Functional changes in regulatory T cells during an experimental infection with sparganum (plerocercofid of Spirometra mansoni)

Abstract

Regulatory T (Treg) cells are important in the regulation of immune response, but the exact regulation of Treg-cell function in vivo is still not well known. In the present study, we investigated the functional activity of CD4⁺ CD25⁺ Treg cells as well as the frequency and number of CD4⁺ CD25⁺ FoxP3⁺ Treg cells in the spleens of experimentally infected mice with a tissue-migrating parasite, sparganum (plerocercoid of Spirometra mansoni) for 3 weeks. The results demonstrated fluctuations in the Treg-cell function during the parasite infection, being up-regulated at day 3, down-regulated until day 14, and thereafter up-regulated again at day 21. We also investigated the cytokine-producing capability of the splenocytes to study the pattern of immune response of the mice to the parasite. The results showed decreased capabilities of interleukin-2 (IL-2), interferon-γ (IFN-γ) and IL-17α production, whereas IL-4-producing and IL-10-producing capabilities were increased along with the parasitic infection. Meanwhile, IL-6-producing capability was increased to reach a peak at week 2, and thereafter was decreased to the baseline level. As a regulatory mechanism, we found that Treg-cell function was attenuated in the presence of the crude extracts of sparganum, but was enhanced in the presence of the excretory–secretory products, suggesting that sparganum products were involved in the triggering and regulation of immune response in the acute and chronic phases, respectively. Results show that Treg cells are central in the immune homeostasis in vivo that is maintained by host–parasite interactions during the parasitic infection.

Introduction

Regulatory T (Treg) cells play a key role in the maintenance of immune homeostasis in vivo.¹ Although Treg cells were originally identified in the CD4⁺ CD25⁺ fraction for their critical role in preventing the development of autoimmune diseases, they are also important in the regulation of almost all kinds of immune responses, including parasitic infections.^2,3 Treg cells exert their regulatory effects by suppressing the proliferation and function of immune effector cells, including CD4⁺ helper T cells, CD8⁺ cytotoxic lymphocytes, B cells and natural killer cells.^4–7

Many reports are accumulating on the biological role of Treg cells in parasitic infections. On the basis that depletion of CD4⁺ CD25⁺ cells resulted in decreased parasitic burden, it has been proposed that CD4⁺ CD25⁺ Treg cells may contribute to the immune evasion of Schistosoma japonicum or Plasmodium yoelii.^8,9 In contrast, CD4⁺ CD25⁺ Treg cells seem to be involved in pathogenic responses during Leishmania amazonensis infection, as depletion of CD4⁺ CD25⁺ cells resulted in exacerbated lesions with an increase in parasitic burden.¹⁰ Therefore, the biological role of Treg cells seems not to be consistent, and may vary depending on the state of the host–parasite relationship.

Kinetic change in the frequency of Treg cells during parasitic infections has been investigated in several reports, and accumulation of Treg cells in the lesion or systemic circulation is consistently observed in chronic parasitic infections. Therefore, it can be argued that Treg cells are critical in the maintenance of chronic parasitic infections.^11,12 Meanwhile, functional regulation of Treg cells during parasitic infections, from acute to chronic stage, has not yet been reported.

Sparganum (the plerocercoid of Spirometra mansoni) is a tissue-invading parasite that infects human as an intermediate host.¹³ Human sparganosis occurs worldwide but is frequently found in East and South East Asia. After oral infection, the sparganum penetrates the intestinal wall and migrates into the skin, usually forming a subcutaneous nodule in the chronic phase; it infrequently invades the central nervous system and causes cerebral sparganosis.^13–15 For the diagnosis of sparganosis, ELISA is a useful method for susceptible populations and for screening of neurological patients in endemic areas.^13,16 In addition, some molecules in excretory–secretory products (ESP) such as cathepsin S-like protease and neutral cysteine protease in crude extracts (CE) have been known as immune modulators.^17,18 Therefore, murine sparganosis may be an appropriate model for the study of host–parasite interaction during a chronic parasite infection following acute tissue reactions.

