Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 327-334
doi:10.1111/j.1745-7270.2006.00169.x
Characterization of CD4+ T Cell
Responses in Mice Infected with Schistosoma japonicum
Min-Jun JI, Chuan SU, Yong
WANG, Hai-Wei WU, Xiao-Ping CAI, Guang-Fu LI, Xiang ZHU, Xin-Jun WANG,
Zhao-Song ZHANG, and Guan-Ling WU*
Department
of Pathogen Biology, Nanjing Medical University, Nanjing 210029, China
Received: December
7, 2005
Accepted: March 22,
2006
This work was
supported by the grants from the National Natural Science Foundation of China (No.
30430600 and No. 30100156)
*Corresponding
author: Tel/Fax, 86-25-86863187; E-mail, [email protected]
Abstract To better understand the interaction
between Schistosoma japonicum and its murine host, we characterized the
immune response of CD4+ T cells generated during an experimental
S. japonicum infection based on different key aspects, from gene
expression to cell behavior. Mouse oligonucleotide microarrays were used to
compare gene expression profiles of CD4+ T cells from spleens of mice
at 0, 3, 6 and 13 weeks post-infection. Flow cytometry analysis was used to
determine type 1 and type 2 cytokine-secreting CD4+ T cells, to test apoptosis of
CD4+
T cells and to count CD4+CD25+ T cells, a kind of regulatory
subpopulation of CD4+ T cells. The percentage of
interleukin-4-producing CD4+ T cells was found to be much higher than
that of g-interferon-producing cells,
especially after stimulation with S. japonicum egg antigen, which
was consistent with type 1 and type 2 cytokine gene expression in the genechip.
Microarray data also showed that S. japonicum induced the increased
expression of Th2 response-related genes, whereas some transcripts related to
the Th1 responsive pathway were depressed. Flow cytometry analysis showed a
marked increase in the apoptotic CD4+ T cells from 6 weeks
post-infection and in the ratio of CD4+CD25+ to CD4+ T cells
in infected mice after 13 weeks. We therefore concluded that experimental
infection of mice with S. japonicum resulted in a Th2-skewed immune
response, which was to a great extent monitored by the immune regulatory
network, including cytokine cross-modulation, cell apoptosis and the
subpopulation of regulatory cells.
Key words Schistosoma japonicum; CD4+ T cell; immune deviation;
immune regulatory network
Schistosoma japonicum continues to pose
a public health problem in Asia, particularly in parts of China [1] and the
Philippines, despite extensive control efforts and the availability of
praziquantel. In the past five years, China witnessed a large-scale outbreak of
acute S. japonicum infection along the Yangtze River [2]. The question
of how to deal effectively with schistosomiasis became the focus of attention
and research throughout the world. A vaccine that reduces parasite or egg
burdens would be a valuable tool to complement existing disease prevention
programs and could offer a more practical approach than repeated chemotherapy
[3]. Even though a wide range of potential vaccine candidate antigens is
available, the specific protective reactions evoked by a vaccine against
schistosomes, either through eliciting strong cellular immunity or
preferentially inducing humoral immunity, are still uncertain. Most
importantly, it is well recognized that a schistosomiasis vaccine will depend
on the generation of an antigen-specific CD4+ T cell
response.
The significant conceptual revolution in immunology to divide mouse
CD4+ T cells into two major populations, Th1 and Th2, with contrasting
and cross-regulating cytokine profiles [4], deeply influenced our understanding
of immunity to schistosome infection. Th1 cells are important for macrophage
activation and the generation of strong cell-mediated immunity, and are
involved in resistance against many intracellular microorganisms, whereas Th2
cells primarily activate the humoral defense mechanisms, classically associated
with resistance to many extracellular helminths. Many previous studies on mice,
rats and humans infected with Schistosoma mansoni suggested that a vaccine
might best exploit Th2-biased effector mechanisms against the parasite.
However, the role of Th2 responses in regulating susceptibility/symbiosis or
resistance to schistosome infections has long been a subject of dispute,
thus schistosomiasis vaccination strategy has not been clearly defined till now
[5].
In order to further understand the immunological basis of a
vaccine-induced protective mechanism, we explored the genetic and immune
elements involved in the natural progression of S. japonicum infection.
We investigated the characteristics of CD4+ T cells
isolated from mice without schistosomiasis, and mice infected with S.
japonicum at 3, 6 and 13 weeks post-infection, corresponding to early,
acute and chronic infection, respectively [6], using flow cytometry and DNA
microarray techniques. This work focused on T helper cell responses and their
relevant downstream effector molecules in the course of S. japonicum
infection, which might provide new insights to elucidate the relationship
between the host and the parasite, and contribute to the development of an
anti-schistosome vaccine through preferential induction of specific Th
polarization or a balance of Th1/Th2 type response.
