Serum Cytokine Levels As Critical Parameters in Early Diagnosis of Disease Progression in COVID-19: A Pilot Study

Background: The severity of Coronavirus disease 2019 (COVID-19) has been proposed to be associated with cytokine dysregulation. A signiﬁcant number of patients become serious and need intensive care in hospitals. Methods: The concentrations of cytokines interleukin (IL-6, IL-10) and tumor necrosis factor (TNF) were estimated using enzyme-linked immunosorbent assay (ELISA) in serum samples of 60 adult patients infected with SARS-CoV-2 along with 50 healthy controls of the same age. The mean age of the subjects was 50-52 years and included an equal number of males and females. The patients were further grouped as severe (38 patients) and non-severe cases (22 patients). Results: The mean serum cytokine levels were signiﬁcantly higher in the COVID-19 patients than in the healthy controls. IL-6 was excessively elevated in comparison to IL-10 and TNF. Comparative analysis of severe versus non-severe cases revealed only slight alterations in the cytokine levels: IL-6 being the most elevated in severe cases. The concentration of the liver enzyme ALT was higher than AST in both severe and non-severe cases. The mean concentration of serum electrolytes (Na, K, and Ca) did not vary much between the patients and healthy controls. Conclusion: There was a signiﬁcant positive correlation between the levels of cytokines serum biomarkers in COVID-19 patients. It may be suggested that early detection of cytokines, especially IL-6 and serum biomarkers can help predict disease prognosis and severity in COVID-19 patients.


Introduction
The SARS-CoV-2 infection (COVID-19) has been wreaking havoc since its outbreak in December 2019.More than 660 million infections and 6.7 million deaths have been reported to date.The virus has not only spread worldwide but has also mutated several times [1].The disease has pneumonia-like symptoms affecting the lungs with varying degrees of severity.From the infected lungs, it may spread to other parts of the body, sometimes leading to multiple organ failure [2].Acute lung injury in COVID-19 patients may be triggered by the release of inflammatory molecules, activated immune cells, overproduction of cytokines, and the presence of adhesins [3].The damage caused to the lungs and other body organs has been linked to the abnormally strong proinflammatory host immune response.Described as the 'cytokine storm syndrome,' it plays an important role in the pathophysiology of COVID-19.The clinical symptoms vary among individuals from asymptomatic and non-specific to more serious, where patients need hospitalization [4].The severity of COVID-19 disease depends on several factors imparting poor prognoses, such as high viral load, old age, and comorbidities (cancer, coronary heart disease, diabetes, and hypertension) [5,6].The disease pathogenesis is not clearly understood and comprises several intricate and complex physiological processes.Cytokines are released by immune cells to regulate inflammatory responses during tissue damage and infections.It has been shown that some proinflammatory cytokines, namely, tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), interferon gamma (IFN-γ), interleukins (IL-1, IL-8, and IL-6), play a crucial role in several pathological conditions [7,8].The involvement of TNF-α is an intermediary in the pathogenesis of certain viral infections like influenza [9].Some other cytokines, like IL-4 and IL-10, help reduce inflammation [10].The role of cytokines in disease pathogenesis is still unclear, but reports suggest that their excessive outburst may be responsible for disease progression and sometimes death of the patient [11,12].The serum cytokine levels must be regulated, but whether anti-cytokine therapy has any benefit needs further evaluation [13].The liver, heart, and kidneys are vital organs of the human body that perform some critical functions.Detection of specific biomarkers and their appropriate concentrations in the blood helps investigate these organs' health status [14].A strong connection has been observed between the severity of COVID-19 and chronic liver diseases accompanied by inflammation and dysregulation of immune responses.Patients suffering from underlying conditions carry a greater risk of adverse consequences after contracting COVID-19.Moreover, when infected, healthy individuals exhibit abnormal liver function, directly implicating the virus in damaging the liver and DOI 10.18502/sjms.v18i2.13603metabolism [15].Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are biomarkers for hepatocellular damage.Bilirubin and albumin levels assess the liver's secretory and synthetic capacities, respectively [16].Increased levels of serum creatinine can indicate renal dysfunction.Serum ferritin, creatine kinase (CK), lactate dehydrogenase (LDH), inflammatory factors, and electrolytes (sodium, potassium) can be used as indicators for COVID-19 [17].As observed with other viral infections, increasing cytokine levels have emerged as early parameters in COVID-19 disease advancement [18].Hence, estimating increased serum cytokine levels and abnormalities in the hepatic, renal, and cardiac biomarkers can be important in predicting poor outcomes in COVID-19 patients.The levels of IL-6, IL-10, and TNF in the serum can also predict the severity of the disease, as cytokines play an important role in host immunity and immunopathology during the infection.The present study thus attempts to correlate cytokine levels with biomarkers of vital body organs in COVID-19 patients.