In the present study, we investigated the functional activity of CD4⁺ CD25⁺ Treg cells in association with the numerical changes in CD4⁺ CD25⁺ FoxP3⁺ cells in the spleens of mice experimentally infected with spargana. The results suggested that Treg cells were central to the in vivo immune regulation mediated through host–parasite interactions.

Materials and methods

Animals and parasites

Male BALB/c mice (23–27 g) at 6–8 weeks of age were obtained from Daehan Biolink (Eumsung, Korea). All mice were maintained on a 12 : 12 hr light : dark cycle at a constant temperature of 24 ± 3° and were maintained in specific pathogen-free conditions. This study was performed according to Korean Food and Drug Administration guidelines and was specifically approved by the Institutional Animal Care and Use Committee of Ewha Womans University Graduate School of Medicine (Permit Number: 10-0133). All the surgeries were performed under isoflurane anaesthesia, and every effort was made to minimize animal suffering.

Spargana were collected from snakes (Rhabdophis tigrina) caught in Korea in 2002 and were maintained in the laboratory through animal passage.¹⁹ After collecting worms, spargana were washed with sterile PBS (pH 7·4) containing antibiotics to prevent bacterial contamination. The BALB/c mice were grouped (n = 4) and each mouse was infected with two spargana. Mice were killed on days 3, 7, 14 and 21, and blood was collected for the measurement of serum antibody specific to sparganum. In addition, infection was confirmed by collecting worms from subcutaneous nodules of the infected mice on each experimental day.

ELISA for the measurement of specific antibody to sparganum

Sera were prepared from blood drawn from the hearts of the control or infected mice, and were kept frozen at −70°. Sparganum-specific antibody was assayed by micro-ELISA (Kim et al.²⁰). Briefly, a total of 2·5 μg/ml crude extracts were coated on flat-bottom polystyrene 96-well plates and incubated overnight at 4°. Sera were diluted to 1 : 100 and peroxidase-conjugated anti-mouse IgG (H&L specific goat serum; ICN Pharmaceuticals, Costa Mesa, CA) was diluted at 1 : 4000. After incubation, all the procedures were stopped by addition of 2 m H₂SO₄ and optical density was measured at 492 nm.

Crude extracts and excretory–secretory products of sparganum

A total of 1·0 g spargana was collected from naturally infected snakes and washed three times with sterile PBS (pH 7·4) to remove host-tissue particles. Worms were homogenized in a Teflon pestle homogenizer with 10 × volume of PBS at 4°. After centrifugation at 10 000 g for 1 hr at 4°, the resultant supernatant was regarded as CE of sparganum. The ESP was prepared according to the method described by Cho et al.²¹ Briefly, five spargana were incubated in 4 ml sterile PBS at 37° for 30 min and the supernatant was regarded as ESP. After centrifugation to remove particles from spargana, the protein contents were measured by Micro BCA protein assay kit (Intron, Seoul, Korea) and stored at −70° until used.

Flow cytometry

For flow cytometric measurements, spleen cells were prepared from the control or infected mice by squeezing the cells on a cell strainer (70 μm; BD Biosciences, San Jose, CA) and lysing the erythrocytes using ACK lysis buffer. After Fc receptors were blocked using anti-mouse CD16/CD32 (2.4G2) for 15 min at 4°, cells were stained for surface antigens with anti-CD3 (145-2C11), anti-CD4 (H129.19), anti-CD8 (53-6.7), anti-CD19 (1D3) or anti-CD25 (PC61) for 30 min at 4°. All the reagents were purchased from BD Biosciences. For intranuclear staining for FoxP3, cells were fixed and permeabilized using a mouse regulatory T-cell staining kit (eBiosciences, San Diego, CA) and were stained with FJK-16s-PE-Cy5. Each sample was acquired with a FACSCalibur (BD Biosciences) and was analysed using winlist software (Verity, Topsham, ME).

