical Center Presbyterian and Shadyside hospitals with SARS
-
CoV
-
2 infection as
documented by RT
-
PCR. Patients were enrolled within 72 hours of intubation whenever possible. Upon
the
informed consent of the patient’s legally authorized representative, patients were enrolled
in the University of
Pittsburgh Acute Lung Injury Registry
and Biospecimen Repository
(
IRB# PRO10110387
), which has been
described elsewhere.
10,11
Longitudinal samples were collected on days 5 and 10 after enrollment.
Sample
c
ollection
, handling, and virus inactivation
Endotracheal aspirates were collected from mechanically ventilated patients after pre
-
oxygenation with
100% oxygen for 2
minutes. 5
-
10 milliliters of 0.9% sodium chloride solution were instilled via the instillation
port on the closed suction system of the endotracheal tube to lavage the airspaces. De
-
identified samples were
placed in biohazard bags and decontaminated per lo
cal protocol then transported on ice for further processing in
a Bio
-
Safety level 2+ laboratory space.
12
Raw endotracheal aspirates were diluted 1:1 in 8% (volume/volume)
paraformaldehy
de and stored at 4°C overnight to inactivate virus.
Following viral inactivation with 4%
paraformaldehyde overnight fixation, endotracheal aspirates were washed twice in sterile phosphate buffered
saline (PBS) and pelleted at 600 g.
Electron
tomography
The washed cell pellet was re
-
suspended in 0.5 mL 1% glutaraldehyde then pelleted at 600 g and stored
at 4°C.
ETA samples were further centrifuged to concentrate the pellet, then transferred to brass planchettes
(Ted Pella, Inc.) and ultra
-
rapid
ly frozen with a HPM
-
010 high pressure freezer (Bal
-
Tec/ABRA, Switzerland).
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As previously described,
6
t
he vitrified samples were transferred under liquid nitrogen to cryotubes (Nunc)
containing a frozen solution of 2.5% osmium tetroxide, 0.05% uranyl acetate in acetone. Tubes were loaded into
an AFS freeze
-
substitution
machine (Leica Microsystems) and processed at
-
90°C for 72 hr, warmed over 12 hr
to
-
20°C, held at that temperature for 8 hr, then warmed to 4°C for 1 hr. The fixative was removed and the
samples rinsed 4 x with cold acetone, following which they were infi
ltrated with Epon
-
Araldite resin (Electron
Microscopy Sciences, Port Washington
,
PA) over 48 hr.
Samples were
flat
-
embedded between two Teflon
-
coated glass microscope slides and the resin polymerized at 60°C for 48 hr. Flat
-
embedded ETA was observed
with a
stereo dissecting microscope to ascertain and select well
-
preserved samples. Suitable
regions
were
extracted with a scalpel and glued to the tips of plastic sectioning stubs. Semi
-
thick (
300
-
400 nm) serial sections
were cut with a UC6 ultramicrotome (Leic
a Micro
-
systems) using a diamond knife (Diatome, Ltd. Switzerland).
Sections were placed on Formvar
-
coated copper
-
rhodium slot grids (Electron Microscopy Sciences) and stained
with 3% uranyl acetate and lead citrate. Gold beads (10 nm) were placed on both
surfaces of the grid to serve as
fiducial markers for subsequent image alignment. Grids were placed in a dual
-
axis tomography holder (Model
2040, E.A. Fischione Instruments, Export, PA) and imaged with a Tecnai TF30ST
-
FEG trans
-
mission electron
microscope
(300 KeV; ThermoFisher Scientific) equipped with a 2K CCD camera (XP1000; Gatan, Inc,
Pleasanton CA). Tomographic tilt
-
series and large
-
area montaged over
-
views were acquired using the SerialEM
software package (Mastronarde, 2005). For tomography, samples
were tilted + /
-
64° and images collected at 1°
intervals. The grid was then rotated 90° and a similar series taken about the orthogonal axis. Tomographic data
were calculated, analyzed and modeled using the IMOD software package (Kremer et al., 1996; Mast
ronarde,
2008) on MacPro
and iMac Pro
computers (Apple, Inc, Cupertino, CA
).
