of the proinflammatory cytokines IL-6, TNF-a,
and IL-1bproduced by monocytes and mDCs
upon TLR stimulation (Fig. 2B). This was con-
sistent with the lack ofor diminished expres-
sion of the genes encoding IL-6 and TNF in
the CITE-seq analysis (Fig. 5C). These results
suggest an impaired innate response in blood
leukocytes of patients with COVID-19. This
concept was further supported by the CyTOF
and flow cytometry data that showed decreased
HLA-DR and CD86 expression, respectively,
in myeloid cells (Fig. 5, D and E, and fig. S16).
To obtain deeper insight into the mechanisms
of host immunity to SARS-CoV-2, we performed
CITE-seq single-cell RNA-seq and bulk RNA-seq
analysis in COVID-19 patients at various stages
of clinical severity. Our data demonstrate that
SARS-CoV-2 infection results in an early wave
of IFN-ain the circulation that induces an ISG
signature. Although the ISG signature shows
a strong temporal dependence in our data-
sets, we also find that the ISG signature is
strongly induced in patients with moderate
COVID-19 infection (Fig. 4G). Consistent with
this, Hadjadjet al.( 5 ) have reported an en-
hanced expression of ISGs in patients with
moderate disease compared with those with
severe or critical disease. Taken together, these
data suggest that SARS-CoV-2 infection induces
an early, transient type I IFN production in the
lungs that induces ISGs in the peripheral blood,
primarily in patients with mild or moderate
disease. Additionally, we observed reduced
expression of genes encoding proinflamma-
tory cytokines, as well as HLA-DR expression
in myeloid cells, which was consistent with the
CyTOF and flow cytometry data showing re-
duced HLA-DR and CD86 expression, respec-
tively, in myeloid cells.
Our multiplex analysis of plasma cytokines
revealed enhanced levels of several proin-
flammatory cytokines, as has been observed
previously ( 35 ), and revealed a strong associ-
ation of the inflammatory mediators EN-RAGE,
TNFSF14, and OSM with the clinical severity
of the disease. Notably, the expression of genes
encoding both TNFSF14 and OSM were down-
regulated in the PBMCs from COVID-19 pa-
tients with severe disease in the analysis of
CITE-seq data (Fig. 5C), which suggests a tis-
sueoriginforthesecytokines.Thegeneen-
coding EN-RAGE, however, was expressed at
high levels in blood myeloid cells in patients
with severe COVID-19 (Fig. 5, C to F) (although
it is also possible that EN-RAGE is expressed
in the lungs too). Of note, these three cytokines
have been associated with lung inflamma-
tory diseases. In particular, EN-RAGE has been
shown to be expressed by CD14+HLA-DRlo
cells, the myeloid-derived suppressor cells, and
it is a marker of inflammation in severe sepsis
( 21 , 25 , 36 ). Additionally, its receptor, RAGE,
is highly expressed in type I alveolar cells in
the lung ( 24 ). Notably, we observed that the
classical monocytes and myeloid cells from
severe COVID-19 patients in the single-cell
RNA-seq data expressed high levels of S100A12,
thegeneencodingEN-RAGE,butnotthetyp-
ical inflammatory molecules IL-6 and TNF-a.
These data suggest that the proinflammatory
cytokines observed in plasma likely originate
from the cells in lung tissue rather than from
peripheral blood cells. Taken together with
the mass cytometry data, the plasma cytokine
data may be utilized to construct an immu-
nological profile that discriminates between
severe versus moderate COVID-19 disease
(fig. S20).
These results suggest that SARS-CoV-2 infec-
tion results in a spatial dichotomy in the innate
immune response, characterized by suppres-
sion of peripheral innate immunity in the face
of proinflammatory responses that have been
reported in the lungs ( 37 ). Furthermore, there
is a temporal shift in the cytokine response
from an early but transient type I IFN response
to a proinflammatory response during the later
and more severe stages, which is similar to that
observed with other diseases such as influenza
( 38 ). Notably, there were enhanced levels of
bacterial DNA and LPS in the plasma, which
were positively correlated with the plasma
levels of EN-RAGE, TNFSF14, OSM, and IL-6,
which suggests a role for bacterial products—
perhaps of lung origin—in augmenting the pro-
duction of inflammatory cytokines in severe
COVID-19. The biological consequence of the
impaired innate response in peripheral blood
is unknown but may reflect a homeostatic
mechanism to prevent rampant systemic hyper-
activation, in the face of tissue inflammation.