Materials and Methods
Parasites, experimental
animals and infections
A Chinese mainland strain of S. japonicum was maintained in Oncomelania
hupensis snails as intermediate hosts and mice as definitive hosts.
Cercariae for experimental infections were used within 1 h of collection. Female
BALB/c mice, 8 weeks old, were obtained from the Center of Experimental Animals
(Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai, China). BALB/c mice were percutaneously infected with S. japonicum
by covering the abdomen for 20 min with a glass slide carrying approximately 20
cercariae.
Intracelluar interferon (IFN)-g and interleukin (IL)-4 levels of CD4+ T cells
At 3, 6 and 13 weeks post-infection, groups of four randomly chosen
mice were killed. Intracellular cytokine detection was carried out by flow
cytometry on spleen cells from uninfected mice and mice with 3-, 6- or 13-week old
schistosome infections. Spleens were aseptically removed and single cell
suspensions were prepared by gently teasing them through sterile stainless
steel screens into complete RPMI 1640. Splenocytes were incubated (1?106 cells/ml/well; 4 h, 37 ?C, 5% CO2) in the presence of medium alone or of soluble S. japonicum egg
antigen (SEA; 50 mg/ml). Prior to performing intracellular flow cytometry, cells were
treated with Brefeldin A (10 mg/ml; Pharmingen, San Diego, USA). Cells were
washed by centrifugation at 300 g for 10 min with 1% fetal calf serum in
phosphate-buffered saline (PBS), and blocked for 15 min at 4 ?C with 50% fetal
calf serum in PBS. Cells were surface-stained for 45 min at 4 ?C with
Cy-Chrome-conjugated anti-mouse CD4 (Pharmingen), then fixed and permeabilized
in Cytofix/Cytoperm medium (Pharmingen) for 20 min at 4 ?C. After washing,
cells were incubated in 50 ml of 1?perm/wash solution, mixed with
0.5 mg of fluorescein-isothiocyanate (FITC)-conjugated anti-mouse IFN-g and
phycoerythrin (PE)-conjugated anti-mouse IL-4 (Pharmingen) for 30 min at 4 ?C.
After incubation, the cells were washed and fixed with 1% paraformaldehyde.
Flow cytometry was carried out with FACSCalibur cytometer (Becton Dickinson,
San Jose, USA), and the data were analyzed with the CellQuest software program
(version 1.22; Becton Dickinson). In all experiments, unstained cells and cells
stained separately with each fluorochrome were included to optimize
compensation setting. The addition of irrelevant isotype-matched monoclonal
antibody in parallel with experimental samples was used to confirm that the
cytokine signals detected were specific. Lymphocytes were gated according to
their forward and side-scatter characteristics. Cy-Chrome–stained CD4+ lymphocytes were gated and 15,000 events were analyzed per sample.
IFN-g and IL-4 positive CD4+ T cells were analyzed as
dot-plots. FACS quadrants were set with cells from infected mice using
negative controls. The percentage of CD4+ T cells
containing intracytoplasmic IL-4 or IFN-g was determined in
appropriate quadrants.
Isolation of CD4+ T cells
and array hybridization
At each time point (0, 3, 6 and 13 weeks post-infection), CD4+ T cells were isolated from spleens of five mice, pooled and used as
starting material for DNA microarray detection. For purification of CD4+ T cells from the splenocytes, a magnetic activated cell sorter
system (Miltenyi Biotec, Bergisch Gladbach, Germany) was used according to the
manufacturer’s instructions. Antisense cRNA of purified CD4+ T cells was prepared following Affymetrix (Santa Clara, USA) recommendations.
Briefly, total RNA was extracted from the purified CD4+ T cells
with the Trizol procedure. Double-stranded cDNA was retro-transcribed with a
special oligo(dT)24 primer with a T7 RNA polymerase promoter
sequence and the Superscript Choice System for cDNA synthesis (Life
Technologies, Gaithersburg, USA). Double-stranded cDNA (1 mg) was
transcribed to biotin-labeled anti-sense cRNA with an ENZO kit (Affymetrix).
cRNA was purified on an affinity column (RNeasy; Qiagen, Hilden, Germany), and
then fragmented to an average size of 50–200 bp by incubation for 35
min at 94 ?C in 40 mM Tris-acetate at pH 8.1. Samples were diluted in the
hybridization solution to a final concentration of 0.05 mg/ml and heated at
94 ?C for 5 min. Analysis of the samples was done by hybridizing the fragmented
cRNAs to the Affymetrix U74A genechip array, representing approximately 6000
murine genes and 6000 Expressed Sequence Tags (ESTs), at 45 ?C for 16 h in a
rotisserie oven set at 60 rpm. After hybridization, the chips were rinsed with
non-stringent wash buffer, then stringent wash buffer, stained by incubation
with 2 mg/ml of PE-streptavidin (Molecular Probes, Eugene, USA), washed
again, and read by a confocal scanner (GeneArray 2500; Agilent, Palo Alto, USA)
and analyzed with the Microarray Suite 5.0 gene expression analysis program
(Affymetrix).
