Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 299-304
doi:10.1111/j.1745-7270.2006.00168.x
Dynamics of CD4+CD25+ T Cells
in Spleens and Mesenteric Lymph Nodes of Mice Infected with Schistosoma
japonicum
Xiao-Ping CAI#, Hui
ZHANG#,
Yong-Chen ZHANG#,
Yong WANG*, Chuan SU, Min-Jun JI, Hai-Wei WU, Xiang ZHU, Zhao-Song ZHANG, and
Guan-Ling WU*
Department
of Pathogenic Biology, Jiangsu Province Key Laboratory of Modern Pathogenic
Biology, Nanjing Medical University, Nanjing 210029, China
Received: January
10, 2006
Accepted: March 8,
2006
This work was
supported by the grants from the National Natural Science Foundation of China (No.
30371344) and Jiangsu Provincial Academic Research Program of Natural Science
(No. 03KJB310075)
# These authors
contributed equally to this work
*Corresponding
authors:
Yong WANG: Tel,
86-25-86862774; Fax, 86-25-86862774; E-mail, [email protected]
Guan-Ling
WU: Tel, 86-25-86863187; Fax, 86-25-86863187; E-mail, [email protected]
Abstract
CD4+CD25+ T cells
play a major role in modulating immune response, but few reports have been published
about schistosomiasis. Here, we investigated the changes in CD4+CD25+ T cell
populations in spleens and mesenteric lymph nodes of mice infected with Schistosoma
japonicum. The proportions of CD4+CD25+ T cells in total CD4+ T cells
were analyzed by flow cytometry. CD25 and Foxp3 expression was
measured by real-time quantitative polymerase chain reaction. The suppressive
activities of CD4+CD25+ T cells were detected by in
vitro proliferation of splenocytes. Evidence showed that the percentage of
CD4+CD25+ T cells
was the same as controls 3 weeks post-infection. At the acute stage of
infection, the percentage decreased significantly. However, at the chronic
stage of infection, it rebounded to normal levels or even higher. The
expression of the CD25 and Foxp3 showed gradual increase along with the
infection progress. In vitro experiment also showed the strong
suppressive effect of CD4+CD25+ T cells, isolated during the
chronic stage, on proliferation of the CD25– splenocytes.
This is the first time that the dynamics of CD4+CD25+ T cell populations was
demonstrated in mice infected with schistosomiasis. In conclusion, our data
indicated that CD4+CD25+ cells might be involved in
the immune modulation during S. japonicum infection, which enhances
current knowledge of the mechanisms of the immuno-downregulation and
re-infection in schistosomiasis.
Key words Schistosoma japonicum; CD4+CD25+ T cells; chronic infection; Foxp3
Schistosomiasis is a parasitic disease affecting approximately 200
million people in 74 tropical and subtropical countries worldwide. It is a
significant public health problem. Chronic infection is the main impact of
schistosomiasis on health due to repeated infection and development of
non-fatal but debilitating sequelae, such as granulomatous inflammation and
fibrosis. Schistosoma japonicum, one of the schistosome species, mainly
exists in China and Philippines. At present, one of the most important
challenges to people in endemic areas is the long-term persistence of the
pathogen in the host [1]. Despite nearly four decades of effort, there is still
no effective vaccine against schistosomiasis [2].Our previous investigations into human populations showed that the
specific responses of cytokines and antibodies elicited by the infection of S.
japonicum was gradually downmodulated with the development of the disease
[3,4]. In experimental rodent models, our studies determined interferon-g mediated
resistance against S. japonicum in chronic status, showing the
downregulation of Th1 response [5]. Moreover, some published reports showed the
reduced incidence and progression of experimental autoimmune encephalomyelitis
during schistosomiasis [6]. This apparently paradoxical effect of persistent
infection and downregulation can be observed in other chronic infections,
including hepatitis C virus chronicity [7]. CD4+CD25+ T cells are arguably one
of the best characterized regulatory T cell subsets to date, which play major
roles in autoimmunity and transplantation immunity, as well as in tumor
immunity [8–10]. Recently, more evidence has been found to support the
association between chronic infections and CD4+CD25+ T cells [11,12]. With respect to S. japonicum infection,
little was known about the constituents and functions of CD4+CD25+ T cells. Several studies have also provided
evidence that the Foxp3 gene, which encodes a transcription repressor,
is specifically expressed in CD4+CD25+ T cells.