Preparation of cells for in vitro proliferation assay

The CD4⁺ CD25⁺ fraction was separated from the splenocytes of the control or infected mice for Treg cells by immunomagnetic selection using a Treg-cell isolation kit obtained from Miltenyi Biotech (Auburn, CA). The flow cytometric analysis revealed the purity of CD4⁺ CD25⁺ cells to be between 90% and 95%. The CD4⁺ CD25⁻ fraction was separated for the effector T (Teff) cell. CD4⁻ fraction was also isolated and used for antigen-presenting cells (APC) after treatment with mitomycin C. Parts of CD4⁺ CD25⁻ Teff cells were labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA) as described elsewhere, to trace the proliferative response.²²

In vitro proliferative assay

For the proliferative assay of Teff cells, 10⁴ CFSE-labelled Teff cells were stimulated with 1 μg/ml anti-CD3e (eBiosciences) in the presence of 10⁵ APC. To assess the suppressive function of Treg cells, equal numbers or half the number of unlabelled Treg cells were added to the co-culture of 10⁴ CFSE-labelled Teff cells and 10⁵ APC, and the cells were stimulated with 0·1 μg/ml of anti-CD3e and anti-CD28 (eBiosciences). When necessary, various concentrations of CE or ESP were added to the cultures of APC and Teff cells in the absence or presence of half or equal numbers of Treg cells, to investigate the modulatory effects of sparganum products on the function of Teff and Treg cells. The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Hyclone, Logan, UT) in round-bottomed 96-well plates. On day 3 of culture, the cells were harvested for staining with phycoerythrin-conjugated anti-CD25 (BD Biosciences) and peridinin chlorophyll protein-conjugated anti-CD4 (BD Biosciences). Whole cells were acquired for analysis using winlist software (Verity). For analysis of the proliferative response of the Teff cells, the proliferative fraction was estimated for the cells exclusively gated for CFSE⁺ CD4⁺ live cells according to the scattering characteristics.

Functional assessment of Teff cells, APC and Treg cells during the parasitic infection

The functional activities of Teff cells, APC and Treg cells of the infected mice were assessed by comparison with those from the control mice before infection (uninfected or day 0). For the functional assessment of the Teff cells during the parasitic infection, the proliferative responsiveness, represented by the proliferative fraction, of Teff cells from the infected mice and from the control mice were compared in the presence of the control APC (Fig. 3a). Likewise, for the functional assessment of APC, the proliferative responsiveness of the control Teff cells in the presence of the APC from the control and the infected mice were compared (Fig. 3b). For the functional assessment of Treg cells, the suppressive activities of Treg cells were calculated by comparing the proliferative fraction of the control Teff cells in the co-culture with the control APC in the absence and presence of the Treg cells from the control or infected mice, to obtain percentage suppression (% suppression) by Treg cells (Fig. 3c).

Figure 3.

Functional changes in effector T (Teff) cells, antigen-presenting cells (APC) and regulatory T (Treg) cells during experimental infection with sparganum. In terms of proliferative fraction, the proliferative responsiveness of CD4⁺ CD25⁻ Teff cells from the spleens of mice infected with the parasite did not show any significant change during the experimental infection with sparganum until day 21 (a). In contrast, the values of the proliferative fraction of the control Teff cells in the presence of the APC from infected mice changed remarkably during the experimental infection, reflecting that the feeder function of the APC changed remarkably in a parallel way (b). The feeder function of the APC was decreased from day 3 to day 7, and was increased at day 14, thereafter it had decreased again at day 21. The suppressive activity of CD4⁺ CDC25⁺ Treg cells, expressed as % suppression, was increased at day 3 and began to decrease from day 7 and was significantly decreased at day 14, thereafter it had significantly increased again at day 21 (c). Data were pooled from three repeated experiments, each containing four mice per group. Error bars show standard errors. Data of the infected mice were compared with those of the control mice using independent Student's t-test. *P < 0·05.