Apoptosis of CD4+ T cells
The apoptosis of CD4+ T cells was examined in mouse
spleens during S. japonicum infection. To determine early membrane and
DNA changes, splenocytes were stained with FITC-conjugated Annexin V and
propidium iodide (PI) according to the manufacturer’s instructions (BioVision,
Mountain View, USA). Spleen cells were prepared as described above. Cells (1?106) were first surface-labeled with
Cy-Chrome-conjugated anti-CD4 for 15 min at room temperature. After washing,
cells were suspended in 400 ml Annexin V binding buffer in the presence of 5 ml Annexin V-FITC
and PI for 5 min at room temperature and immediately processed by flow cytometry.
Cy-Chrome-stained CD4+ lymphocytes were gated and 10,000 events were
analyzed per sample.
Numeration of spleen CD4+CD25+ T cells
The ratio of CD4+CD25+ T cells
to CD4+ T cells was also analyzed using flow cytometry with spleen cells from
uninfected and schistosome-infected mice. Splenocyte suspension was aseptically
prepared from each mouse with specific checking points. FITC anti-mouse CD4 and
PE anti-CD25 antibodies (eBioscience, San Diego, USA) were simultaneously added
to 1?106 splenocytes,
and co-cultured for 15 min at 37 ?C in the dark to investigate the expression
of cell surface markers. After they were washed twice with PBS, labeled cells
were suspended in 500 ml PBS and analyzed using a FACSCalibur cytometer and CellQuest software.
Viable lymphocytes were gated based on forward scatter/side scatter profiles,
after which CD4+CD25+ T cells were gated based
on expression of CD4 and CD25. In each experiment, FITC-conjugated anti-rat
immunoglobulin (Ig) G2b antibody and PE-anti-rat IgG1 antibody were used as
isotype controls.
Statistical analysis
The data from flow cytometry were expressed as mean+/–SD for the
control and experimental groups. The statistical significance (P<0.05) was determined by Student's t-test.
Results
Frequency of type 1 and type 2
cytokine-secreting CD4+ T cells during S. japonicum
infection
Three specific time points were selected to determine the cytokine
production by CD4+ T cells from mice spleens infected with
S. japonicum, and uninfected mice spleens as controls. We used
intracellular cytokine staining technology to specially investigate if there
were characteristic changes in the frequency of type 1 (IFN-g-producing) and
type 2 (IL-4-producing) CD4+ T cells as the infection
progressed. As shown in Table 1, from 3 weeks post-infection to 13 weeks
post-infection, the frequency of IL-4-producing CD4+ T cells
showed a remarkable upregulation tendency, in media alone or in SEA
stimulation. A significant increase of intracellular IL-4-stained CD4+ T cells in 13-week-infected mice, compared to those in uninfected
mice, is shown in Fig. 1. In contrast, IFN-g-producing CD4+ T cells underwent a slight increase from the early to the chronic
stage of infection, and numbered less than IL-4-producing CD4+ T cells after 6 weeks post-infection. The results suggested that
Th2 type response, centering on IL-4-producing CD4+ T cells,
developed to a dominance after the start of S. japonicum egg laying, the
beginning of the acute stage of S. japonicum infection.
Imbalance of Th1/Th2 type
responsive pathway
Compared with the protein level of two representative cytokines,
IL-4 and IFN-g, the gene transcripts of the counterpart should be investigated. To
further explore the general pattern of immune response, the expression profiles
of CD4+ T cells from uninfected mice and mice infected with S. japonicum
for 3, 6 and 13 weeks were observed on a genome scale. A major part of
differential genes belonging to Th2 responsive genes indicated an increase. As
shown in Table 2, chemokines (ECF-L, MIP1a), costimulatory molecules
(OX40, CD28, inducible T cell costimulator [ICOS]), the Janus kinase-signal
transducer and activator of transcription (Jak-STAT) signaling pathway (Jak3,
SH2 domain protein 2A) and transcription factors (Jun B, NFATc, MAIL) kept an
upregulating tendency in the course of S. japonicum infection,
especially after numerous egg laying. In addition, many antibody genes, such as
IgA, IgG1, IgG3, IgM and IgE, had the same trend as the above Th2 related
genes. Of these, IgM showed a higher level than any other antibody in the
chronic stage of infection and IgE expression rapidly increased with large
numbers of schistosome eggs depositing in the tissue. However, Fig. 2
shows that IgG2b, the representative antibody of Th1 response, was
downregulated in the chronic course of S. japonicum infection. In
contrast to the upmodulation of Th2 response, the gene transcripts of the
representative cytokines and the trend towards declining expression of
IFN-inducible genes have been discussed previously [7], suggesting the Th1
response and IFN downstream pathway were suppressed. Thus, a marked Th2
response-bias imbalance was observed.
