Cytokine Storm May Not Be the Chief Culprit for the Deterioration of COVID-19

Abstract

COVID-19 is spreading and ravaging all over the world, and the number of deaths is increasing day by day without downward trend. However, there is limited knowledge of pathogenesis on the deterioration of COVID-19 at present. In this study we aim to determine whether cytokine storm is really the chief culprit for the deterioration of COVID-19. The confirmed COVID-19 patients were divided into moderate group (n = 89), severe group (n = 37), and critical group (n = 41). Demographic data were collected and recorded on admission to ICU. Clinical data were obtained when moderate, severe, or critical COVID-19 was diagnosed, and then compared between groups. The proportion of enrolled COVID-19 patients was slightly higher among males (52.5%) than females (47.5%), with an average age of 64.87 years. The number of patients without comorbidities exceed one third (36.1%), and patients with 1, 2, 3, 4 kinds of comorbidities accounted for 23.0%, 23.0%, 13.1%, and 4.9%, respectively. IL-6, IL-10, TNF, and IFN-γ, including oxygenation index, sequential organ failure assessment score, white blood cell count, lymphocyte count, lymphocyte percentage, platelet, C-reaction protein, lactate dehydrogenase, creatine kinase isoenzyme, albumin, D-Dimer, and fibrinogen showed significant difference between groups. Some, but not all, cytokines and chemokines were involved in the deterioration of COVID-19, and thus cytokine storm maybe just the tip of the iceberg and should be used with caution to explain pathogenesis on the deterioration of COVID-19, which might be complex and related to inflammation, immunity, blood coagulation, and multiple organ functions. Future studies should focus on identification of specific signaling pathways and mechanisms after severe acute respiratory syndrome coronavirus 2 infections (IRB number: IRB-AF/SC-04/01.0).

Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) owns high infectivity with efficient human-to-human spread among close contacts (20,32) and thus has posed a terrible threat to a large number of people's lives and health care system worldwide. As the first country with outbreak, Chinese medical staff has extensive experiences in prevention, identification, management, and intervention of COVID-19. According to Chinese experiences, an estimated 15–30% of COVID-19 patients developed into severe and critical cases, usually manifested as acute respiratory distress syndrome and required ventilatory support (5,13,23). Moreover, the 28-day mortality of critically ill adult COVID-19 patients even exceeded 60% (36). However, pathogenesis on the deterioration of COVID-19 is relatively unknown at present.

First of all, previous studies on severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) had shown that virus-induced dysregulated immune response and cytokine storm caused extensive lung damage and fatal pneumonia (4). The genome structures, pathogenesis, and clinical presentations of the above two were similar, characterized by accumulation of inflammatory monocyte/macrophages and neutrophils in the lungs, which were the predominant source of delayed but elevated low-level expression of interferon (IFN-α and IFN-γ) and moderate increase of proinflammatory cytokines (tumor necrosis factor [TNF], Interleukin-1β [IL-1β], IL-6, IL-12, and transforming growth factor-β) as well as significant upregulation of inflammatory chemokines (CC-chemokine ligand 2 [CCL2], CCL3, CCL5, interferon-inducible protein-10 [CXCL-10], CXCL-9, IL-2, and IL-8) (2,7,14,17,18,27,31,37,39), especially in critically ill patients (15,22). On the contrary, the anti-inflammatory cytokine IL-10 had a significantly lower level (8). In addition, a similar pattern was repeated in other animal model experiments (24,26). Lastly, higher plasma levels of some cytokines and chemokines have been found in COVID-19 patients (13,34). Accordingly, cytokine storm is also suspected to be the chief culprit for the occurrence and deterioration of COVID-19. But is that really the case?

To better address this issue, we attempt to explore the pathogenesis on the deterioration of COVID-19 by comparing serum cytokine and chemokine levels, and other clinical data in different disease severity groups. The findings of our study will be conductive to understand the pathogenesis on the deterioration of COVID-19, and thus improve clinical intervention strategies.

Methods

Study design

A total of 61 COVID-19 patients from COVID-19 treatment center of the First Affiliated Hospital of Harbin Medial University in Heilongjiang province from February 14 to March 26 were included in this retrospective study. These patients were divided into moderate group (n = 89), severe group (n = 37), and critical group (n = 41) according to the Diagnosis and Treatment of New Coronavirus Pneumonia (the seventh edition) on the 1st, 5th, 10th, 15th, 20th, and 25th after ICU admission. Demographic data were collected and recorded on admission to ICU. Clinical data, including oxygenation index (OI), sequential organ failure assessment (SOFA) score, white blood cell count (WBC), lymphocyte count (LYMPH), lymphocyte percentage (LYM%), platelet (PLT) C-reaction protein (CRP), lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB), creatinine (CRE), albumin (ALB), D-Dimer, fibrinogen (FIB), IL-2, IL-4, IL-6, IL-10, TNF, and IFN-γ were obtained when moderate, severe, or critical COVID-19 was diagnosed, and then compared between groups. This study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (IRB number: IRB-AF/SC-04/01.0).

