​Association Of Interleukin-6 Levels With Morbidity And Mortality in Patients With Coronavirus Disease 2019 (COVID-19)

Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791

Jiali Zhout,Wenbo Het', Jingyu Liang, Lang Wang, Xiaomei Yu, Mingwei Bao,and Huafen Liu*

Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, People's Republic of China

SUMMARY: The prognostic value of interleukin-6(IL-6) in coronavirus disease 2019 (COVID-19)needs to be clarified. In this retrospective study, COVID-19 patients treated at Renmin Hospital of Wuhan University from January 7 to February 8. 2020 with measurements of serum IL-6 levels within 1 week after admission were included. Data regarding demographics, clinical characteristics, laboratory tests, complicat1ons.and outcomes were collected and analyzed S1xty-s1x patients d1agnosed with COVID-19 were included in this study(31 patients were females). They were divided into a normal group (serum IL-6<10 pg/mL, n= 35)and an abnormal group (serum IL-6<10 pg/mL, n = 31). Compared with the normal group, the incidence of critical cases (P<0.001), acute respiratory distress syndrome(ARDS)(P =0.001), acute cardiac injury (P= 0.002), cardiac insufficiency (P= 0.039), mechanical ventilation rate(P = 0.002), and mortality (P = 0.021) was significantly increased in the abnormal group. Serum IL-6 concentration was an independent predictor of fatal outcomes (P= 0.04). The optimal cutoff value of serum IL-6 concentration for predicting fatal outcomes was 26.09 pg/mL(P<0.001). In COVID-19, elevated serum IL-6 levels were associated with critical illness, use of mechanical ventilation and complications, including heart injury and ARDS.and could predict a fatal outcome. Early detection of serum IL-6 levels after admission should be necessary for COVID-19 patients.

cistanche extract can help people improve their immunity to get against Coronavirus Disease

INTRODUCTION

Coronavirus disease 2019(COVID-19) caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)occurred in December 2019 and has become an ongoing global health threat(1). As of October, 16.2020,39.023,292 patients have been infected, and 1.099,586 deaths have been reported worldwide(2). Previous studies on SARS have suggested that several factors influence the pathogenesis of coronavirus infections, such as titers of viral infection, host immunity, and induction of cytokines(3). It has been suggested that a dysregulated immune response accompanied by cytokine storms is involved in the pathogenesis of human coronavirus disease, such as SARS and the Middle East respiratory syndrome(3). A prospective study on SARS indicated that the progression of SARS is associated with immunopathological responses, rather than uncontrolled viral infections(4). Increased levels of chemokines and cytokines, such as interleukin-6(IL-6), have been detected in SARS patients (5). The increase in serum chemokine/cytokine levels was by their elevated expression in the lung tissues(6). Recent evidence from autopsies has suggested the involvement of aberrant host immune response and inflammatory cytokine storm in alveolar gas exchange damage(7). Since these chemokines/cytokines are involved in the chemotaxis and activation of neutrophils and monocytes (8), which could cause damage to the target organs, such as the lungs, the levels of these chemokines/cytokines could be related to the severity of the disease. IL-6 is an upstream mediator of the inflammatory pathway(9,10)and is associated with both the acute immune response and chronic inflammation(11,12). IL-6 can be used as a quantitative marker of systemic inflammation(13)and has been used for prognosis evaluation (14). This study aimed to investigate the association of IL-6 levels with morbidity and mortality in patients with COVID-19, which might provide evidence for the utility of IL-6 in risk stratification and clinical decision.

PARTICIPANTS AND METHODS

Study design and participants: In this retrospective. the single-center study, we reviewed all confirmed cases of COVID-19 admitted from January 7 to February 8, 2020, at the isolation ward of Renmin Hospital (Wuhan University, Wuhan, China). All enrolled patients were diagnosed according to Interim guidance for novel coronavirus pneumonia published by the National Health Commission of the People's Republic of China(15).

