In December 2019, in Wuhan, Hubei province, China, several patients with atypical pneumonia were hospitalized (1). A new virus from the group of coronaviruses was identified and called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the disease called Coronavirus Disease (COVID-19) (2). SARS-CoV-2 is highly infectious and asymptomatic patients are the main source of infection (3). Because of its easy and quick transmission and interhuman spread, the WHO declared it as a pandemic on March 11, 2020 (1). Recent studies have shown that different biochemical parameters are altered in patients with COVID-19, and some of them are useful as indicators of disease severity (4–6). Patients can have various forms of the disease, from asymptomatic to acute respiratory distress syndrome (ARDS) and respiratory insufficiency (7, 8). The most common manifestations are fever, cough, dyspnea, fatigue, chest pain, loss of sense of smell, headache, nausea or vomiting, and pharyngalgia (7, 9). Chest X-rays (CXR) is not considered a sensitive method for the detection of lung abnormalities in the early stage of the disease, but it is useful for monitoring the lung progression in later stages of the disease (10, 11). Immunopathogenic mechanisms during the disease vary from immunosuppression to uncontrolled immune-inflammatory response (12, 13). While the immunosuppression is conveyed through well-known immunosuppressive cytokines such as transforming growth factor β (TGF-β) and interleukin (IL)-10, the uncontrolled immune response is manifested by the rapid production and excretion of proinflammatory cytokines, such as cytokines of the IL-1 family, tumor necrosis factor α (TNFα), and IL-6. (12, 14) The rapid secretion of cytokines, chemokines, and other proinflammatory molecules, often referred to as cytokine storm, can be clinically manifested as ARDS with consequent respiratory failure (15, 16). Even though ARDS and cytokine storm are well-established entities in modern medicine, the exact mechanisms of respiratory failure and impeding proinflammatory cascade in COVID-19 are still challenging but might be of great significance when it comes to predicting the outcome of the disease and treatment (17).

One of the cytokines that have not been sufficiently studied in the immunopathogenesis of COVID-19 is IL-33. As a member of the IL-1 family, IL-33 induces an inflammatory response within the tissue (18–20). In the lungs, it is secreted mainly by injured epithelial alveolar cells (10, 19). As a previous study showed that the virus can trigger the secretion of IL-33 through activation of SARS CoV-2-derived papain-like protease (PLpro), IL-33 is considered as a predictor of severe forms of COVID-19. However, the potential role of IL-33 in disease genesis and progression has not been studied sufficiently (18, 19).

In this study, we analyzed the correlation of IL-33 and other innate immunity cytokines such as TNF-α, IL-1β, IL-6, IL-12, and IL-23 with disease severity. Also, we analyzed interdependence between disease severity and clinical, biochemical features, and CXR lung findings in 220 patients with COVID-19.

In this study, we analyzed the relationship between clinical features, laboratory features, radiographic findings, and innate immunity cytokine profile (IL-33, TNF-α, IL-1β, IL-6, IL-12, and IL-23) with disease severity. Patients with COVID-19 were divided into two groups (mild/moderate and severe/critical) according to the criteria described in the material and methods. We found that gender and age correlate with disease severity. Most patients in the severe/critical group were male (81.8%) and older (over 64.5 years). This is in accordance with the study of Hu et al., where the authors suggested that age and gender may be risk factors for severe clinical outcomes (24). In our study, patients in the severe/critical group have a significantly higher frequency of dyspnea, fatigue, and auscultatory attenuated breathing sound and diffuse cracks in comparison to the mild/moderate group (Table 1). Also, the higher values of the neutrophil count, CRP, D dimer, PCT, LDH, urea, creatinine, CK, Ferritin, TBIL, AST, as well as lower values of lymphocyte and monocyte count, and albumin were detected in the group of patients with severe COVID-19 (Table 2). The same phenomenon was reported in the studies of Wang et al. (25) and Sánchez et al. (26). We detected higher values of white blood cell count, ALT, and lower values of red blood cells, platelets count, K⁺, and Na⁺ concentrations in patients with severe COVID-19, but the differences did not reach statistical significance (Table 2). Given the vast range of immune, biochemical, and metabolic disruptions during severe COVID-19, multiple organ failure is inevitable. As far as it is known, the thrombosis of various organs, renal, hepatic, cardiac, and neurological injury occurs more frequently as the severity of COVID-19 increases (27–29). In addition, our study revealed that PCT sera levels were significantly higher in the severe/critical group in comparison to the mild/moderate group. Since PCT level is not elevated in viral infections per se, this finding might be a marker of amplified systemic inflammatory response (e.g., cytokine storm) or secondary bacterial or fungal infections (27, 30).

