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\begin{document}

\title{Fatal outcome of Severe Fever With Thrombocytopaenia Syndrome (SFTS) and
Severe and Critical COVID-19 is associated with the hyperproduction of
IL-10 and IL-6 and the low production of TGF-\selectlanguage{greek}β\selectlanguage{english}}



\author[1]{Keun Hwa Lee}%
\author[1]{Su Yeon Kang}%
\author[2]{Jeong Rae Yoo}%
\author[3]{Yejin Park}%
\author[4]{So-Hee Kim}%
\author[2]{Sang Taek  Heo}%
\author[1]{Seong Hyeon Park}%
\author[2]{Misun Kim}%
\author[5]{Tae-Jin Kim}%
\author[4]{Songhyeok Oh}%
\author[6]{Moo-Seung Lee}%
\author[1]{Jung Mogg Kim}%
\author[4]{Nam-Hyuk Cho}%
\author[3]{Kyung-Mi Lee}%
\affil[1]{Hanyang University College of Medicine}%
\affil[2]{Jeju National University College of Medicine and Graduate School of Medicine}%
\affil[3]{Korea University College of Medicine}%
\affil[4]{Seoul National University College of Medicine}%
\affil[5]{Korea Institute of Radiological and Medical Sciences Library}%
\affil[6]{Korea Research Institute of Bioscience and Biotechnology}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Severe fever with thrombocytopaenia syndrome virus (SFTSV) and Severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause the
hyperproduction of inflammatory cytokines, which have pathological
effects in patient including severe or fatal cytokine storms. To
characterize the effect of SFTSV and SARS-CoV-2 infection on the
production of cytokines in SFTS and COVID-19 patients, we performed an
analysis of cytokines in SFTS and COVID-19 patients and also
investigated the role of IL-10 \emph{in vitro} studies: LPS-induced
THP-1-derived macrophages, SFTSV infection of THP-1 cells, and
SARS-CoV-2 infection of THP-1 cells. In this study, we found that levels
of both IL-10 and IL-6 were significantly elevated, the level of TGF-\selectlanguage{greek}β
\selectlanguage{english}was significantly decreased and IL-10 was elevated earlier than IL-6 in
severe and critical COVID-19 and fatal SFTS patients, and inhibition of
IL-10 signalling decreased the production of IL-6 and elevated that of
TGF-\selectlanguage{greek}β. \selectlanguage{english}Therefore, the hyperproduction of IL-10 and IL-6 and the low
production of TGF-\selectlanguage{greek}β \selectlanguage{english}have been linked to cytokine storm-induced mortality
in fatal SFTS and severe and critically ill COVID-19 patients and that
IL-10 can play an important role in the host immune response to severe
and critical SARS-CoV-2 and fatal SFTSV infection.%
\end{abstract}%



\sloppy


\textbf{1. INTRODUCTION}

Severe fever with thrombocytopaenia syndrome virus (SFTSV, officially
named \emph{Dabie bandavirus} ) an emerging tick-borne virus in Asia,
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and avian
influenza A (H5N1) viruses can cause severe and often fatal disease that
is characterized by hyperinflammation with features of cytokine
storm.\textsuperscript{{[}1-5{]}}

The cytokine storm is a common pathogenic characteristic, namely,
imbalanced immune responses with an exaggerated inflammatory cytokine
reaction, excessive activation of immune cells, and life-threatening
systemic inflammatory syndromes, which can cause serious pathological
changes and result in multiorgan
dysfunction.\textsuperscript{{[}1-11{]}}

Interleukin 6 (IL-6) is a proinflammatory cytokine with pivotal roles in
inflammation and a key cytokine in cytokine syndrome-induced
mortality.\textsuperscript{{[}5-11{]}}

IL-10 is a regulatory cytokine with pleiotropic roles in the immune
system and is known to be an important immunoregulatory
cytokine.\textsuperscript{{[}12{]}} However, fatal severe fever with
thrombocytopaenia syndrome (SFTS) and severe and critical coronavirus
disease 2019 (COVID-19) had significantly higher serum levels of IL-10
than those of patients with mild to moderate and nonfatal illness; in
addition, systemic hyperproduction of both IL-6 and IL-10 can generate a
cytokine storm, which contributes to their pathology and is more
strongly correlated with the outcome of death.\textsuperscript{{[}5. 7.
9.10{]}}

