Since December 2019, the outbreak of Coronary Virus Disease 2019 (COVID-19) occurred in Wuhan, and rapidly spread across the country, and the world. As the number of diagnoses and deaths continues to rise, this has become the focus of international public health. COVID-19 is highly contagious, and there is no effective treatment. New treatment strategies are urgently needed to improve the treatment success rate of severe and critically ill patients. Increasing evidence has shown that cytokine storm plays an important role in the progression of COVID-19. The artificial liver blood purification system (ALS) is expected to improve the cytokine storm. In the present study, the levels of cytokines and chemokines were detected in 12 COVID-19 patients before and after ALS, and exciting results were obtained. The present study has directly proven that ALS can block the cytokine storm, rapidly remove the inflammatory mediators, and hopefully suppress the progression of the disease, providing a new idea for the clinical treatment of COVID-19.

The present study included 12 critically ill patients with COVID-19, who received treatment with the ALS from January 15, 2020 to March 31, 2020. Each patient’s gender, age, symptoms, complications and disease severity were recorded in detail. A written informed consent was obtained from each patient, or their legally authorized representative. The present study was approved by the Institutional Review Board of the First Affiliated Hospital, School of Medicine, Zhejiang University .

Each patient received three treatment courses with the ALS, and peripheral blood was collected before and right after each treatment course (T1B, T1A, T2B, T2A, T3B and T3A). The EDTA anticoagulated blood was centrifuged at 3,000 rpm for five minutes, and stored at −80°C until assayed.

The magnetic bead-based multiplex immunoassays were developed using the Bio-Plex ProTM Human 48-plex Cytokine Screening Panel, according to manufacturer’s instructions, using the Bio-Plex 200 suspension array system (Bio-Rad, Hercules, CA, USA) in a BSL-2 laboratory. The primary data were analyzed using the Bio-Plex Manager Software, version 6.1.1.

Statistical analysis

The variables were expressed in medians for the statistical analysis. The paired serum cytokine/chemokine results generated at different time points for the same patient were analyzed using the Wilcoxon signed rank test. The statistical analyses were performed using the GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). Statistical analyses of the data were performed using the SPSS software.

Results

Patient information

A personalized ALS was formulated for each patient. The clinical data of the 12 patients are presented in Table 1. There were 10 males and two females, and their median age was 60 years old (range: 36-90 years old). The patient’s coexisting diseases included hypertension (9/12), diabetes (3/12), coronary heart disease (1/12), and arrhythmia (2/12). The incidence of ARDS and renal dysfunction was 12/12 and 4/12, respectively. All 12 patients were admitted to the ICU, seven patients received mechanical ventilation, and three of these patients received extracorporeal membrane oxygenation (ECMO) treatment.

Cytokine/chemokine changes

Each patient received three courses of ALS. Three pairs of blood sample were collected from each patient (T1B-T1A, T2B-T2A and T3B-T3A), and 36 pairs of samples were collected from the 12 patients. It was found that 34 of 48 cytokines and chemokines exhibited significant changes by paired analysis, before and after treatment with the ALS, which included 32 that significantly decreased and two (HGF and IL-12 [p40]) that were significantly upregulated (Figure 1).

The pairwise comparison of cytokines and chemokines levels (T1B vs. T1A, T2B vs. T2A, and T3B vs. T3A) demonstrated that 33 of the cytokines and chemokines exhibited significant changes in response to the ALS, which included 31 that significantly decreased and two (HGF and IL-12 [p40]) that were significantly upregulated. Notably, HGF was significantly upregulated after each treatment (Figure 2).

By pairing analysis, 15 cytokines and chemokines were found to significantly decrease after the first course of treatment with the ALS (T1A vs. T1B, Figure 2), while merely nine remained at lower levels before the second course (T2B vs. T1B, Figure 3). A total of 16 cytokines significantly decreased after the second course of treatment with the ALS (T2A vs. T2B, Figure 2). Compared with T2B, three cytokines significantly increased at T3B. This rapid rebound of cytokine levels indicate the strong cytokine storm in these patients. A total of 20 cytokines significantly decreased after three courses of treatment (T3A vs. T1B). These data indicate that the treatment with the ALS significantly alleviated the cytokine storm, and improved the inflammatory status of the body.

Discussion

The lung is the most common target organ of 2019-nCoV, and most of the infected patients exhibited changes in the images of the lungs, while 29% of these patients were complicated with ARDS^[7]. In the recent study conducted by the investigators, significant cytokine storms have been observed in the COVID-19 patients, and 32 cytokines and chemokines were significantly elevated, compared to healthy controls. The immunologic injury resulted from hypercytokinemia, which may be the critical link in the pathogenesis and deterioration of COVID-19. Hence, the timely control of cytokine storms to reduce inflammatory cell infiltration in the lungs is the key to improve the treatment success rate, and reduce the mortality caused by 2019-nCoV^[8]. The safe and effective elimination of cytokine storms is an important strategy in the treatment of COVID-19. Although a number of studies have shown that blood filtration can eliminate inflammatory cytokines, its effect on the blood level remains limited. The experience of the investigators in treating H7N9-infected patients suggested that the ALS can significantly alleviate the cytokine storm, and plasma exchange（PE） was more effective in removing cytokines/chemokines, when compared to continuous veno venous hemofiltration（CVVH）. PE can effectively remove certain chemical components, even those with relatively high molecular weight, replacing toxic plasma with fresh healthy plasma. Fresh frozen plasma also supplements some important substances of the blood that are consumed during systemic inflammation, and these are of certain benefits for the cytokine network^[14] . Hence, the investigators attempted the ALS in treatment of hypercytokinemia in critically ill COVID-19 patients.

