of 4
Lower Respiratory Tract
Myeloid Cells Harbor SARS-
Cov-2 and Display an
In
fl
ammatory Phenotype
To the Editor:
Severe acute respiratory syndrome-coronavirus 2
(SARS-CoV-2) pneumonia may induce an aberrant
immune response with brisk recruitment of myeloid
cells into the airspaces.
1
Although the clinical
implications are unclear, others have suggested that
in
fi
ltrating myeloid cells may contribute to morbidity
and mortality rates during SARS-CoV-2 infection.
1-3
However, few reports have characterized myeloid cells
from the lower respiratory tract, which appears to be the
primary site of viral-induced disease, during severe
SARS-CoV-2 pneumonia.
Methods
Endotracheal aspirate (ETA) samples were collected prospectively from
seven patients whose condition required mechanical ventilation for
severe pneumonia due to SARS-CoV-2 infection, which was
documented by reverse transcriptase polymerase chain reaction from
April to June 2020. All patients were enrolled in a University of
Pittsburgh lung injury registry and biospecimen repository (IRB#
PRO10110387). ETA were
fi
xed in 4% (volume/volume)
paraformaldehyde overnight then processed for subsequent imaging.
Brie
fl
y, ETA samples were washed twice then pelleted at 600
g
.
Samples for electron microscopy were resuspended in 1% (volume/
volume) glutaraldehyde, repelleted at 600
g
, and processed as
described in a previous report.
4
Samples for light microscopy and
immuno
fl
uorescence were resuspended in phosphate-buffered saline
solution then prepared as cytospins by spinning at 300 rpm onto a
Superfrost plus microscope slide (Fisher Scienti
fi
c). Manual cell
counts of ETA samples were performed after Diff-Quick (Siemens;
Healthcare Diagnostics, Inc) staining, and representative images were
obtained with the use of an Olympus Provis I microscope (Olympus
Corporation). For immuno
fl
uorescence, cytospin slides were placed
in 70% (volume/volume) ethyl alcohol followed by 90% (volume/
volume) ethyl alcohol for 10 minutes each, then allowed to air dry.
Antibodies that were used include SARS-CoV-2 nucleocapsid protein
(NB100-56576; Novus Biologicals), CD14 (#347490; BD Biosciences),
CD16 (MA1-84008; Invitrogen), CD142 (Tissue Factor; BD 550252;
BD Biosciences), and IL-6 (Novus NBP2-44953; Novus Biologicals).
Subsequent staining was performed, and slides were imaged using a
Nikon A1 confocal scanning
fl
uorescence microscope (Nikon Inc).
Preembed immune-electron microscopy was performed after
cytospin preparation with colloidal gold-conjugated secondary
antibodies (CD14, 18 nm; SARS-CoV-2, 6 nm nucleocapsid; Jackson
ImmunoResearch) using a JEOL JEM 1400 transmission electron
microscope (JEOL USA, Inc) at 80 kV with image capture via an
Advanced Microscopy Techniques 2K digital camera. RNAscope was
performed per manufacturer instructions. Quantitative imaging
analysis was performed with the use of object-based area overlap
analysis via Nikon Elements software. Statistical comparisons of co-
expression of CD14, CD16, IL-6, and tissue factor in cells with or
without SARS-CoV-2 nucleocapsid protein were performed with
non-parametric testing in GraphPad Prism version 7.05 (GraphPad
Software).
Results
The median age of the seven patients was 58 years
(range, 56-77 years), and
fi
ve patients (71.4%) were
men. The median duration of reported symptoms
prior to initiation of mechanical ventilation was
7 days (range, 3-11 days). Samples were collected
within a median of 5 days (range, 1-14 days) after
initiation of mechanical ventilation. Notably, three
patients required extracorporeal membrane
oxygenation, and two patients were dead by 60 days of
follow up from ICU admission. There were no
patients with known immune de
fi
cits in this cohort as
de
fi
ned by history of immuno
suppressive therapy,
which included chemotherapy or chronic systemic
steroids, or known immune de
fi
ciency. ETAs were
composed primarily of mononuclear and
polymorphonuclear leu
kocytes (range, 70.6%-
97.5% of nucleated cells) (
Fig 1
A). Electron
tomography of ETAs revealed intracellular
localization of presumpt
ive SARS-CoV-2 virions in
mononuclear leukocytes (
Fig 1
B) and
polymorphonuclear leukocytes (
Fig 1
C). The
identi
fi
cation of SARS-CoV-2 virions by electron
tomography was consistent with immune-electron
microscopy with an antibody
against the nucleocapsid
protein of SARS-CoV-2 that con
fi
rmed the presence
of virus in CD14
þ
cells in the lower airways (
Fig 1
D).
