COVID-19 has quickly spread across the globe, affecting over 600 million people with more than 6.4 million deaths worldwide [1, 2]. Most SARS-CoV-2-infected patients develop only a mild, self-limiting disease. However, approximately 15–20% of COVID-19 patients develop severe pneumonia, and 5–10% need intensive care treatment [3,4,5].
Severe COVID-19 induces a broad range of immunological events, leading to the overproduction of pro-inflammatory cytokines and the alteration of normal antiviral immune responses [6]. This pathological state triggered by the SARS-CoV-2 virus, termed cytokine release syndrome (CRS), is characterized by the rapid and prolonged elevation in the serum levels of more than 20 inflammatory cytokines and chemokines [7]. CRS often induces acute respiratory distress syndrome (ARDS) and secondary hemophagocytic lymphohistiocytosis, which can lead to extensive tissue damage, multi-organ failure, and death [8,9]. Pro-inflammatory cytokines and hyperinflammation are believed to be key factors in this abnormal systemic immune response, which can be associated with significant morbidity and even death [9]. CRS may also be caused by a complex cellular immune dysregulation that is associated with lymphopenia, decreased numbers of circulating T, B, and NK cells, and drastic changes in myeloid cell lines [10,11,12]. Identifying the biomarkers produced during this state of hyperinflammation could be very helpful for understanding and early identification of this phenomenon, for outcome prediction and appropriate management [7,13].
Studies have shown that serum levels of interleukin (IL)-1b, IL-1RA, IL-2, IL-6, IL-7, IL-8, IL-9, IL-10, IL-17, IL-18, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) are significantly increased in COVID-19 [9,14,15,16], and some of them (IL-6, IL-8, and TNF-α) are independently correlated with disease severity [4].
IL-6 is a critical cytokine in COVID-19 associated with CRS [8]. This glycoprotein, secreted by macrophages, is a pro-inflammatory cytokine with an important role in the regulation of homeostasis [8,17]. CRS seems to be associated with IL-6 dysregulation, elevated IL-6 levels being associated with respiratory failure and poor prognosis [18,19]. Another study showed that IL-6 and C-reactive protein levels on the first day of admission were predictors of mortality in severe COVID-19 [20].
Another pro-inflammatory cytokine is IL-8, a potent chemotactic factor that attracts neutrophils, basophils, and T-cells during the inflammatory process. IL-8 is released from several cell types in response to inflammation [21,22,23], and it is believed to serve as a biomarker to indicate the prognosis of COVID-19 [22]. It was also demonstrated that in severe COVID-19 with ARDS, IL-8 may contribute to the formation of neutrophil extracellular traps, which were found in postmortem pulmonary specimens of patients with COVID-19 [23,24].
IL-10 is traditionally classified as an anti-inflammatory and immunosuppressive cytokine, produced by various myeloid- and lymphoid-derived immune cells. Studies suggest that it may also have prognostic utility regarding COVID-19 outcomes [1,25]. Other studies revealed that elevated serum IL-10 levels in patients with COVID-19 can be both an anti-inflammatory mechanism and an immunosuppressive biomarker [26,27]. In COVID-19, a higher level of IL-10 was found to be associated with a more severe disease [28].
A study that analyzed cytokine levels in patients with COVID-19 found that the serum levels of IL-6, IL-8, and IL-10 were significantly higher in patients with a more severe form of the disease. Furthermore, a score combining the levels of these three cytokines was highly predictive of progression to severe COVID-19 [29].
The aim of this study was to investigate the relationship between the serum levels of a panel of pro-inflammatory cytokines consisting of IL-6, IL-8, IL-10, IL-12, TNF-α, IFN-γ and disease severity, need for oxygen therapy, ICU transfer and fatal outcome in patients with confirmed COVID-19.
We performed a single-center prospective cohort study including 181 adult patients admitted to the 1st Infectious Diseases County Hospital of Târgu Mureș, Romania between December 2020 and September 2021. All patients were diagnosed with COVID-19, confirmed by positive real-time polymerase chain reaction (RTPCR). Patients under the age of 18 years, pregnant women, patients with HIV infection or AIDS, different types of cancer, or connective tissue disorders, transplant recipients and patients with incomplete or missing data were excluded from the study.
