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Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, Ithaca, NY, 14853
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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5 wk postinfection ( 1). Th2 cytokines control granuloma formation, fibrosis, and tissue eosinophilia ( 2). Chronic schistosomiasis is characterized by a stage of elevated immune suppression. Infection experiments with cytokine-deficient mice revealed severe consequences for hosts with unbalanced cytokine responses to schistosome eggs ( 3). Long-term survival of persistently infected hosts relies upon a carefully balanced and tightly regulated immune response.
The anti-inflammatory cytokine IL-10 is pivotal for the generation of host-protective homeostatic conditions in schistosomiasis ( 3). Recently, CD4+CD25+ T cells were identified as a major source for IL-10 in schistosome-infected mice ( 4, 5). These CD4+CD25+ T cells were able to inhibit the proliferation of stimulated naive effector CD4+ T cells in vitro, indicating the presence of regulatory T cells (Tregs)2 in this particular T cell population. The in vitro suppression was, in part, reversible by blocking the IL-10 receptor ( 4). In adoptive cell transfers, CD4+CD25+ T cells reduced egg-mediated pathology in schistosome-infected lymphopenic recipient mice ( 4). They also diminished detrimental Th1 cytokine production after transfer into infected IL-10-deficient animals ( 5). All these data suggest the presence and functional importance of Tregs in schistosomiasis. An important regulatory role for CD4+CD25+ T cells also was demonstrated in a model of filariasis, another chronic helminth infection ( 6). However, because CD25, the 
-chain of the IL-2 receptor, also is expressed by activated effector T cells ( 7, 8) and different populations of Tregs (9, 10), the identity of the Treg population in schistosomiasis could not be determined by these experiments.
Intensive research in recent years provided evidence for two general types of regulatory CD4+ T cells. Based on their origin, Tregs are categorized into naturally occurring (naTregs) and induced (inTregs) ( 11). NaTregs arrive in the periphery as functional suppressor T cells after passing the selection process in the thymus ( 9, 12). Their development depends strictly upon the expression of the transcriptional repressor forkhead box P3 (Foxp3) ( 13, 14, 15). Suppressor T cells also can be generated in the periphery from activated effector T cells in various, Foxp3-expression independent, ways ( 16). These inTregs are distinct from naTregs by origin, Ag specificity, and mechanisms of suppression.
Intriguingly, the CD4+CD25+ T cell population from lymphoid tissue of schistosome-infected mice displayed significantly higher expression of Foxp3 mRNA, compared with CD4+CD25– T cells ( 5). This indicates the presence of naTregs within the CD4+CD25+ T cell population, which could be responsible for the immune-suppressive capacity of CD4+CD25+ T cells in schistosome-infected mice ( 4, 5). Therefore, we analyzed the distribution, phenotype, and functional role of naTregs in murine models of schistosomiasis. We were particularly interested to determine whether egg-mediated inflammation might change the ratio between effector CD4+ T cells and naTregs, leading to increased immune suppression in chronic schistosomiasis as recently proposed ( 17). Using the more reliable marker Foxp3, we also investigated whether naTregs are the source for IL-4 and IL-10 production by granulomatous CD4+CD25+ T cells ( 4). Finally, we tested how depletion of naTregs would affect the egg-induced immune response in vivo. Based on previously published data, showing inhibition of Th1 cytokine production by CD4+CD25+ T cells from schistosome-infected mice ( 5), we speculated that naTregs would primarily prevent detrimental Th1 cytokine production and promote a polarized Th2 response.
In our experiments, we detected significant frequencies of naTregs in egg-induced granulomas and draining lymphoid tissue during all stages of schistosomiasis. The natural ratio between effector T cells and naTregs is remarkable stable. However, we found evidence that the phenotype of naTregs changes with disease progression. We also discovered that naTregs do not contribute to Th2 effector cytokine production. Strikingly, they are also no major source for IL-10 in our model. Finally, depletion of naTregs significantly enhances the frequency of effector cytokines-producing T cells in acute schistosome egg-immunized mice, but not the clonal expansion of effector T cells. Thereby we demonstrate an important, IL-10-independent regulatory function for naTregs in schistosome egg-induced inflammation.
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Materials and Methods |
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Female 8–10 wk old C57BL/6Ai (Taconic Farms), B6.SJL-Ptprca Pep3b/BoyJ, C57BL/10SgSnAi-knockout (KO)-RAG2, C.129-Il4tm1Lky/J, C57BL/10SgSnAi-(KO) IL-10 mice (all bred at Cornell University) and outbred National Institutes of Health Swiss mice (Harlan Breeders) were maintained at the College of Veterinary Medicine by Laboratory Animal Services under specific-pathogen-free conditions and kept according to the criteria set by the American Association for the Accreditation of Laboratory Animal Care. Animal use protocols were approved by Cornell University’s institutional animal care and use committee. Biomphalaria glabrata snails infected with the Puerto Rican strain of Schistosoma mansoni were obtained from Biomedical Research Institute (Rockville, MD) and maintained in the laboratory. Infective-stage cercariae were harvested from infected snails. Schistosome eggs for Ag production and immunizations were isolated and enriched for mature eggs from livers of National Institutes of Health Swiss mice 6–7 wk postinfection with 200 cercariae. Soluble egg Ag was prepared from homogenized eggs as described previously ( 2).