Assessment of the holistic immune responsiveness of infected mice

We assessed the immune responsiveness of the infected mice by comparing the proliferative responsiveness of the Teff cells from the infected mice in the presence of APC (and Treg cells) from the cognate infected mice with those of the cells from the control mice (Fig. 4a,b). For the combination of APC + Teff cells, the cells were stimulated with 1 μg/ml anti-CD3e, whereas for the combination of APC + Teff cells + Treg cells, the cells were stimulated with with 0·1 μg/ml anti-CD3e and anti-CD28.

Figure 4.

Immune responsiveness of the mice infected with sparganum. The values of the proliferative fraction of the effector T (Teff) cells in the co-culture with the cognate antigen-presenting cells (APC + Teff), isolated from the same infected mice, was decreased at day 3, thereafter recovered to the control level from day 7 to day 14, and was decreased again at day 21 (a). The proliferative fraction of the Teff cells in the co-culture with the cognate APC and regulatory T cells (APC + Teff + Treg), isolated from the same infected mice, was decreased from day 3 to day 7, and was increased to reach a peak at day 14, thereafter decreasing again at day 21 (b). The serum level of specific antibody was increased steeply from day 7 to day 14 (c). Data were pooled from three repetitive experiments, each containing four mice per group. Error bars show standard errors. Data of infected mice were compared with those of control mice using independent Student's t-test. *P < 0·05.

Restimulation assay for assessing cytokine-producing capability of splenocytes

Briefly, splenocytes, prepared as mentioned above, were stimulated in vitro with anti-CD3e (1 μg/ml) and anti-CD28 (1 μg/ml) for optimal periods. For the detection of IL-2, the supernatants were harvested at 24 hr, and for other cytokines, they were harvested 5 days after stimulation. The supernatants were kept frozen at −70°, and were analysed for each cytokine by Cytometric Bead Array (CBA). The quantitative detection of cytokines was performed using a CBA mouse T helper type 1 (Th1)/Th2/Th17 cytokine kit (BD Biosciences), according to the manufacturer's instructions.

Statistical analysis

Data were pooled from three repeat experiments, each experiment comprising four mice per group. Error bars represent standard error. Data of the infected mice were compared with those of the control mice by using independent Student's t-test. P-values < 0·05 were considered statistically significant.

Results

Temporal changes of the proportion of CD25⁺ FoxP3⁺ fraction in CD4⁺ splenocytes and the absolute counts of CD4⁺ CD25⁺ FoxP3⁺ cells of mice infected with sparganum

The proportion of CD25⁺ FoxP3⁺ fraction in CD4⁺ splenocytes of the infected mice was decreased until day 7, and increased to reach a peak at day 14, and thereafter decreased to the baseline until day 21 (Fig. 1 and see Supplementary material, Fig. S1). Meanwhile, the absolute count of the CD4⁺ CD25⁺ FoxP3⁺ fraction was decreased at day 3, increased to reach a peak at day 7, and thereafter decreased to the baseline level from day 14.

Figure 1.

Temporal changes in the proportion of CD25⁺ FoxP3⁺ fraction in the CD4⁺ splenocytes and the absolute counts of CD4⁺ CD25⁺ FoxP3⁺ cells of mice infected with the plerocercoid of Spirometra mansoni. A representative series of FACS plots showing the temporal change in the CD25⁺ FoxP3⁺ fraction in the CD4⁺ splenocytes of the infected mice (a). The proportion of CD25⁺ FoxP3⁺ fraction in the CD4⁺ splenocytes of infected mice was decreased until day 7, and then increased to reach a peak at day 14; thereafter it decreased again until day 21 (b). The absolute count of the CD4⁺ CD25⁺ FoxP3⁺ fraction was decreased at day 3, peaked at day 7, and then decreased to maintain the baseline level from day 14 (c). Data were pooled from three repeat experiments, each containing four mice per group. Error bars show standard error. Data of infected mice were compared with those of control mice using independent Student's t-test. *P < 0·05.