Further on, we analyzed the CXR findings of 220 patients divided in the mild/moderate and severe/critical groups. As mentioned before, CXR findings are divided into five groups, where CXR I, II, III are expected findings in the mild/moderate group, and CXR IV and V are expected to be found in severe/critical forms of the disease. Radiographic findings such as multifocal consolidation and diffuse alveolar damage, denoted as CXR IV and V, respectively, are significantly more frequent in severe forms of COVID-19 that require supportive modes of ventilation, such as high-frequency ventilation (HFV), non-invasive (NIV), or invasive mechanical ventilation.

It is believed that immunopathology is the main mechanism in the genesis and progression of COVID-19 (12, 14). The cellular composition of the lung infiltrates in patients with COVID-19 pneumonia changes with the progression of the disease. Infiltrates in patients with moderate pneumonia include mainly lymphoid and dendritic cells, while the severe form of the disease is characterized by massive infiltration of macrophages and neutrophils (31, 32). In line with this finding, our results revealed an increment in neutrophil count in patients with severe COVID-19 (Table 2). Interestingly, we detected a decrement in monocyte count in the same patients (Table 2), which we believe may be due to the accumulation of these cells in the lungs and differentiation into macrophages. The destruction of the alveolar epithelium, which is directly related to the severity of the disease, is due to an intense immune response (33, 34). There is ample evidence that both, innate and acquired immunity participate in the immunopathogenesis of COVID-19 (12, 14, 35). However, the main pro-inflammatory mediators that play a key role in tissue destruction belong to the innate immune response (36, 37). In order to clarify the correlation between COVID-19 and innate immunity cytokines of interest, we analyzed serum cytokines in patients with COVID-19. The increased systemic values of TNF-α, IL-1β, IL-6, IL-12, IL-23, and IL-33 (Figure 1) support the prevalence of innate pro-inflammatory mediators in the serum of subjects with a severe form of COVID-19. The obtained results are in line with similar studies, which confirm the strong correlation between disease severity and increased values of some innate immunity-associated proinflammatory cytokines (36–39).

Interleukin-33 is a member of the IL-1 cytokine family that is expressed in barrier tissues and exerts pleiotropic functions. Its role in immune response regulation after cellular stress or damage is crucial and is involved in the pathogenesis of COVID-19 (40). The analysis of the published scRNAseq data of bronchoalveolar lavage fluid (BALF) from patients with mild to severe COVID-19 revealed a population of IL-33-producing cells that increases with the disease. These findings show that IL-33 production is linked to SARS-CoV-2 infection (41). Our result revealed a significantly higher systemic IL-33 concentration in patients with severe COVID-19, positive association between IL-33 and innate immunity mediators, and clinical parameters of COVID-19 (Figure 1, Table 3, Supplementary Table 2), suggesting the important role of this cytokine in the progression of COVID-19. In line with these are the results of other studies, which showed that disease severity is associated with an increased level of IL-33 (41–43).

The almost unchanged ratio between IL-33 and innate immunity mediators of interest during COVID-19 progression (Figure 2) indicates the stable dynamics of cytokine growth. Our results revealed a stronger positive correlation between serum values of IL-33 and innate immunity mediators TNF-α, IL-1β, IL-6, IL-12, and IL-23 in the severe form of COVID-19 (Table 3). Advanced stages of COVID-19 are characterized by high circulating and pulmonary concentrations of innate immunity cytokines (33, 44, 45). Signaling downstream of IL-1 family receptors, including IL-1β and IL-33 receptors, can activate MyD88 and elicit inflammation via production of high amounts of IL-1β, and NF-κB-induced cytokines such as TNF-α, IL-6, IL-12, and IL-23 (38, 46). In line with this, we found a positive mutual relation between the serum values of IL-33 and IL-12, IL-33 and IL-6, and IL-6 and IL-12 (Supplementary Table 1). The revelation of the positive correlation between the systemic IL-33, IL-6, and IL-12 levels in serum of COVID-19 patients suggested the synergistic effect of these cytokines in the pathogenesis of COVID-19 disease.

