Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2014  |  Volume : 27  |  Issue : 4  |  Page : 775--779

Interleukin-17-producing CD4 + T cells in patients with chronic hepatitis B


Maha A El-Basuoni1, Mohamed A Soliman1, Hassan E Zaghla2, Maha M Allam1, Abeer A El-Gazzar1,  
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Hepatology, National Liver Institute, Menoufia University, Menoufia, Egypt

Correspondence Address:
Abeer A El-Gazzar
National Liver Institute, Menoufia University, Yassin Abdel-Ghaffar St, Shebin El-kom, Menoufia
Egypt

Abstract

Objective Our aim is to evaluate the expression of interleukin-17-producing CD4 + T cells (Th17) in peripheral blood of patients with chronic hepatitis B (CHB) and whether these cells show a potential to exacerbate liver damage during chronic hepatitis B virus (HBV) infection. Background Interleukin-17-producing CD4 + T cells (Th17)-mediated immune response has been shown to play a critical role in inflammation-associated disease; however, its role in chronic HBV infection remains unknown. Here, we characterized peripheral Th17 cells and analyzed their association with liver injury in HBV-infected patients. Participants and methods This study included 15 patients with CHB without cirrhosis (11 men and four women), 15 patients with CHB with cirrhosis (eight men and seven women), and 15 healthy controls (10 men and five women). Laboratory investigations including complete blood picture, liver function tests, hepatitis viral markers (HBsAg, anti-HCV-Ab), a-fetoprotein, and HBV DNA PCR were performed for all participants. Evaluation of the level of Th17 cells in the peripheral blood was carried out using the flow cytometry technique. Results The data showed that the frequency of circulating Th17 cells was increased in patients with CHB viral infection compared with the healthy controls, and was significantly higher in patients with CHB-associated cirrhosis. In addition, the increasing levels of circulating Th17 cells were correlated positively with serum alanine transaminase levels. Conclusion Our results showed that Th17 cells were highly enriched in the peripheral blood of CHB patients, and their levels were related to disease progression, suggesting its role in exacerbation of liver damage during chronic HBV infection.



How to cite this article:
El-Basuoni MA, Soliman MA, Zaghla HE, Allam MM, El-Gazzar AA. Interleukin-17-producing CD4 + T cells in patients with chronic hepatitis B.Menoufia Med J 2014;27:775-779


How to cite this URL:
El-Basuoni MA, Soliman MA, Zaghla HE, Allam MM, El-Gazzar AA. Interleukin-17-producing CD4 + T cells in patients with chronic hepatitis B. Menoufia Med J [serial online] 2014 [cited 2020 May 26 ];27:775-779
Available from: http://www.mmj.eg.net/text.asp?2014/27/4/775/149753


Full Text

 Introduction



Hepatitis B virus (HBV) infection is a health problem worldwide. The clinical consequences of HBV infection are highly variable, including clinical syndromes such as fulminant, acute and chronic hepatitis, hepatocellular carcinoma, and the asymptomatic carrier state [1].

Healthy HBV carriers include patients with normal serum aminotransferase levels [alanine transaminase (ALT)] and normal hepatic histology despite persistent viremia. HBV carriage is important as infected carriers are the main reservoir from which HBV disseminates in a population along with the risk of development of life-threatening liver diseases, including hepatocellular carcinoma [2].

HBV itself is noncytopathic, but immune-mediated liver damage often occurs in patients with both acute and chronic HBV infection. Such damage has conventionally been attributed to killing of infected hepatocytes by virus-specific cytotoxic CD8 + T cells [3],[4].

A large number of immune cells, including myeloid dendritic cells, plasmacytoid dendritic cells, and FoxP3-positive regulatory T cells, can be observed in the livers of mildly and severely affected chronic hepatitis B (CHB) patients [5],[6]. These findings suggest that multiple types of immune cells may actively participate in HBV-associated liver pathogenesis. Understanding which types of immune cells contribute toward liver damage during chronic HBV infection is a prerequisite for the discovery of effective treatment strategies [7].

Human interleukin-17 (IL-17)-producing CD4 + T cells (Th17) comprise a newly identified proinflammatory T-cell subset [7]. Th17 cells can also produce a cocktail of cytokines such as IL-17A, IL-17F, IL-21, IL-22, IL-6, and tumor necrosis factor-a, of which IL-17A is characterized as a major effector cytokine. IL-17A can mobilize, recruit, and activate neutrophils, leading to massive tissue inflammation, and promote the progression of autoimmune disease [8].