For each patient, clinical and demographical parameters (sex and age) were obtained from the medical records and introduced into a database. We collected data on comorbidities, serum cytokine levels, clinical severity of COVID-19, need for oxygen therapy, length of stay, ICU transfer and outcome. Cytokine (IL-6, IL-8, IL-10, IL-12, TNF-α and IFN-γ) serum levels were measured in the first 1–3 days of admission. The blood samples were collected in the first 24 h after hospitalization and, where appropriate, in the first 24 h after reclassification into another severity group, at any time during hospitalization. In the statistical analysis we used the blood samples collected in the first 24 hours after admission.
Patients were split into two age groups:
We compared cytokine serum levels between cases with COVID-19 of different severities to evaluate their predictive value of in relation to the disease severity, need for oxygen therapy, ICU transfer and outcome. We also compared serum cytokine levels according to sex, age, comorbidities and length of stay.
The severity of COVID-19 was defined based on the World Health Organization’s guidance on the clinical management of COVID-19 published on November 23, 2021. Accordingly, ‘mild stage’ was defined as a disease with few symptoms (low fever, cough, fatigue, anorexia, shortness of breath, and myalgias), without evidence of viral pneumonia or hypoxia. ‘Moderate stage’ was defined as a disease with fever and respiratory symptoms, associated with pulmonary imaging findings but no signs of severe pneumonia and an oxygen saturation (SpO2) of ≥94% on room air. ‘Severe stage’ was defined as the presence of severe pneumonia, plus one of the following: respiratory rate of >30 breaths/min, severe respiratory distress, or SpO2 <94% on room air. Based on this classification, study cases were divided into two categories: 1) non-severe (mild and moderate stage); 2) severe (severe stage).
The blood samples were collected by venous puncture into vacuum collectors with clot accelerator, were centrifuged and the serum was cryopreserved until analysis at −20 °C. The samples were transported to the Laboratory of Humoral Immunology of the Center for Advanced Medical and Pharmaceutical Research, where serum cytokine levels were measured using xMAP technology with a customized Human Magnetic beads panel for cytokine detection (EMD Millipore Corporation) on a Flexmap 3D analyzer. The laboratory procedures were performed following the manufacturers’ recommendations. For all cytokines, the measuring intervals ranged between 0.64 and 10,000 pg/ml, and the intra-assay coefficient of variation was below 3.5%.
We performed a descriptive analysis, expressing categorical variables with numbers and percentages; numerical variables with mean, median, range and inter-quartile range (IQR). The distribution of the data was assessed using the Kolmogorov-Smirnov test and analyzed using the non-parametric Mann-Whitney U test. Each parameter was estimated using a 95% confidence interval (95% CI). Multivariate logistic regression analysis was performed for disease severity, ICU transfer and outcome. The predictive ability of the studied parameters regarding ICU transfer and outcome was estimated using the receiver operating characteristic curve (ROC) and the area under the curve (AUC), where an AUC 0.5 indicated no predictive ability, a value of 0.8 was considered good, and a value of 1.0 was considered perfect. All statistical analyses were performed using IBM SPSS Statistics for Windows version 26 (Armonk, NY: IBM Corp). A
The study was approved by the ethics committees of the “George Emil Palade” University of Medicine, Pharmacy, Science and Technology of Târgu Mureș (1237/08.01.2021) and of the Mureș County Clinical Hospital (19038/21.12.2020). Written informed consent was obtained from each patient before inclusion, and full anonymity was preserved for all participants.
A total of 181 hospitalized patients were enrolled in the study. At admission, 87 (48.06%) patients had severe COVID-19, and 48 (26.51%) were reclassified from moderate to severe COVID-19 during hospital stay. The clinical and demographic characteristics of the patients are presented in Table 1.
