key: cord-0760702-ylqt9d1p
authors: Al‐bassam, Wasan W.; Al‐Karaawi, Ibtihal A.; Sharquie, Inas K.; Ad'hiah, Ali H.
title: Evaluation of interleukin‐38 levels in serum of patients with coronavirus disease 2019
date: 2022-04-14
journal: J Med Virol
DOI: 10.1002/jmv.27762
sha: 084cf008594e9a5285c463614c99dfb1bdfac7b8
doc_id: 760702
cord_uid: ylqt9d1p

Interleukin‐38 (IL‐38) has recently been considered as a cytokine with anti‐inflammatory properties in viral respiratory infections, particularly coronavirus disease 19 (COVID‐19), but the evidence has not been well elucidated. Therefore, a case‐control study was conducted to determine IL‐38 serum levels in 148 patients with COVID‐19 (45 moderate, 55 severe, and 48 critical) and 113 controls. Results demonstrated that IL‐38 levels did not show significant differences between patients and controls (68.7 [interquartile range: 62.7–75.6] vs. 67.7 [58.0–82.6] pg/ml; probability = 0.457). Similarly, patients stratified by disease severity, age group, gender, or chronic disease showed no significant differences between IL‐38 levels in each stratum. Whereas, overweight/obese patients had a significantly lower median of IL‐38 compared to normal‐weight patients. Further, IL‐38 showed significantly higher levels in the age group ≥50 years of patients with critical illness than in the age group <50 years. Female patients with severe disease also showed significantly elevated levels of IL‐38 compared to male patients. In conclusion, the study indicated that serum IL‐38 levels were not affected by COVID‐19 infection, but the distribution of patients according to disease severity, age, gender, and body mass index may better reveal the role of IL‐38 in disease pathogenesis.

| 1 in cases of ARDS, where the risk of death is increased. 6 Thus, investigation of the mechanisms by which the human body balances the activity of immune system (proinflammatory and anti-inflammatory cytokines) to enable an appropriate response in viral clearance is a fundamental issue. 7 One group of cytokines that has been addressed for its role in immunopathogenesis of COVID-19 and/or disease severity is the IL-1 family of cytokines. Interestingly, this family includes cytokines with both proinflammatory (IL-1α, IL-1β, IL-18, IL-33, and IL-36) and anti-inflammatory (IL-37 and IL-38) effects, some of which are linked to damaging inflammation, while others mediate resistance to viral and bacterial infections. 8 In COVID-19 infection, the IL-1 cytokine family has been identified to play a pivotal role in the induction of cytokine storm due to dysregulated immune responses. 9 Further, the anti-inflammatory cytokine IL-37 showed reduced levels in serum of severely ill COVID-19

patients. 10 A recent study also demonstrated for the first time that IL-38 is an additional cytokine of this family that showed significantly elevated circulating levels in COVID-19 patients. The study concluded that IL-38 may exert a protective role against immune system hyperactivity, and may alleviate induced lung inflammation. 11 Besides, it has been indicated that dysregulated serum levels of IL-38 are linked to immunopathogenesis of several inflammatory and immune-mediated disorders, as well as lungrelated diseases. 12 Due to limited data on IL-38 and COVID-19, the current study examined serum levels of IL-38 in patients with COVID-19 and healthy controls to evaluate the potential role of this cytokine in pathogenesis of COVID-19 infection. Further, disease severity (moderate, severe, and critical), as well as patient-related parameters (age, gender, body mass index and chronic disease status) were taken into account in the evaluation.

A case-control study was conducted on COVID-19 patients admitted to COVID- 19 severe (<94% oxygen saturation and ≥30 breaths/minute respiratory rate) and critical (respiratory failure) illness. 13 Patients were also characterized for body mass index (BMI; normal-weight and overweight/obese) and chronic disease (present and absent).