Numerical changes in lymphocyte subsets in the spleens of mice infected with sparganum

The proportions of CD19⁺ B cells and CD3⁺ T cells tended to decrease from day 3 and increase again from day 7, but without any statistical significance (Fig. 2a). Meanwhile, the proportion of CD4⁺ cells was decreased significantly at day 7 and that of CD8⁺ cells was increased significantly at day 21. In terms of absolute counts, all the subsets, CD19⁺, CD3⁺, CD4⁺ and CD8⁺ cells, were significantly decreased at day 3 and were increased at day 7 (except CD4⁺ cells), in parallel with the temporal change in total splenocytes (Fig. 2b).

Figure 2.

Quantitative changes in lymphocyte subsets during experimental infection with plerocercoid of Spirometra mansoni. The proportions (a) and absolute counts (b) of CD19⁺ cells, CD3⁺ cells, CD4⁺ cells, and CD8⁺ cells in the spleens of infected mice. Data were pooled from three repeated experiments, each containing four mice per group. Error bars show standard error. Data of infected mice were compared with those of control mice using independent Student's t-test. *P < 0·05.

Functional changes in Teff cells, APC and Treg cells during experimental infection with sparganum

In terms of proliferative fraction, no significant change in the proliferative responsiveness of CD4⁺ CD25⁻ Teff cells from the spleens of the mice infected with the parasite was observed during the experimental infection with sparganum until week 3 (Fig. 3a). On the other hand, a remarkable change in the values of the proliferative fraction of control Teff cells in the presence of APC from infected mice was observed during the experimental infection, reflecting that the feeder function of the APC changed remarkably in a parallel manner (Fig. 3b). It decreased from day 3 to day 7, was increased at day 14, and thereafter had decreased again at day 21. Meanwhile, the suppressive activity of CD4⁺ CDC25⁺ Treg cells, expressed as % suppression, was increased at day 3 and began to decrease from day 7 and was significantly decreased at day 14, thereafter it had significantly increased again at day 21 (Fig. 3c and see Supplementary material, Fig. S1). The patterns of temporal change in the functional activities of APC and Treg cells seem to contradict each other.

Immune responsiveness of mice infected with sparganum

The proliferative responsiveness of Teff cells in co-culture with the cognate APC (APC + Teff), isolated from the same infected mice, was decreased at day 3, recovered thereafter to the control level from day 7 to day 14, and had decreased again at day 21 (Fig. 4a). The proliferative responsiveness of Teff cells in co-culture with the cognate APC and Treg cells (APC + Teff + Treg), isolated from the same infected mice, was decreased from day 3 to day 7 and increased to reach a peak at day 14, but had decreased again at day 21 (Fig. 4b). To evaluate the immune responsiveness of the infected mice, we measured the serum level of specific antibody as an objective parameter of immune response to the parasite. The result showed an exponential pattern of increase, exhibiting a steep increase from day 7 to day 14 (Fig. 4c). The serum level of specific antibody at a specific time-point reflected the amount of produced antibody accumulated until then, considering the short experimental period when compared with the long half life of the serum antibody. Therefore, the result suggested that specific immune response was weak until day 7, and began to increase from day 7 to reach a peak at day 14, and thereafter became weak again. This interpretation corresponds well with the proliferative responsiveness of Teff cells in the presence of both APC and Treg cells (APC + Teff + Treg, Fig. 4b). Taken together, it seems inappropriate to evaluate immune responsiveness without Treg cells, and it is proposed that Treg cells play a critical role in the regulation of immune responsiveness in vivo.