Quantitative imaging of ET
A cells revealed SARS-
CoV-2 nucleocapsid protein expression (n
¼
6; patient
7didnothavesuf
fi
cient ETA available for imaging)
(
Fig 2
A), many of which were also positive for CD14,
IL-6, and tissue factor immunostaining (
Fig 2
B).
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ResearchLetters
]
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963
Myeloid cells that expressed SARS-CoV-2
nucleocapsid protein were more likely to express the
in
fl
ammatory markers CD14 and CD16 (
P
<
.01 by
Mann-Whitney test) (
Fig 2
C) compared with myeloid
cells without viral co-locali
zation within each sample.
Similarly, lower respiratory
tract cells that expressed
SARS-CoV-2 nucleocapsid protein were more likely to
express IL-6 (
P
<
.01) (
Fig 2
D) and tissue factor (
P
<
.05) (
Fig 2
D) compared with cells without co-
localization of viral pro
tein within each sample.
Finally, we noted that ETA myeloid cells showed
IL6,
F3,
and
CD14
transcripts (representative image of
polymorphonuclear leukocyte) (
Fig 2
E).
Discussion
Taken together, our
fi
ndings suggest that lower
respiratory tract myeloid cells found in ETA samples
harbor SARS-CoV-2 virus and display an in
fl
ammatory
phenotype marked by expression of CD14, CD16, IL-6,
and tissue factor. Although others have shown co-
localization of SARS-CoV-1 and H1N1 in
fl
uenza virus
with human monocyte/macrophages in autopsy
studies,
5
,
6
we believe this to be the
fi
rst description and
con
fi
rmation of the presence of SARS-CoV-2 virions
inside lower respiratory tract myeloid cells, including
polymorphonuclear leukocytes, from human samples.
Although the clinical implications of our
fi
ndings are
Figure 1
A-D, Lower respiratory tract myeloid cells can harbor SARS-CoV-2 virions. A, Representative images of cytospins prepared from endo-
tracheal aspirate samples; patient 3 is on the left and patient 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 overview of lower respira
tory
tract mononuclear leukocyte (presumptive macrophage) from patient 4: the upper inset shows the region indicated by the square that shows a
tomographic slice with presumptive SARS-CoV-2 virion in a smooth-walled compartment or surface invagination; the lower inset shows a higher
magni
fi
cation tomographic view of presumptive virion with apparent spike proteins indicated by red arrowheads. C, Polymorphonuclear leukocyte
(presumptive neutrophil) from patient 7; the inset shows the region indicated by the square in the overview that contained presumptive SARS-CoV-2
virions (red arrowheads). D, Immunotransmission electron microscopy of lower respiratory tract mononuclear leukocyte from patient 6 with CD14
(18 nm gold colloid; open arrowhead) surface immunostaining and internal immunostaining of SARS-CoV-2 Nucleocapsid protein (6 nm gold colloid;
black arrowheads at clusters of staining). SARS-CoV-2
¼
severe acute respiratory syndrome coronavirus 2.
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unclear, we speculate that the bene
fi
ts of dexamethasone
in patients with SARS-CoV-2 pneumonia whose
condition requires mechanical ventilation
7
potentially
result from modulation of in
fl
ammatory myeloid cells
recruited to lung airspaces, which are deleterious in
mouse models of SARS-CoV-1 pneumonia.
8
Notably,
we found that lower respiratory tract myeloid cells can
harbor virus as long as 14 days after initiation of
mechanical ventilation.