Patients and data collection
A total of 60 COVID-19 patients were randomly selected from the Ohud Hospital, Madinah, Saudi Arabia.A healthy control group of similar age (50 subjects) was selected from Taibah University, Madinah, Saudi Arabia.The clinical and laboratory data of the patients were analyzed and documented.All the cases were assessed according to the inclusion and exclusion criteria.Individuals with a history of endocrine diseases were excluded.Patients with confirmed COVID-19 were categorized into two groups (severe and non-severe) based on clinical characteristics, symptoms, and results obtained from chest radiography [19].
Blood samples (3 ml) were collected in serum separator tubes and allowed to clot for 2 h at room temperature before centrifugation at 1000×g for 15 min.Serum was collected and stored at -80 ∘ C. The serum concentrations of TNF-α, IL-10, and IL-6 were determined in the subjects.Biochemical assays (kidney and liver function tests) were performed to estimate the concentration of creatinine, urea, Na, K and Ca, troponin I, C-reactive protein (CRP), LDH, CK, ALT, AST, bilirubin, albumin, and ferritin using the automated ARCHITECT c4000, as per the manufacturer's guidelines.DOI 10.18502/sjms.v18i2.13603

Enzyme-linked immunosorbent assays (ELISA)
Cytokine concentrations were determined using ELISA development kits (CUSABIO Technology LLC, Houston, USA) in adherence with the manufacturer's instructions for TNF-α, IL-10, and IL-6.The antibody specific for each cytokine was pre-coated onto a microplate.The optical density was verified using a Mindray mr-96a/Mindray ELISA reader microplate reader (Mindray, Guangdong, China) set at 450 nm.The experiment was carried out twice, with duplicate samples within each experiment.

Statistical analysis
Quantitative data were normally distributed and expressed as mean with standard deviations (± SD).Student's t-test evaluated differences between groups.The Spearman correlation coefficient calculations were used to investigate the association between the different parameters.All statistical analyses were performed using the Statistics Package of Social Science (SPSS 20, SPSS Inc., Chicago, IL) and two-tailed p-values.

Patient characteristics and cytokine levels
The mean age of the COVID-19 patients was comparable (50 ± 10.6 years) to that of the healthy controls (52 ± 12.8 years) and included subjects from both genders equally.The serum concentrations of liver enzymes ALT and AST in the patients were high (60.44 ± 33.07 U/L and 49.20 ± 25.17 U/L) in comparison to healthy controls, where the values were 10.8 ± 4.6 U/L and 15.7 ± 7.3 U/L, respectively.Similarly, in COVID-19 patients the concentrations of albumin and bilirubin were 45 ± 15.35 g/L and 50.34 ± 21.80 mg/dL, respectively, while healthy controls were 40.7 ± 31.9 g/L and 1.4 ± 0.8 mg/dL, respectively.Thus, the serum concentrations of liver function biomarkers in COVID-19 patients were much higher than in healthy controls.although not troponin (0.5 ± 0.20 ng/L), were significantly increased in the patients in comparison to healthy controls where these values were respectively, 110.57± 27.6 U/L, 50.44 ± 40.17 U/L, 6.9 ± 3.35 mg/L, 107.69 ± 55.80 ng/mL, and 0.2 ± 0.10 ng/L (Table 1).Also, the levels of cytokines IL-6 (0.8 ± 0.75 pg/mL), IL-10 (3.2 ± 2.05 pg/mL), and TNF (6.1 ± 3.32 pg/mL) in healthy controls were found to increase to 30.27 ± 13.35 pg/mL, 17.6 ± 7.2 pg/mL and 15.4 ± 6.4 pg/mL in the diseased individuals, p < 0.001**.

