|Year : 2017 | Volume
| Issue : 1 | Page : 249-254
Role of interleukin-33 in patients with chronic hepatitis C in Menoufia University Hospitals, Egypt
Ahmed A Salama1, Nahed A El-Ragehy1, Atef A Ali2, Esraa E Elmahdy MBBCh 1
1 Department of Microbiology and Immunology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Tropical Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||08-Jan-2016|
|Date of Acceptance||16-Feb-2016|
|Date of Web Publication||25-Jul-2017|
Esraa E Elmahdy
Tala City, Menoufia Governorate, 32653
Source of Support: None, Conflict of Interest: None
The aim of this study was to determine the serum levels of interleukin-33 (IL-33) in chronic hepatitis C (CHC) patients in Menoufia University Hospitals and to detect its relation with degree of liver fibrosis.
IL-33 is a novel member of the IL-1 family, which has been shown to play an important role in T helper 2-associated immune responses. Recent studies have suggested the role for IL-33 in the pathogenesis of liver damage during acute and chronic hepatitis; furthermore, IL-33 may be involved in the development and progression of liver fibrosis.
Materials and methods
The study was conducted on 60 patients with CHC and 20 healthy controls who were examined and compared for serum IL-33 levels using enzyme-linked immunosorbent assay technique. Post-treatment IL-33 serum concentrations were evaluated in 20 patients. All studied groups were subjected to history taking, complete medical examination, thorough laboratory investigations, and fibroscan to assess the degree of liver fibrosis.
Serum IL-33 levels were significantly higher among CHC patients in comparison with healthy controls (P < 0.001). IL-33 levels were correlated positively to the increase in liver enzymes and degree of liver fibrosis (P < 0.001). CHC patients after antiviral therapy had lower post-treatment IL-33 levels in comparison with baseline values (P < 0.001).
Serum IL-33 was significantly elevated in CHC patients. IL-33 concentrations were significantly reduced following a successful course of antiviral treatment.
Keywords: chronic hepatitis C, interleukin-33, liver fibrosis
|How to cite this article:|
Salama AA, El-Ragehy NA, Ali AA, Elmahdy EE. Role of interleukin-33 in patients with chronic hepatitis C in Menoufia University Hospitals, Egypt. Menoufia Med J 2017;30:249-54
|How to cite this URL:|
Salama AA, El-Ragehy NA, Ali AA, Elmahdy EE. Role of interleukin-33 in patients with chronic hepatitis C in Menoufia University Hospitals, Egypt. Menoufia Med J [serial online] 2017 [cited 2019 Aug 23];30:249-54. Available from: http://www.mmj.eg.net/text.asp?2017/30/1/249/211480
| Introduction|| |
Hepatitis C virus (HCV) is one of the major causes of chronic liver diseases in the world . Approximately 180 million people are currently infected with HCV, and an alarming number of new infections occur annually. The prevalence of hepatitis C varies significantly worldwide, imposing an important burden in highly endemic countries . Egypt has the highest prevalence of HCV in the world, estimated nationally at 14.7% .
Among those who are infected with chronic HCV, only 15–25% spontaneously clear the infection. The remaining proportion of patients continue to have persistent viremia. About 15–30% of people with chronic infection would progress to cirrhosis over the duration of two to three decades. In the patients who develop HCV-related cirrhosis, the risk for development of hepatocellular carcinoma (HCC) has been shown to be 1–4% per year .
Interleukin (IL)-33 was discovered as a novel cytokine belonging to the IL-1 family in 2005. It belongs to the IL-1 family and is also known as a nuclear factor of high endothelial venules . IL-33 plays an important role in T helper 2 (Th2)-associated immune responses; in fact, IL-33 expression has been shown to drive in vitro the production of Th2 cytokines from polarized Th2 cells and to induce in vivo the expression of IL-4, IL-5, IL-10, and IL-13 .
IL-33 is a ligand for the IL-1 receptor family member suppression of tumorigenicity 2 (ST2), whose signal pathway involves myeloid differentiation primary response gene 88 (MyD88) and nuclear factor-κB . ST2 has a transmembrane form of ST2 (ST2 or ST2L) and a soluble form of ST2 (sST2). ST2 is expressed on Th2 and mast cells and functions as a mediator of IL-33 bioactivities, whereas sST2 acts as a decoy receptor for IL-33.
Biologically, IL-33 induces Th2 cell differentiation and activates mast cells, leading to Th2 cytokine production and Th2 response as well as pulmonary and mucosal Th2 inflammation . It acts as a proinflammatory and chemoattractant cytokine, enhancing massive cellular infiltration in the sites where inflammation takes place . IL-33 is involved in the pathogenesis of liver damage during acute and chronic hepatitis. In addition, IL-33 levels have been positively correlated with liver fibrosis in mice and humans . IL-33 overexpression is associated with the development and progression of HCV-related liver fibrosis .