Chronic disease was defined as cardiovascular disease (CVD), diabetes, or both. Patients were also profiled for the following laboratory parameters: hemoglobin (Hb), platelet count, white blood cell count (WBC), erythrocyte sedimentation rate (ESR), random blood glucose (RBG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total cholesterol, triglycerides, creatinine, blood urea nitrogen (BUN) and uric acid.

The control group included blood donors and health service personnel, who were healthy and did not suffer from respiratory and chronic diseases. They were seronegative for anti-COVID-19 IgG and

IgM antibodies and C-reactive protein, and their ESR was below 20 mm/h.

Human IL-38 enzyme-linked immunosorbent assay (ELISA) kit was used to determine IL-38 level in serum of participants (Cat. No E3276Hu Bioassay Technology Laboratory, China), and instructions of manufacturer were followed. Standard curve range of the kit was 0.5-200 pg/ml.

Categorical variables were expressed as number and percentage, and significant differences were assessed using Pearson chi-square test. Continuous variables were subjected to two tests of normality; Kolmogorov-Smirnov and Shapiro-Wilk tests. Normally distributed variables were given as mean and standard deviation (SD), and significant differences were assessed using one-way analysis of variance (ANOVA) test. Skewed variables were presented through median and interquartile range (IQR), and significant differences were assessed using Mann-Whitney U (to compare two groups) or Kruskal-Wallis H (to compare more than two groups) test. Receiver operating characteristic (ROC) curve was applied to calculate area under the curve (AUC). All participants were distributed into two groups in relation to median levels of IL-38 in controls; low and high production groups (< and ≥ median, respectively), and then logistic regression analysis was performed to calculate odds ratio (OR) and 95% confidence interval (CI). A probability (p) value ≤ 0.05 was taken statistically significant. 

The 148 patients enrolled in this study were categorized according to severity of COVID-19. Forty-five patients had moderate disease, while remaining patients experienced either severe or critical illness (55 and 48 patients, respectively). The latter two groups of patients had a significantly higher mean age compared to patients with moderate disease (58.1 ± 12.6 and 58.8 ± 14.9 vs. 52.2 ± 14.8 years, respectively; p = 0.048). In fact, more than 50% of moderate cases were classified under the age group < 50 years (53.3%), while 69.1% and 75% of severe and critical cases were above the age of 50 years, respectively. These differences were significant (p = 0.011). Distributions of gender (males and females), BMI (normal-weight and overweight/obese) and chronic disease (present and absent) subgroups in the three disease severity groups showed no significant differences. With regard to chronic diseases (CVD, diabetes, or both), it is noteworthy that patients with moderate, severe, or critical disease shared an increased incidence of these diseases (53.3%, 63.6%, and 50.0%, respectively). Patients were also defined by the laboratory parameters listed in Table 1 . Means of these parameters showed no significant differences between the three disease severity groups. WBC, ESR, and RBG were exceptions and positively correlated COVID-19 severity. Their means were significantly higher in patients with severe or critical illness than in patients patients in the three disease severity groups, but no significant differences were found. In the case of chronic disease, there were no significant differences between the IL-38 levels in patients with and without chronic disease in the three disease severity groups ( Figure 2 ).

ROC curve analysis demonstrated that IL-38 was a poor predictor of COVID-19 as the estimated AUC was 0.522 (p = 0.546).

A similar predicting power was indicated when the patients 

Based on median levels of IL-38 in controls, all subjects were classified as low and high production groups (<67.7 and ≥67.7 pg/ml, respectively), and then logistic regression analysis was performed to calculate OR and 95% CI. Although the analysis did not reveal a significant association between IL-38 and the risk of developing 

A previous study has demonstrated that serum levels of IL-38 increased in patients with SARS-CoV-2 infection and these levels were declined after recovery from the infection. This increase was more pronounced in nonsevere cases than in severe cases.

