Parasite Immunology, 2014, 36, 658–667
DOI: 10.1111/pim.12135
Immune response of cdT cells in Schistosome japonicum–infected C57BL/6 mouse liver H. XIE,1,† D. CHEN,2,† L. LI,3,† X. YU,3 C. WU,4,5 H. GU,6 X. TANG,7 A. PENG8 & J. HUANG3 Functional Experiment Centre, Guangzhou Medical University, Guangzhou, China, 2Department of Laboratory Medicine, Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, China, 3Department of Pathogenic Biology and Immunology, institute of immunology, Guangzhou Medical University, Guangzhou, China, 4Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China, 5Key Laboratory of Tropical Disease Control Research of Ministry of Education, Sun Yat-sen University, Guangzhou, China, 6Department of Pharmacology, Vanderbilt University, Nashville, TN, USA, 7Department of Infectious Diseases, Affiliated No.8 Guangzhou Peoples Hospital, Guangzhou Medical University, Guangzhou, China, 8Clinical laboratory, Traditional Chinese Medicine Hospital of Guangdong province, Guangzhou, China 1
SUMMARY Systematic evaluation of the role of cdT cells during the Schistosoma japonicum infection has not been reported, despite the fact that cdT cells contribute to many infectious diseases in innate immunity. Therefore, the aim of this study was to observe the properties of cdT cells in the liver of C57BL/6 mice infected by S. japonicum. In this report, using immuno-fluorescent histological analysis, cdT cells were found around hepatic granulomatous. Moreover, the flow cytometry results revealed that the percentage of hepatic cdT cells increased significantly after S. japonicum infection. More interestingly, a subset of CD3 cdTCR+ cells were found and markedly increased after infection. Furthermore, expression of activation markers (CD25 and CD69) and cytokine profiles were detected in these hepatic CD3+cdTCR+and CD3 cdTCR+ cells. The significantly higher level of CD69, IL-4 and IL-17 were observed in CD3+cdTCR+ cells after infection, suggesting that CD3+cdTCR+ cells instead of CD3 cdTCR+ cells might play a predominant role during the infection. Finally, our results indicated that the expression of NKG2D on CD3+cdTCR+ cells was higher than that on CD3 cdTCR+ cells. Collectively, cdT cells could play an important role in the liver of C57BL/6 mouse during japonicum infection.
Correspondence: Jun Huang, Department of Pathogenic Biology and Immunology, Guangzhou Medical College, 510182 Guangzhou, China (e-mail: [email protected]) and Anping Peng, Clinical laboratory, Traditional Chinese Medicine Hospital of Guangdong province, 510120 Guangzhou, China (e-mail: penganping @21cn.com). † These authors share equal first authorship. Received: 8 April 2014 Accepted for publication: 29 July 2014
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Keywords cytokines, NKG2D, Schistosoma japonicum, cdT cell
INTRODUCTION Schistosomiasis is a chronic parasitic disease caused by extracellular parasite Schistosoma, such as Schistosoma japonicum, and widespread in vertebrates including humans (1). A key feature of the immune response in S. japonicum-infected mice is triggered by parasite eggs that are gradually deposited in host tissues, particularly in the liver (2, 3). Infection of S. japonicum, a multicellular parasite which has an extremely diverse repertoire of antigens, induces the production of bulk cytokines to induce immune cells that play important roles in the immune response to infection (1). The main adaptive immune response against schistosomes is mediated by MHC class II-restricted CD4+T cells (4). Moreover, cdT cells, CD1restricted NKT cells and CD8+T-cell responses are also induced, although their effects are less clear (5). T cells are subdivided into two large populations distinguished by their surface expression of ab and cd T-cell receptors (TCR) (6). T cells undergo extensive DNA rearrangements at the a, b, c and d TCR loci aiming to express functional TCR chains and make a selection between two developmental programs during the DN3 stage. Thus, two distinct characteristics and functions of T-cell subsets are generated (7, 8). Cells with the ab TCR generally express CD4 or CD8 lineage markers and mostly fall into helper or effector/cytotoxic subsets. Whereas cd cells in humans usually do not express these lineage markers. They usually do not require conventional antigen presentation in the context of MHC (8). In fact, the ab and cdT-cell populations recognize different types of antigens. © 2014 John Wiley & Sons Ltd
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abT cells recognize nonself-peptide fragments restricted by MHC molecules. However, cdT cells recognize unconventional antigens including stress molecules and nonpeptidic metabolites of isoprenoid biosynthesis, and so on (9). Although the cdT cells comprise a very small percentage of hepatic immune cells, they seem to be the first line of immune response against invading pathogens (10). cdT cells display a number of effective functions leading to cells proliferation, release of pro-inflammatory cytokines (e.g. IFN-c) and anti-inflammatory cytokines (e.g. IL-4 and IL-10) (11, 12). They also express a high level of cytolytic molecules, such as granzyme A, granzyme B and Fas ligand and may help defense against pathogens via targeted killing (13). Recently, data have been obtained suggesting that cdT cells acted as early responders that promoted inflammation through the production of interleukin-17 (IL-17) (14). In some cases, cdT cells but not Th17 cells have been shown to be the primary source of IL-17A production, such as in the early phase of Mycobacterium tuberculosis (15) and Escherichia coli infections (16). cdT cells could also act as antigen-presenting cells (APC) to initiate immunity, hence acting as a bridge between innate and adaptive immunity (17). It also has an important role in regulating the initial immune response by influencing the migration and activity of neutrophils, macrophages, T and NK cells (18–20). It has been reported that the liver was probably an important organ for activation and expansion of cdT cells in humans and mice (21). In previous study, we found that the largest proportion of cdT cells produced IL-17 among three cell types (CD4+ T cells, NKT cells and cdT cells) in the S. japonicum-infected mouse liver (22). However, there is limited information about the immune response of cdT cells in Schistosomiasis. The purpose of this study is to identify the potential role of cdT cells during S. japonicum infection in C57BL/6 mice liver.
