Changes of Treg and Th17 cells balance in the development of acute and chronic hepatitis B virus infection.

Abstract

Many studies suggest that in chronic hepatitis B virus (HBV) infection regulate T (Treg) cells and interlukin-17-producing T help cells (Th17) are mutually antagonistic in the immune response. This study is aimed to reveal the cell differentiation environment and the significance of Treg and Th17 balance in the development of acute and chronic HBV infection.
Ten patients with acute HBV infection (AHB) and forty-eight patients with chronic HBV infection, including 12 asymptomatic HBV carriers (HBV carriers), 18 chronic hepatitis B patients (CHB) and 18 acute-on-chronic HBV-related liver failure (ACHBLF) were enrolled. Treg and Th17 cells differentiation related cytokine levels were detected by using ELISA. Flow cytometry was employed to count the Treg and Th17 frequency in peripheral blood.
Compared to health controls both AHB and ACHBLF patients favoured Th17 cell differentiation, accompanied by a higher proportion of peripheral Th17 cells (P < 0.01) and high level of interleukin-17A (IL-17A) (P < 0.01). However, asymptomatic HBV carriers and CHB were conducive to Treg cell differentiation. In AHB and ACHBLF, peripheral blood IL-17A + CD4 + T cell frequency increased significantly compared with healthy controls. Changes of Treg and Th17 cell frequency were not completely consistent. Both CHB and ACHBLF had lower level of Treg/Th17 ratio than in health control (P < 0.05). Both plasm IL-17A levels (r = -0.72, p<0.001) and Th17 frequency(r = -0.49, p = 0.0003) negatively correlated with plasma HBV DNA load in patients with chronic HBV infection. In addition, both Th17 frequency and plasm IL-17A levels positively correlated with ALT (r = 0.33,p = 0.01 Vs r = 0.29, p = 0.04) and total bilirubin levels (r = 0.72,p<0.0001 Vs r = 0.53, p = 0.0001) in these chronic HBV-infected subjects. However, for AHB there were positive correlation between both Th17 frequency (r = 0.64, p = 0.04) and plasm IL-17A levels (r = 0.69, p = 0.02) with serum ALT levels, but no significant correlation between both HBV DNA level and total bilirubin level with Th17 frequency or plasm IL-17A levels were found. Furthermore, Treg/Th17 ratio was negatively correlated with total bilirubin levels (r = -0.41, p = 0.004) in chronic HBV-infected patients, especially in patients with ACHBLF (r = -0.69, p = 0.001) and positively correlated with viral load in these chronic HBV-infected subjects (r = 0.55, p<0.0001).
Th17 cells are involved in acute and chronic HBV infection, especially in AHB and ACHBLF. CHB and ACHBLF patients manifested obvious Treg/Th17 ratio imbalance, which might be linked to disease progression and the continuous HBV infection.

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RESEARCH ARTICLE Open Access
Changes of Treg and Th17 cells balance in the
development of acute and chronic hepatitis B
virus infection
Liang Xue-Song1*, Li Cheng-Zhong1, Zhou Ying2and Wan Mo-Bin1*
Abstract
Background: Many studies suggest that in chronic hepatitis B virus (HBV) infection regulate T (Treg) cells and
interlukin-17-producing T help cells (Th17) are mutually antagonistic in the immune response. This study is aimed to
reveal the cell differentiation environment and the significance of Treg and Th17 balance in the development of
acute and chronic HBV infection.
Methods: Ten patients with acute HBV infection (AHB) and forty-eight patients with chronic HBV infection,
including 12 asymptomatic HBV carriers (HBV carriers), 18 chronic hepatitis B patients (CHB) and 18 acute-on-chronic
HBV-related liver failure (ACHBLF) were enrolled. Treg and Th17 cells differentiation related cytokine levels were
detected by using ELISA. Flow cytometry was employed to count the Treg and Th17 frequency in peripheral blood.
Results: Compared to health controls both AHB and ACHBLF patients favoured Th17 cell differentiation,
accompanied by a higher proportion of peripheral Th17 cells (P<0.01) and high level of interleukin-17A (IL-17A)
(P<0.01). However, asymptomatic HBV carriers and CHB were conducive to Treg cell differentiation. In AHB and
ACHBLF, peripheral blood IL-17A+CD4+T cell frequency increased significantly compared with healthy controls.
Changes of Treg and Th17 cell frequency were not completely consistent. Both CHB and ACHBLF had lower level of
Treg/Th17 ratio than in health control (P<0.05). Both plasm IL-17A levels (r=−0.72, p<0.001) and Th17 frequency
(r=−0.49, p=0.0003) negatively correlated with plasma HBV DNA load in patients with chronic HBV infection. In
addition, both Th17 frequency and plasm IL-17A levels positively correlated with ALT (r=0.33,p=0.01 Vs r=0.29,
p=0.04) and total bilirubin levels (r=0.72,p<0.0001 Vs r=0.53,p=0.0001) in these chronic HBV-infected subjects.