Clinical and demographic characteristics of the cohort, grouped by disease severity
Female sex | 44 | 35 | 0.229 |
Male sex | 50 | 52 | |
Age (median) | 64 (21–86) | 64 (24–90) | 0.575 |
Length of stay (days) | 12 (1–32) | 14 (4–36) | <0.001 |
Hypoxemia requiring oxygen therapy | 49 (52.12%) | 85 (97.7%) | |
Arterial hypertension | 56 (59.57%) | 54 (62%) | 0.424 |
Cardiovascular diseases | 22 (23.4%) | 18 (20.68%) | 0.398 |
Insulin-dependent diabetes | 6 (6.38%) | 15 (17.24%) | 0.230 |
Non-insulin-dependent diabetes | 14 (14.89%) | 11 (12.64%) | 0.413 |
COPD | 3 (3.19%) | 2 (2.29%) | 0.537 |
Asthma | 4 (4.25%) | 4 (4.59%) | 0.596 |
Hyperlipidemia | 30 (31.91%) | 25 (28.73%) | 0.381 |
Obesity | 25 (26.59%) | 38 (43.67%) | |
Chronic renal disease | 3 (3.19%) | 8 (9.19%) | 0.083 |
ICU transfer | 8 (8.51%) | 27 (31.03%) | |
Discharged | 87 (92.55%) | 70 (80.45%) | |
Died | 7 (7.44%) | 17 (19.54%) |
COPD= chronic obstructive pulmonary disease; ICU=intensive care unit.
The mean age of the patients was 64 years (range 21–90). There were 57 severe and 57 non-severe cases among patients above 60 years; 30 severe and 37 non-severe cases among patients below 60 years (
We found a significant difference in the distribution of sexes between patients above and below 60 years, with a male predominance among younger patients (
Population distribution by ethnicity: 177 (97,7%) white patients and 4 (2,2%) Indo-Aryan (gipsy). We found no correlation between ethnicity and disease severity, outcome or ICU transfer requirement.
The patients’ comorbidities included arterial hypertension, diabetes mellitus, chronic obstructive pulmonary disease (COPD), asthma, pulmonary fibrosis, hyperlipidemia, obesity and chronic renal and liver disease. IL-6 (p = 0.035), IL-8 (p <0.0001), IL-10 (p = 0.009), and TNF-α (
The average length of hospital stay was 13 days, with an equal distribution between sexes. Hospital stay was significantly longer in patients over 60 years (
With the exception of IL-12 (
In total, 134 out of 181 (74.03%) patients needed oxygen therapy, 90 (67.16%) of which were over 60 years. We found a positive correlation between the need for oxygen therapy and age (r =0.163;
In total, 35 (19.33%) patients required ICU transfer, 25 (71.42%) from the >60 age group and 10 (28.58%) from the
Serum cytokine levels in patients with COVID-19 according to the need for ICU transfer
IFN-γ (pg/ml) | 0.78 (0.64–2.98) | 0.64 (0.64–1.84) | 0.403 |
IL-6 (pg/ml) | 29.08 (4.69–189.1) | 0.64 (0.64–7.7) | <0.0001 |
IL-8 (pg/ml) | 11.99 (2.41–37.81) | 1.15 (0.64–5.54) | <0.0001 |
IL-10 (pg/ml) | 30.45 (13.16–70.25) | 6.29 (1.16–23.78) | <0.0001 |
IL-12 (pg/ml) | 0.64 (0.55–1.48) | 0.64 (0.61–1.21) | 0.591 |
TNF-α (pg/ml) | 21.72 (15.47–33.69) | 19.73 (11.78–30.77) | 0.147 |
ICU=intensive care unit, IFN-γ=interferon gamma, IL=interleukin, TNF=tumor necrosis factor
Of the 114 patients over 60 years, 94 (82.46%) were discharged and 20 (17.54%) have died, while of the 67 patients below 60 years, 63 (94.03%) were discharged and 4 (5.97%) have died (
Serum cytokine levels in survivors vs. non-survivors
IFN-γ (pg/ml) | 0.64 (0.64–1.87) | 0.94 (0.57–2.97) | 0.662 |
IL-6 (pg/ml) | 0.64 (0.64–7.85) | 38.56 (15.24–714.3) | <0.0001 |
IL-8 (pg/ml) | 1.1 (0.64–5.41) | 18.89 (7.49–54.89) | <0.0001 |
IL-10 (pg/ml) | 6.76 (0.64–23.82) | 42.45 (27.19–80.85) | <0.0001 |
IL-12 (pg/ml) | 0.64 (0.59–1) | 0.93 (0.57–2.54) | 0.054 |
TNF-α (pg/ml) | 18.98 (12.29–29.47) | 25.8 (18.65–35.97) | 0.013 |
We performed a multivariate regression analysis, in which the dependent variables were represented by severity (non-severe vs. severe), survival (deceased vs. alive), and ICU transfer (transferred to the ICU vs. not transferred to the ICU) and the independent variables were represented by the different interleukins. The aim of the multivariate regression analysis was to assess the prognostic utility of interleukins based on the dependent variables. An estimated risk value of >1, expressed through the odds ratio (OR), signifies a risk or unfavorable prognosis.