Antibodies
The following Abs were used for flow cytometry analysis in this study: whole rat IgG (Rockland); anti- CD16/CD32 (clones CT-17.1, CT-17.2), anti-mouse CD4 (clones CT-CD4, RM4-5), and anti-mouse CD25 (clone PC61) (all from Caltag); anti-mouse CD45.1 (clone A20), anti-mouse CD103 (clone 2E7), anti-mouse Foxp3-PE staining kit, and anti-BrdU (clone PRB-1) (all from eBioscience); and anti-mouse CD4 (clone RM4-5) and anti-mouse IL-10 (clone JES5-16E3) (both from BD Pharmingen).
Preparation of Abs from hybridoma supernatant
Abs for in vivo depletion of CD25+ T cells (Clone PC61) and isotype control (clone GL113) were produced from hybridoma supernatants. Briefly, the pH of the sterile-filtered hybridoma supernatant was adjusted to pH 8, before processing. Then the supernatant was run over a protein G column with a flow rate <0.5 ml per minute. The column was washed with PBS (pH 8), and the bound Abs were eluted with 0.1 M glycine/HCl (pH 3). Immediately, the collected fractions were neutralized with Tris buffer (pH 9) and analyzed for their protein contents by measuring the absorption at 280 nm. Ab-containing fractions were pooled and dialyzed against PBS. Ab concentrations were determined by a bicinchoninic acid Bradford protein test (Pierce), and the purity was tested by standard SDS-PAGE. Ab preparations were kept frozen at –70°C.
Infection, immunization, cell transfer, and naTreg depletion
Mice were infected percutaneously by exposing their tail for 40 min to 25–30 cercariae. At designated time points, mice were sacrificed by injection of pentobarbital/heparin solution. Livers were perfused, and infection rates and egg burden were determined by worm and egg counts. In immunization experiments, mice were challenged i.p. with 5000 S. mansoni eggs in 0.5 ml of PBS. In cell transfer studies, infected RAG2–/– mice were reconstituted i.p. with a mix of freshly isolated CD4+CD25+ T cells from C57BL/6 donors (5 x 104 cells per mouse) and CD4+CD25– T cells from B6.SJL-Ptprca Pep3b/BoyJ (5 x 105 cells per mouse) at day 13 after infection. For depletion of CD25+naTregs, naive C57BL/6, C57BL/10SgSnAi-(KO) IL-10 or C.129-Il4tm1Lky/J mice were immunized i.p. with 0.5 mg/mouse anti-CD25 Ab (clone PC61). Control mice were treated similarly with the corresponding isotype Ab (clone GL113) or PBS. Depletion was verified by flow cytometry analysis of tail blood samples for CD4+CD25+ T cells 5 days later. Only mice with CD25+ T cell frequencies <0.5% of total CD4+ T cells were included in the experiment. One day later, all mice were immunized with 5000 schistosome eggs i.p. Mice were sacrificed 1 wk after egg immunization, and cell isolates were prepared from spleen and peritoneal cavity for further analysis. For detection of CD4+ T cell proliferation in vivo, mice were injected daily with 1 mg of BrdU (ICN Biomedicals) in 0.5 ml of PBS i.p. 3–6 days after egg immunization, according to a published protocol ( 18).
Cell purification
Spleen and mesenteric lymph nodes were removed and mechanically disrupted. Single-cell suspensions were filtered through 100-µm cell strainers. RBCs in splenocyte isolates were lysed with ACK buffer (BioWhittaker). Granuloma cells were isolated from liver tissue of infected mice as described ( 4). Briefly, livers were perfused with sterile PBS to remove blood cells. The tissue was passed through a sterile stainless sieve and washed twice with cold PBS. The pellet of granulomas was resuspended in RPMI 1640 medium containing 1 mg/ml collagenase D (Roche) and 4 U/ml DNase I (Invitrogen Life Technologies) and digested for 20–30 min at 37°C. The digested material was passed through a cell strainer (100 µm) and washed twice with RPMI 1640 medium. Erythrocytes were lysed with ACK buffer, and cells were washed twice with RPMI 1640 medium. Peritoneal cells from egg-immunized mice were isolated by lavage of the peritoneal cavity with 5 ml of ice-cold PBS. For cell transfers, CD4+CD25+ and CD4+CD25– T cells were isolated from splenocytes of naive donors by flow cytometry sorting. Cell suspension were stained with specific Abs for CD4 and CD25 and then sorted for the two target populations on a FACS Aria (BD Biosciences). Purity of the cell isolates was higher than 98%. For detection of IL-4 production by Foxp3+CD4+ T cells, splenocytes and liver cells from infected C.129-Il4tm1Lky/J were enriched by a magnetic beads assay for CD4+ cells according to the manufacturer’s protocol (MACS; Miltenyi Biotec). Then cells were stained with an Ab for CD4 and sorted with high purity (> 97%) for CD4+GFP+ and CD4+GFP– cells on a FACSAria cell sorter (BD Biosciences). For all intracellular cytokine stainings, CD4 cells were purified by MACS.