However, for AHB there were positive correlation between both Th17 frequency (r=0.64, p=0.04) and plasm IL-17A
levels (r=0.69, p=0.02) with serum ALT levels, but no significant correlation between both HBV DNA level and total
bilirubin level with Th17 frequency or plasm IL-17A levels were found. Furthermore, Treg/Th17 ratio was negatively
correlated with total bilirubin levels (r=−0.41, p=0.004) in chronic HBV-infected patients, especially in patients with
ACHBLF (r=−0.69,p=0.001) and positively correlated with viral load in these chronic HBV-infected subjects (r=0.55,
p<0.0001).
Conclusions: Th17 cells are involved in acute and chronic HBV infection, especially in AHB and ACHBLF. CHB and
ACHBLF patients manifested obvious Treg/Th17 ratio imbalance, which might be linked to disease progression and
the continuous HBV infection.
Keywords: HBV, Treg, Th17, Immune
* Correspondence: liangxuesong2000@163.com; mobinwan@yahoo.com.cn
1Department of Infectious Diseases, Changhai Hospital, Second Military
Medical University, Shanghai, 200433, China
Full list of author information is available at the end of the article
© 2012 Liang X-S et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Liang X-S et al. BMC Gastroenterology 2012, 12:43
http://www.biomedcentral.com/1471-230X/12/43

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Background
Hepatitis B virus (HBV) can cause acute and chronic infec-
tion. Chronic infection is closely related with liver cirrhosis
and hepatocellular carcinoma [1]. More than 2 billion
people have been infected with HBV globally and there are
still approximately 350 million chronic HBV infection vic-
tims or HBV carriers [2]. HBV do not directly cause liver
cell injury. The outcomes after infection are closely related
to the host immune response. Appropriate immune re-
sponse can lead to viral clearance and recovery, excess im-
mune response can lead to liver failure and inadequate
immune response will result in sustained HBV infection
[3-6].
Foxp3+CD4+CD25+regulatory T cells (Foxp3+Treg)
play an anti-inflammatory role mainly through contact
dependent suppression or releasing anti-inflammatory fac-
tors on other immune cells such as CD4+ and CD8+T
cells, natural killer (NK) cells and NKT cells, B cells and
dendritic cells (DC) [7-9]. Thus, Foxp3+Treg cells are
considered to be of great importance in maintaining self-
tolerance, the immune balance and preventing auto-
immune diseases, allergies and other immune pathological
conditions. In chronic hepatitis B (CHB), Foxp3+Treg
cells are closely related with the development and progress
of the disease [10-12].
CD4+T cells secreting interleukin-17 (IL-17) are a
newly established T helper cell subset (Th17), defferent
from Th1 and Th2 cells. Th17 cells originate from the
same naive cells with Foxp3+Treg cells, mainly secreting
pro-inflammatory cytokines IL-17A, which is linked to in-
flammation and host antimicrobial immunity [13,14]. Evi-
dence has shown that circulating IL-17+Tcells are largely
accumulated in the livers of CHB patients and that their
frequency increases with progression from CHB to acute-
on-chronic liver failure (ACLF) [15-19].
In general, Foxp3+Treg and Th17 cells are both
involved in the pathogenesis of CHB. Foxp3+Tregs and
Th17 cells are closely associated with each other, to tie
Foxp3+Treg cells with Th17 cells, Zhang JY et al. used
the ratio of Foxp3+Treg cells to Th17 cells as an index
and found that Treg/Th17 ratios were decreased in CHB
patients compared with HCs, and entecavir-induced sup-
pression of HBV replication lead to a significant reduction
of Treg/Th17 ratios [20]. Zhai S et al. also used Treg/Th17
ratios to characterize Th-17 and Treg cells in the blood of
HBV-associated ACLF patients and found that the ratio of
Th17 to Treg cells is associated with survival of patients
with HBV-associated ACLF [21]. However, the relationship
of these two types of cells in the development of acute and
chronic HBV infection is still not clearly studied. To better
illustrate these two closely related immune cells in the de-
velopment of acute and chronic HBV infection, we
detected the frequency of peripheral blood Foxp3+Treg
and Th17 cells and related cell differentiation factors in 58
cases of acute and chronic HBV infection. Compared with
the healthy controls, Th17
increased in patients with acute HBV infection (AHB) and
acute-on-chronic HBV-related liver failure (ACHBLF),
slightly increased in the CHB patients and not changed in
asymptomatic HBV carrier. CHB and ACHBLF patients
manifested obvious Foxp3+Treg and Th17 immune im-
balance, which was negatively correlated with the liver in-
jury and positively correlated with HBV DNA levels in
chronic HBV related subjects.