Cytokine-producing capabilities of splenocytes from mice infected with sparganum

To understand the pattern of immune response and its relationship with the functional change in Treg cells during the parasitic infection, we investigated the cytokine-producing capabilities of the splenocytes by restimulation assay in vitro. The capabilities of the splenocytes to produce interferon-γ (IFN-γ) and IL-17α were decreased from day 3 to day 21 during the parasitic infection, except for a temporary recovery of IL-17α-producing capability at day 14 (Fig. 5a,b). On the other hand, IL-4 and IL-10-producing capabilities were increased from day 14 and day 21, respectively (Fig. 5c,d). These results suggested that the immune response of the infected mice was polarized from Th1 or Th17 to Th2 along the progression of the parasitic infection. Meanwhile, the IL-6-producing capability of the splenocytes was significantly increased at day 7 (Fig. 5e). Considering the contra-suppressive action of IL-6 that inhibits the suppressive function of Treg cells, the increased IL-6-producing capability at day 7 coincides with the decrease in Treg-cell function from day 7 (Fig. 3c). Immune responsiveness of the infected mice as reflected by the proliferative responsiveness of Teff cells in the co-culture with APC and Treg cells also began to increase from day 7 (Fig. 4b). On the other hand, IL-2-producing capability was decreased from day 3 until day 21 during the parasite infection (Fig. 5f). The reduced IL-2-producing capability of the splenocytes may be related to an increase in the proportion of activated T cells in the spleens after the parasitic infection on the basis that activated T cells secrete IL-2 in the early phase usually within 24 hr after activation, and T cells that have been already activated would not secrete more IL-2 by restimulation.²³

Figure 5.

Cytokine-producing capabilities of the splenocytes in the mice infected with sparganum. The capabilities of the splenocytes to produce interferon-γ (IFN-γ) and interleukin-17α (IL-17α) were decreased from day 3 to day 21 during the parasitic infection, except for a temporary recovery of IL-17α-producing capability at day 14 (a,b). In contrast, IL-4-producing and IL-10-producing capabilities were increased from day 14 and day 21, respectively (c,d). The IL-6-producing capability of the splenocytes was significantly increased at day 7 (e) whereas the IL-2-producing capability was decreased from day 3 until day 21 during the parasitic infection (f). Data were pooled from three repetitive experiments, each containing four mice per group. Error bars show standard error. Data of the infected mice were compared with those of the control mice using independent Student's t-test. *P < 0·05.

Immunomodulatory effects of crude extracts and excretory–secretory products of sparganum

To understand the mechanism underlying the functional changes in immune cells during experimental infection with sparganum, we investigated the immunomodulatory effects of sparganum products on Teff and Treg cells in vitro. The CE tended to increase the proliferative responsiveness of Teff cells, but without any significance, whereas ESP decreased it in a dose-dependent manner (Figs 6 and Supplementary material, Fig. S3). Both CE and ESP exerted their effects on the function of Treg cells significantly in vitro, but in an opposite way; Treg-cell function was attenuated in the presence of CE, but enhanced in the presence of ESP in a dose-dependent manner.

Figure 6.

Immunomodulatory effects of the crude extracts (CE) and excretory–secretory products (ESP) of sparganum. Addition of CE slightly increased the proliferative responsiveness of effector T (Teff) cells, and decreased the suppressive function of regulatory T (Treg) cells in a dose-dependent manner. In contrast, addition of ESP decreased the proliferative responsiveness of Teff cells, and increased the suppressive function of Treg cells in a dose-dependent manner. Data were pooled from three repetitive experiments, each containing four mice per group. Error bars show standard error. Data of the infected mice were compared with those of the control mice using independent Student's t-test. *P < 0·05.

Discussion

In the present study, we traced the functional activities of Treg cells in association with APC and Teff cells in a quantitative manner during the experimental infection with sparganum. The results showed only minor functional changes in Teff cells, suggesting that Teff cells made only a minor contribution to the regulation of immune responsiveness during experimental infection with sparganum (Fig. 3a). In contrast, the functional activities of APC and Treg cells changed substantially, suggesting that APC and Treg cells might be the major regulators of immune responsiveness during the process of infection (Fig. 3b,c). Meanwhile, the patterns of temporal changes of the functional activities of APC and Treg cells were opposite to each other. Considering the opposite functions of APC and Treg cells, as enhancer and suppressor of the proliferation of the Teff cells, respectively; APC and Treg cells may act in synergy in the regulation of immune responsiveness during experimental infection.