These findings collectively indicate that Foxp3 is a critical control
gene for the development and function of CD4+CD25+ T cells [13]. Therefore, in the present study, we observed the dynamical patterns
of the proportion of CD4+CD25+ T cells
in total CD4+ T cells, the expression of the CD25 gene and Foxp3
molecules in CD4+ T cells in different stages of S. japonicum
infection, as well as the in vitro suppressive functions of the CD4+CD25+ T cells. The results provide information about
the importance of CD4+CD25+ T cells in
schistosomiasis and suggest possibilities for investigating its downregulation
and re-infection.
Materials and Methods
Mice and parasites
Female 6–8-week-old BALB/c mice were purchased from the Center of
Experimental Animals, Yangzhou University (Yangzhou, China). Each mouse was
infected with 14 cercariae of S. japonicum (Chinese mainland strain)
through its abdominal skin. At 3, 6 and 13 weeks post-infection, six to eight
mice were randomly chosen from each group and killed. Each group had normal
mice as controls.
Preparation of CD4+ T cells
At a series of time points post-infection, mesenteric lymph nodes
and spleens were taken from six selected mice to prepare single cell
suspensions. After lysis of the red blood cells of spleens, the CD4+ T cells were enriched by positive selection using magnetic
microbeads (MACS; Miltenyi Biotech, Bergisch Gladbach, Germany) according to
the manufacturer’s instructions. The purity of CD4+ T cells
was analyzed by flow cytometry and found to be 93.01% (data not shown). Each
experimental group had normal mice as control.
Preparation of CD4+CD25+ T cells
At 13 weeks post-infection, mesenteric lymph nodes and spleens were
harvested from six infected mice and single cell suspensions were prepared. CD4+CD25+ T cells were purified using magnetic
microbeads (Miltenyi Biotech) according to the manufacturer’s protocol. In
brief, non-CD4+ T cells were depleted using biotin-conjugated
antibodies and anti-biotin microbeads with LD columns (Miltenyi Biotech). Then
CD4+CD25+ T cells were enriched from CD4+ T cells by positive selection using anti-CD25 antibody conjugated
to phycoerythrin (PE) and anti-PE microbeads with MS columns (Miltenyi
Biotech). CD4+CD25+ T cells were additionally
stained with fluorescein-isothiocyanate (FITC)-conjugated anti-CD4 antibody
(eBioscience, San Diego, USA). The purity of isolated cells was 93.57% (data
not shown). Normal mice of the same age were used as controls.
Flow cytometric analysis
At each time point during the longitudinal study (3, 6 and 13 weeks post-infection),
the percentage of CD4+CD25+ T cells in total CD4+ T cells was analyzed in each mouse. Cells (1?106) from mesenteric lymph nodes or
spleens were co-cultured with FITC-conjugated anti-CD4 antibody and
PE-conjugated anti-CD25 antibody (eBioscience) for 45 min at 4 ?C in the dark.
After they were washed twice with phosphate-buffered saline, the labeled cells
were analyzed on a FACScan cytofluorometer (BD Biosciences, Mountain View, USA)
using CellQuest 1.22 software (Bector Dickinson, Mountain view, USA). In each
experiment, FITC-rat-immunoglobulin G2b antibody and PE-rat-immunoglobulin G1
antibody were used as isotype controls.
Gene expression analysis by
real-time quantitative polymerase chain reaction (PCR)
Total RNA extraction from purified CD4+ T cells
was carried out with Trizol (Invitrogen, San Diego, USA). Individual samples of
RNA (1 mg) were reverse-transcribed using avian myeloblastosis virus reverse
transcriptase in the presence of oligo-d(T)15 primer (Promega, Madison, USA). A
target cDNA sample was added to TaqMan universal PCR master mix (Applied
Biosystems, Foster City, USA) to generate sensitive quantitative gene
expression data on ABI Prism 7000 sequence detection systems (Applied
Biosystems). Primers and FAM dye-labeled TaqMan MGB probes for CD25 and Foxp3
(Table 1) were designed using PrimerExpress software (Applied
Biosystems). Relative quantities of PCR products were determined using the
comparative threshold cycle method described by Applied Biosystems.