Generally, confirmed cases were diagnosed based on epidemiological history(e.g., cluster transmission), clinical manifestations(fever and/or respiratory symptoms, lung imaging, normal or decreased counts of white blood cells and lymphocytes at the early stage of disease onset), and pathogenic or serological evidence for SARS-CoV-2 infection (SARS-CoV-2 nucleic acid detection and serum-specific antibodies). The diagnostic criteria of suspected cases were: patients who had epidemiological history and 2 of the 3 clinical manifestations, or individuals who had all 3 clinical manifestations when there was no epidemiological history. Patients suspected of COVID-19 with a positive nucleic acid test or serum-specific antibody test were diagnosed as confirmed cases. Only patients with measurements of serum IL-6 levels during the first week after admission were included. According to the criteria reported by the World Health Organization-China Joint Mission on COVID-19(16), patients were classified into 4 groups: mild(laboratory-confirmed, without pneumonia), moderate(laboratory-confirmed and with pneumonia), severe(dyspnea, respiratory frequency ≥30/min, blood oxygen saturation <93 %, oxygen partial pressure(PaO,)/oxygen inspiration fraction (FiO,)ratio<300, and/or lung infiltrates >50 % of the lung field within 24-48 h)and critical (respiratory failure requiring mechanical ventilation, shock or another organ failure that requires intensive care). The PaO,/FiO, the ratio was obtained by dividing the PaO, by the FiO, expressed as decimals. This study (No. WDRY2020-K032) was approved by the Ethics Committee of Renmin Hospital of Wuhan University. Written informed consent was waived by the same committee.

Procedures: Real-time PCR targeting SARS-CoV-2 specific nucleic acid was used for the diagnosis of COVID-19. Serum IL-6 levels were determined by enzyme-linked immunosorbent assay according to the manufacturer's instructions(ET Healthcare Inc., Palo Alto, CA, USA)and the normal range was<10 pg/mL based on the test manual and reagent description of Renmin Hospital of Wuhan University. Serum cardiac troponin I(can)levels were measured using a standard assay kit and Siemens ADVIA CENTAUR XP automatic chemiluminescence immunoassay analyzer (Siemens Healthcare Diagnostics [Shanghai], Walpole, NJ, USA). N-terminal pro-B-type natriuretic peptide (NT-proBNP)levels were measured using a Cobas E601 immune analyzer(Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. The patient's medical records were carefully reviewed and analyzed by3 trained physicians. Patient data regarding demographics, comorbidities, signs and symptoms, laboratory results, and complications were collected. The occurrence of medical complications was confirmed by 3 physicians according to the following criteria: acute respiratory distress syndrome(ARDS)was defined according to the Berlin definition(17); the acute cardiac injury was defined as a serum level of the can above the 99th percentile upper reference limit or new abnormalities were shown on electrocardiography and echocardiography(1); cardiac insufficiency was defined as an increased level of NT-proBNP over the normal range with the presence of associated symptoms, such as dyspnea, orthopnea, and edema of the lower extremity; acute kidney injury was identified according to the Kidney Disease: Improving Global Outcomes of Definition (18).

Outcomes: The primary outcome was mortality as of April 9,2 months from the last admission. Patients who met the following criteria were discharged: significantly improved symptoms, no fever for at least 3 days, obvious absorption of inflammation in pulmonary imaging, and negative results for at least 2 consecutive tests of SARS-CoV-2 nucleic acid. Patients who did not meet the discharge criteria continued hospitalization for treatment and observation.

Statistical analysis: Statistical analyses were performed using SPSS version 26.0. The normality of continuous variables was tested using the Kolmogorov-Smirnov test. The patients were divided into 2 study groups (with normal or elevated serum IL-6 levels)Continuous and categorical variables were summarized as counts (percentage)and median (interquartile range), respectively. Differences between groups were tested by hypothesis testing using the Mann-Whitney U test and Chi-squared test, respectively. Bivariate correlations between IL-6 and other variables were evaluated using Spearman's correlation coefficient. Univariate and multivariate logistic regressions were used to assess the associations between elevated IL-6 levels and outcomes, and odds ratios(ORs) and 95% confidence intervals (CIs) were calculated. The following hierarchical regression models were used: Model 1 was unadjusted. Model 2 was adjusted for age and sex. Model 3 was adjusted for age, sex, comorbidities including hypertension, cardiovascular disease, cerebrovascular disease, diabetes, chronic kidney disease, and chronic obstructive pulmonary disease. Model 4 was adjusted for variables in Model 3 plus procalcitonin.