Transforming growth factor-\selectlanguage{greek}β \selectlanguage{english}(TGF-\selectlanguage{greek}β\selectlanguage{english}) is a regulatory cytokine with
pivotal functions in the control of inflammation. SARS-CoV-2 induces a
TGF-\selectlanguage{greek}β-\selectlanguage{english}dominated chronic immune response in severe COVID-19 while TGF-\selectlanguage{greek}β
\selectlanguage{english}was downregulated in SFTS patients compared with healthy
controls.\textsuperscript{{[}12-15{]}}

In this study, we found that the levels of IL-6 and IL-10 were
significantly higher and produced at robust levels in fatal SFTS
patients and severe and critically ill COVID-19. In contrast, TGF-\selectlanguage{greek}β \selectlanguage{english}was
significantly lower in fatal SFTS and severe and critical COVID-19
patients than in patients with nonfatal SFTS patients and mild to
moderate COVID-19. Namely, elevated levels of IL-6 and IL-10 and
decreased levels of TGF-\selectlanguage{greek}β \selectlanguage{english}have been linked to severe inflammation
and~fatality SFTS and in COVID-19 patients.

We also found that IL-10 is elevated earlier than IL-6 and TGF-\selectlanguage{greek}β, \selectlanguage{english}and
the blocking of IL-10 signalling using an antibody against the IL-10
receptor can reduce IL-6 production and increase TGF-\selectlanguage{greek}β \selectlanguage{english}production in
LPS-induced, SFTSV and SARS-CoV-2 -infected immune cells, respectively,
suggesting that IL-10, IL-6 and TGF-\selectlanguage{greek}β \selectlanguage{english}may contribute to disease severity
in SFTS COVID-19 and patients.

Therefore, IL-10 plays an important role in the host immune response to
severe and critical SARS-CoV-2 and fatal SFTSV infection, and these
results demonstrated that targeting IL-10 signalling using a monoclonal
antibody against the IL-10 receptor is a potential immune-based
intervention against fatal SFTS and severe and critically ill COVID-19
disease.\textsuperscript{{[}7, 10{]}}

\textbf{2. MATERIAL and METHODS}

\textbf{2.1 SFTS patients}

We performed a retrospective study on eligible patients with SFTS from
May 2013 to April 2022. During the study period, 84 patients were
confirmed to be positive for partial small (S) and large (L) segments of
SFTSV RNA using real-time PCR.\textsuperscript{{[}16{]}}

Of these patients, 65 confirmed patients were analysed in the present
study (\textbf{Supplemental table 1} ). The study was approved by the
Institutional Review Board (IRB) at the Jeju National University
Hospital (IRB file no. 2021-03-012) and the study design and baseline
characteristics of SFTS patients are available in
the\textbf{Supplemental Data 1} .

\textbf{2.2 COVID-19 patients}

We performed a retrospective study on eligible patients with COVID-19
from August 2020 to July 2021.

During the study period, 188 confirmed patients were admitted, and 109
of these patients were analysed in the present study
(\textbf{Supplemental table 2} ). The study was approved by the
Institutional Review Board (IRB) at the Jeju National University
Hospital (IRB file no. 2020-10-019), and the study design and baseline
characteristics of COVID-19 patients are available in
the\textbf{Supplemental Data 2} .

\textbf{2.3 Analyses of cytokines in SFTS and COVID-19 patients}

To characterize the effect of SFTSV and SARS-CoV-2 infection on the
production of serum cytokines in SFTS and COVID-19 patients, Interleukin
(IL)-2, IL-4, IL-6, IL-10, IL-17A, interferon-\selectlanguage{greek}γ \selectlanguage{english}(IFN-\selectlanguage{greek}γ\selectlanguage{english}), and tumour
necrosis factor (TNF-\selectlanguage{greek}α\selectlanguage{english}) were measured using human Th1/Th2/Th17 CBA kits
(BD Bioscience, San Diego, CA) according to the manufacturer's
instructions, with minor modifications. Sample acquisitions were
performed with a FACS Canto II flow cytometer and analysed by FCAP Array
software version 3.0 (BD Bioscience). All statistical analyses were
performed using SPSS 22.0 (SPSS, an IBM Company). TGF-\selectlanguage{greek}β \selectlanguage{english}was measured in
the collected serum using a TGF-\selectlanguage{greek}β-\selectlanguage{english}1 Human ELISA Kit (Thermo Fisher
Scientific) according to the manufacturer's protocols.

To compare the mean difference between patients with fatal and nonfatal
SFTS disease and between patients with severe and critical and mild to
moderate COVID-19 disease, we usually used a two-sample t-test. When
using this method, we checked some assumptions, such as normality, equal
variance, and independence. When these assumptions were not met, we used
a nonparametric two-sample t-test called the Wilcoxon-Mann-Whitney
test.\textsuperscript{{[}7{]}} \emph{P} values\textless{}0.05 indicated
statistical significance.