Cell stainings and flow cytometry
Cells were generally blocked with anti-CD16/CD32 Abs (Fc-Block) and rat IgG before incubation with specific staining Abs. Every intracellular staining was controlled by matched isotypes. For analysis of Foxp3-expressing T cells, cell suspensions were stained for CD103 and then fixed overnight according to the manufacturer’s protocol (eBioscience). After washing, cells were stained for CD4 and CD25. Then cells were permeabilized and stained intracellularly for Foxp3 or isotype control, using the Foxp3 staining kit (eBioscience). In dedicated experiments, the Foxp3 staining was combined with an intracellular staining for IL-10, using the Foxp3 staining kit (eBioscience). For all intracellular cytokine stainings (IL-4, IFN-
, IL-10), purified CD4+ T cells were activated with ionomycin (IOM) and PMA (both from Sigma-Aldrich) according to standard protocols for 6 h in complete culture medium after isolation. For the last 4 h of their stimulation, the cells were treated with GolgiPlug (BD Pharmingen) according to the manufacturer’s recommendation. Then they were fixed and prepared for the Ab stainings. Anti-BrdU stainings were performed according to a modified protocol from Lucas et.al ( 19). Briefly, isolated cells were stained against CD4, washed in PBS, and fixed for 12 h in 1% formaldehyde and 0.01% Tween 20 in PBS. After fixation cells were washed first with PBS and then in 0.15 M NaCl, 4.2 mM MgCl2, pH 5. Thereafter cells were incubated for one hour in the same buffer containing 50 U of DNase I and then washed in PBS. Finally, cells were incubated in PBS containing 0.5% Tween 20 and anti-BrdU Ab or the corresponding isotype control at 1 µg/100 µl for 30 min at room temperature. After washing in PBS, cells were analyzed for their specific stainings. Detecting proliferating thymocytes from BrdU-treated mice tested the protocol.
Cytometric data acquisition was performed on a FACSCalibur using CellQuest software (BD Biosciences), and data analysis was performed using FlowJo software (version 4.6.2 for MacOS X; Tree Star).
Statistical analysis
Differences between variables were evaluated by unpaired Student’s t test with Welch’s correction using GraphPad Prism software (version 4.0b; GraphPad). Results were considered significant for p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Previous studies demonstrated the presence of CD4+CD25+ Tregs in liver, draining lymph nodes, and spleen of C57BL/6 mice infected with S. mansoni ( 4, 5). At the time the lack of definitive markers for naTregs in inflammatory responses prevented any further quantitative or qualitative evaluation of the Treg population in schistosome-infected mice. Therefore, we established an adoptive transfer model in lymphopenic RAG–/– mice ( 20) to identify naTregs and effector T cells in egg-induced inflammation. It allowed us to investigate the distribution of naTregs in schistosomiasis and to determine whether the ratio between naTregs and effector T cells changes in favor of higher suppression when the disease progresses to the chronic stage, as it has been proposed ( 17).
CD4+CD25+ and CD4+CD25– T cells were sorted to high purity (>98%) from naive mice and mixed at the natural ratio (1:10) of CD4+CD25+ naTregs to CD4+CD25– effector T cells ( 9). The two populations were isolated from two different C57BL/6-donor strains (C57BL/6 and B6.SJL-Ptprca Pep3b/BoyJ). Thereby, the transferred CD4+ T cells were genetically identical, except for the gene encoding the surface molecule CD45. The cell mix was transferred i.p. into RAG–/– mice 2 wk after their infection with 30 cercariae. A specific Ab for the isoform CD45.1 was used to distinguish the transferred CD4+ T cell populations by flow cytometry after isolation from hepatic granulomas, mesenteric lymph nodes (MLN), and spleen of recipients at three different time points postinfection. The gating strategy and exemplary results for our analysis are shown in Fig. 1A. By in vitro suppression assays, we confirmed a similar suppressive capacity for naTregs when they were mixed with effector T cells from syngenic or from congenic donors (data not shown).
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In secondary lymphoid organs of infected and noninfected recipient mice, most naTregs coexpress CD25 and CD103
NaTregs constitutively express CD25 ( 9, 12), which is widely used to define these cells in naive and also inflammatory settings, although activated effector T cells ( 7, 8) and perhaps certain inTregs also express CD25 ( 10). Recently, it was discovered that activated/memory subsets of naTregs express the integrin E(CD103)
7 ( 21, 22, 23). CD103 deficiency by naTregs causes resistance of otherwise susceptible BALB/c mice against Leishmania major-infection by preventing naTreg retention at the inflammatory site ( 24).
In our congenic transfer model, we analyzed the expression of CD25 and CD103 in defined populations of naTregs and effector T cells according to the strategy in Fig. 1A. Thereby we explored the expression pattern for both surface molecules by effector T cells and naTregs in vivo, analyzed the impact of egg-induced inflammation on expression of these markers, and determined whether low expression of CD103 by naTregs prevents their accumulation in chronic granulomas.