Significantly, disease severity was associated with age, neutrophil count, glucose, urea, creatinine, BIL T, BIL D, AST, LDH, CK, D dimer, CRP, PCT, TNF-α, IL-1β, IL-6, IL-12, IL-23, and IL-33 (Tables 1, 2; Supplementary Table 2). Among all the analyzed innate immunity cytokines, the strongest correlation was between IL-33 and disease severity (Supplementary Table 2). Serum levels greater than 332.08 pg/ml of IL-33 are associated with increased severity of COVID-19, indicating that IL-33 could be used as a prognostic biomarker in patients with a diagnosis of COVID-19. Interestingly, when CXR findings are compared with the sera levels of IL-33, a significant increase of IL-33 was detected in patients with CXR III, IV, and V findings. Focal consolidation, denoted as CXR III, is a CXR sign of mild to moderate cases of COVID-19. However, having in mind that elevated IL-33 is a marker of severe COVID-19 (19, 40), our result might imply that focal consolidation, alongside elevated IL-33 in the serum of patients might predict the progression of mild/moderate to severe/critical forms of the disease (Figure 3). As it is known, inflammation is closely related to the severity of COVID-19 (37). Increased inflammation consequently leads to more extensive tissue damage, and increased externalization of heat-shock proteins and alarmins. As IL-33 functions as both, alarmin and cytokine, it is possible that at first, as an alarmin, IL-33 facilitates inflammation, and then perpetuates it via interaction with other innate immunity cytokines or other immune cells, forming a circulus vitiosus of inflammation in the lungs.

Taking into account the results of this and similar studies, we believe that SARS-CoV-2 infection triggers IL-33 release from damaged epithelial cells in the lung (47). Released IL-33 initiates type 2 immune response that further leads to impaired antiviral activity and dysregulation of neutrophils (18). Dampened antiviral immunity results in delayed viral clearance and COVID-19 progression. In a more progressive stage of the disease, extensive injury of alveolar epithelial cells caused by strong interactions between the airway epithelium and activated immune cells is followed by overproduction of IL-33 (47). Under these circumstances, increased IL-33 facilitates lung inflammation by inducing the production of various innate proinflammatory cytokines (IL-1β, IL-6, TNF-α, IL-12, IL-23) in several target cells leading to the most severe forms of the disease (20, 48). Keeping in mind obtained data that IL-33 portrays an important role during severe form of COVID-19 followed by augmented inflammation, IL-33 might act as a promising therapeutic target which hinders COVID-19 severity.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The study involving human participants was reviewed and approved by Ethics Committees of the University Clinical Center of Kragujevac, Serbia (Approval Number 01/20-406) Faculty of Medical Sciences, University of Kragujevac, Serbia (Approval Number 01-6776). The patients/participants provided their written informed consent to participate in this study.

SM and IJ: conceptualization. SM, NG, and MilenaJ: methodology. MilenaJ: software and formal analysis. IJ and VM: validation. SM, MarinaJ (6th author), NG, and MilenaJ: investigation. NA: resources. SL, NZ, and MiodragJ: data curation. SM: writing—original draft preparation. IJ and MarinaJ (2nd author): writing—review and editing. VV, MilanJ, and JS: visualization. IJ: supervision. IJ and NA: project administration. NA and ZM: funding acquisition. All authors have read and agreed to the published version of the manuscript.

This work was supported by a grant from the Science Fund of the Republic of Serbia (CIBIRDS), Serbian Ministry of Education, Science and Technological Development (175069), project with PR China (06/2018).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.