Changes in peripheral blood cytokines in patients with severe fever with thrombocytopenia syndrome

Abstract Severe fever with thrombocytopenia syndrome (SFTS) is recognized as an emerging infectious disease. This study aimed to investigate the pathogenic mechanism of SFTS. A total of 100 subjects were randomly included in the study. Cytokine levels were detected by enzyme‐linked immunosorbent assay and the viral load was detected by micro drop digital PCR. The results showed that levels of interleukin‐6 (IL‐6), IL‐8, IL‐10, IFN‐inducible protein‐10 (IP‐10), monocyte chemoattractant protein‐1 (MCP‐1), macrophage inflammatory protein‐1α (MIP‐1α), transforming growth factor‐β1 (TGF‐β1), and regulated upon activation normal T cell expressed and secreted factor (RANTES) differed significantly among the SFTS patient group, healthy people group, and asymptomatic infection group (p < .05). Compared to the healthy people group, the patient group had increased cytokine levels (IL‐6, IL‐10, IP‐10, MCP‐1, and IFN‐γ) but reduced levels of IL‐8, TGF‐β1, and RANTES (p < .0167). IL‐6, IL‐8, IL‐10, IP‐10, MCP‐1, MIP‐1α, TGF‐β1, and the RANTES levels had different trends after the onset of the disease. IL‐6, IL‐10, IP‐10, and MCP‐1 levels in severe patients were higher than those in mild patients (p < .05). There was a positive correlation between viral load and IL‐6 and IP‐10 but a negative correlation between viral load and RANTES. SFTSV could cause a cytokine change: the cytokine levels of patients had different degrees of fluctuation after the onset of the disease. The levels of IL‐6 and IL‐8 in the asymptomatic infection group were found between the SFTS patients group and the healthy people group. The levels of IL‐6, IL‐10, IP‐10, and MCP‐1 in the serum could reflect the severity of the disease, and the levels of IL‐6, IP‐10, and RANTES were correlated with the viral load.


| INTRODUCTION
Severe fever with thrombocytopenia syndrome (SFTS) is a novel emerging infectious disease caused by the SFTS virus (SFTSV), a tick-borne phlebovirus of the family Phenuiviridae in the order Bunyavirales. However, according to the nomenclature by the International Committee of Taxonomy of Viruses (ICTV), SFTSV has been classified into the Genus Banyangvirus, Family Phenuiviridae, and re-named as the Huaiyangshan banyangvirus. In this article, we have referred to it as "SFTSV". 1,2 The clinical symptoms of SFTS patients are characterized by fever, thrombocytopenia, leukopenia, gastrointestinal and neurological symptoms, as well as abnormal concentrations of laboratory parameters. 3,4 As the case-fatality rate of SFTS ranges from 5% to over 40%, with 0.3-11.3% seroprevalence of anti-SFTSV antibodies in healthy individuals living in endemic areas (China, Japan, Korea) and no effective therapies or vaccines, understanding the pathogenic mechanism of SFTS is important for this disease. [5][6][7][8] Cytokines are small molecular polypeptides with immunological activity synthesized and released by activated lymphocytes or some tissue cells. Complications or, ultimately, death arising from virus infections are often associated with hyperinduction of proinflammatory cytokine production, which is also known as a "cytokine storm". 9 The outcomes in SFTS patients include multiple organ dysfunction syndrome (MODS), shock, disseminated intravascular coagulation, and death. 10,11 Cytokine storm has been confirmed to be associated with MODS, and the correlation of cytokine levels between SFTS patients relative to a healthy population was reported in previous studies. 12 -15 In this study, the serum cytokine profile of patients with SFTS infection with symptomatic disease, those with SFTS antibodies, but no history of clinical illness, and healthy individuals were examined to explore whether there are differences in serum cytokine. The correlation between cytokine levels and viral load was measured for the serum samples to investigate the patterns of changes and clinical significance.

