IL-6 and IL-10 Levels, Rather Than Viral Load and Neutralizing Antibody Titers, Determine the Fate of Patients With Severe Fever With Thrombocytopenia Syndrome Virus Infection in South Korea

Severe fever with thrombocytopenia syndrome (SFTS) is a new tick-borne viral disease, and most SFTS virus (SFTSV) infections occur via bites from the tick Haemaphysalis longicornis; however, SFTSV transmission can also occur through close contact with an infected patient. SFTS is characterized by acute high fever, thrombocytopenia, leukopenia, elevated serum hepatic enzyme levels, gastrointestinal symptoms, and multiorgan failure and has a 16.2 to 30% mortality rate. In this study, we found that age, dyspnea rates, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase, multiorgan dysfunction score (MODS), viral load, IL-6 levels, and IL-10 levels were higher in patients with fatal disease than in patients with nonfatal disease during the initial clinical course of SFTS. In addition, we found that IL-6 and IL-10 levels, rather than viral load and neutralizing antibody titers, in patients with an SFTSV infection strongly correlated with outcomes (for severe disease with an ultimate outcome of recovery or death).

Most SFTSV infections occur via bites from the tick Haemaphysalis longicornis; however, SFTSV transmission can also occur through close contact with an infected patient (9). SFTS is characterized by acute high fever, thrombocytopenia, leukopenia, elevated serum hepatic enzyme levels, gastrointestinal symptoms, and multiorgan failure and has a 16.2 to 30% mortality rate (1,3,6,10).
In this study, we report the clinical and laboratory variables and clinical outcomes of confirmed SFTS patients with nonfatal and fatal disease from 2013 to 2019 in South Korea and show that age, dyspnea rates, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase, multiorgan dysfunction score (MODS), viral load, IL-6 levels, and IL-10 levels were higher in patients with fatal disease than in patients with nonfatal disease during the initial clinical course of SFTS.
In addition, we found that systemic IL-6 and IL-10 levels in patients with an SFTSV infection more strongly correlated with outcomes (for severe disease with an ultimate outcome of recovery or death) than did viral load and neutralizing antibodies.

MATERIALS AND METHODS
We confirmed 62 SFTS patients treated at a single tertiary hospital on Jeju Island from April 2013 to December 2019 (case fatality rate (CFR = 11.2%), and 54 SFTS patients were enrolled in the study ( Table 1).
To investigate demographic, clinical, and laboratory variables, including SFTS viral loads (Ct value) and the levels of cytokines (obtained during the first visit to the hospital), we collected 155 serum samples from 54 patients (patients with nonfatal disease: n = 47, mean age: 61.4 ± 14.4; patients with fatal disease: n = 7, mean age: 72.0 ± 9.9, CFR = 12.96%). Laboratory variables were confirmed in the Laboratory Department of Jeju National University Hospital ( Table 1). This study was approved by the Institutional Review Board (IRB) of Jeju National University Hospital (IRB file no. 2018-11-002).
For molecular diagnosis of SFTSV and measurements of viral load, RNA was extracted from stored patient serum (155 serum samples from 54 patients) using a QIAamp Viral RNA Mini kit (QIAGEN, Hilden, Germany). Real-time one-step RT-PCR was performed using an Ezplex ® SFTS virus Real-time PCR Kit ( S M L GE N E T R E E , S o u t h Ko r e a ) ac c o r d i n g t o t h e manufacturer's instructions. The patients were confirmed within one day in the hospital.
To characterize the effect of SFTSV infection on the production of serum cytokines in SFTS patients, interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor (TNF-a), and interferon-g (IFN-g) 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 analyzed by FCAP Array software version 3.0 (BD Bioscience, San Diego, CA).
All statistical analyses were performed using SPSS version 20.0 (IBM Corp., Armonk, NY, USA). P values < 0.05 indicated statistical significance. To compare the mean difference between patients with fatal and nonfatal disease, we usually used a twosamples t-test. When using this method, we checked some assumptions, such as normality, equal variance, and independence. In this case, the two groups had quite different sample sizes (n = 47 and n = 7), and the normality assumption for each group did not hold. We used a nonparametric twosample t-test called the Wilcoxon-Mann-Whitney test (Tables 1 and 2).
We also studied the kinetics of the viral load and cytokine levels and compared them with the titer of neutralizing antibodies, which was previously shown to differ between patients with fatal severe disease and patients with nonfatal severe disease (11).
In patients with nonfatal severe disease, the levels of IL-6 and IL-10 were lower, and the viral load was higher than those of patients with fatal severe disease and decreased over time. In our previous paper, we showed that the titer of neutralizing antibodies in patients with nonfatal severe disease increased over time, although one patient with nonfatal severe disease did not produce neutralizing antibodies, similar to patients with fatal severe disease (Tables 3-1, 3-2).

DISCUSSION
IL-6, a proinflammatory cytokine, is essential for escalating the cell response to control persistent viral infection, and expression of IL-10, an important anti-inflammatory cytokine, is significantly elevated in SFTS patients, especially in patients with fatal disease.
The overproduction of IL-6 and IL-10 can create a cytokine storm, which is considered to contribute to the pathology of SFTS (12,13).
In this study, high levels of IL-6 and IL-10 and high viral loads were found to coexist in patients with fatal and nonfatal disease.
In addition, IL-6 and IL-10 levels were higher in patients with fatal severe disease than in patients with nonfatal severe disease, and the levels of these cytokines were both decreased in patients with nonfatal severe disease.
The viral load was higher in patients with nonfatal severe disease than in patients with fatal severe disease at the first visit to the hospital and decreased over time. The titers of neutralizing antibodies for some patients with nonfatal severe disease was lower than that of patients with fatal severe disease at the first visit to the hospital but increased over time. However, one patient did not produce neutralizing antibodies such as a patient with fatal severe disease.
Therefore, we suggest that IL-6 and IL-10 determine the fate of patients (for severe disease with an ultimate outcome of recovery or death) more than viral load and the titer of neutralizing antibodies.
The limitations of our study include the relatively small number of patients studied (n = 54). However, this is a rigorous prospective study that took 7 years (from 2013 to 2019) in a representative hospital for the treatment of SFTS on Jeju Island, South Korea.
In summary, we reported that the levels of serum IL-6 and IL-10 were elevated in patients with fatal severe disease, while the  levels these cytokines decreased in patients with nonfatal severe disease. This suggests that IL-6 and IL-10, rather than viral load and the titer of neutralizing antibodies, play an important role in determining the fate of patients.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
This study was approved by the Institutional Review Board (IRB) of Jeju National University Hospital (IRB file no. 2018-11-002). The patients/participants provided their written informed consent to participate in this study. Unit: pg/ml, *The hospitalization and sampling date; P, positive; N, negative; FRNT50, 50% focus reduction neutralization test.