- This study aimed at determining the serum levels of IL-33 in patients with chronic hepatitis C (CHC) and detects the correlation between biochemical liver parameters [alanine transaminase (ALT) and aspartate aminotransferase (AST) serum levels] and serum levels of IL-33 in CHC patients.
| Materials and Methods|| |
Study population and selection of patients
The study was conducted during the period from December 2014 to December 2015 at Menoufia University Hospitals, Egypt. The study protocol was approved by the local ethics committee of the Menoufia University. All participants provided written informed consent before inclusion into the study.
- This study included 60 CHC patients – 20 patients of Child–Pugh class A, 20 patients of Child–Pugh class B, and 20 patients of Child–Pugh class C (eight of them with proved HCC) – and 20 healthy volunteers as a control group. The patients were selected from outpatient clinics and inpatient departments of Tropical Medicine in Menoufia University. The 20 patients of child A class were subjected to post-treatment evaluation of IL-33 after completing antiviral therapy with sofosbuvir and ribavirin for 6 months.
- Inclusion criteria were age more than or equal to 18 years and the presence of CHC. CHC was proved by both positive HCV antibodies and serum HCV RNA for at least 6 months. Individuals with a history of or currently having hepatitis B or D or HIV infections, with autoimmune hepatitis or metabolic liver diseases who received immunosuppressive therapy were excluded. In addition, individuals who had received antiviral therapy during the past 12 months and had other conditions known to be associated with elevated serum levels of IL-33, such as rheumatoid arthritis, psoriasis, and inflammatory bowel diseases were excluded.
All patients were subjected to clinical assessment including full history and clinical examination, abdominal ultrasonography, and fibroscan. Laboratory investigations including liver function tests (except for prothrombin time) and kidney function tests were carried out on Integra 400 autoanalyzer (Roche Diagnostics Corporation, Hague Road, Indiana Polis, Indiana, USA). Prothrombin time and concentration were evaluated using Thrombrel-S (human thromboplastin containing calcium) from Behring Diagnostic Inc. (San Jose, California, United States). Complete blood count was determined using autoanalyzer ADVIA 2120 Hematology System (Siemens Healthineers, Germany). HCV antibodies were detected using enzyme-linked immunosorbent assay (ELISA) third generation, using kit from the BioChem ImmunoSystems Inc. (catalog number 163000), and HCV quantitative assay based on real-time PCR was carried out using automated cobas amplicore analyzer (Roche Diagnostic Systems, Tokyo, Japan).
Collection of blood samples
About 10 ml of venous blood was aseptically drawn from all participants. A volume of 3 ml was transferred slowly into a plain tube. The serum was allowed to separate in a serum separator tube (about 4 h) at room temperature and the centrifugation at ~1000 g for 15 min was carried out. The serum samples were aliquoted and stored at −80°C until further use for IL-33 quantification, and repeated freeze-thaw cycles were avoided. In addition, the rest of the blood samples were subsequently used for clinical chemistry.
Quantification of interleukin-33 serum levels
Serum IL-33 levels were quantified using Boster's human IL-22 ELISA kits according to the manufacturer's instructions (Boster Biological Technology Co. Ltd., Pleasanton, California, USA). Boster's human IL-33 ELISA kit was based on standard sandwich ELISA technology. A monoclonal antibody from mouse specific for IL-33 has been precoated onto 96-well plates. Standards (Escherichia coli, S112–T270) and test samples were added to the wells, a biotinylated detection polyclonal antibody from goat specific for IL-33 was added subsequently, followed by washing with PBS or TBS buffer. Avidin–biotin–peroxidase complex was added and unbound conjugates were washed away with PBS or TBS buffer. horseradish peroxidase (HRP) substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed with HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow color was proportional to the amount of human IL-33 sample captured in plate.
The data collected were tabulated and analyzed using statistical package for the social sciences computer program version 20 (SPSS; SPSS Inc., Chicago, Illinois, USA). Descriptive data were presented as percentage, and quantitative data as mean ± SD and median. Variables of not normally distributed data between two groups were compared using the Mann–Whitney test. The Kruskal–Wallis test was used for quantitative variables of not normally distributed data. The Post-hoc test was used after the Kruskal–Wallis test to show any significant difference between the individual groups. The Spearman correlation test was used to assess the relationship between variables in the same group. Paired sample t-test was applied for comparison between the normally distributed quantitative data at interval for the same group. P value is considered significant if less than 0.05. Receiver operating characteristic curve was also used to compare diagnostic tests by drawing a curve for each. The area under a curve represents the overall accuracy of a test; the larger the area, the better the test. The curve closest to the upper left corner has the greater sensitivity and specificity and hence more accurate than others.
| Results|| |
This study included 60 CHC patients (20 patients of child A class, 20 patients of child B, and 20 patients of child C) with a mean age of 52.28 ± 11.60 years, and the majority of patients were male. The majority of patients presented with liver fibrosis stage 3. The patient characteristics are shown in [Table 1].