| Inclusion criteria
SFTS patients: epidemiological history, clinical signs, and laboratory tests were diagnosed; patients met one or more of the following criteria: (1) the virus was isolated from the patient's samples; (2) SFTSV RNA was detected in the patient's serum; (3) a fourfold or greater increase in antibody titers was detected between paired patient serum samples collected from the acute and convalescent phases of infection. 16 Mild SFTS cases: Low fever (37.2-39°C), mild fatigue, and gastrointestinal symptoms, the platelet count was (50-130) × 10 9 /L, and the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), and lactate dehydrogenase (LDH) were less than two times the upper limit of normal (ULN) values.
Severe SFTS cases: high fever (39-40°C), fatigue, obvious gastrointestinal symptoms, neurological symptoms, the platelet count was (30-50) × 10 9 /L, and sharply elevated (more than five times ULN) of LDH, ALT, AST, and CK. 17 Healthy people: according to the epidemiological history, clinical manifestations, and laboratory test results, the diagnosis should meet one or two of the following criteria: (1) laboratory tests showed that SFTSV nucleic acid; (2) SFTSV immunoglobulin G (IgG) or IgM antibodies were negative in serum samples.
Asymptomatic infected persons: there were no clinical symptoms (such as fever, cough, and sore throat) and the serum samples were positive for SFTSV or serum specific IgM antibody.  | 4705 necrosis factor-α (TNF-α), and regulated upon activation normal T cell expressed and secreted factor (RANTES) were tested. For this ELISA kit, 50 µl of serum samples, twofold diluted standard, and diluted detection antibody were added. Samples were incubated at room temperature for 2 h on a microplate shaker set at 300 revolutions per minute (rpm) and washed with washing buffer, and 100 µl of diluted streptavidin-horseradish peroxidase (HRP) was added. Plates were developed by using a substrate solution after samples were incubated and washed. The detection of antibodies (IgG and IgM) to SFTSV is previously described. 18 2.5 | Detection of viral load by reversetranscriptase droplet digital polymerase chain reaction (RT-ddPCR) One-step RT-ddPCR was performed in a 20 µl reaction volume containing 4 µl of RNA sample, 5 µl of supermix for probes, 2 µl of reverse transcriptase, 0.5 µl of probes, 1.6 µl of target primers, 1 µl of DTT, and 4.3 µl of RNase-free water. The 20-µl reaction volume and 70 µl droplet generation oil for probes were, respectively, loaded into the sample wells and oil wells of a disposable droplet generator cartridge. Then, droplets were generated by a QX200 droplet generator device, and 40 µl of mixed liquid was carefully transferred to a 96-well PCR plate (Bio-Rad). One-step RT-ddPCR amplification was performed with the following conditions: 48°C for 60 min, 95°C for 10 min, 40 cycles of 95°C for 30 s, and 58°C for 1 min followed by 98°C for 10 min.

| Sample and data collection
Additionally, no template control was included in this assay. 19

| Statistical analysis
Statistical analyses were performed using SPSS 21.0 software (SPSS, Inc.). Means and standard deviations (SDs) were described as continuous variables with normal distribution; medians and interquartile (IQ) ranges were used for abnormal distribution. The Shapiro-Wilk test was conducted to test the normal distribution. If data met the criteria for the abnormal distribution, then a nonparametric test was performed to evaluate the differences in cytokine levels. Otherwise, the analysis of variance was used. The categorical variables were calculated with the χ 2 test. The correlations between variables were assessed using the Pearson test. A p value of < .05 was considered to be significant. Besides this, when we used the Bonferroni method for multiple comparisons, the adjusted α value was 0.0167, then p < .0167 was considered to be statistically significant. 20

| Ethics approval and consent to participate
The study was approved by the Ethical Committee of Henan CDC, and the committee's reference number is 2016-KY-002-02. All participants provided written informed consent for use of their samples in research.

| Respondent characteristics
For the SFTS patient group, the mean age was 65.45 ± 9.22 years, and 50 were female. The mean age of the healthy people group was 66.57 ± 10.18 years, with 44 males and 56 females. For the asymptomatic infected person group, the mean age was 64.24 ± 8.98 years, and 39 were males. The results of the analysis of variance showed that there were no significant differences in age, gender, occupation, and marital status among the SFTS patients' group, healthy people group, and asymptomatic infection group (p > .05) ( Table 1).

| Cytokine levels in serum
The levels of 11 cytokines were quantitatively determined in serum samples of each respondent on admission. We analyzed cytokine levels  Horizontal bars indicate the group median. *p < .0167 between the patient and asymptomatic infected person groups. **p < .0167 between the healthy people and patient groups, ***p < .0167 between the healthy people and asymptomatic infected person groups. IFN-γ, interferon-γ; IL-6, interleukin-6; IP-10, IFN-inducible protein-10; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein-1α; RANTES, regulated upon activation normal T cell expressed and secreted factor; SFTS, thrombocytopenia syndrome; TGF-β1, transforming growth factor-β1 highest, and IL-6 levels were the lowest in this group. Compared to the asymptomatic infected group, MCP-1 levels were higher than the control group. Otherwise, TGF-β1 levels were lower than those in the recessive-infected person group (p < .0167).
For the asymptomatic infected person group, serum TGF-β1 levels were the highest among the three groups. RANTES levels were increased in this group compared to the patient group but exhibited no significant difference in the healthy people group (p > .0167) ( Figure 1).