The peripheral and intrahepatic Th17 cells are preferentially increased in CHB patients, which might activate myeloid dendritic cells and monocytes to release inflammatory cytokines during chronic HBV infection. Thus, Th17 cells may participate in the immunopathogenesis of chronic HBV infection [9].

 Participants and methods



Study population

This study was carried out on 30 patients: 15 patients diagnosed with noncirrhotic chronic HBV and 15 patients diagnosed with chronic HBV-associated cirrhosis. They presented to the Hepatology Department, National Liver Institute, Menoufia University, in the period from April 2012 to April 2013. The diagnosis was made on the basis of clinical examination, laboratory tests, and ultrasound. Fifteen apparently healthy individuals age and sex matched with the patients were included in the study as a control group.

A written consent was obtained from each individual and the protocol was approved by the ethical committee of the National Liver Institute, Menoufia University.

All patients and control groups were subjected to the following:

(1) Full assessment of history.

(2) Complete clinical examination.

(3) Abdominal ultrasonography.

(4) Laboratory investigations including:

(a) Complete blood picture.

(b) Liver function tests: Aspartate transaminase (AST), ALT, serum bilirubin, serum total proteins, serum albumin, alkaline phosphatase, and serum g-glutamyltransferase.

(c) Viral markers (HBsAg, HCV-Ab).

(d) Serum a-fetoprotein.

(e) Quantitative HBV DNA by PCR.

(f) The level of peripheral Th17 by flow cytometry.

Sample collection

Sixteen milliliters of venous blood samples were taken by sterile venipuncture after minimal venous stasis using sterile disposable syringes. The blood samples were distributed as follows: 5 ml of venous blood was delivered in a vacutainer plain test tube. Blood was left for a sufficient time to clot, and serum was then separated after centrifugation at 3000 rpm/min for 10 min. Liver function tests, hepatitis viral marker, and a-fetoprotein were performed. Five milliliters of venous blood was delivered in a vacutainer plain test tube, and the serum was extracted and stored at −80°C until HBV DNA PCR was performed.

Three milliliters of venous blood was delivered in a vacutainer plastic tube containing EDTA for complete blood count and the remaining 3 ml was placed in a vacutainer heparinized plastic used for flow cytometric analysis.

Frequency of Th17 by flow cytometry

Assay procedure.

Cell culture

Fresh peripheral blood mononuclear cells were separated from heparinized peripheral blood by density gradient centrifugation (1800 rpm for 20 min) using Ficoll-Paque solution.Cells were washed twice by PBS by centrifugation at 4000 rpm for 5 min and then the supernatant was aspirated completely.Cells were resuspended in culture medium RPMI 1640 supplemented with 5% autologous serum at a density of 10 7 cells/ml.Peripheral blood mononuclear cells were cultured in nonstick polypropylene round-bottom tubes to avoid attachment of monocytes to the tubes.Cells were cultured in two tubes: one was a negative control, where the sample was treated in the same way as the stimulated sample, except for the addition of cytoStim.Twenty microliters of cytoStim per milliliter cell suspension was added.Cells were mixed carefully and incubated at 37°C, 5% CO 2 , for 2 h.Monensin (concentration 2 mmol/l) cells were added and incubated for a further 4 h.

Immunostaining

Cells were harvested and washed once by ice-cold PBS, suspended in 100 ml PBS per tube, and then 5 ml of monoclonal antibodies CD4-PE was added (20 min at 4°C).After one wash, the supernatant was aspirated, 100 ml of IC fixation buffer was added, followed by 100 μl of permeabilization solution, and finally 5 ml of anti-human IL-17 FITC was added and then treated for 20 min at 4°C.Analysis was carried out on a FACS brand flow cytometer. The number of events to be analyzed was set to 50 000.

Statistical analysis

Data were analyzed statistically using the SPSS (Statistical Package for Social Science) program version 13 for Windows for all the analyses (Kirkwoodm, 1990). Qualitative data are show as frequency and percent and quantitative data are shown as mean ± SD. The Student t-test was used for comparison between two groups with quantitative normally distributed data, and the Mann-Whitney U-test was used for abnormally distributed data. The one-way analysis of variance test was used for comparison between three or more groups with quantitative normally distributed data. The c2 -test was used to compare the qualitative variables. Pearson correlation coefficient was used to determine the correlation between variables. P-value was considered statistically significant when it was less than 0.05.

