Comparison of the serum level of interleukin‐4 in patients with brucellosis and healthy controls

Abstract Introduction Evaluation of cytokines such as interleukin‐4 (IL‐4) can be an important tool in examining immune responses to brucellosis. Also, determining the response rate to treatment is necessary for controlling and eradicating of disease. The review of previous studies reveals contradictory results that require further research in this regard. The aim of this study was to compare the serum level of IL‐4 in patients with brucellosis and healthy controls. Material and Methods In this descriptive‐analytical study for comparison of two groups, a total of 165 participants, including 83 patients with brucellosis and 82 non‐infected people, were evaluated after matching of sex and age in Hamadan (northwest of Iran) in 2017 and the serum level of IL‐4 was compared by ELISA method. The collected data were analyzed by SPSS software version 21 at 95% significant level. Results Mean of age in the case and control groups were 50.25 ± 16.01 and 43.26 ± 15.6 years, respectively. The serum levels of IL‐4 in the case and control groups were 1.42 ± 0.51 pg/mL and 1.31 ± 1.02 pg/mL, respectively. Based on the non‐parametric Mann‐Whitney test, the IL‐4 level was significantly higher in the case group, compared with the control (P < .001), but no statistically significant relationship was found between serum levels of IL‐4 with age, sex, and serologic titers of Wright and 2ME. Conclusion In patients with brucellosis, the level of IL‐4 increases independently of the duration and severity of the disease, which indicates the role of this cytokine of immune system in this infectious disease.

IL-4 was discovered first in 1980. This cytokine is produced by basophils, mast cells, eosinophils, and Th2 lymphocytes.  contributes to the regulation of cell division, gene expression, and prevention of apoptosis in many cells, such as lymphocytes, macrophages, epithelial cells, fibroblasts, and endothelial cells.
Moreover, IL-4, along with other cytokines (eg, IL-10, IL-13, and IL- 15), contributes to the differentiation and transformation of CD 4+ lymphocytes into Th2 cells; it also inhibits Th1 and reduces the production of IFN-γ by Th1 cells. Another role of IL-4 is regulation of immunoglobulin class changes and production of IgE and IgG-4 in B-cells. ( 14 ) In a study by Akbulut et al( 15 ) investigating intracellular cytokines produced by Th1 and Th2 lymphocytes in brucellosis via flow cytometry, despite the lack of a significant difference in the level of CD 4+ -IL 4+ cells between the brucellosis and healthy controls, the levels of CD 3+ -IL 4+ cells in patients with brucellosis were lower than healthy subjects.
The review of previous studies reveals contradictory results that require further research in this regard. In the study by Ahmed et al, the levels of IL-4, IL-12, TNF-α, and IFN-γ were investigated in patients with brucellosis and healthy subjects. In this study, the serum levels of IL-4 and TNF-α were not detectable in patients, whereas In a study by Demirdag et al, the serum levels of IFN-γ, TNF-α, and IL-4 were measured in healthy subjects recruited as the control group and patients with brucellosis before and after treatment.
The serum levels of IFN-γ and TNF-α increased in the brucellosis group compared with the controls, and a significant reduction was observed after treatment, but the serum level of IL-4 was not significantly different between the control and patient groups and also after treatment.

| MATERIAL S AND ME THODS
In this descriptive-analytical study for comparison of two groups, patients with a diagnosis of brucellosis, who were admitted to the infection ward of Sina Hospital or the outpatient clinic, were recruited using the available sampling method. The inclusion criteria were a Wright test titer ≥ 1/80 and a 2ME test titer ≥ 1/40 in symptomatic patients. Written consent forms were obtained from all the participants, and their demographic characteristics were recorded.
The blood samples (5 cc) were taken before starting the treatment, and the sera were stored at −70°C in the hospital laboratory until further analysis.
After reaching the desired number of samples, the serum level of IL-4 was measured using the Human IL-4 High-Sensitivity ELISA Kit (Invitrogen), with a sensitivity of 0.1 pg/mL, assay range of 0.25-16.0 pg/mL, and intra-assay CV of 8.8%, based on the ELISA method using a Stat Fax 3200 ELISA reader (wavelength, 450 nm). The control group was selected after matching for age and sex. The history of healthy participants, who were referred to the laboratory for checkups, was also taken for evaluation of exclusion criteria. Blood samples were collected from these participants similar to the patients for further tests.
Moreover, the history of collagen vascular disease, anti-inflammatory or corticosteroid drugs consumption, immunodeficiency, use of immunosuppressive drugs, prior antibiotic therapy that was effective for brucellosis, and recent history of infectious diseases other than Brucella were considered as the exclusion criteria in the both groups. Patients with complications of brucellosis (such as encephalitis and orchitis) were also excluded due to the probable potential effect of complications on the serum level of IL-4. It should be noted that the person who performed the tests was completely blind to the group of subjects being tested.
The sample size was estimated at 45 subjects per group, based on the results of a study by Ahmed et al( 16 ) on the evaluation of IL-4 in patients with brucellosis and healthy subjects, using the sample size formula for detecting a difference between two populations, at 95% confidence level and statistical power of 90%. However, in order to increase the study power, 82 subjects were enrolled in each group.
In this study, SPSS version 21 was used to analyze the data.

| RE SULTS
In this study, a total of 165 participants, including 83 patients with brucellosis (case group) and 82 healthy people without brucellosis (control group), were evaluated. In terms of sex, the case group consisted of 49 (59%) men and 34 (41%) women, while the control group included 40 (48.8%) men and 42 (51.2%) women; there was no significant difference in terms of gender between the groups (P = .186). Also, the mean age of subjects in the case and control groups were 50.25 ± 16.01 and 43.26 ± 15.6 years, respectively; however, the difference between the groups was significant in terms of age (P < .001), despite the matching of decades of age.
The serum levels of IL-4 in the case and control groups were 1.42 ± 0.51 pg/mL and 1.31 ± 1.02 pg/mL, respectively (Table 1).

Based on the non-parametric Mann-Whitney test, the IL-4 level was
significantly higher in the case group, compared with the control group (P < .001).
According to the results, the serum levels of IL-4 in men and women with brucellosis were 1.32 ± 0.27 pg/mL and 1.55 ± 0.72 pg/ mL, respectively. Based on the results of Student's t test in patients with brucellosis, there was no significant difference between male and female patients regarding the serum level of IL-4 (P = .089).

Meanwhile, based on the results of Spearman's correlation test,
there was no significant correlation between the age of patients with brucellosis and the serum level of IL-4 (Spearman's ρ = .089, P = .422).
The mean duration of brucellosis in the case group was Meanwhile, the results of the present study showed that in patients with brucellosis, there was no significant relationship between the serum level of IL-4 and the patient's sex, age, duration of disease, and also serologic titers of Wright and 2ME tests; this finding indicates the independence of this cytokine from the mentioned factors.
The independence of serum level of IL-4 from the Wright and 2ME titers suggests the autonomy of cellular pathways of the immune Due to the changes in serum level of IL-4 following the onset of brucellosis, based on the results of the present study and previous researches, along with its changes after treatment of brucellosis as emphasized also in numerous previous studies,( 13,18 ) IL-4 can be used to monitoring treatment process of brucellosis. For this purpose, larger studies are required in different populations.

| CON CLUS ION
In patients with brucellosis, regardless of age, sex, and duration of