Although our results show cells of all four possible CD25/CD103 combinations in transferred naTreg and effector T cell populations, we found major differences between the two populations (Fig. 2). The vast majority of effector T cells do not express either marker or only CD103 (Fig. 2, B, D, and F). Only a small minority of effector T cells express CD25 alone or in combination with CD103. In contrast, in lymphoid tissue of naive and infected animals, a majority of transferred naTregs coexpressed CD25 and CD103 (Fig. 2, A and C). In spleen and MLN, we did not detect a significant impact of egg-induced inflammation on the expression patterns for CD25 and CD103 by naTregs or effector T cells. The same was found for effector T cells in liver (Fig. 2F). We detected inflammation-dependent changes only for hepatic naTregs. In liver tissue of naive mice and in early acute infection most naTregs did not express CD25 or CD103. When the inflammation became more chronic, the frequency of CD25/CD103-coexpressing naTregs increased significantly (Fig. 2E), indicating the possibility of enhanced naTreg activity at the inflammatory site with disease progression. Our data also exclude that general low expression of CD103 by naTregs in schistosomiasis prevents naTreg accumulation at the inflammatory site.
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Egg-induced inflammation causes a modest increase in the frequency of CD25/CD103-coexpressing CD4+ T cells in infected wild-type (WT) mice
Homeostatic expansion in lymphopenic mice alters expression of CD25 and CD103 by transferred naTregs ( 20, 24). Therefore, the validity of our transfer results had to be controlled in WT mice, where similar homeostatic expansion does not take place.
In two separate experiments, cell isolates from liver, MLN, and spleen of infected mice (7, 9, and 12 wk postinfection) and naive controls were stained with specific Abs for CD4, CD25, and CD103 and then analyzed by flow cytometry. Conformity of the infections was verified by individual worm and egg counts (data not shown). We found that egg-induced inflammation induced only modest changes in expression patterns of total CD4+ T cells (Fig. 3). In all tissues, the vast majority of CD4+ T cells did not express CD25 and/or CD103. These cells should be mostly effector T cells (Fig. 2). Their frequency decreased slightly with disease progression. Of specific interest was the frequency of CD25+CD103+CD4+ T cells. In our transfer model, these cells were predominantly of naTreg origin (Fig. 2). Again, we detected only modest changes, mainly a small increase with ongoing inflammation (Fig. 3). Their frequency decreased in chronic disease only in liver (Fig. 3C). We found no evidence for a dramatic accumulation of CD4+CD25+ T cells at the inflammatory site or in draining lymphoid tissue as reported in L. major-infected mice ( 26). These data demonstrate clearly that infection with S. mansoni and subsequent egg-induced inflammation does not cause major changes in frequencies of CD25- and/or CD103-expressing cells when analyzed for total CD4+ T cells. We speculated that, similar to our transfer model, in infected WT mice, the ratio of naTregs and effector T cells is relatively stable.
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So far, the most reliable marker to identify naTregs is the transcription factor Foxp3 ( 14, 15). Elevated Foxp3-expression has been demonstrated in schistosome-infected mice by PCR for the sorted CD4+CD25+ T cell population ( 5, 17). This approach does not exclude contaminations by Foxp3– CD25+ T cells. Thus, it shows that CD4+CD25+ T cells contain a population of Foxp3+ T cells but not that all CD4+CD25+ T cells are Foxp3+ naTregs in schistosome-infected mice.
Therefore, when recently the first Ab for intracellular Foxp3 staining became available, we analyzed the expression of Foxp3 by individual CD4+ T cells in schistosome-infected C57BL/6 mice. The analysis of Foxp3 expression was combined with a staining for CD25 and CD103. The gating strategy is exemplarily shown in Fig. 4A. Our aim was to validate the existence of a stable ratio between naTregs and effector T cells in schistosomiasis and to determine the expression pattern of CD25 and CD103 by individual naTregs in infected WT mice for the first time.
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We also calculated the absolute numbers of Foxp3+ and Foxp3– CD4+T cells. In granulomatous livers, we see a rapid 20- to 25-fold increase of naTreg and effector T cell numbers after the onset of egg-induced inflammation. The numbers start to decline after the peak of acute inflammation (Table I). In spleen and MLN, the changes are more modest. In MLN, the numbers rise and peak at the height of acute inflammation before they slowly start to decline. In spleen, the numbers for both populations increase when the inflammation becomes more chronic. This increase likely reflects the development of splenomegaly in chronic schistosome-infected mice. Importantly, the numbers display that Foxp3+ and Foxp3– CD4+T cells behave similarly regarding migration and/or expansion.
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Foxp3– effector T cells did not change similarly in their CD103/CD25– expression patterns (Fig. 5B). Most CD4+Foxp3– T cells do not express CD25 or CD103 in all three tissues and at all chosen time points. The low frequency and inability to detect strong up-regulation of CD25 expression by effector T cells in infected animals was of interest. We had expected that egg-induced T cell activation would increase CD25 expression by effector CD4+ T cells, particularly because IL-2 is controlling schistosome egg-induced Th2 inflammation in various models ( 27, 28, 29, 30).
In contrast with our transfer model (Fig. 2), we did not detect a substantial population of CD25–CD103+ effector CD4+ T cells in most tissues (Fig. 5B). We observed an increase in the frequency of CD25–CD103+ effector T cells in liver only (Fig. 5B). Nevertheless, their frequency was still much lower than was recently reported for CD4+CD25–CD103+ T cells in skin lesions of L. major-infected mice ( 24).