cells weresignificantly
Results
Foxp3+Treg and Th17 cell differentiation factor levels in
the HBV infected patients
In order to learn about the Foxp3+Treg and Th17 cells
differentiation environment in HBV infected patients, we
selected five cytokines primarily related to Foxp3+Treg
and Th17 cell differentiation: interleukin-1beta (IL-1β),
interleukin-23(IL-23), interleukin-6(IL-6),
growth factor-beta1 (TGF-β1) and interleukin-2 (IL-2). As
shown in Figure 1, compared with the healthy controls,
the differentiationenvironment
ACHBLF was conducive to Th17 cell differentiation, which
was demonstrated by significantly elevated IL-23 and IL-6
levels (Figure 1A, B). In CHB, the environment benefited
Foxp3+Treg cells differentiation, demonstrated by higher
IL-2 and TGF-β1 levels (Figure 1C, D). Asymptomatic
HBV carrier had similar Foxp3+Treg and Th17 cells dif-
ferentiation environment with healthy controls. For all
HBV infected patients, the level of IL-1β not changed sig-
nificantly (Figure 1E).
transforming
inboth AHBand
Peripheral blood Foxp3+Treg and Th17 cells levels and
their effectors
Based on the knowledge of Foxp3+Treg and Th17 cells
differentiation factors in acute and chronic HBV infection,
we further evaluated the Foxp3+Treg and Th17 cells level
in different stages of HBV infection in peripheral blood,
and the results were shown in Figure 2. In both AHB and
ACHBLF patients, peripheral blood IL-17A+CD4+T cell
frequency increased significantly compared with the
healthy controls.(AHB:2.18±0.42%;ACHBLF:2.38±1.32%;
Control:1.07±0.23%) (Figure 2B). Peripheral blood IL-17A
level consistent with Th17 cell frequency in AHB and
ACHBLF, and the IL-17A level was significantly higher
than that in healthy controls and asymptomatic HBV car-
riers or CHB (Figure 2C). Despite Foxp3+Treg cells gen-
erate from the same naive T cell poll that generates Th17
cells, we found changes of the frequency of Foxp3+Treg
cells and Th17 cells were not completely synchronous dur-
ing the development of acute and chronic HBV infection
except in AHB stage, increased significantly accordingly
(AHB, 8.83±3.65% Vs healthy controls 5.30±1.82%, P
<0.001)(Figure 2D).
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Given the nonsynchronous changes of Foxp3+Treg and
Th17 cells in different stages of HBV infection, to better
correlate these two types of immune cells closely in func-
tions and differentiation, we used Foxp3+Treg/Th17 ratio
to describe their relationship. The results showed that
compared with the control group (5.41±1.21), Treg/Th17
ratios in ACHBLF group (1.74±0.25, P <0.001) and CHB
group (3.25±0.63, P <0.05) were significantly decreased
and the ratios in the AHB group (4.97±0.78, P>0.05) and
HBV carrier group (6.29±0.94, P>0.05) did not change
significantly (Figure 2E).
Relationship between Th17 population and liver injury in
acute and chroinc HBV infection
To further detect the relationship between the Th17 cells
frequency and liver injury, we analyzed correlation between
either Th17 frequency or its main effector IL-17A and
plasma HBV DNA load or ALT levels and total bilirubin
levels in these acute and chronic HBV infected patients. For
chronic HBV infected patients, there were some significant
negative correlations between plasma HBV DNA load and
both plasma IL-17A levels (r=−0.72,p<0.001; Figure 3A)
and Th17 frequency (r=−0.49,p=0.0003; Figure 3B). Fur-
ther analysis indicated that these negative association did
not occur in different separate chronic HBV infected
cohorts (Figure 3A, B). In addition, we found that both
Th17 frequency and plasma IL-17A levels positively corre-
lated with ALTand total bilirubin levels (Figure 3C, D, E, F)
in these chronic HBV-infected subjects, but further analysis
found that these positive association existed only between
plasma IL-17A levels and total bilirubin levels in patients
with ACHBLF(r=0.70,p=0.001) (Figure 3F). However,
there were positive correlation between both Th17 fre-
quency(r=0.64,p=0.04) and plasma IL-17A levels with
serum ALT levels(r=0.69,p=0.02), but no significant cor-
relation between both HBV DNA level and total bilirubin
level with Th17 frequency or plasma IL-17A levels were
found in AHB(Figure 3G-L). No clear correlation between
Foxp3+Treg cell frequency and liver injury was detected.
