IL-2 and IL-1β Patient Immune Responses Are Critical Factors in SARS-CoV-2 Infection Outcomes

Background: Immune dysregulation has been linked to morbidity and mortality in COVID-19 patients. Understanding the immunology of COVID-19 is critical for developing effective therapies, diagnostics, and prophylactic strategies to control the disease. Aim: The aim of this study was to correlate cytokine and chemokine serum levels with COVID-19 disease severity and mortality. Subjects and Methods: A total of 60 hospitalized patients from the Tabuk region of Saudi Arabia with confirmed COVID-19 were included in the study. At hospital admission, the IL-1 β, IL-2, IL-8, IL-10, LT-B4, and CCL-2 serum levels were measured. The cytokine levels in COVID-19 patients were compared to the levels in 30 healthy matched control subjects. Results: The IL-1 β, IL-2, LTB-4, CCL-2, and IL-8 levels (but not IL-10) were significantly higher in all COVID-19 patients (47 survivors and 13 non-survivors) compared with the levels in the healthy control group. In the non-survivor COVID-19 patients, patients’ age, D-dimer, and creatinine kinase were significantly higher, and IL-1 β, IL-2, and IL-8 were significantly lower compared with the levels in the survivors. Conclusion: Mortality rates in COVID-19 patients are associated with increased age and a failure to mount an effective immune response rather than developing a cytokine storm. These results warrant the personalized treatment of COVID-19 patients based on cytokine profiling.


Introduction
The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in December 2019 resulted in a coronavirus pandemic. By the end of March 2022, 750,910 confirmed cases of COVID-19 and 9047 deaths had been reported in Saudi Arabia [1]. The major components of COVID-19 pathogenesis are pulmonary inflammation with significant lung damage and generalized immune dysregulation, and COVID-19-associated morbidity and mortality have been linked to an altered host immune response [2]. Two competing hypotheses explain the severity and mortality of COVID-19: a cytokine storm and the failure of the host to mount a protective immune response, resulting in uncontrolled viral dissemination and organ injury.
Paradoxically, the second theory for the morbidity and mortality caused by COVID-19 is an "immunologic collapse" of the host's immune system, manifested as unrepressed viral replication and spreading with direct host cytotoxicity. This opposing theory is supported by progressive and profound lymphopenia, which is comparable to the levels in AIDS patients. Several lymphocyte subsets, including CD4 + T, CD8 + T, and natural killer cells, which play important antiviral roles, and B cells, which are required for viral-neutralizing antibody production, are lost [7]. IL-2 deficiency appears to be a critical factor contributing to severe manifestations of COVID-19 infection, such as acute respiratory distress syndrome, multiorgan failure, and death [8].
Divergent immunotherapy avenues have emerged based on these two divergent hypotheses. Systemic corticosteroids are typically the first line of treatment to control the severity of systemic inflammation [9]. However, while initially beneficial, corticosteroid therapy has been linked to increased viral loads [10]. Widespread inflammation develops in patients with COVID-19. Therefore, inhibitors of proinflammatory cytokine signaling may have therapeutic value [11]. On the other hand, recombinant interleukin-2 stimulates lymphocyte recovery in patients with severe COVID-19 [12]. Age, obesity, pregnancy, and multiple comorbidities are significantly associated with worse outcomes in patients with COVID-19. In addition to the immunological response, other parameters were linked to the cytokine storm and mortality risk, including creatine kinase (CK), lactose dehydrogenase (LDH), C-reactive protein (CRP), and D-dimer.
The effectiveness of antiviral therapies is unclear, and national COVID-19 management protocols are updated based on the available literature [13]. Thus, understanding the immunology of COVID-19 is critical for developing effective therapies and diagnostic and prophylactic strategies for this disease. Our study aimed to develop a predictive model of COVID-19 mortality based on patient clinical features and immunological responses to guide diagnostic and therapeutic intervention and efficiently utilize the available resources during the pandemic.

Patients and Controls
A total of 60 patients who were diagnosed with COVID-19 and 30 healthy age-and gender-matched control subjects were included in the study. The COVID-19 diagnosis was confirmed by RT-PCR using nasopharyngeal and oropharyngeal samples from all study participants. All eligible participants were enrolled consecutively during the study. Patients under the age of 18 or taking immunosuppressive or immunomodulatory drugs (corticosteroids and cytokine antagonists) were excluded from the study.