Despite the novelty of these
fi
ndings, the mechanisms by
which virions enter lower respiratory tract myeloid cells
and survive phagocytic degradation are unclear. Others
have demonstrated SARS-CoV-1 virions within
monocytes/macrophages without productive replication
in vitro,
9
,
10
and previous reports have shown survival of
HIV virions in bone marrow macrophages in a
humanized mouse model.
4
Therefore, it remains to be
determined whether productive replication of SARS-
CoV-2 can occur in these cells. A limitation of our study
is that ETAs may not re
fl
ect fully the distal airspaces; we
suggest that these
fi
ndings should be validated in BAL
samples. Further work in preclinical models of SARS-
CoV-2 pneumonia are needed to determine the
protective or maladaptive role of viral uptake by myeloid
cells and the associated in
fl
ammatory phenotype to help
guide future clinical research strategies.
William G. Bain, MD
Hernán F. Peñaloza, PhD
Pittsburgh, PA
Mark S. Ladinsky
Pasadena, CA
Rick van der Geest, MS
Mara Sullivan, BS
Mark Ross, MS
Georgios D. Kitsios, MD, PhD
Barbara A. Methé, PhD
Bryan J. McVerry, MD
Alison Morris, MD
Alan M. Watson, PhD
Simon C. Watkins, PhD
Claudette M. St Croix, PhD
Donna B. Stolz, PhD
Pittsburgh, PA
Pamela J. Bjorkman, PhD
Pasadena, CA
Janet S. Lee, MD
Pittsburgh, PA
Median % cells + for
Nucleocapsid protein
0
Pt 1
Pt 2
Pt 3
Pt 4
Pt 5
Pt 6
30
20
10
A
% Cells
0
N-
14+
N-
16+
N+
14+
N+
16+
100
**
50
C
**
% Cells
0
N-
IL6+
N-
TF+
N+
IL6+
N+
TF+
100
**
50
D
*
Figure 2
A-E, Lower respiratory tract myeloid cells that harbor SARS-CoV-2 virions display an in
fl
ammatory phenotype. A, Quantitative immuno-
fl
uorescence with median percentage (n
¼
3 slides per patient) of total endotracheal aspirate cells that expressed SARS-CoV-2 nucleocapsid protein (n
¼
6
patients; patient 7 did not have suf
fi
cient endotracheal aspirate for immuno
fl
uorescence staining). B, Representative montage from a single poly-
morphonuclear cell shows co-localization by immuno
fl
uorescence. Panels from left to right show merge, CD14 (green), IL-6 (red), SARS-CoV-2 nucleocapsid
protein (white), and Imaris (Bitplane) surface-rendered image of the overlapping areas of labeling. The blue nuclear stain in all panels is DAPI; the
white
scale bar is 10 microns. C, Comparison of endotracheal aspirate cells that co-expressed CD14 (14) or CD16 (16) with (N
þ
) or without (N-) SARS-CoV-2
nucleocapsid protein in each sample (n
¼
5 patients; patient 2 was removed due to low number of cells with nucleocapsid protein). Statistical comparison by
Mann-Whitney test. The double asterisks indicate a probability value of
<
.01. D, Comparison of endotracheal aspirate cells that co-expressed IL-6 (IL-6
þ
)
or tissue factor (TF
þ
)with(N
þ
) or without (N-) SARS-CoV-2 nucleocapsid protein in each sample (n
¼
5 patients; patient 2 was removed due to low
number of cells with nucleocapsid protein). Statistical comparison by Mann-Whitney test; the single asterisk indicates a probability value of
<
.05; the double
asterisks indicate a probability value of
<
.01. E, Representative in situ localization of CD14 (green), IL6 (white), and tissue factor or F3 (red) transcript and
DAPI nuclear staining (blue) in an endotracheal aspirate myeloid cell. Pt
¼
patient; SARS-CoV-2
¼
severe acute respiratory syndrome coronavirus 2.
chestjournal.org
965
AFFILIATIONS:
From the Acute Lung Injury Center of Excellence,
Division of Pulmonary, Allergy, and Critical Care Medicine, (W.