Furthermore, each sample was normalized to b-actin and expressed as a
-fold increase or decrease versus uninfected controls of the same age.
In vitro proliferation assays
Proliferation assays were performed by culturing 2?104 CD4+CD25+ T cells or CD25– splenocytes
from the mice (13 weeks post-infection) in 96-well U-bottom plates (0.2 ml;
Nunc, Rochester, USA). Concanavalin A (ConA; 2 mg/ml) or soluble egg
antigen (SEA) (20 mg/ml) were used as stimulators. Cells were incubated for 72 h in
RPMI 1640 medium (Gibco BRL, Gaithersburg, USA) with penicillin (100 U/ml),
streptomycin (100 U/ml) and 10% heat-inactivated fetus calf serum. During the
last 8 h of the culture period, [3H]thymidine was added (1 mCi/well) and the
proliferation responses were determined by the mean [3H]thymidine
incorporation of triplicate wells.
Statistical analysis
Data were analyzed using Student’s t-test and P<0.05 was considered statistically significant.
Results
Proportion of CD4+CD25+ T cells during
the infection progress
To further investigate changes in the number of CD4+CD25+ T cells as the infection progressed,
quantitative and dynamic analysis of the proportion of CD4+CD25+ T cells was carried out. At 3 weeks post-infection,
the average ratio of CD4+CD25+ T cells
in CD4+ T cells in the infected group was approximately the same as in the
uninfected group. But at the acute stage of infection (6 weeks post-infection),
the ratio was significantly decreased in the infected group compared to the
uninfected mice. In the chronic stage (13 weeks post-infection), the ratio
rebounded to the normal level in cells from mesenteric lymph nodes, but was
dramatically higher than the uninfected control in cells from spleens (Fig.
1). The profiles of CD4+CD25+ T cells
in the lymph nodes and spleens were the same during the infection progress.
Expression of CD25 and Foxp3
genes
For the quantitation of gene expression, b-actin was used as the endogenous control. The results were analyzed using
arithmetic formulas to achieve the amount of target, which were normalized to
an endogenous reference and relative to a calibrator. The results were
expressed as the -fold increase compared to normal mice, as shown in Fig. 2.
In general, at the acute stage, the expression of CD25 [Fig. 2(A)]
was increased significantly (P<0.05, for cells from lymph nodes; P<0.01, for cells from spleens). At the chronic stage, the mRNA level of CD25
was significantly higher than the normal level. The expression of Foxp3
in infected groups [Fig. 2(B)] was increased from the acute stage to the
chronic stage, to significantly higher levels than that of the control groups.
In particular, the mRNA level of Foxp3 in the cells from spleens was
much higher (P<0.05).
CD4+CD25+ T cells inhibited
proliferation of CD25–
splenocytes
When the splenocytes from chronic infected mice were stimulated with
ConA or SEA, the average proliferation in cultures, where CD4+CD25+ T cells were co-cultured, was significantly
reduced compared with that in cultures without CD4+CD25+ T cells (Fig. 3). Low inhibition rates were observed without
antigen stimulation. CD4+CD25+ T cells
alone displayed little proliferation when stimulated with ConA or SEA (data not
shown).