Study Design and Sample Collection
The following data were collected at admission: demographic, clinical characteristics, radiological findings, laboratory values for CRP, ferritin, D-dimer, and CK, and routine laboratory tests, including hemogram and liver and renal function tests. Patients were followed up prospectively with a standardized clinical approach until hospital discharge or demise. In addition to the routine tests, 3-5 mL of peripheral blood was collected from patients on admission and from the healthy control patients. Serum was separated from whole blood by centrifugation at 2500 rpm for 15 min and stored at −80 • C. Patients were clinically followed, and outcomes (improvement or death) were determined for the hospital stay period.

Statistical Analyses
Data are presented as frequencies (percentages), means ± standard deviations, or medians (interquartile ranges), as appropriate. Linearity of all quantitative data was assessed using Kolmogorov Smirnov analyses and groups were compared using Student's unpaired t-tests, Mann-Whitney-U tests, or Kruskal-Wallis tests followed by post hoc Dunnet's tests as appropriate for the type of data. ROC curve analyses were used to assess the ability of various parameters at admission to predict mortality. Odd's ratios were calculated considering 95% confidence intervals. SPSS software, version 19.0 (Statistical Package for the Social Sciences Inc., Chicago, IL, USA) was used for all statistical analyses. A significance level of <0.05 was considered statistically significant.

Results
The 60 patients were grouped based on their outcomes. A total of 47 (78.33%) subjects survived and improved, and 13 (21.67%) succumbed. The comparisons of clinical characteristics are shown in Table 1. Age was significantly higher in the non-survivor group compared to age in the survivor group (p = 0.002). No significant differences in the frequency of symptoms were noted between the two study groups. A history of diabetes mellitus (Odd's ratio: 6.25 95% C.I.:1.24-31.32, p = 0.015) and ischemic heart disease (Odd's ratio: 3.625, C.I. 0.972-13.52, p = 0.047) were significantly more common in the non-survivors compared with the frequency in the survivors. Histories of smoking, hypertension, or chronic kidney disease were not significantly different between the two groups. The D-dimer (p < 0.0001) and CK (p = 0.015) levels were significantly higher in the nonsurvivors compared with the levels in the survivors. The treatments, immunomodulators, antiviral medications, and steroids, were similar between the two groups. However, significantly more non-survivors required oxygen therapy (p = 0.003), mechanical ventilation (p = 0.007), and ICU admission (p < 0.0001). The duration of hospital stay (p = 0.03) and O 2 therapy (p = 0.019) were significantly longer in the non-survivors compared with the duration for the survivors.
The levels of IL-1 β and IL-2 were significantly lower in the non-survivors compared to the levels in the survivors (p = 0.001 and 0.011, respectively) ( Table 2). LTB-4 also tended to be lower in the non-survivors compared to the survivors (p = 0.056). No significant differences were detected in the other measured cytokines and chemokines.
All cytokine and chemokine levels except for IL-10 were higher in both of the groups of COVID-19-positive subjects compared with the levels in the healthy controls. Post hoc analyses revealed that IL-1 β, IL-2, LTB-4, CCL-2, and IL-8 were significantly higher in COVID +ve subjects from both the survivor and non-survivor groups compared to the control group. IL-1 β, IL-2, and IL-8 levels were significantly lower in the non-survivors compared with the levels in the COVID +ve survivors (Table 3). The predictive potentials of various parameters were assessed using ROC curve analysis. Age (sensitivity = 84.6% and specificity = 66.0% above 60 years), D-dimer (sensitivity = 92.3% and specificity = 66.0% above 151 ng/mL), and creatinine kinase (sensitivity = 84.6% and specificity = 61.7% above 142.5 U/L) predicted mortality. In addition, IL-1 β had the best area under the curve (80.2%) with a sensitivity of 84.6%. The IL-1 β sensitivity was 68.1% below the cut-off value of 109.46 (pg/mL). IL-2 (sensitivity = 84.6% and specificity = 70.2% below 85.54 pg/mL) also predicted poor prognosis (Table 4, Figure 1).

Discussion
COVID-19 affected millions of people globally since it was declared a pandemic by the World Health Organization on March 11, 2020 [14]. Cytokine levels are important indicators of clinical disorders, and accurate quantification of cytokines provides valuable information for monitoring patient immune status in various diseases [15]. In this study, distinct cytokine and chemokine profiles and their correlation with disease outcome shed light on those factors linked with COVID-19 severity and mortality. In this study, IL-1 β, IL-2, LTB-4, CCL-2, and IL-8 levels were higher in all COVID-19 patients (survivors and non-survivors) compared with the levels in the healthy control group. However, succumbed patients generally showed lower levels of IL-1 β, IL-2, and IL-8 ROC curve analysis for the prognostic accuracy of the various parameters revealed that higher mortality risk was associated with increasing age and higher D-dimer and CK levels ( Figure 1) and decreased levels of IL-1 β, IL-2, and LTB-4 ( Figure 2).