| Dynamic changes of serum cytokines in patients
The cytokine levels in serum samples of 22 mild patients were measured during hospitalization at Days 3, 5, 7, and 9 due to most patients dropping below the measurable level after the tenth day.
IL-8 and IL-10 gradually increased during our observation period, but IP-10 gradually decreased. MIP-1α did not significantly fluctuate after hospitalization. IL-6, MCP-1, TGF-β1, and RANTES had different trends, either increasing first and then decreasing or vice versa.
IFN-γ and TNF-α were not described due to incomplete data ( Figure 2).

| Comparison of cytokine levels in the SFTS patient group
As shown in (Table 3)  Note: Unit, pg/ml.

| Viral load in SFTS patients
The SFTSV viral load in the blood of 53 patients on admission ranged from 0.11 × 10 3 to 6.81 × 10 6 copies/ml. The exponential average RNA

| DISCUSSION
To date, the pathogenesis of SFTSV infection has not been clearly defined. As we know, the immune response plays an important role in the prognosis of infectious diseases, especially viral infections. 21 After consulting the relevant studies, we found that: disease severity of hemorrhagic fever with renal syndrome caused by hantavirus was correlated with some cytokines, including IL-2, IL-6, IL-8, IL-10, TGF- survivors and uninfected controls. 26 Based on these previous studies, we analyzed eleven cytokines associated with SFTSV infection.
IL-2 levels were not detected due to the very low content of IL-2 in SFTS patients, levels of 7-8 pg/ml in healthy people, and lower F I G U R E 3 Correlation between viral load and cytokines. Correlations between variables were assessed using the Pearson test. p < .05 was considered significant. VL, viral load; Lg RNA copies/ml, Log 10 RNA copies/ml than 1 pg/ml in SFTS patients. 14 In the acute phase, both IL-6 and IL-10 were increased in the patient group compared to the healthy people group, possibly because IL-6 is an important factor in inducing B cells to differentiate into plasma cells to produce antibodies upon virus infection, and IL-10 has an anti-inflammatory effect. 27,28 The above results were similar to previously reported studies. 13,14,28 Serum levels of IL-8 were lower in the SFTSV-infected patient group than the control group, possibly because IL-8 functions as an immunological inflammatory regulatory factor in complex cytokine networks of body systems, the result was similar to that of Zhang et al. 29,30 After the onset of the disease, IL-6 had a gradual increase between three and seven days, and IL-8 and IL-10 had the same trends between three and nine days. We inferred that levels of these cytokines saw dramatic changes in the infection process.
Except for RANTES and MIP-1α, the other cytokines were increased in the patient group compared to the healthy people and asymptomatic infected person groups. The significant role of MCP-1 and IP-10 in virus-infected disease progression has been reported. 31 In this study, serum MCP-1 and IP-10 levels in the SFTS patient group were increased compared to the healthy people group. In addition to IP-10, MCP-1 and RANTES had large fluctuations after onset, suggesting that dynamic levels of cytokines in plasma might be closely related to the condition of this disease. Nevertheless, the limitation was that the severe patients in dynamic changes of serum cytokines were not observed due to the short time in hospital and fewer serum samples collected. IP-10 and MCP-1 had significant increases in severe patients compared with mild patients; these observations might be somewhat different from those of a previous study. 3 The correlations between viral load and cytokines suggested that higher numbers of the virus were capable of inducing higher levels of IP-10 and MCP-1 while repressing the production of RANTES to a large extent.
IFN-γ had a remarkable change among the three groups and was significantly elevated in patients with severe symptoms, the result was similar to that of the previous study. 21 TNF-α showed no significant differences among the three groups and the patients with severe and mild symptoms. However, some studies reported that high levels of TNF-α were associated with the severity of the disease, and TNF-α is a pro-inflammatory cytokine known to have a key role in the pathogenesis of chronic immune-mediated diseases. 32,33 Determining the reasons underlying this difference requires our indepth study and analysis.