There was no significant difference in the distribution of age and sex among these groups of participants, but the concentrations of serum ALT and AST in CHC were significantly higher compared with healthy control (HC). Although high levels of virus RNA were detected in CHC patients, there was no detectable viremia in HC.
There was a highly statistically significant difference between the two studied groups as regards IL-33 serum levels (P < 0.001). Serum levels of IL-33 in the studied group of patients ranged from 60 to 600 pg/ml with a mean value of 261.93 ± 152.155, whereas in the control group IL-33 serum level ranged from 32.0 to 72.0 pg/ml with a mean value of 44.0 ± 9.96 [Table 2] and [Figure 1].
|Table 2 Comparison between studied groups of cases and controls as regards serum levels of interleukin-33 in pg/ml|
Click here to view
|Figure 1: The mean value of interleukin-33 serum levels among the case and control groups.|
Click here to view
There was a highly significant positive correlation between fibrosis stage and serum levels of IL-33 (P < 0.001). Patients exhibiting fibrosis score of F1–F2 had a significantly lower IL-33 concentration compared with patients with a more advanced fibrosis score (F3–F4) [Table 3].
|Table 3 Relationship between serum levels of interleukin-33 and fibrosis stage in the studied group of cases (n=60)|
Click here to view
In addition, the concentrations of serum IL-33 in CHC patients were positively correlated with the levels of serum ALT and AST (P < 0.05 and P = 0.05, respectively). The concentrations of serum IL-33 in CHC patients were positively correlated with the levels of serum HCV RNA (P < 0.001) [Table 4] and [Figure 3].
|Table 4 Correlation between serum levels of interleukin-33 in pg/ml and other assessed laboratory parameters in the studied group of cases|
Click here to view
|Figure 2: Relationship between serum levels of interleukin-33 among the studied group of cases and fibrosis stage.|
Click here to view
|Figure 3: Correlation between serum levels of interleukin-33 in pg/ml among the studied group of cases and alanine transaminase serum levels.|
Click here to view
The Model for End-Stage Liver Disease score includes the laboratory parameters for creatinine, bilirubin, and international normalized ratio for prothrombin time. In the present study, there was a significant positive correlation between Model for End-Stage Liver Disease score and IL-33 serum levels (P < 0.001) [Table 5].
|Table 5 Relationship between serum levels of interleukin-33 in pg/ml and prognosis score (Model for End-Stage Liver Disease) in the studied group of cases (n=60)|
Click here to view
Furthermore, IL-33 serum levels have a positive correlation with Child–Pugh score (P < 0.001). The highest level of IL-33 was observed among patients with class C, whereas the lowest level of IL-33 was observed among patients with class A. Moreover, CHC patients with HCC showed a significantly higher IL-33 serum level concentrations compared with CHC patients without HCC (P < 0.001) [Table 6] and [Figure 4].
|Table 6 Relationship between serum levels of interleukin-33 and Child-Pugh classification in the studied group of cases (n=60)|
Click here to view
|Figure 4: Interleukin-33 serum levels among different classes of Child–Pugh score.|
Click here to view
Moreover, overall pretreatment IL-33 concentrations were higher compared with overall post-treatment concentrations with a high statistically significant difference (P < 0.001) [Table 7] and [Figure 5].
|Table 7 Effect of antiviral treatment with sofosbuvir (Sovaldi) on interleukin-33 levels among the child A cases (n=20)|
Click here to view
|Figure 5: Effect of antiviral treatment on interleukin-33 levels among the studied cases.|
Click here to view
| Discussion|| |
This study was performed at Menoufia University Hospitals to study serum levels of IL-33 in patients with CHC, and to detect its correlation with the HCV virus activity and the degree of liver fibrosis.
Data obtained in this study showed that, compared with healthy individuals, IL-33 serum levels were significantly elevated in patients with CHC. This is in agreement with the study conducted by Wang et al. , who documented that serum IL-33 was significantly upregulated in CHC compared with controls. Hegazy and Henawy  in Egypt also documented that IL-33 serum levels were significantly elevated in patients with CHC.
In the present study, we observed that there was a highly significant positive correlation between fibrosis stage and serum levels of IL-33. This is in agreement with the results obtained by Cacopardo et al. , who showed that the patients with fibrosis score (F1–F2) had a significantly lower IL-33 concentration compared with patients with a more advanced fibrosis score (F3–F4). Marvie et al.  alsorevealed that IL-33 overexpression is associated with the development of HCV-related liver fibrosis and showed that IL-33 is upregulated in human and murine fibrosis, and is mainly produced by sinusoidal endothelial cells and activated hepatic stellate cells.