Further analysis of Treg/Th17 ratio with the degree of
liver injury and viral load showed that Treg/Th17 ratio
positively correlated with viral load in these chronic
HBV-infected subjects (r=0.55, P<0.0001) (Figure 4A)
and negatively correlated with total bilirubin levels
Figure 1 Serum level of Th17 and Treg-related cytokines in patients with HBV infection. (A) Interleukin IL-23, (B) IL-6, (C) IL-2,(D)
transforming growth factor (TGF)-β1,and (E) (IL)-1β were tested. Data are presented as mean ± standard deviation and analyzed by one-way
ANOVA analysis. CHB, chronic hepatitis B; NC, normal control, AHB, acute hepatitis B, and ACHBLF, acute-on-chroni HBV-related liver failure.
** P < 0.01;*P < 0.05.
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(r=−0.41, P =0.004) in chronic HBV-infected patients,
especially in patients with ACHBLF (r=−0.69, p=0.001)
(Figure 4B). There was no significant correlation between
Treg/Th17 ratio and serum ALT levels in patients with
chronic HBV infection (Figure 4C). Furthermore, there
were no significant correlation between Treg/Th17 ratio
and liver injury or viral load in AHB (Figure 4D, E, F).
Discussion
Immune-mediated liver injury is an important pathogen-
esis of HBV infection. Recent studies focused on the
roles of Treg cells in persistent HBV infection [10-12].
Newly discovered Th17 cells shared the same naive T
cells with Treg cells and the same signaling pathways of
cell differentiation. Considerable development plasticity
has attracted great attention [22-24]. In this study, Treg
and Th17 cell frequency and related differentiation cyto-
kine environment in acute and chronic HBV infected
patients were detected. We found that in AHB and
ACHBLF, peripheral blood Th17 cell percentage of the
total CD4+ T cells increased significantly compared with
the healthy controls. We found no significant difference
between the healthy controls and asymptomatic HBV
carriers or CHB. In addition, change of peripheral blood
IL-17A levels consistent with Th17 cells and increased in
AHB and ACHBLF. Besides, peripheral IL-17A level and
Th17 cell frequency positively correlated with liver injury
in AHB or chronic HBV infected subjects. For AHB IL-
17A level and Th17 frequency positively correlated with
ALT level and for chronic HBV-infected subjects espe-
cially ACHBLF they were positively correlated with total
bilirubin level. Ge et al. [18] 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. And Ye
et al. [17] and Wang et al. [25] demonstrated that 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 inflamma-
tion degrees. All these findings strongly suggest that
Th17 cells participate in liver injury process and viral
clearance after HBV infection. Furthermore,we found
that both Th17 frequency and plasma IL-17A levels were
reversely correlated with viral load in total chronic HBV-
infected subjects. These results were in contrast to the
findings of Zhang et al.[15]. The difference may be due
to the different study groups and detection methods
used.
Differentiation processes of Th17 and Treg cells cross
with each other [26-28]. In the mouse model, studies
Figure 2 Distribution of Th17 and Treg cell subset population in each group. (A) Representative dotplots of intracellular IL-17 and Foxp3
staining in different HBV infected patients; (B) Percentage of IL-17A+CD4+T cells on total CD4+T cells; (C) Serum level of IL-17A; (D) Percentage
of FoxP3+CD25+CD4+T cells on total CD4+T cells; (E) Ratio of Treg cells to Th17 cells. CHB, chronic hepatitis B; AHB, acute hepatitis B, and
ACHBLF, acute-on-chronic HBV-related liver failure.** P<0.01;*P<0.05.
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found that inflammatory cytokines IL-6 could completely
block the FoxP3+Treg cells differentiation and IL-6 with
TGF-β could induce Th17 cell differentiation [22]. In
view of the connection between Treg and Th17 cells, we
examined the Treg and Th17 cells differentiation micro-
environment in different stages of HBV infection and
found that IL-23 and IL-6 levels in AHB were signifi-
cantly higher than those in chronic HBV infection and
the healthy controls. On the contrary, TGF-β and IL-2
levels were significantly elevated in patients with CHB.
All these results suggest that change of Th17 and Treg
cell level mainly originate from the host immune micro-
Figure 3 The relationship between peripheral Th17 frequency/serum IL-17A level and virological/biochemical characters of patients
with HBV infection. Peripheral Th17 frequency or serum IL-17A level negatively correlated with plasma HBV loads (A,B) and positively correlated
with serum ALT or total bilirubin level (C,D,E,F) in total chronic HBV infected subjects. In AHB, Th17 frequency and serum IL-17A level only
positively correlated with ALT (I, J), but not significantly correlated with HBV DNA level and total bilirubin level (G,H,K,L). Solid line, linear growth
trend; r, correlation coefficient. P-values are shown.
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