Discussion
The equilibrium of the immune response is maintained by positive and
negative events, which is a dilemma for the host that needs to trigger its
immune system not only for defensing against invading parasites but also for
minimizing the pathological effects. The character of the immune response in
the host of schistosomes is chronic inflammation, but this is not enough to
eliminate the parasites, resulting in the persistence of chronic infection. In
the study of the leishmaniasis model, Xu et al. showed that CD4+CD25+ T cells could suppress both Th1 and Th2
responses and contribute to the maintenance of chronic infection [14]. Hisaeda et
al. found that CD4+CD25+ T cells
played a crucial role in immune suppression during the chronic infection of
malaria parasites, as well as the escape of parasites from host immune
surveillance [15]. In the present study, we investigated the proportion of CD4+CD25+ T cells both from mesenteric lymph nodes and
spleens during different stages of infection. At 3 weeks post-infection (before
eggs were produced), the results were the same in both the infected and the
control groups. Then at 6 weeks post-infection (acute stage), the ratios of CD4+CD25+ T cells in CD4+ T cells
were significantly decreased compared to the normal ratios. At 13 weeks
post-infection (chronic stage), the ratios rebounded to the same or even higher
than normal levels. However, the results of real-time PCR indicated that the
mRNA of CD25 increased along with the development of infection. In
addition, in vitro experiment demonstrated that the CD4+CD25+ T cells could clearly suppress the
proliferation of CD25– splenocytes. This is the
first study to probe the dynamics of CD4+CD25+ T cell populations in mice infected with S. japonicum. We
speculate that CD4+CD25+ T cells might be induced
posterior to the inflammatory effect cells. Until the chronic stage of
infection, they would exert a suppressive effect on inflammatory cells, which
might result in the ratios of CD4+CD25+ T cells
relatively increasing. The results also showed that little proliferation of CD4+CD25+ T cells was observed when they were stimulated
using ConA or SEA alone, which was similar to the report of McHugh et al.
[16]. So in our study, the change in the ratios of CD4+CD25+ T cells might be caused by the inhibitory effect, which led to the
decrease of inflammatory cells.Although the role of CD4+CD25+ T cells in the suppression of autoimmune and transplant immune
responses are generally acknowledged, the mechanism of these cells exerting
regulatory effects on infective diseases is still a matter of debate. Several
mechanisms have been suggested for the regulatory effect of CD4+CD25+ T cells. The causative gene Foxp3, as
one of the markers exists mainly in CD4+CD25+ T cells, encodes a member of the forkhead/winged helix
family and acts as a transcriptional repressor. It has been shown that Foxp3
is expressed exclusively in CD4+CD25+ T cells
in the thymus and periphery and is not induced upon activation of CD25– T cells [17]. This is best illustrated by immune dysregulation,
polyendocrinopathy, enteropathy, X-linked syndrome, a rare monogenic disease of
male children that is accompanied by autoimmune disease (such as type 1
diabetes), inflammatory bowel disease and severe allergies similar to those
produced in mice by depletion of CD4+CD25+ T cells [18]. Our data showed that the Foxp3 gene expression
changes were synchronous with those of CD25 and were elevated more than
3.5-fold at 13 weeks post-infection compared to the normal levels. The results
illustrated that, at the chronic stage, the Foxp3 gene was in a
condition of significantly high expression. The Foxp3 gene is necessary
and sufficient for the development and function of CD4+CD25+ T cells. Retroviral gene transfer of Foxp3 can convert na?ve
T cells to regulatory T cells whose phenotype and function are similar to
naturally arising CD4+CD25+ T cells [14]. Thus our
findings give a strong clue to the development and activation of CD4+CD25+ T cells at the chronic stage of
schistosomiasis. However, how CD4+CD25+ T cells
mediate immune suppression has not been elucidated. Differences were shown
disaffinity in different models. Studies suggest interleukin-10 and
transforming growth factor-b possess important effects in the function of CD4+CD25+ T cells [19,20]. Therefore, further
experiments will be needed to explore the real mechanism.Our study demonstrated that in mice infected with S. japonicum,
the proportion of CD4+CD25+ T cells in CD4+ T cells had regular changes, that is, at the acute stage the
percentages of CD4+CD25+ T cells decreased
significantly and at the chronic stage they increased to higher than normal
levels. Schistosomes actually cause little damage at the beginning of
infection. The eggs are trapped in tissues of the host but elicit powerful and
potentially damaging immune responses that are responsible for the pathological
consequences. Thus the acute stage is important to divide the immune response.
In summary, this study provided insight into the change patterns of CD4+CD25+ T cell populations of mice during S.
japonicum infections and, together with more comprehensive study in the
future, might better understanding of the mechanisms of immuno-regulation in
schistosomiasis.
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