Correlation between cytokine levels and biochemical markers in COVID-19 patients
The relationship between serum cytokines and other biochemical markers in COVID-19 patients is in Table 3.The values indicated that serum IL-6 showed significant positive correlations with ALT (r = 0.750, p = 0.02), AST (r = 0.659, p = 0.04), CK (r = 0.569, p = 0.04), Ferritin (r = 0.704, p = 0.02), and CRP (r = 0.859, p = 0.001).On the other hand, IL-10 showed significant positive correlations with only ALT (r = 0.636, p = 0.04) and AST (r = 0.672, p = 0.5).Interestingly, TNF showed a positive correlation with ALT (r = 0.726 p = 0.05), AST (r = 0.682 p = 0.03), CK (r = 0.619 p = 0.04), Ferritin (r = 0.604 p = 0.02) and CRP (r = 0.784, p = 0.03).Our results showed no significant positive correlations between serum cytokines with biological parameters like bilirubin, creatinine, Na, and LDH.infected.Cytokine storm generation is not a universal feature of all infections.It has been observed to be triggered by the influenza A virus and may have been the cause of death during the 1918 influenza pandemic [20].Immunotherapy, organ transplantation, cancer therapy, AIDS, and sepsis are some conditions that can trigger the overproduction of cytokines and inflammatory molecules, affecting multiple organs and become lifethreatening [21].In the case of COVID-19, the selective behavior of the host response toward the virus needs to be investigated.Although unclear, the expression of specific genes may generate a cytokine storm besides other factors [22].The severity of the disease depends on underlying health conditions like diabetes, heart disease, hypertension, and other comorbidities.Early diagnosis can be made by estimating the level of cytokines before the body organs are affected, specifically the heart, liver, and kidneys.Elevated cytokine levels and anomalies in the blood profiles of patients can be helpful indicators of disease progression [23] .Previous reports have implicated excessively high levels of IL-6 in disease severity and high mortality in COVID-19 patients [24].The available clinical data have indicated that moderate and severe COVID-19 patients display abnormal liver function with increased levels of AST and ALT [25].However, the impact of COVID-19 on liver and kidney function is yet to be completely understood.Studies have shown that cytokine storms generated in non-COVID-19 cases also lead to acute liver injury [26][27].Early measurement of serum cytokines is thus a reliable predictor of disease outcome and, hence, is critical for early diagnosis and treatment.A model based on cytokine levels can help optimize therapeutic strategies and design clinical trials to modulate the released inflammatory molecules [28].Increased amounts of LDH and CK enzymes in the blood indicate cellular damage.Similarly, the release of cardiac biomarkers (CK, troponin, myoglobin) in the blood indicates a damaged heart.Their levels can be estimated in diagnosing cardiac ischemia, a condition common in SARS-CoV2 infection.The protein troponin is the most commonly used biomarker, with very high sensitivity as it remains in the blood for longer periods.Although not specific, the concentration of CK often doubles when the heart is damaged [25].In the present study, the serum concentrations of cytokines IL-6, IL-10, and TNF were all found to be elevated in COVID-19 patients.The concentration of IL-6 was found to be elevated 37-fold, which is extremely high in comparison to the other two cytokines.Ferritin and IL-10 were elevated by 5.5-fold, while TNF was only 2.5-fold higher compared to the healthy controls.TNF is a proinflammatory cytokine largely released by activated macrophages, T lymphocytes, and natural killer cells.It displays a complex network of interactions with other cytokines and further stimulates their release.IL-10, on the other hand, is an inhibitor of pro-inflammatory DOI 10.18502/sjms.v18i2.13603cytokine production and antigen presentation in activated monocytes/macrophages [29].Along with elevated serum cytokine levels, COVID-19 patients displayed increased levels of liver enzymes (ALT and AST).Interestingly, ALT, a more specific indicator of liver inflammation [15], was found in higher concentrations than AST.Compared to healthy individuals, the serum of COVID-19 patients showed elevated levels of AST, ALT, bilirubin, creatinine, urea, K, CK, LDH, troponin, ferritin, and CRP.Elevation of serum AST and ALT levels (3-5-fold) can be due to hepatocellular damage, but a 36-fold higher bilirubin level in COVID-19 patients suggests obstruction of bile flow (cholestasis).We also found the patients' CK and CRP levels to be 7-8-fold elevated.Although there was no change in the Na and Ca ion concentrations, the level of K was slightly lower, which may be due to its loss in urine [30].Ferritin is a cytosolic protein that is released into the serum.Usually, it is used as a diagnostic marker for anemia, but high serum ferritin levels may indicate acute inflammatory reactions.Ferritin, CRP, and IL-6 have been considered possible immunological biomarkers for severe COVID-19 and probable tools for screening and early diagnosis [23].As expected, the comparison of severe and nonsevere COVID-19 cases revealed that the serum cytokine levels and biomarkers were slightly higher in the former cases.Severe cases showed around 7.5 times higher levels of ferritin.The liver enzyme ALT and CK concentrations were 5-6 times higher in severe cases indicating greater liver damage here.

Conclusion
Managing the host's immune status and other biological parameters can significantly combat the SARS-CoV-2 infection.Screening patients for hyperinflammation and managing cytokine storms in immunocompromised patients, along with other biomarkers of vital organs, can improve the mortality rate.Therapeutic strategies may include a combination of antibiotics, immunoglobulins, and inhibition of cytokine overproduction.In order to understand the mechanisms and factors involved in COVID-19 disease progression, a comparative analysis of the serum cytokine levels should be done in patients and healthy individuals.Estimating increased serum cytokine levels and abnormalities in the hepatic, renal, and cardiac biomarkers can play an important role in predicting poor outcomes in COVID-19 patients.The levels of IL-6, IL-10, and TNF in the serum can also predict the severity of the disease, as cytokines play an important role in host immunity and immunopathology during the infection.Since there is no effective treatment for COVID-19 to date, the estimation of cytokine levels and biomarker molecules in patients may be used as indicators for early detection and appropriate treatment.

Table 1 :
Clinical characteristics and cytokine levels in COVID-19 patients and the healthy population.Data are presented as mean ± SD. *P < 0.05, **P < 0.001.

Table 2 :
Alterations in cytokine levels and other biomarkers in severe and non-severe COVID-19 patients.
Data were analyzed using t-test.The values indicate statistical significance (p < 0.001** and p < 0.05*)

Table 3 :
Relationship between serum cytokine levels and other biochemical parameters in COVID-19 patients.
microbial infections.Insufficient cytokines are incapable of modulating inflammation and dealing with pathogenic invasion.Similarly, excessively high amounts can sometimes cause irreparable damage.COVID-19 patients discharged from the ICU seem to have more elevated levels of inflammatory cytokines than individuals who are less severely DOI 10.18502/sjms.v18i2.13603