Accordingly, the significance of IL-38 in predicting the prognosis of SARS-CoV-2 infection has been proposed. 11 The results of the current study were not in favor of these findings and instead showed that serum levels of IL-38 were not affected by COVID-19 as no significant differences were found between patients and controls. COVID-19 infection is known to cause dysregulated production of cytokines with a hallmark of elevated levels of proinflammatory cytokines associated with suppressed antiviral immunity, and this positively correlates with disease severity and increased morbidity and mortality. 18 Elevated levels of proinflammatory cytokines, such as IL-1, IL-6, and TNF-α, trigger an intense inflammatory response by recruiting immune cells such as macrophages to the alveoli, which in turn recruit more inflammatory cells such as monocytes that produce greater amounts of cytokines, thus increasing the inflammatory F I G U R E 2 Scatter dot plots with median (horizontal red line) and interquartile range (vertical red line) of Interleukin-38 (IL-38) serum levels in COVID-19 cases stratified by disease severity (M, moderate; S, severe, and C, critical) and age group (<50 and ≥50 years, gender (♂: male and ♀: female), BMI (body mass index; N: normal-weight and O: overweight/obese) and chronic disease (P: present and A: absent). Significant differences between medians were assessed using Mann-Whitney U test. IL-38 tended to show higher levels in the age group ≥50 years of patients with severe or critical illness than in the age group <50 years, but the difference was only significant in severe cases. Female patients with severe disease also showed significantly elevated levels of IL-38 compared to male patients. IL-38 levels tended to be lower in overweight/ obese patients (O) than in normal-weight patients in the three disease severity groups, but no significant differences were recorded. In the chronic disease condition, there were no significant differences between the levels of IL-38 for patients with and without chronic disease in the three disease severity groups response, particularly in patients with severe COVID-19. 19 The ill patients compared to patients with nonsevere disease. 11 In the current study, increased levels of IL-38 were found in severe/critical patients over 50 years of age. At older ages, cytokine dysregulation was indicated with a progressive tendency toward a proinflammatory phenotype. 23 COVID-19 is also associated with upregulated levels of proinflammatory cytokines. 3 Therefore, increased levels of IL-38 in patients over 50 years of age may counteract these up-regulations of proinflammatory cytokines. It has also been shown that serum IL-38 levels exhibit an age-related modality in healthy aging, while elevated IL-38 levels were reached more rapidly in older patients with type 2 diabetes mellitus, and an increase in IL-38 levels might have occurred to exert an anti-inflammatory effects. 24 Besides age, IL-38 levels were significantly increased in severely ill females compared to male patients. There is no direct evidence to support upregulated levels of IL-38 in females compared to males. In Behçet's disease, a chronic multisystem autoimmune disease, serum levels of IL-38 were higher in female patients with a positive pathergy test than in patients with a negative test and in patients with ocular involvement than in patients without ocular involvement. These differences were not found in male patients. 25 In multiple sclerosis and systemic sclerosis, IL-38 levels showed no significant differences between male and female patients. 26 In the case of COVID-19, IL-10, an anti-inflammatory cytokine, was demonstrated to exhibit a favorable female phenotype, and a more robust anti-inflammatory response was observed in female patients compared to males. 27 Accordingly, gender difference in levels of anti-inflammatory cytokines is an important issue in understanding why female COVID-19

patients have a better prognosis and lower mortality than male patients, but the evidence has not been conclusive and further studies are warranted.