Receiver operating characteristic(ROC) curves were used to assess predictive discriminatory capability(19). The most accurate cutoff value was calculated using the Youden index. All significance levels were computed for 2-tailed testing and the cutoff of significance was set at P<0.05.

cistanche extract can help people improve their immunity to get against Coronavirus Disease

RESULTS

Demographics, clinical characteristics, and laboratory findings: A total of 66 patients diagnosed with coronavirus were included in this study. The median age was 63 years (range 31-92 years). In total, 31 patients were females(47.0%).By April 9,2020,56 patients were discharged, 8 patients died, and 2 patients continued hospitalization. The length of hospital stay was 32 (range 21-43) days. Table 1 shows that common comorbidities included hypertension (24 patients, 36.4 %), diabetes(14 patients. 21.2 %),and cardiovascular disease(1l patients, 16.7%). There were 3 moderate, 46 severe, and 17 critical cases. Patients were classified into elevated and normal groups according to the normal range of serum lL-6 concentrations(<10 pg/mL). The median serum IL-6 concentration in the elevated group was 30.00 pg/mL, while that in the normal group was 1.77 pg/mL. Compared with patients with normal serum IL-6 levels, patients with elevated serum IL-6 levels were older and the proportion of

Association of IL-6 with Morbidity and Mortality in COVID-19

Table 1. Demographics, clinical characteristics, and laboratory findings

Continuous variables are expressed as median (IQR)and categorical variables are expressed as count (percentage). SEO was the lowest value of pulse oxygen saturation documented during a hospital stay, IL-6 concentrations were determined within 1 week after admission while other laboratory results and vital signs were acquired on admission.

ALT, alanine aminotransferase; AST, aspartate aminotransferase; COPD, chronic obstructive pulmonary disease; DBP, diastolic blood pressure; IQR, interquartile range; NT-proBNP, N-terminal pro-B-type natriuretic peptide; SBP, systolic blood pressure.

cistanche extract can help people improve their immunity to get against Coronavirus Disease

critical cases were significantly higher. The interval from onset to admission in the elevated group was significantly shorter than that in the normal group, while no difference in-hospital stay was found between the 2 groups. There were more patients in the elevated group with a history of cerebrovascular disease than in the normal group. The lowest pulse oxygen saturation (SpO,)documented during a hospital stay in the elevated group was significantly lower than that in the normal group. In the laboratory findings on admission, the platelet count was significantly decreased, while serum concentrations of creatinine, can, procalcitonin, and C-reactive protein were all significantly increased in patients with elevated IL-6 levels, compared to those in patients with normal IL-6 levels.

Correlation between serum IL-6 concentrations and other variables: As demonstrated by Spearman's correlation (Table 2), serum IL-6 concentrations were positively correlated with age, urea, creatinine, can, NT-proBNP, C-reactive protein, and procalcitonin.

Positive correlations with IL-6 were also found for white blood cell count and neutrophil counts. In addition, serum IL-6 concentrations were negatively correlated with the lowest SpO, documented during a hospital stay, Lymphocyte counts, and platelet counts.

Complications and outcomes in patients with different serum IL-6 levels: As described in Table 3, worse outcomes and higher incidences of complications were observed in patients with elevated serum IL-6 levels compared with those in the normal group. Complications, including acute cardiac injury, cardiac insufficiency, and ARDS, occurred more frequently in patients with elevated IL-6 levels than in those with normal levels. In comparison to the normal group, the incidence of critical cases and deaths in the elevated group was significantly higher, and more patients in the elevated group received mechanical ventilation. The median time to death was 3(1-37) days after the detection of serum IL-6 levels in the death cases.