Bain, H. Peñaloza, R. van der Geest, G. Kitsios, B. A. Methé, B.
McVerry, A. Morris, and J. Lee), University of Pittsburgh; the Center
for Biologic Imaging, Department of Cell Biology (M. Sullivan, M.
Ross, A. Watson, S. Watkins, C. St Croix, and D. Stolz), University of
Pittsburgh; and Staff Physician (W. Bain), Veterans Affairs
Pittsburgh Healthcare System; and the Division of Biology and
Biological Engineering (M. Ladinsky and P. Bjorkman), California
Institute of Technology.
FINANCIAL/NONFINANCIAL DISCLOSURES:
None declared.
FUNDING/SUPPORT:
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. Bain); the National Heart, Lung, and Blood Institute of
the National Institutes of Health under Award Numbers K23
HL129987 (G. Kitsios); P01HL114453 (J. Lee, B. McVerry); and R01
HL136143, R01 HL142084, K24 HL143285 (J. Lee) and R21
HL143091 (B. A. Methé); and the UPMC Immune Therapy and
Transplant Center (A. Morris). Electron and confocal microscopy at
the University of Pittsburgh Center for Biologic Imaging was
supported by National Institutes of Health Of
fi
ce of the Director
awards S10OD010625 and S10OD019973 (S. Watkins). Electron
tomography at the California Institute of Technology was supported
by a George Mason University Fast Grant (P. Bjorkman), National
Institute of Allergy and Infectious Diseases (NIAID) Grant 2 P50
AI150464 (P. Bjorkman). Electron microscopy was performed with a
TF-30 electron microscope that is maintained by the California
Institute of Technology Kavli Nanoscience Institute.
CORRESPONDENCE TO:
Janet S. Lee, MD; e-mail:
leejs3@upmc.edu
Published by Elsevier Inc. under license from the American College
of Chest Physicians. This is an open access article under the CC BY-
NC-ND license (
http://creativecommons.org/licenses/by-nc-nd/4.0/
).
DOI:
https://doi.org/10.1016/j.chest.2020.10.083
Acknowledgments
Author contributions:
W. Bain takes responsibility for the content of
this manuscript. W. Bain enrolled patients, collected samples,
performed the experiments, designed, analyzed, and interpreted the
data, and wrote the manuscript. H. Peñaloza and M. Ladinsky
performed the experiments and designed and interpreted the data. R.
van der Geest, M. Sullivan, and M. Ross performed the experiments
and revised the work for important intellectual content. G. Kitsios
enrolled patients, collected samples, provided critical input to the
design of the experiments, and revised the work for important
intellectual content. B. A. Methé, B. McVerry, and A. Morris provided
critical input to design of the experiments and revised the work for
important intellectual content. A. Watson and S. Watkins provided
critical input to the design of the experiments, expert imaging input,
and revised the work for important intellectual content. C. St Croix and
D. Stolz performed the experiments, designed and interpreted the data,
and revised the work for important intellectual content. P. Bjorkman
conceived, designed, interpreted the data, and revised the work for
important intellectual content. J. Lee conceived, designed, analyzed,
interpreted the data, and wrote the manuscript.
Role of sponsors:
The content is solely the responsibility of the authors
and does not necessarily represent the of
fi
cial views of the National
Institutes of Health, Department of Veterans Affairs, or any other
sponsoring agency.
Other contributions:
The authors thank the patients and patient
families who have enrolled in the University of Pittsburgh Acute Lung
Injury Registry; the physicians, nurses, respiratory therapists, and other
staff at the University of Pittsburgh Medical Center Presbyterian and
Shadyside Hospital ICUs for assistance with coordination and
collection of endotracheal aspirate samples; Nicole Bensen, BS, and
Caitlin Schaefer, MPH, at the University of Pittsburgh for assistance
with identifying and consenting patients and their families and for
assistance with collection of endotracheal aspirate samples; and
Heather Michael, BS, and Lauren Furguiele, BS, at the University of
Pittsburgh for assistance with processing endotracheal aspirate
samples.
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