\textbf{2.4 THP-1 cells, SFTSV, and SARS-CoV-2}

The human monocytic cell line THP-1 (ATCC TIB-202) was used to model
macrophages. THP-1 cells were cultured in RPMI-1640 medium (Gibco)
supplemented with 10\% foetal bovine serum (FBS) (Gibco), 1\%
penicillin-streptomycin (Gibco), 200 mM L-Glutamin (Gibco) and 55 mM
2-mercaptoethanol (Gibco) and kept in a humidified 5\%
CO\textsubscript{2} incubator at 37\selectlanguage{ngerman}°C.

THP-1 cells were differentiated into a macrophage phenotype at a density
of 2-4 × 10\textsuperscript{5} cells/mL in 100mm Cell Culture Dish
(Corning Ins.), treated with 100 ng/mL phorbol myristate acetate (PMA)
(Sigma-Aldrich) for 24 hours (h), washed and suspended in culture medium
without PMA.

SFTSV (GenBank accession no. MN329148-MN329150) was isolated from a
Korean SFTS patient. The virus was propagated and titrated in Vero E6
cells (ATCC CRL-1586), which were cultured in Dulbecco's modified
Eagle's medium (DMEM) (Gibco) supplemented with 10\% FBS.

SARS-CoV-2 /BA.1.1 was isolated from a nasopharyngeal swab taken from a
patient with COVID-19. The virus was propagated and titrated in Vero E6
cells (ATCC CRL-1586), which were cultured in Dulbecco's modified
Eagle's medium (DMEM) (Gibco) supplemented with 2\% FBS and 1\%
penicillin\selectlanguage{english}-streptomycin (Gibco).\textsuperscript{{[}17{]}}

\textbf{2.5 LPS-induced THP-1-derived macrophages to investigate the
role of IL-10 and IL-6.}

THP-1 cells were divided into three different treatment groups: THP-1
cells treated with LPS (2 \selectlanguage{greek}μ\selectlanguage{english}g/mL) (Sigma-Aldrich) as the control group;
cells treated with LPS (2 \selectlanguage{greek}μ\selectlanguage{english}g/mL) (Sigma-Aldrich) and IL-6R polyclonal
antibody (10 \selectlanguage{greek}μ\selectlanguage{english}g/mL) (Invitrogen); and cells treated with LPS (2 \selectlanguage{greek}μ\selectlanguage{english}g/mL,
Sigma-Aldrich, St Louis, USA) and IL-10RA polyclonal antibody (10 \selectlanguage{greek}μ\selectlanguage{english}g/mL)
(Invitrogen).

The three different treatment groups were stimulated for 6, 12, 24 and
48 h, and the levels of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-\selectlanguage{greek}γ, \selectlanguage{english}and
TNF-\selectlanguage{greek}α \selectlanguage{english}were measured in the collected supernatants using human
Th1/Th2/Th17 CBA kits (BD Bioscience) according to the manufacturer's
instructions, with minor modifications. Sample acquisitions were
performed with a FACS Canto II flow cytometer and analysed by FCAP Array
software version 3.0 (BD Bioscience). TGF-\selectlanguage{greek}β \selectlanguage{english}was measured in the
collected supernatants using a TGF-\selectlanguage{greek}β-\selectlanguage{english}1 Human ELISA Kit (Thermo Fisher
Scientific) according to the manufacturer's
protocols.\textsuperscript{{[}7{]}}

\textbf{2.6 Investigating the role of IL-10 in SFTSV infected
THP-1-derived macrophages.}

To investigate the role of IL-10 in SFTSV-infected THP-1 cells, THP-1
cells were infected with SFTSV at a MOI of 1, and SFTSV-infected THP-1
cells were divided into two different treatment groups: SFTSV-infected
THP-1 cells were used as the control group, and SFTSV-infected THP-1
cells were treated with the IL-10RA polyclonal antibody (10 \selectlanguage{greek}μ\selectlanguage{english}g/ml)
(Invitrogen). The two different treatment groups were incubated for 6,
12, 24 and 48 h, and the levels of IL-2, IL-4, IL-6, IL-10, IL-17A,
TNF-\selectlanguage{greek}α, \selectlanguage{english}and IFN-\selectlanguage{greek}γ \selectlanguage{english}were measured using human Th1/Th2/Th17 CBA kits (BD
Bioscience) according to the manufacturer's instructions, with minor
modifications. Sample acquisitions were performed with a FACS Canto II
flow cytometer and analysed by FCAP Array software version 3.0 (BD
Bioscience). TGF-\selectlanguage{greek}β \selectlanguage{english}was measured in the collected supernatants using a
TGF-\selectlanguage{greek}β-\selectlanguage{english}1 Human ELISA Kit (Thermo Fisher Scientific) according to the
manufacturer's protocols.\textsuperscript{{[}7. 18{]}}