The functional analysis of Teff cells, Treg cells and APC in the present study reflects their potential functions, as they were the results of artificial combination (for example, Teff cells from control mice plus Treg cells from infected mice, and vice versa) as well as natural combinations (i.e. Teff and Treg cells both from the control mice or both from the infected mice). On the other hand, cognate combination of APC, Teff cells and Treg cells may provide a clue to the question of which is more important between APC and Treg cells in the regulation of immune responsiveness in vivo. The results showed that the large functional change in APC was only slightly reflected in the combination of APC plus Teff cells, suggesting that APC contributed only slightly to the functional regulation of Teff cells (Figs 3b and 4a). In contrast, addition of Treg cells to the combination of APC and Teff cells gave rise to an exaggerated functional change, suggesting that Treg cells are more important than APC, and are critical in the regulation of the functional activity of Teff cells in vivo (Figs 3c and 4b).

The combination of APC, Teff cells and Treg cells for functional assay in the present study is arbitrary in the sense that the data were obtained from experiments using an optimized but arbitrary cell ratio of 10 : 1 : 1. Nonetheless, the result of this combination of APC, Teff cells and Treg cells exactly corresponded to the current theory of the temporal change in adaptive immune response: a short inductive period followed by a rapid increase to reach a peak, and thereafter a decrease to the baseline.²⁴ In the present study, the serum level of specific antibody to the parasite also followed the usual pattern of an adaptive immune response (Fig. 4c). Therefore, it might be argued that our quantitative model of functional assay is valuable to gain an understanding of immune regulation during experimental infection with sparganum.

We incidentally observed that immune responsiveness decreased to a nadir at an early stage (day 3) before experiencing a large increase (Fig. 4b). Coincidentally, the suppressive activity of Treg cells was up-regulated, suggesting a temporary tolerance state (Fig. 3c). Previously, Takahashi et al.²⁵ reported that Treg cells were functionally more sensitive than Teff cells. Hence, at the initial stage of parasite infection, the more sensitive Treg cells might respond earlier than the Teff cells, leading to a temporary hyporesponsive state (Fig. 7). However, in terms of quantity, the absolute count of CD4⁺ CD25⁺ Foxp3⁺ Treg cells was decreased at day 3, but increased to a peak at day 7. This suggests a temporal gap between the functional and numerical changes in Treg cells, probably because time is needed for cell division and proliferation. The initial decrease in the cell count of CD4⁺ CD25⁺ Foxp3⁺ Treg cells at day 3 might be related to the decreased cell count of total splenocytes (Fig. 2b). Meanwhile, the proportion of CD4⁺ CD25⁺ Foxp3⁺ Treg cells was also decreased until day 7 (Fig. 1b). Considering the minor fraction that CD4⁺ CD25⁺ Foxp3⁺ Treg cells, ranging from 6% to 13%, comprise among CD4⁺ cells, and the actively proliferating major fractions in the spleens during parasitic infection, including CD4⁺ CD25⁻ cells, CD8⁺ cells, B cells, natural killer cells, the persistent albeit decreased Treg-cell proportion implied that Treg cells were also actively proliferating during the specified period. Otherwise, the proportion of Treg cells would continuously become smaller, and ultimately perish. However, the proportion of CD4⁺ CD25⁺ Foxp3⁺ Treg cells was actually increased at day 14, suggesting that Treg cells had been proliferating vigorously. Taken together, the results of the quantitative analysis of CD4⁺ CD25⁺ Foxp3⁺ Treg cells would be more experimental evidence supporting the hypothesis that Treg cells proliferate well in vivo.²²

Figure 7.