Predictive value of serum IL-6 concentrations for outcomes: Logistic regression analysis for associations between serum IL-6 levels and outcomes is shown in Table 4. Univariate analysis (Model 1)revealed that serum IL-6 levels were associated with critical illness and mortality. After adjusting for age, sex, comorbidities, and procalcitonin (Model 4), serum IL-6 levels were still independently associated with a critical illness. In multivariate logistic regression models(Models 2 to 4), after age, sex, comorbidities,

and procalcitonin levels were adjusted hierarchically and serum IL-6 concentration remained an independent predictor of fatal outcome. In contrast, no significant association with disease severity or outcome was found for C-reactive protein, a classical inflammation marker that was routinely determined. The predictive value of serum IL-6 concentrations for a fatal outcome was further evaluated using the ROC curve(Fig. 1). After optimization, the threshold of serum IL-6 concentrations for predicting death was 26.09 pg/mL, with a sensitivity of 87.5% and a specificity of 77.6 %(area under the curve 0.887,95% CI0.767-1.000, P<0.001).

cistanche extract can help people improve their immunity to get against Coronavirus Disease

DISCUSSION

The present study illustrates the correlations between serum IL-6 levels, complications, and outcomes in patients with COVID-19. We report 3 major findings in this study. First, patients with elevated serum IL-6 levels had a higher incidence of critical illness, complications including heart injury and ARDS, use of mechanical ventilation, and fatal outcome. Second, serum IL-6 concentration was positively correlated with biomarkers for cardiac and kidney injuries. Third, elevated serum IL-6concentration was an independent predictor of fatal outcomes in patients with COVID-19. In the present study, elevated serum IL-6 levels were associated with decreased oxygen saturation, increased disease severity, and frequent use of mechanical ventilation, indicating that serum IL-6 might reflect the severity of inflammation and lung lesions and could have predictive value in patients with COVID-19. There were several confounding factors associated with serum IL-6 levels which may influence disease severity and outcomes,

Association of IL-6 with Morbidity and Mortality in COVID-19

Table 4. Univariate and multivariate logistic regression analysis of serum I-6 and

C-reactive protein for outcomes

Model 1 was crude(unadjusted); Model 2 was adjusted by age and sex; Model 3 was adjusted by age, sex, comorbidities including hypertension, cardiovascular disease, cerebrovascular disease, diabetes, chronic kidney disease, and chronic obstructive pulmonary disease; Model 4 was adjusted by variables in Model 3 plus procalcitonin. CI, confidence interval; OR, odds ratio.

Fig.1. Receiver operating characteristic(ROC)curve of serum lL-6 concentrations for fatal outcomes. The area under the curve was 0.887. The optimal cutoff value was selected for which the point on the ROC curve had the minimum distance to the upper left corner (where sensitivity =1 and specificity =1).

such as age and bacterial infection. A close relationship between serum IL-6 levels and age was observed in the present study, and a similar relationship has been previously reported in patients without the infectious disease(20), indicating a possible inherent relationship between age and inflammation. In addition, ascending serum IL-6 levels may be associated with bacterial infections. A previous study on pneumonia revealed an association between bacterial infection and increased serum IL-6 levels(21). Our data also showed significant positive correlations between serum IL-6 levels and indices of bacterial infection, such as neutrophil count and procalcitonin. To evaluate the independent predictive value of IL-6, age and procalcitonin levels were adjusted using multivariate logistic regression. The results showed that serum IL-6 concentration was independently related to critical illness and was an independent predictor of fatal outcomes.