Apoptosis of CD4+ T cells
during infection
Following the demonstration of an imbalance of Th1 and Th2 response
after egg laying, we determined if apoptosis of CD4+ T
lymphocytes occurred abnormally in infected mice during S. japonicum
infection. Splenocytes were specifically labeled with Cy-Chrome-conjugated
anti-CD4 monoclonal antibody, then dual-stained with FITC-conjugated Annexin V
as a marker for cell membrane changes indicative of early events associated
with apoptosis, and also with PI to determine the level of late apoptosis in the
CD4+ T lymphocyte population. As shown in Fig. 3, by 3 weeks
post-infection there was little difference in CD4+ T cell
apoptosis between the uninfected mice group and the 3-week-infected mice group.
There was a marked increase in the number of apoptotic CD4+ T cells in the spleens of infected mice at 6 weeks post-infection
with S. japonicum. Even more remarkable changes were observed in
13-week-infected mice compared with uninfected mice.
Proportion of CD4+CD25+ T cells
during S. japonicum infection
To further investigate if CD4+CD25+ T cells, a kind of major regulatory T cell, in CD4+ T cells have any relation to the immune deviation to Th2 response
during S. japonicum infection, we made the quantitative and dynamic
analysis on the ratio of CD4+CD25+ T cells
to CD4+ T cells from early to chronic infection. Fig. 4 shows that
the average ratios in the uninfected group and 3-week post-infection group were
approximately the same, but were quite distinct in the 6-week post-infection
and 13-week post-infection groups versus the control group. By 6 weeks
post-infection, there was a significant decrease in the ratios of CD4+CD25+ T cells to CD4+ T cells
in the spleens of infected mice. In contrast, the proportion of CD4+CD25+ T cells in 13-week-infected mice was
dramatically higher than in the uninfected controls.
Discussion
CD4+ T cells play an important role in the
regulation of immune response and in the modulation of the functions of other
cells, including dendritic cells, natural killer cells, macrophages, and
cytotoxic T cells. This modulation is mainly mediated through cytokines, and it
may also involve direct cell-cell interactions through relative surface
molecules. In the present work, to understand the genetic and immune elements
involved in schistosomiasis progression, we investigated the participation of
CD4+ T cells in cell responses during S. japonicum infection in
mice using flow cytometry and DNA microarray techniques. This work will provide
general insights into the immune pattern and immune regulatory network induced
by S. japonicum.
To summarize, our results showed that there were dynamic changes in
the frequency of Th1 and Th2 cells in the spleens of mice during infection. A
dramatic elevation in IL-4-producing Th2 cells was coincident with a
generalized type 2 cytokine gene profile [7], especially after the onset of egg
laying. In contrast, IFN-g-producing Th1 cells had only a small increase as infection
progressed, whereas the IFN-g gene representation peaked at the acute stage of schistosome
infection, then plateaued. Thus, the pattern of Th1/Th2, with continuous, vast
egg depositing in tissues, was gradually out of balance and inclined to a
Th2-predominant response, so-called “Th2 polarization”, which was
mainly led by schistosome egg antigens [8] and their inducible “cytokines
field” [9].
To further clarify this characteristic pattern of Th2 polarization,
we tracked the gene expression of some downstream effector molecules in the Th1
and Th2-responsive pathway. Our previous studies [7] on the downmodulated
outcome of IFN-inducible genes suggested a dramatic inhibition of the IFN
pathway during infection progression. In contrast to the gradual fall of the
Th1 effect, Th2 response was very highly activated, with persistently elevated
expression of Th2-related genes. Our results showed a similar pattern in
chemokine expression in S. japonicum infection as that found in the
course of S. masoni infection [10]. MIP1a and ECF-L, a novel
eosinophil chemotactic cytokine [11], were associated with type 2 egg-induced
responses, which recruited macrophages and eosinophils to local inflammatory
sites and participated in schistosome egg antigen-elicited granuloma formation
and subsequent fibrosis. It was suggested that the pattern of chemokine
expression might determine the character of an inflammatory effect to initiate
a polarized immune response. Of particular note, CD28, Jak3, NFATc, Jun B and
others, in association with signal recognition, the Jak-STAT signaling pathway
and gene transcription, were essential for maintaining the predominance of Th2
cell activation during chronic schistosomiasis. Engagement of CD28 on T cells
provided a costimulatory signal necessary for T cell activation and differentiation.