 Results



[Table 1] shows that all the studied groups were age and sex matched as there is no statistically significant difference between the groups studied.{Table 1}

[Table 2] shows a high statistically significant increase in the level of Th17 in the patients of groups II and III compared with the control group.{Table 2}

[Table 3],[Table 4] and [Table 5] show a statistically significant increase in the frequency of Th17 cells among CHB (cirrhotic and noncirrhotic) patients compared with the healthy controls. The data also showed that the frequency of Th17 cells was significantly higher in CHB cirrhotic patients compared with noncirrhotic groups.{Table 3}{Table 4}{Table 5}

[Table 6] shows no significant correlation between Th17 and HBV DNA levels in both the noncirrhotic and the cirrhotic group.{Table 6}

[Table 7] shows a statistically significant correlation between Th17 and inflammatory markers of the liver, namely, AST, ALT, and serum bilirubin level.{Table 7}

 Discussion



The frequencies of peripheral Th17 cells increased gradually from the asymptomatic chronic group (AsC) to the CHB group and the acute liver failure group (ALF), which were all higher than those in the control group, except that there was no statistical significance between the AsC group and the normal controls. In addition, the frequencies of Th17 cells in the ALF group were also significantly higher than those in the CHB and AsC groups. Moreover, advanced-stage ALF patients had a significantly higher frequency of peripheral Th17 cells than early-stage ALF patients [10].

Zhang et al. [11] found that the percentage of Th17 cells was significantly increased in CHB patients compared with the healthy controls (HC). Particularly in ALF patients, the Th17 frequency increased beyond that in CHB patients [11]. Also, the activity of Th17 cells was further investigated by measurement of IL-17 production from purified CD4 T cells in response to plate-coated anti-CD3 and soluble anti-CD28. CD4 T cells from CHB patients produced more IL-17 than those of HC patients under anti-CD3 and anti-CD28 stimulation. Thus, these data indicate that Th17 cells were preferentially increased in the peripheral blood of CHB patients and simultaneously showed increased activity [11].

Sun et al. [12] confirmed that the percentage of Th17 cells was increased significantly in HBV-cirrhotic patients when compared with the healthy controls. In HBV-cirrhotic patients, the Th17 frequency increased even further with the severity of liver disease, in particular, in Child-Pugh C patients, who showed the highest levels of Th17 cells [12].

Du et al. [13] showed that serum IL-17 protein levels and peripheral blood mononuclear cell IL-17A mRNA levels were found to be significantly higher in HBV-infected patients compared with normal controls. IL-17 expression in the liver tissues of the patients was correlated positively with inflammation grade and fibrosis stage, and positively stained lymphocytes suggested that IL-17 plays a role in chronic HBV infection [13].

The highest IL-17 levels in the serum and liver were observed in our cirrhotic patients, suggesting that IL-17 might contribute toward the pathogenesis and/or progression of liver fibrosis. This finding was in agreement with that of the Du et al. [13], who reported the same result in HBV cirrhotic patients [13].

Liang et al. [14] found that peripheral blood Th17 cell levels in active chronic HBV infection were significantly higher than those in the healthy controls, associated with elevated serum levels of IL-17A. IL-17 secretion cells in the liver of CHB and peripheral Th17 cells increased among patients with CHB compared with healthy controls, associated with fibrosis and inflammation degrees. All these findings strongly suggest that Th17 cells participate in the liver injury process and viral clearance after HBV infection [14]. Du et al. [13] added that a significant elevation in serum IL-17 level was reported in patients with severe acute hepatic injury and fulminate hepatic failure [13].

Consistently, on analyzing the frequency of IL-17-secreting CD4 + T cells, Wu et al. [15] observed that acute hepatitis B and severe CHB patients showed a significant increase in the frequency of Th17 cells compared with patients with mild CHB and healthy controls [15].

To further detect the relationship between the frequency of Th17 cells and liver injury, we analyzed the correlation between Th17 frequency and plasma HBV DNA load or ALT levels and total bilirubin levels in our HBV-infected patient groups.

In the current study, no significant correlation was detected between Th17 and HBV DNA level in both the noncirrhotic and the cirrhotic group.

Bo et al. [10] reported that were in agreement with ours; they found no significant correlation between peripheral Th17 cells frequencies and serum HBV DNA loads [10].

In contrast, Zhang et al. [11] found significant positive correlations between Th17 frequency and both plasma HBV DNA load and serum ALT levels in these HBV-infected patients [11]. Further analysis indicated that these positive associations occurred only in patients with CHB, but not in patients with ACLF. In addition, we also found that CHB patients with high HAI scores (G2-G3) had a greater proportion of Th17 cells than did CHB patients with low HAI scores (G0-G1) [11].

Interestingly, in this study, we found a statistically significant correlation between Th17 and inflammatory markers of the liver, namely, AST, ALT, and serum bilirubin level.