\textbf{3. RESULTS}

\selectlanguage{greek}\textbf{3.1 Levels of serum IL-6, IL-10, and TGF-β in SFTS and COVID-19
patients.}\selectlanguage{english}

Among SFTS patients, serum IL-6 and IL-10 concentrations in those with
fatal disease were significantly higher than those in patients with
nonfatal disease, and TGF-\selectlanguage{greek}β \selectlanguage{english}concentrations in the former were
significantly lower than those in the latter during the initial clinical
course of hospitalization (\textbf{Figure 1} and \textbf{Supplemental
table 3} ).

However, there were no statistically significant differences in serum
levels of IL-2, IL-4, IL-17A, IFN-\selectlanguage{greek}γ, \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}between patients with
fatal disease and those with nonfatal disease (\textbf{Supplemental
figure 1} and \textbf{Supplemental table 3} ).\textsuperscript{{[}7{]}}

In COVID-19 patients, serum IL-6 and IL-10 concentrations in patients
with severe and critical disease were significantly higher than those in
patients with mild to moderate disease, and TGF-\selectlanguage{greek}β \selectlanguage{english}concentrations in
patients with severe and critical disease were significantly lower than
those in patients with mild to moderate disease during the initial
clinical course of hospitalization (\textbf{Figure 2}
and\textbf{Supplemental table 4} ).

However, similar to the results of SFTS patients, there were no
statistically significant differences in plasma levels of IL-2, IL-4,
IL-17A, IFN-\selectlanguage{greek}γ, \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}between patients with mild to moderate and
severe/critical disease (\textbf{Supplemental figure 2}
and\textbf{Supplemental table 4} ).

In this study, we found that the levels of serum IL-6, IL-10, and TGF-\selectlanguage{greek}β
\selectlanguage{english}were significantly associated with the outcomes of patients with SFTSV
and SARS-CoV-2 (\textbf{Figures 1} and\textbf{2}
).\textsuperscript{{[}7, 9, 10, 14, 15{]}.}

\selectlanguage{greek}\textbf{3.2 Kinetics of IL-6, IL-10, and TGF-β in SFTS and COVID-19
patients.}\selectlanguage{english}

We studied the kinetics of the IL-6, IL-10, and TGF-\selectlanguage{greek}β \selectlanguage{english}levels in patients
with nonfatal SFTS and severe COVID-19 patients, and the results showed
that IL-10 is elevated earlier than IL-6 (\textbf{Tables 1}
and\textbf{2} ).\textsuperscript{{[}7{]}} In contrast, TGF-\selectlanguage{greek}β \selectlanguage{english}is
decreased later (\textbf{Tables 1} and \textbf{2} ).

Therefore, we studied the correlation between IL-6, IL-10, and TGF-\selectlanguage{greek}β \selectlanguage{english}in
LPS-induced THP-1-derived macrophages treated with IL-6R and IL-10RA
polyclonal antibodies. The results showed that the level of IL-6 was
decreased and that of TGF-\selectlanguage{greek}β \selectlanguage{english}increased when we treated with the IL-10RA
polyclonal antibody (\textbf{Figure 3} and \textbf{Supplemental table 5}
).

However, there were no significant differences between IL-6, IL-10, and
TGF-\selectlanguage{greek}β \selectlanguage{english}in LPS-induced THP-1-derived macrophages treated with the IL-6R
polyclonal antibody (\textbf{Figure 3} and \textbf{Supplemental table 5}
).

We also treated THP-1-derived macrophages infected with SFTSV and
SARS-CoV-2 with the IL-10RA polyclonal antibody to determine the role of
IL-10 in SFTSV and SARS-CoV-2 infection and the results were similar to
those for LPS-induced THP-1-derived macrophages: the level of IL-6 was
decreased and that of TGF-\selectlanguage{greek}β \selectlanguage{english}increased (\textbf{Figures 4} and
\textbf{5}and \textbf{Supplemental table 6 and 7} ).