Study population

In this retrospective study, the inclusion criteria included: (1) admitted to ICU; (2) confirmed COVID-19 patient; (3) age >18 years; whereas COVID-19 patients who met the following criteria were excluded: (1) pregnant or breastfeeding women; (2) incomplete medical records. All enrolled COVID-19 patients were dealt with by the same experienced intensivists and similar treatment regimens according to the Diagnosis and Treatment of New Coronavirus Pneumonia (the seventh edition).

Diagnosis of COVID-19

All enrolled COVID-19 patients were confirmed by detecting SARS-CoV-2 nucleic acid on oropharyngeal and/or nasopharyngeal swabs.

Data collection

Demographic data, including age, gender, and number of comorbidities, and clinical data, including OI, SOFA score, WBC, LYMPH, LYM%, PLT, CRP, LDH, CK-MB, CRE, ALB, D-Dimer, FIB, IL-2, IL-4, IL-6, IL-10, TNF, and IFN-γ were collected and recorded from medical records through dedicated personnel. The members of our research team were unaware of the patient's personal information beyond the data required for this study.

Statistical analyses

SPSS 23.0 (SPSS, Inc., Chicago, IL) was used for statistical analyses. Descriptive analysis was performed on the enrolled COVID-19 patients' age, gender, and number of comorbidities. Kruskal–Wallis rank sum test was employed for group comparison and pairwise comparison of the clinical data with non-normal distribution. Analysis of variance (ANOVA) and LSD were adopted for group comparison and pairwise comparison of ALB, respectively. p-Values <0.05 were considered to indicate statistical significance.

Results

Descriptive analysis of age, gender, and number of comorbidities

As shown in Tables 1 and 2, the proportion of enrolled COVID-19 patients was slightly higher among males (52.5%) than females (47.5%), with an average age of 64.87 years. The number of patients without comorbidities exceed one third (36.1%), and patients with 1, 2, 3, and 4 kinds of comorbidities accounted for 23.0%, 23.0%, 13.1%, and 4.9%, respectively.

Table 1.

Descriptive Analysis of Age

	Minimum	Maximum	Mean	Standard deviation	Median
Age	23.00	87.00	64.87	12.76	65.00

Table 2.

Descriptive Analysis of Gender and Number of Comorbidities

Variable	Classification	Frequency	Percentage
Number of comorbidities	0.00	22	36.1
	1.00	14	23.0
	2.00	14	23.0
	3.00	8	13.1
	4.00	3	4.9
Gender	Female	29	47.5
	Male	32	52.5

Group comparison and pairwise comparison of the contents of serum cytokines and chemokines

As shown in Table 3, IL-6, IL-10, TNF, and IFN-γ showed significant difference between groups (p = 0.000, = 0.000, = 0.005, = 0.006, respectively), except for IL-2 and IL-4 (p = 0.339, = 0.086, respectively).

Table 3.

Group Comparison and Pairwise Comparison of the Contents of Serum Cytokines and Chemokines

	Moderate group	Severe group	Critical group	X²	p
IL-2	1.20 ± 0.66	1.23 ± 0.81	1.44 ± 0.84	2.163	0.339
IL-4	1.22 ± 1.00	0.81 ± 0.76	1.20 ± 0.83	4.915	0.086
IL-6	22.64 ± 58.38	30.46 ± 70.64	1,680.90 ± 5,038.87^a	20.764	0.000
IL-10	6.54 ± 5.63	5.97 ± 3.04	42.40 ± 100.20^ab	28.332	0.000
TNF	0.99 ± 0.75	0.66 ± 0.61	1.57 ± 2.02^b	10.719	0.005
IFN-γ	1.15 ± 0.48	1.34 ± 0.77	1.74 ± 1.36^a	10.124	0.006

a,b

Represent significant difference compared with moderate group and severe group, respectively.

Group comparison and pairwise comparison of other clinical data

As shown in Table 4, OI, SOFA score, WBC, LYMPH, LYM%, PLT, CRP, LDH, CK-MB, ALB, D-Dimer, and FIB showed significant difference between groups (p = 0.000, = 0.000, = 0.000, = 0.000, = 0.000, = 0.000, = 0.000, = 0.000, = 0.000, = 0.004, = 0.000, = 0.001, respectively), except for CRE (p = 0.532).

Table 4.

