Fatal outcome of severe fever with thrombocytopenia 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‐β

Severe fever with thrombocytopenia 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 severe fever with thrombocytopenia syndrome (SFTS) and COVID‐19 patients, we performed an analysis of cytokines in SFTS and COVID‐19 patients and also investigated the role of interleukin‐10 (IL‐10) in vitro studies: lipopolysaccharide‐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 transforming growth factor‐β (TGF‐β) 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 signaling decreased the production of IL‐6 and elevated that of TGF‐β. Therefore, the hyperproduction of IL‐10 and IL‐6 and the low production of TGF‐β 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.

fatal disease that is characterized by hyperinflammation with features of cytokine storm. [1][2][3][4][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. [1][2][3][4][5][6][7][8][9][10][11] Interleukin 6 (IL-6) is a proinflammatory cytokine with pivotal roles in inflammation and a key cytokine in cytokine syndromeinduced mortality. [5][6][7][8][9][10][11] IL-10 is a regulatory cytokine with pleiotropic roles in the immune system and is known to be an important immunoregulatory cytokine. 12 However, fatal severe fever with thrombocytopenia 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, is more strongly correlated with the outcome of death, and viral load is not strongly correlated with outcomes (for severe disease with an ultimate outcome of recovery or death) than IL-6 and IL-10 levels in patients with an SFTSV infection and the relationship between mean cycle threshold (C t ) values of real time RT-PCR and severity of disease remain disputable and not clearly defined in COVID-19. 5,7,9,10 Transforming growth factor-β (TGF-β) is a regulatory cytokine with pivotal functions in the control of inflammation. SARS-CoV-2 induces a TGF-β-dominated chronic immune response in severe COVID-19 while TGF-β was downregulated in SFTS patients compared with healthy controls. [12][13][14][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-β 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-β 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-β, and the blocking of IL-10 signaling using an antibody against the IL-10 receptor can reduce IL-6 production and increase TGF-β production in lipopolysaccharide (LPS)-induced, SFTSV and SARS-CoV-2-infected immune cells, respectively, suggesting that IL-10, IL-6, and TGF-β 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 signaling 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. 7,10 2 | MATERIAL AND METHODS

| 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 RT-PCR. 16 Of these patients, 65 confirmed patients were analyzed in the present study (Table S1). 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 Data S1.

| 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 analyzed in the present study (Table S2). The study was approved by the 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 Data S2. 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. 7 p < 0.05 indicated statistical significance.

| Levels of serum IL-6, IL-10, and TGF-β in SFTS and COVID-19 patients
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-β concentrations in the former were significantly lower than those in the latter during the initial clinical course of hospitalization ( Figure 1; Table S3).
However, there were no statistically significant differences in serum levels of IL-2, IL-4, IL-17A, IFN-γ, and TNF-α between patients with fatal disease and those with nonfatal disease ( Figure S1; Table S3). 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-β 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 ( Figure 2; Table S4).
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-γ, and TNF-α between patients with mild to moderate and severe/critical disease ( Figure S2; Table S4).
Therefore, we studied the correlation between IL-6, IL-10, and TGF-β 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-β increased when we treated with the IL-10RA polyclonal antibody (Figure 3; Table S5).
F I G U R E 4 IL-6 concentration in SFTSV-infected THP-1 cells is suppressed by IL-10RA polyclonal antibody and TGF-β 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). TGF-β was measured in the collected supernatants using a TGF-β-1 Human ELISA Kit (Thermo Fisher Scientific) according to the manufacturer's protocols [10].
We also treated THP-1-derived macrophages infected with Serum IL-10 is an important anti-inflammatory cytokine. 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 (Figures 1 and 2). 5,7,9,10 In this study, we found that TGF-β concentrations were significantly lower in fatal SFTS and severe and critical COVID-19 patients (Figures 1 and 2). Namely, the hyperproduction of IL-6 and IL-10, which is a feature of cytokine storms, and the low production of TGF-β have been linked to cytokine storm-induced mortality in fatal SFTS and severe and critically ill COVID-19 patients (Figures 1   and 2).
When we blocked IL-10 signaling using an antibody against the IL-10 receptor, the production of IL-6 was decreased, and the production of TGF-β was increased (Figures 3-5; Table S5-S7).
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. 5,7,[9][10][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-β 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-β was elevated in the THP-1-cell study (Figures 3-5).
Therefore, we suggest that IL-10 can induce the production of IL-6 and inhibit the production of TGF-β 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.

| 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 signaling using monoclonal antibodies against the IL-10 receptor is a promising therapeutic for treating fatal SFTS and severe and critically ill COVID-19 patients. 7

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data used to support the results of this study are available from the corresponding author (Keun Hwa Lee) upon reasonable request.

This study was reviewed and approved by the Local Research Ethics
Committee of the Jeju National University Hospital. Informed consent was obtained from all patients following the principles of the Helsinki Declaration.