The results of the multivariate regression analysis showed that depending on the ‘severity’ variable, IL-8 had a prognostic value for disease severity, increased levels being associated with severe forms (Table 4).
Results of the logistic regression analysis regarding the disease severity
IFN-γ | 0.9505 | 0.8830 to 1.0232 | 0.1769 |
IL-10 | 1.0070 | 0.9980 to 1.0161 | 0.1297 |
IL-12 | 1.0395 | 0.9661 to 1.1184 | 0.2995 |
IL-6 | 0.9994 | 0.9974 to 1.0015 | 0.5799 |
IL-8 | 1.0364 | 1.0008 to 1.0743 | 0.0414 |
TNF-α | 1.0072 | 0.9797 to 1.0355 | 0.6103 |
Depending on the ‘survival’ variable, IL-8, IL-6 and IL-10 had prognostic value, increased levels being associated with death. Of note, these three interleukins had higher values in deceased patients even at the bivariate analysis (Table 5).
Results of the logistic regression analysis regarding the outcome
IFN-γ | 0.8594 | 0.7494 to 0.9856 | 0.0302 |
IL-10 | 1.0122 | 1.0026 to 1.0219 | 0.0126 |
IL-12 | 1.0513 | 0.9849 to 1.1223 | 0.1330 |
IL-6 | 1.0046 | 1.0008 to 1.0093 | 0.0399 |
IL-8 | 1.0430 | 1.0175 to 1.0693 | 0.0009 |
TNF-α | 1.0054 | 0.9626 to 1.0501 | 0.8085 |
Depending on the ‘ICU transfer’ variable, IL-8, IL-6 and IL-10 had prognostic value, increased levels being observed in those transferred to the ICU. Similarly, to the ‘survival’ variable, these three interleukins had higher levels in those transferred to the ICU even at the bivariate analysis (Table 6).
Results of the logistic regression analysis regarding ICU transfer
IFN-γ | 0.9212 | 0.8257 to 1.0278 | 0.1419 |
IL-10 | 1.0114 | 1.0018 to 1.0210 | 0.0196 |
IL-12 | 0.9977 | 0.9287 to 1.0719 | 0.9503 |
IL-6 | 1.0080 | 1.0006 to 1.0164 | 0.0409 |
IL-8 | 1.0353 | 1.0121 to 1.0590 | 0.0026 |
TNF-α | 0.9871 | 0.9508 to 1.0248 | 0.4959 |
ROC curve analysis for IL-8 regarding disease severity yielded an AUC of 0.632 (95% CI 0.550–0.714;
ROC curve analysis also showed a good predictive ability for IL-6 (AUC 0.853; 95% CI 0.759–0.946;
Cut-off levels and AUC values for IL-6 and IL-10 regarding patient outcome are shown in Table 7.
Cut-off values and ROC analysis for biomarkers with statistical significance in predicting outcome in COVID-19 patients
IL-6 | 20.14pg/ml | 0.853 (0.793–0.901) | <0.0001 | 75.00 | 87.26 |
IL-10 | 18.00pg/ml | 0.831 (0.768–0.882) | <0.0001 | 91.67 | 70.70 |
Outcome prediction for the appropriate management of patients with COVID-19 is a serious challenge. The severity of COVID-19 depends on several factors, but the pathogenesis of CRS and hyperinflammation remains in center of the interest. In these settings, a bio-marker with high sensitivity and specificity can guide the clinician to identify high-risk patients for close follow-up and appropriate management.
Hormonal differences between men and women may cause differences in ACE-2 expression and the production of cytokines, and may also affect mortality [30,31]. In women, estrogen has a protective effect on the immune system [31]. In a retrospective study conducted on 548 inpatients with COVID-19, Qin
Studies have shown that age may be the most important risk factor for severe COVID-19 and its complications [31,33]. Furthermore, older patients can have an increased production of pro-inflammatory cytokines, which can lead to CRS [34]. Although in our study IL-6, IL-8, IL-10 and TNF-α serum levels were significantly higher in subjects >60 years, we found no correlation between age and disease severity.