\textbf{4. DISCUSSION}

Fatal SFTS and severe and critically ill COVID-19 patients develop a
pathological state termed cytokine release
syndrome.\textsuperscript{{[}1, 3-11{]}}

Cytokine release syndrome can be triggered by infections and is
characterized by rapid and prolonged systemic elevation of inflammatory
cytokines and chemokines, and IL-6 is a proinflammatory cytokine and a
key cytokine in cytokine release syndrome-induced
mortality.\textsuperscript{{[}1, 3-11{]}}

Serum IL-10 is an important anti-inflammatory
cytokine.\textsuperscript{{[}12{]}} However, the serum IL-10
concentration was significantly higher in fatal SFTS, severe and
critically ill COVID-19, and H5N1 patients and, like IL-6, can predict
poor outcomes in SFTS and COVID-19 patients (\textbf{Figures 1}
and\textbf{2} ).\textsuperscript{{[}5, 7, 9, 10{]}}

In this study, we found that TGF-\selectlanguage{greek}β \selectlanguage{english}concentrations were significantly
lower in fatal SFTS and severe and critical COVID-19 patients
(\textbf{Figures 1} and \textbf{2} ). Namely, the hyperproduction of
IL-6 and IL-10, which is a feature of cytokine storms, and the low
production of TGF-\selectlanguage{greek}β \selectlanguage{english}have been linked to cytokine storm-induced mortality
in fatal SFTS and severe and critically ill COVID-19 patients
(\textbf{Figures 1}and \textbf{2} ).

Furthermore, IL-10 was elevated earlier than IL-6, and TGF-\selectlanguage{greek}β \selectlanguage{english}was
decreased later than IL-10 in SFTS and COVID-19 patients (\textbf{Tables
1} and \textbf{2} ).

When we blocked IL-10 signalling using an antibody against the IL-10
receptor, the production of IL-6 was decreased, and the production of
TGF-\selectlanguage{greek}β \selectlanguage{english}was increased (\textbf{Figures 3, 4, and 5}
and\textbf{Supplemental table 5, 6,} and \textbf{7} ).

IL-10 is usually known as an anti-inflammatory cytokine {[}12{]}.
However, IL-10 can also be an immune-activating and proinflammatory
cytokine in some autoimmune diseases, cancers, and severe and critically
ill COVID-19 patients. Patients with fatal SFTS and H5N1 present with
dramatically elevated serum IL-10 concentrations that correlate with
disease severity.\textsuperscript{{[}5, 7, 9-11{]}}

Fatal SFTS and severe and critically ill COVID-19 patients present with
dramatically elevated serum levels of IL-10 and IL-6 and dramatically
decreased serum levels of TGF-\selectlanguage{greek}β \selectlanguage{english}that correlate with disease severity.

When we blocked the signal of IL-10 using an antibody against the IL-10
receptor, IL-6 was decreased and TGF-\selectlanguage{greek}β \selectlanguage{english}was elevated in the THP-1-cell
study (\textbf{Figures 3-5} ).

Therefore, we suggest that IL-10 can induce the production of IL-6 and
inhibit the production of TGF-\selectlanguage{greek}β \selectlanguage{english}in cytokine storms and might play a
pathological role in SFTS and COVID-19 disease progression and also
propose that IL-10 may be a potential target for reducing SFTS and
COVID-19 mortality.

\textbf{5. CONCLUSION}

In conclusion, our findings demonstrated that IL-10 is a potential
target for the treatment of SFTSV and SARS-CoV-2-related
immunopathology.

Therefore, blockade of IL-10 signalling using monoclonal antibodies
against the IL-10 receptor is a promising therapeutic for treating fatal
SFTS and severe and critically ill COVID-19
patients.\textsuperscript{{[}7, 10{]}}

\textbf{ACKNOWLEDGEMENTS}

This study was supported by grants from the National Research Foundation
of Korea (NRF) and the Ministry of Science, ICT, and Future Planning
(grant number: NRF-2021R1A2C2091578).

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\textbf{Figure legends}

\textbf{Figure 1.} The serum levels of IL-6, IL-10, and TGF-\selectlanguage{greek}β \selectlanguage{english}in SFTS
patients with nonfatal and fatal diseases. The serum concentrations of
IL-6, IL-10, and TGF-\selectlanguage{greek}β \selectlanguage{english}from SFTS patients (total 65; nonfatal (\emph{n}
=58) and fatal (\emph{n} =7)) were analysed immediately after hospital
admission. Each dot shows the cytokine concentration in an individual,
and the horizontal bars indicate the respective group median.

\textbf{Figure 2.} The serum levels of IL-6, IL-10, and TGF-\selectlanguage{greek}β \selectlanguage{english}between
mild to moderate patients and severe and critically ill COVID-19
patients. The serum concentrations of IL-6, IL-10, and TGF-\selectlanguage{greek}β \selectlanguage{english}from
COVID-19 patients (total 109; mild (\emph{n} =40), moderate (\emph{n}
=40) and severe (\emph{n} =27) and critical (\emph{n} =2)) were analysed
immediately after hospital admission. Each dot shows the cytokine
concentration in an individual, and the horizontal bars indicate the
respective group median.