Discussion
COVID-19 affected millions of people globally since it was declared a pandemic by the World Health Organization on March 11, 2020 [14]. Cytokine levels are important indicators of clinical disorders, and accurate quantification of cytokines provides valuable information for monitoring patient immune status in various diseases [15]. In this study, distinct cytokine and chemokine profiles and their correlation with disease outcome shed light on those factors linked with COVID-19 severity and mortality. In this study, IL-1 β, IL-2, LTB-4, CCL-2, and IL-8 levels were higher in all COVID-19 patients (survivors and non-survivors) compared with the levels in the healthy control group. However, succumbed patients generally showed lower levels of IL-1 β, IL-2, and IL-8

Discussion
COVID-19 affected millions of people globally since it was declared a pandemic by the World Health Organization on March 11, 2020 [14]. Cytokine levels are important indicators of clinical disorders, and accurate quantification of cytokines provides valuable information for monitoring patient immune status in various diseases [15]. In this study, distinct cytokine and chemokine profiles and their correlation with disease outcome shed light on those factors linked with COVID-19 severity and mortality. In this study, IL-1 β, IL-2, LTB-4, CCL-2, and IL-8 levels were higher in all COVID-19 patients (survivors and non-survivors) compared with the levels in the healthy control group. However, succumbed patients generally showed lower levels of IL-1 β, IL-2, and IL-8 than the levels in patients who survived COVID-19. Furthermore, significant differences were detected between the non-survivors and survivors regarding age and the levels of D-dimer and CK; the values for the non-survivors were significantly higher than the values for the survivors. Lower levels of IL-1 β, IL-2, and LTB-4 and higher levels of D-dimer and CK and advanced age predicted a higher risk of death.
The results of this study confirm that immune system changes due to aging generally result in a decline in immunity to pathogens. The senescent immune system's diminished capacity is clinically evident; aging is associated with high morbidity and mortality rates for various infections and lower vaccine efficacy in older patients [16]. T cells play an important role in the body's response to COVID-19. Our results show that a multi-layered immune response is important for controlling the virus during the acute phase of infection and reducing disease severity. Evidence suggests that T cells play a more significant role than antibodies [17]. Niessl et al., reported that T cells are important for the viral clearance of SARS-CoV-2 and limit the severe cytokine storm associated with COVID-19 [18]. With aging, the senescent immune system's supply of different T cell subsets decreases, and fewer cells are available to respond to a new virus. The Jolla Institute explained why older COVID-19 patients are much more vulnerable to the disease. With increasing age, the reservoir of T cells that can be activated against a specific virus decline, and the body's immune response becomes less coordinated, contributing to an increased susceptibility to severe or fatal COVID-19 [17]. IL-2 deficiency appears to be a critical factor in SARS-CoV-2 infection, leading to more severe manifestations, such as a weak immune response to the virus, acute respiratory distress syndrome, multiorgan failure, and death [8]. This explanation is supported by our results showing that non-survivors express lower levels of IL-2, IL-1β, and IL-8 but higher levels of D-dimer and CK, denoting excessive tissue damage. The results from the study done by Remy et al., (2020) strongly suggest that the primary endotype of COVID-19 is one of immunosuppression rather than hyperinflammation. They concluded that COVID-19 causes a profound defect in host immunity rather than hypercytokinemia-induced organ injury, and the defect in host immunity includes the profound depletion of effector immune cells and severe functional defects in the T cells and monocytes [7]. Our results agree with these conclusions; COVID-19 patients with severe illness and poor prognosis had lower levels of IL-1B, IL-2, and IL-8 than COVID-19 patients with good prognosis. Thus, broadly inhibiting the host inflammatory response with steroids or biological inhibitors of the cytokines or cytokine receptors may worsen clinical outcomes in some COVID-19 patients by further impairing the compromised host's protective immune response.

Conclusions
Understanding the immunological response to COVID-19 is critical for developing effective therapies and diagnostic and prophylactic strategies to control the disease. According to our results, different cytokine profiles are associated with COVID-19 severity and can differentially predict mortality. These results warrant the personalized treatment of COVID-19 patients based on cytokine profiling. Our study also confirms the effects of age on immunity to pathogens; aging is linked to high morbidity and mortality rates for various infections, as well as vaccine efficiency.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Local Research Ethical Committee, University of Tabuk, UT-122-16-2020 on 5 May 2020.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The datasets used and/or analyzed in this study are available from the corresponding author on reasonable request.