Comorbidities, such as hypertension and diabetes, can also affect the production of pro-inflammatory cytokines including IL-2R, IL-10 and TNF-α [32]. Our findings are in agreement with these reports, as serum levels of IL-6, IL-8, IL-10, TNFα were significantly elevated in patients with arterial hypertension.
Studies suggest that obesity-induced inflammation can modify innate and adaptive immune responses, resulting in a greater vulnerability to infection [35,36]. In our study, we found a significant association between obesity and disease severity (
Hypoxemia, longer hospital stays and ICU transfer were significantly associated with severe COVID-19. However, there were no significant differences between patients with severe and non-severe disease regarding hypertension and diabetes mellitus.
A longer hospital stay was associated with the presence of respiratory failure and ICU transfer, and serum levels of IL-6, IL-8, and IL-10 were higher in these patients.
Guo
In our study, elevated IL-6, IL-8, and IL-10 serum levels were correlated with the presence of respiratory failure. An injured lung can be a major source of IL-6 production, which may explain correlations observed between cytokine serum levels and the need for oxygen therapy [4,39]. TNF-α is linked to bronchial hyperresponsiveness and is involved in the deterioration of respiratory epithelium by cytokines [8]. We found TNF-α serum levels to be correlated with the presence of hypoxemia. However, our results suggest that cytokines have a poor predictive value regarding the need for oxygen therapy.
Studies suggest there is a positive correlation between ICU transfer and elevated serum levels of IL-6, IL-8 and TNF-α. Also, higher TNF-α levels are associated with a longer ICU stay in men [13,18]. We found significantly increased serum levels of IL-6, IL-8, IL-10 and TNF-α in patients who required ICU transfer.
In previous studies, increased TNF-α, IL-1Ra, IL-6, IL-8, IL-15 and IL-10 serum levels were associated with higher mortality in COVID-19 [14]. Furthermore, IL-1, IL-6 and TNF-α produced by macrophages have been reported as pathogenic factors in the excessive inflammatory response in COVID-19. Research suggests that the inflammatory cascade and increased secretion of pro-inflammatory cytokines in the lower airways may be responsible for tissue destruction in various organs [18,40,41]. In this study, we found elevated IL-6, IL-8, IL-10 and TNF-α serum levels in patients with fatal outcome and who required oxygen therapy. Of the studied cytokines, IL-6 had the best predictive ability for outcome (AUC 0.853; 95% CI 0.759–0.946;
IL-6 is reported to have a unique role in the cytokine storm related to COVID-19. Increased IL-6 serum levels can modulate the activity of natural killer cells and were found to be associated with other immune dysregulations. Previous studies have explored its predictive value regarding disease severity, ICU transfer and outcome [1,3,42]. In this study, IL-6 had a good predictive ability for ICU transfer (AUC = 0.801) and outcome (AUC = 0.853).
IL-8 is involved in the activation and recruitment of neutrophils in COVID-19, and studies suggest that it may be a biomarker of ARDS [14,43]. In our study, IL-8 demonstrated a poor predictive ability for outcome, ICU transfer or the need for oxygen therapy. These findings suggest that compared to the other cytokines we studied, IL-8 is a poor predictor of COVID-19 severity.
IL-10, an anti-inflammatory cytokine, amplifies viral sepsis in severe COVID-19, probably through overactivation and proliferation [14,44]. Some studies have found that IL-10 can be used to predict poor outcome in COVID-19 [1,13]. Our study confirms these findings, as IL-10 showed a good predictive capacity for outcome.
Similarly, to our study, Zhou
Our findings indicate that IL-6, IL-8, and IL-10 serum levels are significantly increased in COVID-19 but are associated with poor predictive ability regarding disease severity and the need for oxygen therapy. From the studied cytokines, IL-6 and IL-10 seem to be independent predictors for ICU transfer and outcome, with IL-6 having a better predictive performance. A limitation of the study is the small number of patients and a rather limited level of originality when addressing the cytokine role in the COVID-19 severity. We believe though that by diversifying the endpoints it provides a useful addition to the literature on this subject.