\textbf{Figure 3.} The lipopolysaccharide (LPS)-induced IL-6
concentration in THP-1 cells is suppressed by the IL-10RA polyclonal
antibody, and the LPS-induced TGF-\selectlanguage{greek}β \selectlanguage{english}concentration in THP-1 cells is
induced by the IL-10RA polyclonal antibody. Human monocyte THP-1 cells
were treated with LPS (10 \selectlanguage{greek}μ\selectlanguage{english}g/mL), LPS plus IL-10RA polyclonal antibody
(10 \selectlanguage{greek}μ\selectlanguage{english}g/mL) or LPS plus IL-6R polyclonal antibody for 6, 12, 24 and 48 h,
and the levels of IL-6 and IL-10 were measured using human Th1/Th2/Th17
CBA kits (BD Bioscience, San Diego, CA). TGF-\selectlanguage{greek}β \selectlanguage{english}was measured in the
collected supernatants using a TGF-\selectlanguage{greek}β-\selectlanguage{english}1 Human ELISA Kit (Thermo Fisher
Scientific) according to the manufacturer's protocols {[}10{]}.

\textbf{Figure 4.} IL-6 concentration in SFTSV-infected THP-1 cells is
suppressed by IL-10RA polyclonal antibody and TGF-\selectlanguage{greek}β \selectlanguage{english}concentration in
SFTSV-infected THP-1 cells is induced by IL-10RA polyclonal antibody.
Human monocyte THP-1 cells were infected with SFTSV with IL-10RA
polyclonal antibody for 6, 12, 24 and 48 h, and the levels of IL-6 and
IL-10 were measured using human Th1/Th2/Th17 CBA kits (BD Bioscience,
San Diego, CA). TGF-\selectlanguage{greek}β \selectlanguage{english}was measured in the collected supernatants using a
TGF-\selectlanguage{greek}β-\selectlanguage{english}1 Human ELISA Kit (Thermo Fisher Scientific) according to the
manufacturer's protocols {[}10{]}.

\textbf{Figure 5.} IL-6 concentration in SARS-CoV-2-infected THP-1 cells
is suppressed by IL-10RA polyclonal antibody and TGF-\selectlanguage{greek}β \selectlanguage{english}concentration in
SARS-CoV-2-infected THP-1 cells is induced by IL-10RA polyclonal
antibody. Human monocyte THP-1 cells were infected with SARS-CoV-2 or
SARS-CoV-2 with IL-10RA polyclonal antibody for 6, 12, 24 and 48 h, and
the levels of IL-6 and IL-10 were measured using human Th1/Th2/Th17 CBA
kits (BD Bioscience, San Diego, CA). TGF-\selectlanguage{greek}β \selectlanguage{english}was measured in the collected
supernatants using a TGF-\selectlanguage{greek}β-\selectlanguage{english}1 Human ELISA Kit (Thermo Fisher Scientific)
according to the manufacturer's protocols {[}10{]}.

\textbf{Supplemental Figure 1.} The levels of IL-2, IL-4, IL-17A, IFN-\selectlanguage{greek}γ,
\selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}between nonfatal patients and fatal SFTS patients. The serum
concentrations of IL-2, IL-4, IL-17A, IFN-\selectlanguage{greek}γ, \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}from SFTS
patients were analysed immediately after hospital admission. Each dot
shows the cytokine concentration in an individual, and the median with
its range is presented. *The concentrations of IFN-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}in all
fatal patients were similar to those in nonfatal patients except one
fatal patient. This fatal patient had the highest concentration of IFN-\selectlanguage{greek}γ
\selectlanguage{english}(3258 pg/mL) and TNF-\selectlanguage{greek}α \selectlanguage{english}(51.3 pg/mL). IL-10 (145 pg/mL) and IL-6 (4106
pg/mL) levels in this fatal patient were the highest, and TGF-\selectlanguage{greek}β \selectlanguage{english}(51.3
pg/mL) levels were lower than those in nonfatal patients. Therefore, the
IFN-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}data of this fatal patient were not included in the
IFN-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}data shown here.

\textbf{Supplemental Figure 2.} The levels of IL-2, IL-4, IL-17A, IFN-\selectlanguage{greek}γ,
\selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}between mild to moderate patients and severe and critically
ill COVID-19 patients. The serum concentrations of IL-2, IL-4, IL-17A,
IFN-\selectlanguage{greek}γ, \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}from COVID-19 patients were analysed immediately after
hospital admission. Each dot shows the cytokine concentration in an
individual, and the median with its range is presented.