MATERIALS AND METHODS Mice, parasites and infection Sixty female C57BL/6 mice, 6–8 weeks old, were purchased from Zhongshan University Animal Center (Guangzhou, China) and maintained in a specific-pathogen-free facility at the Guangzhou Medical University. Schistosoma japonicum cercariae were shed from naturally infected Oncomelania hupensis snails, which were purchased from Jiangsu Institute of Parasitic Disease (Wuxi, China). Mice were infected percutaneously with 40 5 cercariae, and they were sacrificed at different times after infection. Animal experiments were performed in strict accordance with the regulations for the Administration of © 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 658–667
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Affairs Concerning Experimental Animals, and all efforts were made to minimize suffering.
Antibodies FITC-conjugated anti-mouse cdTCR (17A2), PE-conjugated anti-mouse CD8 (53-6.7), PerCP-cy5.5-conjugated anti-mouse CD4 (RM4-5), PE-conjugated anti-mouse CD25 (3C7), APC-conjugated anti-mouse CD69 (H1.2F3), APC-cy7-conjugated anti-mouse CD3 (145-2C11), PE-conjugated anti-mouse NKG2D(CX5), APC-conjugated antimouse IFN-c (XMG1.2), PE-conjugated anti-mouse IL-4 (11B11), APC-conjugated anti-mouse IL-9 (D9302C12), PE-conjugated anti-mouse IL-17 (TC11-18H10) and isotype-matched rat IgG2a mAb were purchased from BD/ Pharmingen (San Diego, CA, USA).
Immuno-fluorescence histology To detect cdT cells, fixed livers were embedded in OCT compound (Sakura Finetec Japan, Tokyo, Japan), frozen in liquid nitrogen and stored at 70°C. Frozen tissues were cut (7 lm), mounted on slides, fixed in ice-cold acetone for 20 min, and permeabilized with 01% Triton/PBS. Slides were then treated with the blocking buffer (10% foetal calf serum in PBS) for 1 h. Slides were incubated with FITC conjugated anti-mouse cdTCR (BD, Inc., San Diego, CA, USA) in a 1 : 20 dilution overnight at 4°C. Finally, samples were washed with PBS three times. Fluorescent staining patterns were detected and acquired by serial imaging on a CARL ZEISS Axio Imager confocal microscope.
Isolation lymphocytes The precava was cut and sterile normal saline was injected to remove blood from the liver through the ventriculus sinister. The livers were then harvested. Single cell suspensions were prepared by digesting tissue in Hanks balanced salt solution (HBSS). Digested tissue was passed through both gauze and glass wool filters to remove clumped cells. Lymphocytes were isolated by Ficoll-Hypaque (DAKEWE) density-gradient centrifugation. Isolated cells were washed twice in HBSS and resuspended at 2 9 106 cells/ mL in complete RPMI 1640 medium supplemented with 10% heat inactivated foetal calf serum (FCS), 100 U/mL penicillin, 100 lg/mL streptomycin, 2 mM glutamine and 50 lM 2-mercaptoethanol.
Cell surface and intracellular cytokine staining (ICS) Single cell suspensions from liver of control mice and mice infected with S. japonicum were stimulated with 20 ng/mL
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Figure 1 Detection of cd T cells in livers of C57BL/6 mice by immuno-fluorescence and flow cytometry. Female C57BL/6 mice were infected with 40 5 Schistosome japonicum cercariae per mouse. Six weeks after the infection, the mice were sacrificed. (a) cdTCR is identified by green fluorescence (FITC), whereas nuclei are visualized using DAPI staining (blue).cd T cells are found around granulomatous (white arrows). (b) Lymphocytes isolated either from livers of normal controls or infected C57BL/6 mice were stained with anti-cdTCR-FITC mAbs and analyzed by flow cytometry. The cells were gated on cd T lymphocytes via their forward and side scatter properties and their cdTCR expression profile. Representative flow cytometry dot plots were shown. (c) Results represent mean percentages (SD) of cd T cells within hepatic lymphocytes isolated either from liver of normal controls or infected mice. The result represents for four independent experiments. **, P < 001.