Serum IL-6 levels might also be related to complications associated with COVID-19. Our data showed significant positive correlations of IL-6 with several biomarkers for kidney function and cardiac injury, such as urea, creatinine, can, and NT-proBNP As described in Table 3, the incidence of several complications, such as ARDS, acute cardiac injury, and cardiac insufficiency, were significantly higher in patients with elevated levels of serum IL-6 than in those with normal IL-6 concentrations. Heart injuries in COVID-19 might be caused by several factors, such as virus invasion and systemic inflammation. It has been reported that the invasion sites of SARS-coronavirus (SARS-CoV)correspond to the presence of angiotensin-converting enzyme 2(ACE2)(22), which is abundantly expressed in the lung and small intestine(23). ACE2 is also expressed in the endothelium and smooth muscles of the vasculature in the heart(24). SARS-CoV RNA was detected in autopsied human hearts obtained from patients infected with SARS-CoV, and macrophage infiltration was observed in these infected hearts(25). However, there is no histological evidence supporting the direct impairment of the SARS-CoV-2 on cardiomyocytes(26). Systemic inflammation under pathophysiological conditions might also cause heart damage. It has been reported that some circulating cytokines, including IL-1, IL-4, IL-6, IL-8, and IL-18, which are related to inflammatory cardiac pathologies, are involved in cardiac dysfunction(27). Our results also found correlations between IL-6 and cardiac injury or cardiac dysfunction, suggesting a possible role of inflammation in heart injury in COVID-19 patients.

The optimal cutoff value of IL-6 for mortality prediction in the present study was 26.09 pg/mL, which was similar to that reported in other studies. For instance, IL-6>25 pg/mL was reported to be an important risk factor for severe COVID-19 and/or in-hospital mortality (28). Severe complications were more likely to occur in COVID patients with IL-6levels>32.1 pg/mL based on a study population of 140 patients with mild to severe diseases (29).In addition, L-6 is a potential biomarker for the progression of COVID-19 (30). Monoclonal antibodies targeting the IL-6 pathway have been used in the treatment of COVID-19, which might block the inflammatory storms. For example, tocilizumab, a monoclonal antibody that blocks IL-6 receptors, has shown encouraging clinical results in COVID-19 treatment(7). Detection of serum IL-6 levels after admission should be necessary for patients with COVID-19, which could help to identify patients at high risk and provide guidance for immune-modulating therapies.

As a retrospective study on severe diseases, IL-6 was not routinely determined for all patients. Only a small portion of patients received measurements of IL-6 within the first week after admission, and most of the measurements were not performed on admission. Serum IL-6 levels determined within the first week after admission were used for the analysis. In addition, other important cytokines involved in systemic inflammation such as IL-10 were not determined at the same time.

In conclusion, in patients with COVID-19, elevated serum IL-6 levels were associated with critical illness, use of mechanical ventilation, and complications including heart injury and ARDS. Serum IL-6 concentration was an independent predictor of fatal outcomes. The optimal cutoff value of IL-6 for mortality prediction was 26.09 ng/mL. Our study suggests that detection of serum IL-6 levels after admission should be necessary for COVID-19 patients.

Acknowledgments This work was supported by the National Natural Science Foundation of China(grant numbers 81570450, 81900455).

Conflict of interest None to declare.

REFERENCES

1. Huang C, Wang Y, Li X, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020;395:497-506.

2. World Health Organization (WHO). Weekly operational update on COVID-19-16 October 2020.Available at <https://www who.int/publications/m/item/weekly-update-on-covid-19---16-october-2020>. Accessed October 20.2020.

3. van den Brand JM, Haagmans BL, van Riel D, et al. The pathology and pathogenesis of experimental severe acute respiratory syndrome and influenza in animal models. J Comp Pathol.2014;151:83-112.

4. Peiris JS, Chu CM, Cheng VC, et al. Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study.Lancet.2003;361:1767-72.

5. Wong CK, Lam CW, Wu AK, et al. Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome. Clin Exp Immunol.2004;136:95-103.

6. Jiang Y, Xu J, Zhou C, et al. Characterization of cytokine/ chemokine profiles of the severe acute respiratory syndrome. Am J Respir Crit Care Med.2005;171:850-7.

7. Fu B. Xu X. Wei H. Why tocilizumab could be an effective treatment for severe COVID-19?J Transl Med. 2020:18:164. 8.Tsushima K, King LS, Aggarwal NR, et al.Acute lung injury review. Intern Med.2009;48:621-30.