Liang et al. [14] reported that, in chronic HBV-infected patients, there were some significant negative correlations between plasma HBV DNA load and both plasma IL-17A levels and Th17 frequency. This negative association did not occur in different chronic HBV-infected cohorts. In addition, they found that both Th17 frequency and plasma IL-17A levels correlated positively with ALT and total bilirubin levels in these chronic HBV-infected patients, but further analysis indicated a positive association between plasma IL-17A levels and total bilirubin levels in patients with acute on top of chronic HBV liver failure. However, there were positive correlations between both Th17 frequency and plasma IL-17A levels with serum ALT levels [14].

Wu et al. [15], in agreement with our study, found that the increase in circulating Th17 cells was correlated positively with serum ALT level. They also found that the elevated proportion of Th17 cells was correlated positively with the serum ALT levels in severe CHB patients [15].

These findings suggest that multiple types of immune cells may actively participate in HBV-associated liver pathogenesis. Understanding which types of immune cells contribute toward liver damage during chronic HBV infection is a prerequisite for discovering effective treatment strategies [7].

 Conclusion



It can be concluded that the expression of IL-17-producing CD4 + T cells (Th17) in peripheral blood of patients with CHB has the potential to exacerbate liver damage during chronic HBV infection. Thus, Th17 cells may participate in the immunopathogenesis of chronic HBV infection. Therefore, IL-17 may be a potential therapeutic target for the prevention of liver tissue damage in HBV-infected patients; large-scale studies are recommended to confirm this.

 Acknowledgements



Conflicts of interest

There are no conflicts of interest.

References

1Kaleli I, Demir M, Cevahir N, Yilmaz M, Demir S. Serum neopterin levels in patients with replicative and nonreplicative HBV carriers. BMC Infect Dis 2006; 6 :157.
2 Hilleman MR. Overview of the pathogenesis, prophylaxis and therapeusis of viral hepatitis B, with focus on reduction to practical applications. Vaccine 2001; 19 :1837-1848.
3 Guidotti LG, Chisari FV. Noncytolytic control of viral infections by the innate and adaptive immune response. Annu Rev Immunol 2001; 19 :65-91.
4 Szabo G, Mandrekar P, Dolganiuc A Innate immune response and hepatic inflammation. Semin Liver Dis 2007; 27 :339-350.
5 Xu D, Fu J, Jin L, Zhang H, Zhou C, Zou Z, et al. Circulating and liver resident CD4 + CD25 + regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B. J Immunol 2006; 177 :739-747.
6 Dunn C, Brunetto M, Reynolds G, Christophides T, Kennedy PT, Lampertico P, et al. Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell-mediated liver damage. J Exp Med 2007; 204 :667-680.
7 Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol 2007; 25 :821-852.
8 O′Quinn DB, Palmer MT, Lee YK, Weaver CT. Emergence of the Th17 pathway and its role in host defense. Adv Immunol 2008; 99 :115-163.
9 Zhang Z, Zou ZS, Fu JL, Cai L, Jin L, Liu YJ, Wang FS. Severe dendritic cell perturbation is actively involved in the pathogenesis of acute-on-chronic hepatitis B liver failure. J Hepatol 2008; 49 :396-406.
10Yang B, Wang Y, Zhao C, Yan W, Che H, Shen C, Zhao M Increased Th17 cells and interleukin-17 contribute to immune activation and disease aggravation in patients with chronic hepatitis B virus infection. Immunol Lett 2013; 149 :41-49.
11Zhang J, Zhang Z, Lin F, et al. Interleukin-17-producing CD4 T cells increase with severity of liver damage in patients with chronic hepatitis B. Hepatology 2010; 51 :81-91.
12Sun HQ, Zhang JY, Zhang H, Zou ZS, Wang FS, Jia JH. Increased Th17 cells contribute to disease progression in patients with HBV-associated liver cirrhosis. J Viral Hepat 2012; 19 :396-403.
13Du W, Zhen J, Zeng Z, et al. Expression of interleukin-17 associated with disease progression and liver fibrosis with hepatitis B virus infection: IL-17 in HBV infection, 2013. Available at: http://www.diagnosticpathology.org/content/8/1/40.
14Liang X, Li C, Zhou Y, Wan M. Changes of Treg and Th17 cells balance in the development of acute and chronic hepatitis B virus infection. Gastroenterology 2012; 1 :12-43.
15Wu W, Li J, Chen F, Zhu H, Peng G, Chen Z. Circulating Th17 cells frequency is associated with the disease progression in HBV infected patients. J Gastroenterol Hepatol 20102:750-757.