Group Comparison and Pairwise Comparison of Other Clinical Data

	Moderate group	Severe group	Critical group	X²/F	p
OI	385.00 ± 168.41	314.89 ± 173.20^a	194.99 ± 134.93^ab	43.877	0.000
SOFA score	1.67 ± 1.43	2.32 ± 1.49	5.67 ± 3.77^ab	51.209	0.000
WBC	5.18 ± 2.47	6.22 ± 2.64	8.40 ± 4.73^ab	32.617	0.000
LYMPH	1.20 ± 0.53	1.07 ± 0.66	0.74 ± 0.48^ab	25.941	0.000
LYM%	24.81 ± 9.20	18.19 ± 8.39^a	10.45 ± 7.44^ab	56.911	0.000
PLT	227.48 ± 84.52	331.81 ± 465.94	149.46 ± 100.13^ab	24.397	0.000
CRP	12.43 ± 14.81	36.89 ± 42.84^a	71.87 ± 61.00^a	54.918	0.000
LDH	438.61 ± 122.23	526.73 ± 122.13^a	791.13 ± 450.70^ab	57.234	0.000
CK-MB	4.93 ± 4.91	5.56 ± 4.02	14.32 ± 14.44^ab	31.541	0.000
CRE	69.37 ± 36.75	62.66 ± 25.06	73.10 ± 37.95	1.262	0.532
ALB	36.03 ± 5.20	33.55 ± 5.74^a	33.29 ± 5.86^a	4.712	0.004
D-Dimer	2.17 ± 1.83	2.51 ± 2.06	9.81 ± 11.00^ab	37.622	0.000
FIB	4.56 ± 1.53	5.09 ± 1.76	3.57 ± 1.45^ab	14.626	0.001

a,b

Represent significant difference compared with moderate group and severe group, respectively.

OI, oxygenation index; SOFA score, sequential organ failure assessment score; WBC, white blood cell count; LYMPH, lymphocyte count; LYM%, lymphocyte percentage; PLT, platelet; CRP, C-reaction protein; LDH, lactate dehydrogenase; CK-MB, creatine kinase isoenzyme; CRE, creatinine; ALB, albumin; FIB, fibrinogen.

Discussion

Human coronavirus (hCoV) was one of the common pathogens of respiratory infection, causing from mild or middle upper respiratory disease to fatal acute respiratory distress syndrome in humans. Highly pathogenic human viruses had a long history of causing pandemics, from 1918 Spanish influenza virus, 1957/1958 Asian influenza virus, and 1968 Hong Kong influenza virus pandemics to the recent outbreak, including avian influenza (H5N1, H7N9), SARS, swine origin influenza (H1N1), MERS, and the ongoing SARS-CoV-2. The ability of virus to modify genetic information for adapting different environments raised grave global attention and concern upon viral infection (10). Moving forward, similar highly pathogenic hCoVs may be persistent threats of public health worldwide and the intervals between virus pandemics tend to shorten gradually.

Cytokine storm, also known as hypercytokinemia, was referred to the massive burst in immunological mediators, which led to self-inflicted organ damage, disease progression, and ultimately high mortality due to exuberant and prolonged immune reactions and lots of cytokines and chemokines. Excessive immune responses and highly upregulated cytokine and chemokine gene expression played a key role in mediating some pathological processes and multiple organ involvement according to previous researches. In clinic, persistent organ damage with negative viral nucleic acids in the lungs demonstrated the presence and effects of cytokine storm once again. That is to say, some critically ill patients with viral infections actually did not die from the fatal viral infection, but uncontrolled host immune response, which was used to guide clinical intervention strategies. Viral infection-induced cytokine storm was characterized by simultaneous activation of proinflammatory and anti-inflammatory responses (38), a vital regulatory role in which disease progression had been revealed (25). However, not all viral infections were associated with cytokine storm, such as H3N2 and other nonpandemic H1N1 influenza viruses, without evidence of hypercytokinemia (6,35). Then, how about SARS-CoV-2 infection?

The identification of cytokine storm was not a simple matter. First, the cascades of cytokine storm would result in upregulation of most cytokines and chemokines, not just a few of them. Inflammatory mediators, involved in cytokine storm, normally contained IFN, TNF, interleukin, colony-stimulating factors, and various chemokines, over 150 in total (28,30), which interacted through a series of intricate and overlapping cytokine or chemokine signaling pathways. Second, a deficiency of anti-inflammatory cytokines and chemokines, as immunoregulatory molecules to control proinflammatory responses, also played a role to some extent. Lastly, although proliferation and production of disparate cytokines and chemokines were different during cytokine storm, most of them appeared to be moderate or show significant upregulation.

First, plasma IL-6 levels increased continually after early response cytokine stimulation (such as TNF-α and IL-1), and were used to assess the intensity of cytokine storm and be an indicator of disease severity. The release of proinflammatory cytokines and chemokines could cause activation of immune responses, and make the disease continue to progress even in the absence of persistent viral replication (30). The findings of our study show that some, but not all, cytokines and chemokines were involved in the deterioration of COVID-19. Second, IL-10, as a marker of anti-inflammatory response, owned anti-inflammatory properties and could lead to immunoparalysis (30), and its level increased significantly with deterioration of COVID-19 in our study. Lastly, significant upregulation was only found in IL-6 and IL-10, whereas the increase of TNF was limited, although significant. Thus, cytokine storm maybe just the tip of the iceberg and should be used with caution to explain pathogenesis on the deterioration of COVID-19.