Previous studies suggested that priming of Th2 cells was more dependent on CD28
activation than Th1 cells. CD28-deficient mice infected with S. mansoni
generated diminished egg antigen-driven IL-4 and IL-5 production and reduced
parasite antigen-specific IgG1 and polyclonal IgE secretion [12]. Analysis of
additional costimulatory molecules (ICOS, OX40) revealed a generally similar
pattern, with a significant indication of T cell activation in S. mansoni-infected
mice [13]. ICOS costimulation led to the induction of Th2 cytokines without
augmentation of IL-2 production [14]. If the ICOS-B7RP-1 costimulatory pathway
was disrupted, hepatic immunopathology was enhanced and IFN-g production by
CD4+ T cells was increased in murine schistosomiasis, suggesting an
important role for ICOS in Th2 cell differentiation and expansion [15]. Jak3
was an essential kinase for the IL-4-induced Jak-STAT signaling pathway [16].
IL-4 used Jak to initiate STAT6, a transcription factor required for many
biological functions. In addition to Jak-STAT, type 2 cytokines also activated
a variety of other signaling molecules that were vital in regulating the
IL-4-induced response. It was demonstrated that T helper cell-specific
transcription factors, such as NFATc, AP-1/Jun B and LRG-21, determined the
commitment of Th2 cells [17]. Finally, Th2-related antibodies, especially
increased IgE and IgM expression levels, were thought to be a well-recognized
feature as a cross-regulatory antibody phenotype of immune response to schistosome
infection [18,19]. Thus, the molecular basis for driving the development of Th2
response could probably be explained by multiple mechanisms, including
differential cytokine signaling, differential chemokine expression,
differential expression of transcription factors and/or differential remodeling
of Th2-specific genes.
Several possible mechanisms involved in the imbalance of Th1 and Th2
responses have been explored. First, it is well known that Th2-derived IL-4 and
IL-10 could cross-regulate the inflammatory activities of cytokines produced by
Th1 cells. There are also mutual modulations between effecting antibodies
(IgG1, IgG3, IgE) and blocking antibodies (IgM, IgG4), mainly involved in the
effect of antibody-dependent cell-mediated cytotoxicity. Second, after
the acute stage of infection, a large number of CD4+ T cells
underwent apoptosis. This phenomenon was also observed in S. mansoni
infection [20]. Combined with the cytokines secreted by Th1 and Th2 subsets, we
speculated that apoptotic CD4+ T cells were probably Th1 cells
[21]. Third, in mice, CD4+CD25+ T cells
represent one of the CD4+ T cell subpopulations with
immunoregulatory properties. It was demonstrated that natural regulatory CD4+CD25+ T cells over-expressed a subset of Th2 gene transcripts
[22], representing a unique form of Th2-like differentiation, which could
monitor the tolerant state. Accumulating evidence suggested that CD25 mediated
by c-maf promoted the production of Th2 cytokines [23]. Our results showed the
increased proportion of CD4+CD25+ T cells
and a high level of Th2 cytokines in the chronic stage of infection, indicating
CD4+CD25+ T cells might contribute to maintaining the
Th2 type preferential immune environment. The inhibiting effect of CD4+CD25+ T cells was observed in the chronicity of S.
japonicum infection in the preparatory experiment (data not shown), but the
inhibitory mechanism was still unknown. Finally, other mechanisms, such as
direct cell contact through cell surface molecules, chemokines and their receptors,
were thought to be the important regulatory elements in CD4+ T cell response to S. japonicum infection.
Th2 polarization during schistosome infection was also observed in
many various animal models and in infected individuals of schistosome endemic
areas. The skewing of Th2 polarization toward low protection and even blocking
effectors provides a unique model of how schistosome can survive and take
advantage of the host? immune response to reach an equilibrium between host defense and
parasitic escape strategies responsible for the chronicity of the infection.
Therefore, medical intervention, including anti-schistosome drugs and vaccines,
could break the deadlock in the course of the host-parasite interaction.
Acknowledgement
We kindly thank Prof. Andreas Ruppel from University of Heidelberg
(Heidelberg, Germany) for his critical review of this manuscript.
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