The depiction of Foxp3-gated populations in Fig. 5 could give the wrong impression that the vast majority of CD4+CD25+ T cells express Foxp3 in all tissues at all time points, promoting CD25 as a reliable marker for naTregs in schistosome-infected mice. The cell numbers in Table I already show the huge numerical advantage of effector T cells over naTregs. When the same CD4+ T cells in Fig. 5 were gated first on CD25 (using similar gate settings) and then analyzed for expression of Foxp3, we found that, in inflamed liver, many CD4+CD25+ T cells do not express Foxp3 (Fig. 6). Even in MLN and spleen, the percentage of Foxp3– cells in the CD4+CD25+ T population is increasing with disease progression and climbs to >25% in chronically infected spleen. These results demonstrate that CD25 is not a reliable marker for Foxp3+naTregs in late acute and chronically infected mice. Particularly at the inflammatory site, the correlation between CD25 and Foxp3 expression is not high in our model.
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The cytokine profile of naTregs in inflammation is still not well defined. Recently, we reported that granulomatous CD4+CD25+ T cells produce similar amounts of Th2 cytokines and high amounts of IL-10, compared with CD4+CD25– T cells ( 4). Analyzing the cytokine profile of naTregs will help to uncover their functional role in schistosomiasis. Production of IL-4 and IL-10 by naTregs would indicate that these cells support the development of a polarized Th2 cytokine response and predominantly suppress egg-induced Th1 cytokine production ( 5). Furthermore, IL-4 in conjunction with IL-10 is very potent in inducing alternative macrophage activation ( 31). Production of IL-4 and IL-10 by naTregs would imply that these cells are very potent inducers of alternatively activated macrophages. In general, thereby naTregs could not only suppress Th1 inflammation but trigger directly tissue repair ( 32). Most macrophages in granulomas of schistosome-infected mice are alternatively activated ( 33, 34).
To investigate whether naTregs contribute to Th2 cytokine production, we used a transgenic mouse model (4get) with a bicistronic reporter gene, where GFP is expressed under the control of the IL-4 promoter ( 35, 36). This model allows the detection of Th2 cytokine-committed, IL-4-mRNA-expressing cells ex vivo, thus avoiding any in vitro restimulation of the analyzed cells.
We infected 4get mice with 30 cercariae and analyzed the expression of Foxp3, CD25, and CD103 by CD4+ T cells, which did or did not express GFP. In preliminary experiments, we noticed that some GFP fluorescence is lost after fixation for the intracellular staining of Foxp3 (data not shown). Therefore, we sorted CD4+ T cell isolates from spleen/MLN and liver ex vivo into CD4+GFP+ and CD4+GFP– T cells by a combination of MACS and flow cytometry sorting. Then the cells were stained and analyzed. Our technical limitation to four-color analysis made it necessary to separate the Foxp3 staining from the CD25/CD103 staining.
The results demonstrate that practically no Foxp3+CD4+ T cells express IL-4 in chronically infected mice (Fig. 7). Similar data were obtained from an acute egg-immunization model (data not shown). In contrast, significant frequencies of CD25/CD103-expressing CD4+ T cells could be identified in both sorted populations. It indicates that previously detected IL-4-production by granulomatous CD4+CD25+ T cells ( 4) comes from contaminating CD25+ effector T cells. Interestingly, only the GFP– cells contained a population of bright CD25+CD103+ T cells, which were previously identified as naTregs (Fig. 5). It supports our conclusion that naTregs do not produce the Th2 effector cytokine IL-4.
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In accordance with previous reports ( 4, 5), our data show that IL-10-producing T cells can be found in both populations (Fig. 8B). Only few Foxp3+ T cells, most of them not producing IL-10, were identified in the CD25– T cell population. Strikingly, we discovered that the majority of IL-10-producing CD4+CD25+ T cells from liver granulomas did not express Foxp3. These data show that the high IL-10 production by granulomatous CD4+CD25+ T cells ( 4) does not originate from naTregs. In lymphoid tissue, too, the majority of Foxp3+CD25+ T cells did not produce IL-10. Nevertheless, in liver and MLN/spleen, we detected a small population of IL-10+CD25+Foxp3+ T cells (10.2% in MLN/spleen; 10.6% in liver granulomas; gated on CD4+CD25+Foxp3+ T cells). It shows that, in both tissues, a few Foxp3+ naTregs have the ability to produce IL-10 in our model. The high frequency of Foxp3+ cells among CD4+CD25+ T cells from lymphoid tissue (Fig. 6) explains why here IL-10 is almost equally produced by CD25+Foxp3+(46.5%) and CD25+Foxp3– T cells (53.5%) when gated on all CD4+CD25+ IL-10+ T cells.
Finally, we determined whether IL-10 is produced by Th2 effector cells or by a separate population of CD4+ T cells (possibly Tr1 cells) in murine schistosomiasis. Using cell isolates from liver and spleen of chronically infected 4get mice, we analyzed expression of IL-10 and GFP by CD4+ T cells. As before (Fig. 7), CD4+ T cells were sorted into GFP+ and GFP– populations ex vivo. Then they were stimulated and stained for IL-10. In our analysis, we detected almost all IL-10-producing T cells in the GFP+ population (Fig. 8C). It demonstrates that the Th2 effector T cells produce IL-10 in our disease model.