In this study, IL-33 was found to be positively correlated with the levels of serum ALT and AST, which is in agreement with study conducted by Wang et al. .
Furthermore, a significant positive correlation was found between serum levels of IL-33 and levels of serum HCV RNA. This is in agreement with the study byHegazy and Henawy , who revealed that the concentrations of serum IL-33 in CHC patients were positively correlated with the levels of serum HCV RNA.
IL-33 serum levels have a positive correlation with Child–Pugh score. This coincides with the study by Sun et al. , who reported thatserum IL-33 levels correlated positively with Child–Pugh scores and were used to determine the prognosis of liver cirrhosis.
In the current study, it was found that the highest level of serum IL-33 was observed among CHC patients with HCC. There was a statistically highly significant elevation of serum levels of IL-33 in CHC patients with HCC, compared with CHC patients without HCC. This finding coincides with the results reported by Bergis et al. , who observed that serum IL-33 levels in HCC patients were significantly higher compared with serum IL-33 levels in liver cirrhotic patients.
| Conclusion|| |
From this study we can conclude that IL-33 may represent a new and easy-to-detect biomarker for the diagnosis of liver damage in CHC patients, as it appears to be modulated in parallel with biochemical and histologic parameters, such as ALT levels and liver fibrosis. Furthermore, considering that serum IL-33 concentration was significantly reduced following a successful course of antiviral treatment, this cytokine may also represent a sensitive indicator of sustained virologic response.
Further large-scale studies are required to better characterize the role of this cytokine throughout the course of CHC.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Lee M, Yang H, Chen C. Long-term health outcomes of chronic hepatitis C patients. Biomedicine 2012; 2:99–107.
Rossi LM, Escobar-Gutierrez A, Rahal P. Advanced molecular surveillance of hepatitis C virus. Viruses 2015; 7:1153–1188.
Montaser LM, Waked IA, Essa ES, Abd Elhameed RM. Evaluation of CD95 in patients with chronic hepatitis C virus. Menoufia Med J 2014; 27:780–784. [Full text]
Khullar V, Firpi RJ. Hepatitis C cirrhosis. New perspectives for diagnosis and treatment. Vikas World J Hepatol 2015; 7:1843–1855.
Baekkevold ES, Roussigne M, Yamanaka T, Johansen FE, Jahnsen FL, Amalric F, et al.
Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules. Am J Pathol 2003; 163:69–79.
Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, et al.
IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005; 23:479–490.
Alves-Filho JC, Sonego F, Souto FO, Freitas A, Verri WA Jr, Auxiliadora-Martins Ml, et al.
Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection. Nat Med J 2010; 16:708–712.
Wang J, Zhao P, Guo H, Sun X, Jiang Z, Xu L, et al.
Serum IL-33 levels are associated with liver damage in patients with chronic hepatitis C. Mediators Inflamm 2012; 2012:1–7.
Seidelin JB, Rogler G, Nielsen OH. A role for interleukin-33 in TH 2-polarized intestinal inflammation? Mucosal Immunol 2011; 4:496–502.
Arshad MI, Rauch M, L'Helgoualc'h A, Julia V, Leite-de-Moraes MC, Lucas-Clerc C, et al.
NKT cells are required to induce high IL-33 expression in hepatocytes during ConA-induced acute hepatitis. Eur J Immunol 2011; 41:2341–2348.
Marvie P, Lisbonne M, L'Helgoualc'h A, Rauch M, Turlin B, Preisser L, et al.
Interleukin-33 overexpression is associated with liver fibrosis in mice and humans. J Cell Mol Med 2010; 14:1726–1739.
Hegazy WA, Henawy MA. Hepatitis C virus pathogenesis. Serum IL-33 level indicates liver damage. Afr Microbiol Res J 2015; 9:1386–1393.
Cacopardo B, Pinzone MR, Palermo F, Nunnari G. Changes in serum interleukin-33 concentration before and after treatment with pegylated interferon alfa-2a plus ribavirin in patients with chronic hepatitis C genotype 1b infection. Hepat Mon 2012; 12:12.
Sun Y, Zhang J, Lv S, Wang H, Gong M, Du N, et al
. Interleukin-33 promotes disease progression in patients with primary biliary cirrhosis. Tohoku J Exp Med 2014; 234:255–261.
Bergis D, Kassis V, Ranglack A, Koeberle V, Piiper A, Kronenberger B, et al
. High serum levels of the interleukin-33 receptor soluble ST2 as a negative prognostic factor in hepatocellular carcinoma. Transl Oncol 2013; 6:311–318.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]