There was a tendency for IL-38 levels to decrease in overweight/obese patients compared to normal weight patients regardless of disease severity. In fact, more than 50% of COVID-19 patients were overweight/obese, and a positive association between disease severity and obesity has been reported in the literature. 14 The relationship between IL-38 and obesity has not been well investigated, but two related studies deserve mention. In the first, a relative deficiency of IL-38 has been reported in healthy, overweight Europeans and was associated with higher systemic inflammation in the elderly, and those with CVD and metabolic disease. 28 In the second, IL-38 has been proposed as a potential target for the treatment of obesityinduced adipose tissue inflammation. The study also indicated that IL-38 could improve insulin resistance in mice with induced obesity. 29 F I G U R E 4 Spearman's rank order correlation coefficient (r s ) between Interleukin-38 (IL-38) and hemoglobin (Hb), platelets, white blood cell count (WBC), erythrocyte sedimentation rate (ESR), random blood glucose (RBG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total cholesterol, triglycerides, serum creatinine, blood urea nitrogen (BUN), and uric acid in COVID-19 patients. Four significant correlations were found. IL-38 showed negative correlation with Hb (r s = −0.173; p = 0.035) and random blood glucose (r s = −0.250; p = 0.002), while it was positively correlated with BUN (r s = 0.210; p = 0.011) and uric acid (r s = 0.177; p = 0.032). p: Two-tailed Fisher's exact probability patients according to disease severity, age, gender and BMI may better reveal the role of IL-38 in the pathogenesis of COVID-19.

All authors contributed equally to conceptualization, visualization, methodology, investigation, validation, and writing-reviewing and editing.

COVID-19: breaking down a global health crisis

Insights to SARS-CoV-2 life cycle, pathophysiology, and rationalized treatments that target COVID-19 clinical complications

Specific cytokines in the inflammatory cytokine storm of patients with COVID-19-associated acute respiratory distress syndrome and extrapulmonary multiple-organ dysfunction

Cytokines in inflammatory disease

SARS-CoV-2 infection: the role of cytokines in COVID-19 disease

Lung under attack by COVID-19-induced cytokine storm: pathogenic mechanisms and therapeutic implications

Role of the immune microenvironment in SARS-CoV-2 Infection

Overview of the IL-1 family in innate inflammation and acquired immunity

Interleukin-1 in COVID-19 Infection: immunopathogenesis and Possible Therapeutic Perspective

Interleukin-37 is down-regulated in serum of patients with severe coronavirus disease

Interleukin-38 ameliorates poly(I:C) induced lung inflammation: therapeutic implications in respiratory viral infections

Immunobiological properties and clinical applications of interleukin-38 for immune-mediated disorders: a systematic review study

Clinical Management of Severe Acute Respiratory Infection When Novel Coronavirus (2019-nCoV) Infection is Suspected: Interim Guidance 28

Severe obesity is associated with higher in-hospital mortality in a cohort of patients with COVID-19 in the Bronx

Association of body mass index and age with morbidity and mortality in patients hospitalized with COVID-19: results from the American Heart Association COVID-19 Cardiovascular Disease Registry

Differential cytokine signatures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza infection highlight key differences in pathobiology

Cytokine profiles in overweight and obese subjects and normal weight individuals matched for age and gender

Cytokine storm in COVID-19: the current evidence and treatment strategies

Role of inflammatory cytokines in covid-19 patients: a review on molecular mechanisms, immune functions, immunopathology and immunomodulatory drugs to counter cytokine storm

Pro-and antiinflammatory responses in severe COVID-19-induced acute respiratory distress syndrome-an observational pilot study

Role of interleukin-38 in chronic inflammatory diseases: a comprehensive review

IL-38: A new player in inflammatory autoimmune disorders

Age and age-related diseases: role of inflammation triggers and cytokines

Plasma levels of interleukin-38 in healthy aging and in type 2 diabetes

IL-38 serum levels in patients with Behcet's disease and the relationship with clinical features

Elevated IL-38 serum levels in newly diagnosed multiple sclerosis and systemic sclerosis patients

Sex differences in the immune response to acute COVID-19 respiratory tract infection

Reduced concentrations of the B cell cytokine interleukin 38 are associated with cardiovascular disease risk in overweight subjects

Hydrodynamic delivery of IL-38 gene alleviates obesity-induced inflammation and insulin resistance

Ad'hiah AH. Evaluation of interleukin-38 levels in serum of patients with coronavirus disease 2019

The authors sincerely appreciate the cooperation of the medical staff at the COVID-19 care units in Baghdad hospitals.

The authors declare that there are no conflicts of interest.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ali H. Ad'hiah http://orcid.org/0000-0002-2445-2242