Cytospin preparation and endotracheal aspirate
cell
differential
Washed cell pellets were
re
-
suspended in sterile PBS prior to spinning at 300 RPM in a Shandon
Cytospin3 onto a S
uperfrost plus microscope slide (Fisher Scientific)
then air dried
. One cytospin slide was
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stained with Diff
-
Quick (Siemens) then evaluated under light microscopy to confirm
acceptable
spacing and
numbers of cells.
Cell differentials were performed by manual counts of 200 nucleated cells by three separate
authors who were blinded to the others’
results
the mean result of the counts was utilized. Representative
images were obtained utilizing an Olympus Provis I fluorescence microscope.
Immunofluorescence
Cytospin slides were prepared as above
then placed in 70% (vol/vol) ethyl alcohol followed
by 90%
(vol/vol) ethyl alcohol for 10 minutes each, then allowed to air dry. Slides were stored at
-
20°C for subsequent
staining. To perform immunofluorescence staining, cells were washed 3 times in PBS, permeabilized in 0.1%
Triton
-
X
-
100 in PBS for 20 mi
n, and then washed once with PBS. Cells were then blocked in 20% normal goat
serum in PBS for 40 minutes then washed once with 0.5% BSA in PBS (BPBS). Primary antibodies
(Supplemental Table 1)
were added to cells in
B
P
BS
at a 1:100 dilution and incubated
the at room temperature
for 1 hour
.
Cells were washed 4 times in BPBS then secondary antibodies at
designated dilutions
were added
for 1 hour at room temperature. Cells were washed 3 times in BPBS then 3 times in PBS. Nuclei were labeled
with 0.1% Hoechs
t’s dye for 30 seconds then rinsed with PBS. Cells were cover slipped using gelvatol (23 g
poly(vinyl alcohol) 2000, 50 ml glycerol, 0.1% sodium azide to 100 ml PBS), then viewed on a confocal
scanning fluorescence microscope (Nikon A1).
Pre
-
embed Immuno
TEM
Cytospins were collected and processed as for immunofluorescence, except primary antibody dilutions
were used at 1:50 and incubated overnight at 4
o
C and secondary antibodies were incubated at a dilution of 1:10
at room temperature for 4 hours. Colloida
l gold conjugated secondary antibodies used were goat anti
-
mouse 18
nm and goat anti
-
rabbit 6 nm (Jackson ImmunoResearch). After the final wash, samples were fixed in 2.5%
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glutaraldehyde in PBS for 1 hr. Monolayers were then washed in PBS three times then
post
-
fixed in aqueous
1% osmium tetroxide, 1% Fe
6
CN
3
for 1 hr. Cells were washed 3 times in PBS then dehydrated through a 30
-
100% ethanol series then several changes of Polybed 812 embedding resin (Polysciences, Warrington, PA).
Cells were embedded by inverting Polybed 812
-
filled BEEM capsules on top of the
cells. Blocks were cured
overnight at 37
o
C, then cured for two days at 65
o
C. Monolayers were pulled off the slides and ultrathin sections
(60
-
70 nm) of the cells were obtained on a Leica EM UC7 ultramicrotome, post
-
stained in 4% uranyl acetate for
10 min
and 1% lead citrate for 7 min. Sections were viewed on a JEOL JEM 1400 transmission electron
microscope (JEOL, Peobody MA) at 80 KV. Images were taken using a side
-
mount AMT 2k digital camera
(Advanced Microscopy Techniques, Danvers, MA).
RNAscope
Tissue
factor (F3)
,
IL
-
6
, and
CD14
transcripts were localized to endotracheal aspirate
cells by
RNAscope Multiplex Fluorescent V2
assay
(ACD)
according to
manufacturer instructions. Briefly, fixed
-
frozen
cytospin sections were incubated with hydrogen peroxide an
d washed with distilled water. Then slides were
placed in 1x target retrieval agent and antigen retrieval was performed at 99ºC for 5 minutes in a steamer, then
slides were rinsed with distilled water, transferred to Ethanol 100% and dried overnight. The n
ext day, slides
were treated with Protease III and immediately treated for RNAscope. CD14 (C1, 520nm), Tissue Factor (C2,
570nm) and IL
-
6 (C3, 690nm) probes
were then
hybridized, amplified and developed. Finally, slides were
stained with DAPI and mounted w
ith a ProLong Gold Antifade mountant, dried overnight at room temperature
in the dark and store
d
at 4ºC until analysis. Slides were viewed and analyzed on a
confocal scanning
fluorescence microscope (Nikon A1).