NaTregs control effector cytokine expression, but not proliferation, by effector T cells in acute egg-induced inflammation
Cytokine production and proliferation are the main traits of activated effector T cells. Therefore, we investigated how naTregs control these parameters in schistosome egg-induced acute inflammation. We depleted the naTregs population in naive WT mice by treating the animals with 0.5 mg of a CD25-specific Ab. In naive animals, naTregs express almost exclusively high levels of CD25 (Figs. 5 and 6) ( 15, 37). This treatment removed efficiently most CD4+CD25+ T cells for 3–4 wk before they started to reappear (data not shown). One week after Ab treatment, CD25-depleted and isotype-treated mice were immunized with 5000 schistosome eggs i.p. After another week, cells were isolated from peritoneum and spleen and subsequently analyzed for cytokine production and proliferation. This approach avoids the pitfalls of previous transfer experiments ( 4, 5), such as homeostatic expansion or the absence of protective B cells ( 38). We decided against a depletion of CD25+ T cells in infected mice, although it prevented us from analyzing the direct impact of naTregs on egg-induced pathology. In infected mice, the likelihood is high to deplete also CD25+ effector T cells (Fig. 6), including CD25+Foxp3– IL-10-producing T cells (Fig. 8B). Previous transfer experiments and in vitro proliferation assays showed that IL-10-producing CD4+CD25+ T cells have immunoregulatory impact in murine models of schistosomiasis ( 4, 5). Therefore, it would be difficult to determine which population has primary responsibility for changes in inflammation and pathology after depletion of all CD25+ cells in infected animals. Although the Ab treatment will not eliminate CD25–Foxp3+ T cells (Fig. 5A) ( 15, 37), previous suppression and transfer experiments with CD4+CD25– T cells did not indicate a substantial regulatory role for this population in schistosomiasis ( 4, 5).
Strikingly, in egg-immunized 4get mice, depletion of CD25+ naTregs increased strongly the frequency of IL-4-producing CD4+ T cells, compared with isotype- and PBS-treated controls (Fig. 9, A and B). These results demonstrate, for the first time, a functional role for naTregs in schistosome-infected WT mice. Isotype treatment also induced a small and nonsignificant increase in GFP frequencies, which we explain by moderate stimulation of Fc receptor-expressing immune cells by the Ab. Successful induction of egg-mediated inflammation is demonstrated by significant elevation of GFP frequencies in all egg-immunized mice, compared with naive controls.
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-expressing CD4+ T cells in the peritoneum of CD25-depleted mice (Fig. 9, C and D). This increase was particularly notable for single IFN--expressing CD4+ T cells and for IL-4/IFN--double producers. It demonstrates that naTregs suppress acute egg-induced effector cytokine production in an IL-10-independent way. It also excludes that the results in Fig. 9, A and B, stem from a depletion of CD25+IL-10-producing T cells. This conclusion also is supported by a significant increase in the frequency of IL-10-producing CD4+ T cells in CD25-depleted mice, when cell aliquots from experiments in Fig. 9, A and B, were analyzed by intracellular cytokine staining for IL-10 (data not shown). Inhibition of proliferation is a main suppressor mechanism of naTregs in vitro ( 39) and in vivo (40). Therefore, naTregs could limit the frequency of cytokine-producing effector T cells after egg immunization by controlling the clonal expansion of CD4+ T cells. However, when we compared the absolute cell numbers of splenic CD4+CD25– T cells in all egg-immunized 4get mice (Fig. 9, A and B), we found no significant change in CD25-depleted mice (data not shown). It suggested that naTregs do not control primarily the T cell proliferation in our experimental model.
To confirm this hypothesis, we investigated egg-induced proliferation in vivo in CD25+ naTreg-depleted C57BL/6, by treating the animals with 1 mg of BrdU for 4 consecutive days according to a published protocol ( 18). The BrdU treatment was started 3 days after egg immunization. BrdU is incorporated into newly synthesized DNA and is readily detected by intracellular staining with a BrdU-specific Ab. Importantly, similar to the 4get mouse model, this approach allowed investigating the proliferation of cells in vivo, without any in vitro stimulation. We could not use 4get mice for these experiments, because anti-BrdU staining interferes with the GFP fluorescence.
In our analysis, we found no significant evidence for elevated T cell proliferation in CD25-depleted mice (Fig. 9, E and F). Induction of proliferation in all egg-immunized groups is demonstrated by significant higher frequencies of BrdU+ cells in peritoneal isolates, compared with naive controls. This result reveals that naTregs do not reduce the frequency of cytokine-producing effector T cells by limiting the expansion of activated CD4+ T cells in acute schistosome egg-induced inflammation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Strikingly, we discovered no major change in the ratio between CD4+ effector T cells and naTregs in response to egg-induced inflammation at any of the analyzed sites in two different experimental models (Figs. 1B and 4B). These results indicate that the natural ratio between effector T cells and naTregs is sufficient for their regulatory function in murine schistosomiasis. It strongly argues against the hypothesis ( 17) that enhanced and systemic immune suppression in chronic schistosomiasis results from a dramatic increase in the frequency of naTregs, although some mouse strain-specific differences in the frequency of CD4+CD25+ T cells are possible.