\textbf{Supplemental data 1}

\textbf{Study design and SFTS patients}

We performed a retrospective study on patients with severe fever with
thrombocytopaenia syndrome at Jeju National University Hospital in Jeju
Island, South Korea. This region had the highest incidence of SFTS in
South Korea. The study was approved by the Institutional Review Board
(IRB) at the Jeju National University Hospital (IRB file no.
2021-03-012).

Patients were admitted for confirmed SFTS during May 2013-April 2022,
and these patients were eligible to participate in the study. During the
study period, a total of 84 patients were confirmed to be positive for
small (S) and large (L) segments of SFTSV RNA using real-time RT-PCR,
based on the recommended diagnosis method in South Korea. Of these
confirmed patients, 65 were analysed in the present study.

The demographic and clinical characteristics data were obtained from the
electronic medical records and included the patient demographics,
activity at the time of exposure, history of tick bites, presence of
initial symptoms, vital signs, past medical history, CCI, multiple organ
dysfunction score (MODS) during hospitalization and after 72 h of
treatment (inotropes, intubation, renal replacement therapy, and plasma
exchange), disposition after hospital admission, and 30-day mortality.
The patients' blood samples were collected at the first visit to the
hospital and then at regular intervals (every two days in the acute
phase, twice a week in the recovery phase during hospitalization, and
every 2 to 3 months after discharge).

Patients were divided into the severe group (SG) and nonsevere group
(NSG). The SG comprised patients with three or more organ dysfunctions
as defined by Sequential Organ Failure Assessment (SOFA) score including
the following: for the lung a PaO2/FiO2 \textless{}400 or patients
requiring mechanical ventilation; for the liver a serum bilirubin [?]1.2
mg/dL; for the kidney a serum creatinine [?]1.2 mg/dL, urine output
\textless{}500 mL/day or patients requiring haemodialysis; for the
cardiovascular system a mean arterial pressure \textless{}70 mmHg or
patients requiring vasopressor; and for the central nervous system a
Glasgow Coma Scale \textless{}15, or deceased patients (reference
https://doi.org/10.3947/ic.2022.0073.).

\textbf{Baseline characteristics of patients with SFTS}

A total of 65 patients were included in the study. Table 2 shows the
demographic and baseline clinical characteristics of the patients with
SFTS. Of the patients, 37 (56.9\%) were male, and the mean age was 63.7
\selectlanguage{ngerman}± 14.0 years. All patients were inhabitants of Jeju Island. The mean CCI
was very low at 0.5 ± 0.8. Most patients (85\%) were exposed to the
virus through occupational and outdoor activities, with agricultural
activities being the most common route of exposure. Some patients did
not know whether they had been bitten by ticks. Most cases occurred in
summer, followed by autumn. The mean time from symptoms to diagnosis of
SFTS was 5.8 ± 3.3 days, and most cases were confirmed within two days
of admission. The mean initial MODS was 2.7 ± 2.5, and the mean SFTS
viral load was 82,115,042 ± 643,558,056. The 30-day mortality rate was
13.4\%, and 30 patients (46.2\%) were admitted to the intensive care
unit. Initial laboratory findings revealed neutropaenia,
thrombocytopaenia, increased liver enzymes, increased creatine kinase
(CK), lactate dehydrogenase (LDH), and activated partial thromboplastin
time (aPTT).

In the comparison of the recovered group and death group with SFTS, male
patients and older ages showed a tendency for inclusion in the death
group. There were no significant differences in comorbidities, exposure
type, tick bite history, or seasonal occurrence in either group.
However, the mortality of SFTS showed an increasing trend in the spring
season. A higher SFTS viral load and MODS were associated with the death
group more than with the recovered group. Vasopressor, mechanical
ventilation, and CRRT were applied in the death group. There were no
significant differences in the application of therapeutic plasma
exchange, immunoglobulin, or anti-IL-6 antagonist between the groups.
Most fatal cases were deceased within one week of admission.

The initial levels of neutropaenia and thrombocytopaenia could not
distinguish whether SFTS patients would be included in the recovered
group or death group. However, liver enzymes, aPTT, CK and LDH were
significantly different between the two groups.

\textbf{Supplemental data 2}

\textbf{Study design and COVID-19 patients}

We performed a retrospective study on patients with COVID-19 at Jeju
National University Hospital in Jeju Island, South Korea. This hospital
was designated the national negative pressure isolation ward responsible
for treating patients with high-risk COVID-19 in South Korea. The study
was approved by the Institutional Review Board (IRB) at the Jeju
National University Hospital (IRB file no. 2020-10-019).