Quantitative Imaging Analysis
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Co
-
expression
of
Nucleo
capsid protein with CD14, CD16, IL
-
6, and tissue factor was assessed using
object
-
based area overlap analysis (NIS Elements, Nikon Inc., Melville NY). In brief, Z stacks were performed
at Nyquist sampling. The data was deconvolved and b
inary masks for each channel were generated based on
emission intensity. Co
-
expression
was assessed using a binary “and” statement was used to determine pixels
containing both Capsid protein “and” the marker of interest. Imaris (Bitplane) was used to gener
ate
a
surface
rendered image
that
illustrated the areas of co
-
localization in this dataset.
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Supplemental Table
1
: Additional description of antibodies
Epitope
Species
Vendor
Catalogue #
IF Dilution
EM Dilution
SARS
-
CoV
-
2
Nucleo
capsid
Rabbit
Novus
NB100
-
56576
5
μ
g/ml
10
μ
g
/ml
CD14
Mouse, Clone: MΦP9
BD Biosciences
347490
0.25
μ
g
/ml
0.5
μ
g
/ml
CD16
Mouse, Clone DJ130c
Invitrogen
MA1
-
84008
1
μ
g
/ml
N/A
CD142 (TF)
Mouse, Clone HTF
-
1
BD Biosciences
550252
5
μ
g
/ml
N/A
IL6
Rat
Novus
NBP2
-
44953
2
μ
g
/ml
N/A
IgG
Goat anti
-
Mouse 18 nm
Jackson
ImmunoResearch
115
-
215
-
146
N/A
1:10
IgG
Goat anti
-
Rabbit 6 nm
Jackson
ImmunoResearch
111
-
195
-
144
N/A
1:10
IgG
Goat
-
anti
-
mouse Alexa
488
Invitrogen
A11029
2
μ
g
/ml
N/A
IgG
Goat
anti
-
Rabbit Cy3
Jackson
ImmunoResearch
111
-
165
-
003
2
μ
g
/ml
N/A
Abbreviations: IF = immunofluorescence, EM = immuno
-
electron microscopy
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Acknowledgements
The authors wish to thank the patients and patient families that have enrolled in the University of
Pittsburgh Acute Lung Injury Registry. We also thank the physicians, nurses, respiratory therapists and other
staff at the University of Pittsburgh Medical
Center Presbyterian and Shadyside Hospital intensive care units for
assistance with coordination and collection of endotracheal aspirate samples. We thank Nicole Bensen and
Caitlin Schaefer at the University of Pittsburgh for assistance with identifying an
d consenting patients and their
families and for assistance with collection of endotracheal aspirate samples. We thank Heather Michael and
Lauren Furguiele at the University of Pittsburgh for assistance with processing endotracheal aspirate samples.
This
work was supported by
Career Development Award Number IK2 BX004886 from the United
States Department of Veterans Affairs Biomedical Laboratory R&D (BLRD) Service (W.B.); the National
Heart, Lung, And Blood Institute of the National Institutes of Health un
der Award Numbers K23 HL129987
(G.D.K); P01HL114453 (J.S.L., B.J.M.);
and
R01 HL136143, R01 HL142084, K24 HL143285 (J.S.L.)
.
Electron and confocal microscopy at the University of Pittsburgh Center for Biologic Imaging was supported by
National Institutes o
f Health Office of the Director awards
S10OD010625
and
S10OD019973
(S.C.W.).
Electron tomography at the California Institute of Technology was supported by a George Mason University
Fast Grant (P.J.B.), National Institute of Allergy and Infectious Diseases
(NIAID)
Grant 2 P50 AI150464
(P.J.B.), and that EM was performed using a TF
-
30 electron microscope maintained by the California Institute
of Technology Kavli Nanoscience Institute
. The content is solely the responsibility of the authors and does not
neces
sarily represent the official views of the National Institutes of Health, Department of Veterans Affairs, or
any other sponsoring agency.
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14
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