Finally, these results highlight molecules such
as EN-RAGE or TNFSF14, and their receptors,
which could represent attractive therapeutic
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ACKNOWLEDGMENTS
We thank all participants as well as the Hope Clinic and Emory
Children Center staff and faculty. We particularly acknowledge
K. Hellmeister, A. Kay, A. Cheng, J. Traenkner, A. M. Drobeniuc,
H. Macenczak, N. McNair, Y. Saklawi, A. Mehta, M. Bower,
T. Girmay, E. Butler, T. Sirajud-Deen, H. Huston, D. Kleinhenz,
L. Hussaini, E. Scherer, B. Johnson, J. Kleinhenz, J. Morales,
V.Karmali, Y. Xu, and D. Wang. We are grateful for the support of
the Emory Department of Medicine and Pediatrics and the
Georgia Research Alliance. We are thankful to the Human Immune
Monitoring Center (HIMC) for assisting with sample shipments.
We thank G. Kim and M. Blanco from the Stanford Functional
Genomics Facility at Stanford University for assistance with single-
cell RNA-seq and the Yerkes Nonhuman Primate (NHP) Genomics
Core [supported in part by National Institutes of Health (NIH) grant
P51 OD011132]. We thank the HIMC and the Parker Institute for
Cancer Immunotherapy (PICI) for maintenance and access to the
flow cytometer. We acknowledge the support of the clinicians
who facilitated this study, including J. Y. H. Chan, D. P.-L. Lau, and
Y. M. Ho, and the dedicated clinical team at the Infectious
Diseases Centre, Princess Margaret Hospital, Hospital Authority
of Hong Kong. We used equipment purchased with NIH grants
(S10OD018220 and 1S10OD021763) to generate the data.
Funding:This work was supported by NIH grants HIPC U19AI090023
(to B.P.), U19AI057266 (to B.P. and principal investigator
R. Ahmed from Emory University), and U24AI120134 (to S.E.B.);
the Sean Parker Cancer Institute; the Soffer endowment
(to B.P.); the Violetta Horton endowment (to B.P.); a Calmette
and Yersin scholarship from the Pasteur International Network
Association (to H.L.); the National Natural Science Foundation of
China (NSFC)–Research Grants Council (RGC) Joint Research
Scheme (N_HKU737/18) (to C.K.P.M. and M.P.); the Guangzhou
Medical University High-level University Innovation Team
Training Program [Guangzhou Medical University released (2017)
no. 159] (to C.K.P.M. and M.P.); the U.S. NIH (contract no.
HHSN272201400006C) (to M.P.); and the RGC of the
Hong Kong Special Administrative Region, China (project no.
T11-712/19-N) (to M.P.). Next-generation sequencing services
were provided by the Yerkes NHP Genomics Core, which is
supported in part by NIH P51 OD 011132, and the data were
acquired on a NovaSeq 6000 funded by NIH S10 OD 026799.
Author contributions:Conceptualization: B.P., P.S.A., and F.W.;
Investigation: P.S.A., F.W., C.K.P.M., M.P., N.S., Y.F., L.B., D.W.,
J.C., K.L.P., G.A., C.H., and M.P.M.; Data curation and analysis:
P.S.A., F.W., M.S., T.H., N.S., Y.F., D.K., A.A.U., H.T.M., and B.P.;
Patient recruitment and clinical data curation: C.K.P.M., M.P.,
L.B., O.T.-Y.T., G.A., W.S.L., J.M.C.C., T.S.H.C., C.Y.C.C., C.H., M.P.M.,
H.L., E.A., S.E., N.R., and M.P.; Supervision: B.P., M.P., N.R., P.K.,
H.T.M., S.E.B., and E.A.; Data visualization: P.S.A., F.W., M.S., and T.H.;
Writing: P.S.A., F.W., M.S., T.H., and B.P.; Funding acquisition:
B.P. All the authors read and accepted the manuscript.Competing
interests:B.P. and P.S.A. are inventors on a provisional patent
application (no. 63/026,577) submitted by the Board of Trustees
of the Leland Stanford Junior University, Stanford, CA, that
covers the use of“Therapeutic Methods for Treating COVID-19
Infections.”Data and materials availability:The CITE-seq data andArunachalamet al.,Science 369 , 1210–1220 (2020) 4 September 2020 10 of 11
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