Patients admitted from August 2020 to July 2021 and who had confirmed
SARS-CoV-2 infection, were eligible to participate in the study. During
the study period, 430 cases were confirmed to be positive for SARS-CoV-2
using real-time reverse transcriptase polymerase chain reaction (RT-PCR)
on Jeju Island. Of these confirmed patients, 188 patients were admitted
to our hospital, and 109 were analysed in the present study (Table 1).

Demographic and clinical characteristic data included the patients'
demographics (age, sex, and body mass index), past medical history and
comorbidity index score (CCI), source of exposure risk to SARS-CoV-2,
symptom presentation, type of oxygen therapy, hospital treatment
(inotropes, extracorporeal membrane oxygenation), duration of
hospitalization, and mortality. Laboratory variables confirmed in the
Laboratory Department of Jeju National University Hospital were
retrospectively obtained from the registry database.

The severity of illness was classified into the following five groups
according to the National Institute of Health's criteria based on the
patient's worst condition during hospitalization: asymptomatic, mild,
moderate, severe, and critical {[}Reference: National Institutes of
Health (NIH). Clinical spectrum of SARS-CoV-2 infection. Available at:
https://www.covid19treatmentguidelines.nih.gov/overview/clinical-spectrum/.
Accessed 25 June, 2022{]}.

\textbf{Baseline characteristics of COVID-19 patients}

A total of 109 patients were included in the study. Table 1 shows the
demographic and baseline characteristics of the patients according to
the severity of COVID-19. Of all patients, 40 (32.5\%) had mild disease,
40 (17.5\%) had moderate disease, 27 (45\%) had severe disease, and 2
(5\%) had critical disease. The mean age of the patients was 51.1 ± 19.9
years, and 53 (48.6\%) were male. No patients were vaccinated because
COVID-19 vaccination was not introduced in South Korea during the study
period. The mean body mass index (BMI) of the patients was 24.7
kg/m\textsuperscript{2}. The mean Charlson Comorbidity Index (CCI) was
as low as 1.8 ± 1.8. The median time from symptom onset to SARS-CoV-2
RT\selectlanguage{english}-PCR testing was 1 day, and the mean SARS-CoV-2 RT\selectlanguage{english}-PCR cycle threshold
(Ct) value was 22.0 +- 6.8. The type of patient oxygen supplementation
received by patients in the study was as follows: 82 (75.2\%) received
no oxygen supplementation, 21 (19.4\%) received low-flow nasal cannula
therapy and a facial mask with a reservoir, 4 (3.7\%) received high-flow
nasal cannula therapy, and 1 (0.9\%) received invasive mechanical
ventilation. Hydroxychloroquine was administered in 1 (0.9\%),
lopinavir/ritonavir in 21 (19.4\%), remdesivir in 18 (16.7\%), and
corticosteroids in 28 (25.9\%) patients. Total case fatalities were
0.9\%, and the mean number of admission days was 12.5 +- 5.2. In the
comparison of the mild/moderate and severe and critical groups, an older
age, a higher mean BMI, and a higher CCI were associated with the
severe/critical group rather than with the mild/moderate group. There
was no significant differences in the mean SARS-CoV-2 RT\selectlanguage{english}-PCR Ct value
between the mild/moderate and severe/critical groups. High flow and
invasive mechanical ventilation, lopinavir/ritonavir, remdesivir, and
steroids were administered in the severe/critical group compared with
the mid/moderate group. There were no significant differences in the
laboratory results between the groups. However, C-Reactive Protein (CRP)
was higher in the severe/critical group than in the mild/moderate group.
Comparisons of laboratory findings between the groups are presented in
Table 1. Only fatal patients were included in the severe/critical group.

\textbf{Hosted file}

\verb`Figures.pptx`  available at \url{https://authorea.com/users/604993/articles/634633-fatal-outcome-of-severe-fever-with-thrombocytopaenia-syndrome-sfts-and-severe-and-critical-covid-19-is-associated-with-the-hyperproduction-of-il-10-and-il-6-and-the-low-production-of-tgf-%CE%B2}

\textbf{Hosted file}

\verb`TablesKH.docx`  available at \url{https://authorea.com/users/604993/articles/634633-fatal-outcome-of-severe-fever-with-thrombocytopaenia-syndrome-sfts-and-severe-and-critical-covid-19-is-associated-with-the-hyperproduction-of-il-10-and-il-6-and-the-low-production-of-tgf-%CE%B2}

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