In contrast with our observations, high accumulation of CD4+CD25+ Tregs was reported in draining lymph nodes and at the dermal inflammatory site after infection of mice with Leishmania major ( 26, 41). The presence of naTregs compromised an effective Th1 immune response against the protozoan parasite and promoted persistence of the infection. In schistosomiasis, the host is not able to eliminate adult worms or prevent egg laying. Apparently, the functional impact of naTregs is very different. They prevent exacerbated egg-induced effector cytokine production (Fig. 9, A, B, and D). It implies that naTregs control cytokine-mediated pathology in schistosomiasis, which is supported by recent transfer data ( 4).
It is tempting to speculate whether these differences in naTreg distribution result from different survival strategies of the two pathogens and thereby possible manipulations of naTregs by pathogenic organisms. The presence and action of naTregs is necessary for the survival of L. major, but not for the host’s survival. Therefore, accumulation of naTregs at the inflammatory site is mostly beneficial for the pathogen. In schistosomiasis, the pathogen is successful only when it can secure the long-term survival of the host by limiting egg- and immune-mediated tissue damage. This goal requires an optimal balanced immune response against the trapped parasite eggs. No regulation or too much regulation has to be avoided.
As summarized in recent reviews ( 42, 43), there is obviously a multitude of factors, such as type of immune response, tissue location, pathogenicity of the infectious organism, chronicity of the infection, etc., that dictate the functional role, migration, and maybe the suppressive mechanism and Ag specificity of naTregs in various infectious and inflammatory diseases. Therefore, it is inevitable to analyze and compare the role of naTregs in different disease models, to develop a broad picture of their biological role in the mammalian immune system.
Importantly, our results show that naTregs change their phenotype in schistosomiasis. We detected a strongly increasing frequency of CD103-expressing naTregs within the CD4+Foxp3+ T cell population when the egg-induced inflammation progresses to the chronic stage (Fig. 5A). Expression of CD103 was first reported for mucosal T lymphocytes ( 44, 45), and in CD103-deficient mice the number of mucosal T cells is selectively reduced ( 46). Also, 15% of splenic T cells express this integrin chain in mice ( 47). Recently, it was discovered that a significant population (25%) of CD4+CD25+ naTregs in naive mice expresses CD103 ( 21, 48). These cells represent an activated or memory T cell phenotype with higher suppressive capacity than CD103– naTregs ( 21, 22, 23). Therefore, we speculate that the higher frequency of CD103+ naTregs increases immunosuppression in chronic schistosomiasis. Thereby immunoregulation by naTregs could adjust to increased regulatory needs in chronic inflammatory disease. We plan to test this hypothesis in future experiments. Whether naTregs acquire this phenotype in schistosomiasis in response to inflammatory mediators or to increased tissue damage remains to be determined.
In L. major-infected mice, CD103-deficient naTregs are unable to accumulate at the dermal inflammatory site, thereby rendering normally susceptible BALB/c mice into disease-protected animals ( 24). As reported, CD103 is necessary for the retention of naTregs in inflamed dermis but not for their migration. The ligand for E(CD103)7 integrin is e-cadherin, a member of the classical cadherin family. It is highly expressed by epithelial cells, and its main function is the facilitation of cell-cell adhesion ( 49). In skin, Langerhans cells also express e-cadherin and thereby adhere to other local cells in the dermis ( 50).
In normal murine liver tissue, epithelial cells and hepatocytes express e-cadherin ( 51). However, increased expression of CD103 by naTregs in chronic schistosomiasis fails to promote naTreg accumulation in hepatic granulomas (Figs. 4B and 5A). Even in our transfer model, with very high frequencies of CD103+ naTregs in lymphoid tissue (Fig. 2, A and C), naTregs did not accumulate in inflamed liver (Fig. 1B). We speculate that low expression of e-cadherin in the specific environment of egg-induced granulomas could prevent the retention of CD103+ naTregs in higher numbers. Thus, regulation of e-cadherin expression might effectively control the ability of activated naTregs to suppress local inflammatory responses. Because there is first evidence that pathogens are able to impact CD103 expression by naTregs ( 24), it also is conceivable that some pathogens can manipulate e-cadherin expression at sites of infection. Thereby they could generate "quasi-immunoprivileged" areas with enhanced densities of highly immunosuppressive naTregs, which would offer a save haven for pathogen survival.
The cytokine profile of naTregs in infection and inflammation is not well defined yet. As our data demonstrate, particular problems raise from the use of CD25 as defining marker for naTregs. Using Foxp3 as definitive marker for naTregs ( 15), we discovered that they do not produce Th2 effector cytokins and also are not a main source for IL-10 in our model. It shows that previously recorded high production of IL-10 ( 4, 5) and normal expression of IL-4 ( 4) by CD4+CD25+ T cells in schistosomiasis models does not stem from naTregs. Nevertheless, it is important to point out that 10% of naTregs expressed IL-10. In our schistosomiasis model, where the frequency of naTregs stays low in the course of the disease (Figs. 1B and 4B), the physiological impact of IL-10-producing naTregs is negligible (Fig. 9, C and D). Only in in vitro assays with artificially normalized cell numbers for CD4+CD25– and CD4+CD25+ T cells, IL-10-producing naTregs from schistosome-infected mice probably affect the results ( 4, 5).