9. Netea MG, Balkwill F, Chonchol M, et al. A guiding map for inflammation. Nat Immunol.2017;18:826-31.

10. Ridker PM. From c-reactive protein to interleukin-6 to interleukin-1: moving upstream to identify novel targets for atheroprotection. Circ Res. 2016;118:145-56.

11. van der Poll T. Keogh CV, Guirao X, et al. Interleukin-6 gene-deficient mice show impaired defense against pneumococcal pneumonia.infect Dis.1997;176:439-44.

12. Landskron G, De la Fuente M, Thuwajit P, et al. Chronic inflammation and cytokines in the tumor microenvironment. J Immunol Res. 2014;2014:149185.

13. Ridker PM, Everett BM, Thuren T, et al. Antiinflammatory therapy with canakinumab for atherosclerotic disease. N Engl J Med.2017;377:1119-31.

14. Antunes G, Evans SA, Lordan JL, et al. Systemic cytokine levels in community-acquired pneumonia and their association with disease severity.Eur Respir J.2002:20:990-5.

15. National Health Commission of the People's Republic of China.Interim guidance for novel coronavirus pneumonia (Trial Implementation of Sixth Edition).

8334a8326dd94d329df351d7da8ae fc2.shtml>.Accessed February 27,2020.Chinese.

16. WHO. Report of the WHO-China Joint Mission on Coronavirus Disease 2019.Available at < https://www.who.int/docs/default-source/coronaviruse/who-china-joint-mission-on-covid-19-fi nal-report>. Accessed March 5.2020.

17. Ferguson ND, Fan E, Camporota L, et al. The Berlin definition of ARDS: an expanded rationale, justification, and supplementary material. Intensive Care Med.2012;38:1573-82.

18. Khwaja A. KDIGO clinical practice guidelines for acute kidney injury. Nephron Clin Pract.2012;120:c179-84.

19. Lasko TA, Bhagwat JG, Zou KH, et al. The use of receiver operating characteristic curves in biomedical informatics. J Biomed Inform. 2005;38:404-15.

20. Hsu TC, Lin CH, Sun FJ, et al. Postoperative serum levels of interleukin-6 are affected by age in patients with colorectal cancer. Int J Gerontol.2017:11:75-9.

21. Menendez R, Sahuquillo-Arce JM, Reyes S, et al. Cytokine activation patterns and biomarkers are influenced by microorganisms in community-acquired pneumonia. Chest. 2012:141:1537-45.

22. To KF. Lo AW, Exploring the pathogenesis of severe acute respiratory syndrome(SARS): the tissue distribution of the coronavirus(SARS-CoV) and its putative receptor, angiotensin-converting enzyme 2(ACE2). J Pathol.2004;203:740-3.

23. Hamming I, Timens W, Bulthuis ML, et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. The first step in understanding SARS pathogenesis. J Pathol. 2004;203:631-7.

24. Donoghue M, Hsieh F, Baronas E, et al. A novel angiotensin-converting enzyme-related carboxypeptidase(ACE2)converts angiotensin I to angiotensin 1-9. Circ Res.2000:87:E1-9.

25. Oudit GY, KassiriZ, Jiang C, et al. SARS-coronavirus modulation of myocardial ACE2 expression and inflammation in patients with SARS.Eur J Clin Invest.2009;39:618-25.

26. XYZ, Shi L, Wang Y, et al.Pathological findings of COVID-19 associated with acute respiratory distress syndrome. Lancet Respir Med.2020; 8:420-2.

27. Bartekova M, Radosinska J, Jelemensky M, et al. Role of cytokines and inflammation in heart function during health and disease. Heart Fail Rev.2018;23:733-58.

28. Grifoni E. Valoriani A, Cei F, et al. Interleukin-6 as a prognosticator in patients with COVID-19.J Infect.2020:81:452-82.

29. Liu F, Li L, Xu M, et al. Prognostic value of interleukin-6, C-reactive protein, and procalcitonin in patients with COVID-19. J Clin Virol.2020;127:104370.

30. Ulhaq ZS, Soraya GV. Interleukin-6 as a potential biomarker of COVID-19 progression. Med Mal Infect.2020;50:382-3.