A hypothetical model of immune regulation during a tissue-migrating helminth infection. Based on the observation made in the present study, we propose a hypothetical model of immune regulation during a helminth infection that is mediated through host–parasite interaction. Immune responsiveness (red) and regulatory T (Treg) cell function (blue) are smoothed curves of Figs 4(b) and 3(c), respectively. At the initial phase of infection, small amounts of exogenous (parasite) antigens begin to be exposed to the host immune system, and Treg cells prevail over effector T (Teff) cells in the immune response, because Treg cells are more sensitive than Teff cells and the former suppress the latter. Therefore, a transient hyporesponsive state is induced (D0–D3). As the parasites invade host tissues, greater amounts of exogenous antigens are exposed to the host immune system and structural antigens of the parasites may inhibit Treg cells directly or indirectly, inducing a contra-suppressive state so that immune response may occur (D7–D14). After accomplishing tissue migration, the parasites reside in subcutaneous nodules where they may hide the structural antigens from the host immune system. Instead, excretory–secretory antigens may be released to enhance Treg-cell function so that immune homeostasis is maintained during the chronic infection (D21).

In the present study, the suppressive activity of Treg cells was down-regulated from day 7 to day 14, and a big leap in immune response was observed at this period (Figs 3c and 4b,c). The mechanism of ‘contra-suppression’ (functional down-regulation of Treg cells) has been reported by others as well as by us.^1,26 Pathogen-derived molecules trigger dendritic cells, through engaging Toll-like receptor 4 or 9, to secrete IL-6 or IL-21, which inhibits (contra-suppresses) the suppressive function of Treg cells.²⁷ In the present study, the capability of the splenocytes to produce IL-6 in the infected mice was increased at day 7, when the suppressive function of Treg cells began to decrease (Figs 3c and 5e). The suppressive function of Treg cells was at a nadir at day 14, but the observed behaviour could not be explained by the IL-6-producing capability of the splenocytes, suggesting that other contra-suppressive factors may be involved in the contra-suppression of Treg cells at day 14. In the present study, Treg-cell function was attenuated in the presence of CE of sparganum in vitro, suggesting that sparganum products may contribute to the contra-suppressive state of Treg cells at day 14 (Fig. 7).

The combination analysis of APC, Teff cells and Treg cells also showed a depressed immune responsiveness at day 21 after the major peak, implying a homeostatic regulation (Fig. 4b). The increase in serum level of specific antibody also dampened during this period, supporting our interpretation of homeostatic regulation (Fig. 4c). Up-regulation of the suppressive activity of Treg cells after the major peak might contribute to the maintenance of immune homeostasis during chronic parasitic infections. At this time, spargana already resided in subcutaneous nodules, having finished their tissue migration. In the present study, all the spargana were collected up to day 14 in the subcutaneous tissues of the neck/back or axilla of infected mice, forming granulomas. Hence, the spargana might hide their structural antigens in the nodules, which may induce contra-suppression. Instead, spargana might secrete or release certain molecules enhancing the suppressive function of Treg cells. The secreted or released molecules may be absorbed into the circulation and contribute to the establishment of a chronic infection state (Fig. 7).

Studies suggest that autoimmune or allergic diseases might be cured or prevented in several chronic parasite infections.^28,29 Furthermore, many investigators are studying worm therapy for intractable autoimmune diseases, such as inflammatory bowel diseases.³⁰ Although not yet proved, the results of clinical trial with Trichuris suis (pig whipworm) suggested a critical role of FoxP3⁺ Treg cells in the clinical improvement of a number of autoimmune diseases.^31,32 Our present study may be an experimental model showing the interaction of parasites and hosts, to which certain parasitic products may contribute, to reach a chronic state of infection.

The present study is the first approach to the quantitative analysis of the functional activity of Treg cells, in association with those of Teff cells and APC, in an experimental infection model. The results suggested that Treg cells played a critical role in the immune homeostasis maintained by host–parasite interactions during parasitic infection.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1. A representative series of FACS data for the analysis of CD4⁺ CD25⁺ FoxP3⁺ cell frequency during the experimental infection with sparganum.

Figure S2. The gating strategy and algorithm for the analysis of suppressive function of regulatory T cells (Tregs).

Figure S3. Immunomodulatory effects of the crude extracts (CE, A) and excretory–secretory products (ESP, B) of sparganum.