In contrast, when infection-induced inflammation causes a strong accumulation of naTregs in certain tissues, as reported for cutaneous leishmaniasis ( 24, 26), IL-10-producing naTregs are probably becoming a relevant source for IL-10. In our disease model of chronic schistosomiasis, the main source for T cell-produced IL-10 is Th2 effector cells. In accordance with our results, it was published recently that, in a bead-induced pulmonary granuloma model, only CD4+CD25+Foxp3– T cells produce high amounts of IL-10, but not CD4+CD25+Foxp3+ T cells ( 52). Our data also argue against the hypothesis that naTregs induce alternative macrophage activation, thereby directly promoting tissue repair and wound healing ( 32).
In a recently published study, we also found no major regulatory role for TGF- in murine schistosomiasis ( 53). Therefore, we speculate that naTregs regulate egg-induced inflammation not by release of the anti-inflammatory cytokines IL-10 or TGF-, but rather via cell-to-cell contact-dependent mechanisms ( 54). Expression of E7 integrin could improve the contact between CD103+ naTregs and e-cadherin-expressing target cells, thereby promoting increased immune suppression. Thus, expression of E7 integrin might not only affect the tissue distribution but also directly the suppressor capacity of naTregs.
Finally, we revealed that naTregs suppress acute egg-induced Th2-cytokine production. Previous data indicated that CD4+CD25+ T cells promote Th2-cytokine polarization in schistosomiasis by IL-10-dependent inhibition of Th1 cytokine production ( 5). Support for this hypothesis came from allergy models, where depletion of CD4+CD25+ T cells decreased Th2 cytokine production ( 55, 56). However, it also was shown that CD4+CD25+ Tregs are able to reduce Th2 cytokine responses ( 57, 58, 59). We demonstrate that naTregs inhibit IL-4 expression in egg-induced inflammation (Fig. 9, A and B). In IL-10-deficient mice, they control also egg-induced Th1 cytokine expression. (Fig. 9, C and D). Therefore, we conclude that naTreg-mediated suppression is not directed against a particular cytokine pattern but targets T cell-activation in general. This model would make naTregs the prime suspect for the cross-regulation of different inflammatory responses in the same host as reported in certain helminth infection models ( 60, 61, 62).
Interestingly, we found no significant evidence that naTregs control the proliferation of effector T cells in acute egg-induced inflammation (Fig. 9, E and F). Although inhibition of T cell-proliferation by naTreg has been shown in vitro and in vivo ( 39, 40), our data demonstrate that naTregs control cytokine production of effector T cells also directly. This ability of naTregs could be particularly important for regulating the effector T cell response at the inflammatory site. Only few cells proliferate in schistosome granulomas ( 63) but, analyzing infected 4get mice, many produce effector cytokines (data not shown). However, we do not exclude that naTregs limit the expansion of effector T cells at later stages in schistosomiasis.
Helminthic parasites are masters of long-term survival inside their vertebrate hosts. There is increasing evidence that skillful manipulation of the host’s immune system, such as exploitation of the host’s immunoregulatory mechanisms, is an important part of their survival strategy ( 64). The immunoregulatory network, which is activated during chronic infections with helminths, is even able to cross-regulate nonrelated inflammatory responses by their hosts ( 60, 61, 62). It also is debated whether the relatively low incidence of allergic inflammation in highly endemic areas for worm infections is caused by infection-induced immunoregulation ( 65). We show that Foxp3+ naTregs are an essential part of this regulatory network. Clearly, they control egg-induced Th2 effector cytokine production. Their suppressor function is independent upon IL-10 in our model. Strikingly, there was no significant change in the ratio between naTregs and effector CD4+ T cells in acute or chronic disease. We conclude that the natural ratio of naTregs/effector T cells is sufficient for their regulatory function in our model. However, there is a change in the phenotypic appearance of naTregs, which indicates that they are potentially more suppressive in chronic disease. Generating a pool of highly suppressive naTregs could be essential for maintaining a balanced immune response against parasite eggs in chronic schistosomiasis and potentially for cross-regulating other inflammation in the same host. At present, we speculate that naTregs are particular important for immune suppression in chronic schistosomiasis, which is characterized by an IL-10-independent down-regulation of Th2 cytokine production ( 66). Therefore, our results could have important implications for the treatment of chronic inflammatory diseases.
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1 Address correspondence and reprint requests to Dr. Matthias Hesse, Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, VMC C5 147, Ithaca, NY 14853. E-mail address: mh325{at}cornell.edu 
2 Abbreviations used in this paper: Treg, regulatory T cell; naTreg, naturally occurring Treg; inTreg, induced Treg; KO, knockout; IOM, ionomycin; MLN, mesenteric lymph node; WT, wild type.
Received for publication October 4, 2005. Accepted for publication February 15, 2006.
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