In our study, OI, SOFA score, WBC, LYMPH, LYM%, PLT, CRP, LDH, CK-MB, ALB, D-Dimer, and FIB showed significant difference in different disease severity groups, suggesting that pathogenesis on the deterioration of COVID-19 was complex and might be related to inflammation, immunity, blood coagulation, and multiple organ functions, not just cytokine storm. Previous studies had demonstrated that the suppressed and delayed IFN response signaling sensitized T cells to apoptosis through TNF-mediated pathway, and further hindered pathogenic virus clearance (3). Furthermore, IFN impaired T cell responses by upregulating the expression of negative immunoregulatory molecules, for example programmed cell death-1 (PD-1) and Lymphocyte activation gene-3 (LAG-3) (29). Meanwhile, weakened T cell responses might cause uninhibited and uncontrolled innate immune responses, and contribute to form inflammatory milieu in infected tissues (3,16). A progressive decrease in lymphocyte counts indicated severity of damage to the immune system and was closely associated with mortal outcome. Thus, future studies should focus on identification of specific signaling pathways and mechanisms after SARS-CoV-2 infection.

At present, there was lack of effective vaccine and targeted treatment for COVID-19, but merely supportive therapy (9). Thus, strategies to restore immunological equilibrium and maintain immune homeostasis after SARS-CoV-2 infection would likely eliminate invading fatal virus on the one and alleviate deleterious tissue or organ dysfunction on the other. Restoring T cell counts and lymphocyte may be conductive to remission of the condition. Some strategies on dampening immune responses may increase susceptibility to secondary infection in clinic. Corticosteroids, as an anti-inflammatory drug, were commonly applied to attenuate inflammatory response and pulmonary lesions caused by viral or bacterial infections in clinical practice. A more cautious choosing of the dosage, timing, and treatment course of systemic corticosteroid administration were advocated for highly pathogenic hCoVs infection, due to significant adverse effects in the middle and late stages of infection, including immunosuppressive effects, higher subsequent plasma viral load, increased risk of fatal superinfections, avascular necrosis, and ultimately increased mortality (1,11,12,19,21,33). The current experience was that low-dose and short-term corticosteroid treatment might be effective for SARS-CoV-2 infection in the acute phase and could avoid adverse events in the middle and late period.

There are several limitations in our study. First of all, the nature of a single-center retrospective study reduces the reliability of our conclusion. Moreover, the results from relatively small sample size require to be explained with caution. Well-designed and large-scale clinical or animal trials are needed in the near future to confirm our conclusion. Lastly, a limited number of cytokines and chemokines were detected and affected by different onset times.

Conclusions

Some, but not all, cytokines and chemokines were involved in the deterioration of COVID-19. Among them, significant upregulation was only found in IL-6 and IL-10, whereas the increase of TNF was limited, although significant. Thus, cytokine storm maybe just the tip of the iceberg and not responsible for pathogenesis on the deterioration of COVID-19, which might be complex and related to inflammation, immunity, blood coagulation, and multiple organ functions. The efficacy of oXiris, a therapeutic strategy of blood purification, to weaken endotoxins and cytokine storm, is the focus of completed clinical trial in our COVID-19 treatment center, which may bring us further answers before long.

Footnotes

Acknowledgments

The authors are grateful to all colleagues who worked with them in the COVID-19 treatment center of Heilongjiang province, and all those who provided selfless advice and help for this article. They pay tribute to the medical staff who lost their lives in the national fight against the COVID-19 epidemic.

Ethics Approval and Consent to Participate

In this single-center retrospective study, written informed consent was duly obtained from all patients. The study was approved by the Ethics Committee of the first affiliated hospital of Harbin Medical University.

Author Disclosure Statement

The authors declare that they have no competing interests.

Authors' Contributions

All authors took part in designing the study. Y.G., C.W., K.K., Y.P., and Y.L. analyzed and interpreted the results and drafted the article. H.L. and W.Y. collected the data. Y.G., C.W., K.K., Y.P., Y.L., M.Z., and K.Y. discussed the results. All authors revised the article critically for important intellectual content. All authors read and approved the final article.

Funding Information

Supported by Novel coronavirus pneumonia emergency treatment and diagnosis technology research project of Heilongjiang Provincial Science and Technology Department, the National Natural Science Foundation of China (Nos. 81770276, 81772045, 81902000), and Nn10 program of Harbin Medical University Cancer Hospital.

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