A total of 12 patients received treatment with the ALS, which seek to interrupt the inflammatory cascade and anti-inflammatory response. Blood samples were taken before and after each course of the ALS. Then, the levels of 48 cytokines and chemokines were measured.

A significant rebound in cytokine levels was observed during the intervals (T1A-T2B and T2A-T3B), indicating a strong cytokine storm in these critically ill COVID-19 patients. A total of 32 cytokines and chemokines significantly decreased, while two cytokines and chemokines (HGF and IL-12 [p40]) were significantly upregulated after treatment with the ALS. The ALS can significantly alleviate the cytokine storm, and improve the inflammatory status of COVID-19 patients.

In the recent study conducted by the investigators (manuscript in preparation) on COVID-19, it was revealed that among the 48 cytokines/chemokines that were studied, IP-10 was highly positively linked to disease severity. In our previous study on H7N9 avian influenza, it was found that IP-10 is significantly elevated in patients, and is independent risk factor for poor prognosis^[5]. In animal model studies, the monoclonal antibody against CXCL-10/IP-10 can improve the acute lung injury induced by influenza a (H1N1) virus^[16]. IP-10 is a 10-kD chemokine induced by IFN-γ, which is mainly derived from monocytes and lymphocytes. IP-10 plays an important role in inflammation by recruiting a variety of cells to the inflammatory site, and interacting with receptor CXCR3^[16]. In the present study, it was found that the ALS can significantly reduce the levels of chemokine IP-10.

The latest study conducted by the investigators also revealed that M-CSF is highly positively linked to disease severity. M-CSF is a chemokine that can regulate the survival, proliferation and differentiation of mononuclear macrophage lines, which is derived from activated macrophages and endothelial cells^[17]. The treatment with the ALS can also significantly reduce the levels of M-CSF.

Early COVID-19 biopsies have revealed pathological changes, such as edema, proteinaceous exudate, focal reactive hyperplasia of pneumocytes with patchy inflammatory cellular infiltration, and ultinucleated giant cells^[18]. In the middle and late stage, inflammatory cell infiltration dominated by mononuclear macrophage and lymphocyte exudation and alveolar epithelial damage were the main features^[2]. Therefore, it was inferred that the production of cytokines and chemokines, such as IP-10 and M-CSF, after virus infection is involved in the transport of immune cells to the inflamed lung, thereby resulting to a cytokine storm.

SCGF is an endogenous growth factor that can inhibit bone marrow inflammation, enhance hematopoietic recovery after bone marrow suppression, and reverse inflammation^[19]. It is possible that the exhaustion of lymphocytes promotes the generation of SCGF-β. After the treatment with the ALS, the level of SCGF-β also significantly decreases. Hence, it may be necessary to supplement SCGF-β after treatment with the ALS, which may increase the level of peripheral blood lymphocytes and improve the condition.

For the first time, it was found that hepatocyte growth factor (HGF) can be significantly increased after treatment with the ALS. HGF is an interstromal source pleiotropic growth factor, which can promote cell mitosis, growth, maturation, movement and the tissue formation process, and induce angiogenesis when combined with the hepatocyte growth factor receptor (HGF-receptor, also known as c-Met). Furthermore, HGF can improve vascular endothelial permeability and inflammation, protect endothelial cells^[20], and improve the ischemia-reperfusion injury of acute lung injury ^{[21, 22]}. The previous research conducted by the investigators on H7N9 revealed that HGF is highly positively linked to disease severity, and also is an independent outcome predictor. It was suspected that the increase in HGF is the compensation of the body. The treatment with the ALS can increase the HGF levels, and play a protective role against lung damage and liver function damage.

Although the present study revealed that therapy strategies with the ALS can significantly reduce a patient’s cytokine storm, the present study is a non-randomized clinical study. Hence, it is difficult to provide clear evidence to determine whether the ALS could reduce the mortality rate. The most significant difference between our hospital and other hospitals was the intervention of the ALS. Furthermore, the fatality rate of patients was significantly lower than reported. Therefore, it was considered that the ALS can significantly improve the prognosis of COVID-19 patients.

The ALS can significantly reduce cytokine levels, and probably improve the survival of critically ill patients. The present study provides preliminary data that supports the application of ALS for the supportive treatment of critically ill COVID-19 patients. It is recommended to provide early assessment for COVID-19 patients and timely artificial liver intervention, in order to improve the prognosis.