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Figure 2 Expression of CD4 and CD8 on hepatic cd T cells. Six weeks after Schistosome Japonicum infection, mice were sacrificed. Cells were stained with anti-cdTCR-FITC, anti-CD4-Percp5.5 and anti-CD8-PE. cd T cells were gated in the FACS analysis (a) The dot plots show staining with PerCP-cy5.5-conjugated anti-CD4 mAbs and PE-conjugated anti-CD8 mAbs of cd T cells. The representative dot plots were shown. (b) Results represent mean percentages (SD) of CD4+cdT cells, CD8+cdT cells, CD4 CD8 cdT cells from normal and infected liver. The results represent for four independent experiments.
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phorbol 12-myristate 13-acetate (PMA) plus 1 lg/mL Ionomycin for 5 h at 37°C under a 5% CO2 atmosphere. Brefeldin A (10 g/mL, Sigma Shanghai, China) was added during the last 4 h of incubation. Cells were washed twice in PBS, fixed with 4% paraformaldehyde, and permeabilized overnight at 4°C in PBS buffer containing 01% saponin (Sigma), 01% BSA and 005% NaN3. Cells were then stained for 30 min at 4°C in the dark with conjugated antibodies specific for the cell surface antigens CD3, cdTCR, CD4, CD8, CD25, CD69 and NKG2D, the cytokines IFN-c, IL-4, IL-9 and IL-17. The expression phenotypes of antibody-labelled lymphocytes were analysed by flow cytometry (BD Calibur and Aria II) and results analyzed with the program FlowJo version 6.0 (Tree Star, Inc. Ashland, OR USA). Isotype-matched controls for cytokines were included in each staining protocol.
Statistics Statistical evaluation of difference between means was performed by unpaired, two-tailed t-tests. P value of 005). CD69 is one of the earliest expressed markers on T cell. Its expression pattern may reflect T-cell activation status early upon stimulation (31). Further, increasing evidence suggests the activation antigen CD69 may be involved in the development or regulation of important T-cell subsets, and may exert an activating function to trigger pro-inflammatory responses (32, 33). CD25, the IL-2 receptor a-chain, is proposed to be a phenotypical marker of regulatory T cells (34) and expressed on both activated T cells and some regulatory T cells (35). Therefore, the upregulation we observed for cdT cells in infected mice (Figure 3) might be either due to activation or increased receptor expression based on the regulatory background of cdT cells. These results indicated that cdT cells were actively involved in immunity respond to infection and the enhanced activation of cdT cells again suggested that these cells were more primed for action at baseline or resting. TCR assembly starts in the endoplasmic reticulum (ER), where cTCR and dTCR subunits are synthesized and CD3ce and TCRff dimers are formed (36). The CD3 dimer then pair with individual clonotypic TCR chains (37). In this study, we found that the liver of normal mice contained a subset of CD3 cdTCR+cells (125 029%), which markedly increased to 847 047% after infection (Figure 4). The invariant CD3 play a role not only in TCR signal transduction but also in TCR assembly and surface expression (38). The role of CD3 cdTCR+ cells may be predictably blocked owing to lack of surface CD3 expression, compared with CD3+cdTCR+ cells. These
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Figure 7 Cytokine profile of hepatic cd T cells. Six weeks after Schistosome japonicum infection, the mice were sacrificed. Single cell suspensions of liver cells were stimulated with PMA, Ionomycinand BFA. Cells were stained with anti-CD3-APC-cy7, anti-cdTCR-FITC, and then intracellular stained with anti-IFN-c-APC, anti-IL-4-PE, anti-IL-9-APC and anti-IL-17-PE for FACS analysis. (a)Intracellular IFN-c, IL-4,IL-9 and IL-17 expression by gated populations of CD3+cdTCR+ cells, CD3 cdTCR+ and CD3+T cells isolated from normal and infected mice, respectively. Flow cytometric analysis from one representative experiment. (b) Statistical analysis of the expression of IFN-c, IL-4, IL-9 and IL-17 by CD3+cdTCR+ cells, CD3 cdTCR+ and CD3+T cells. The results represent for four independent experiments. The error bars indicate SD.*, P < 005; **, P < 001.
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Figure 8 Expression of NKG2D on the CD3+cdTCR+ cells and CD3 cdTCR+ cells. Six weeks after Schistosome japonicum infection, the mice were sacrificed. (a) Single liver cells were stained with anti-CD3 and anti-cdTCR mAbs. Then, cells were gated on CD3+cdTCR+ cells and CD3 cdTCR+ cells population for the analysis of NKG2D in the livers of infected mice by FCM. (b) The percentages of NKG2D on CD3+cdTCR+ cells and CD3 cdTCR+ cells in the hepatic mononuclear cells were calculated from eight independent experiments with similar results. *, P < 005. © 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 658–667
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results indicated that the infection might influence cd cells differentiation and development. A higher level of CD4+cells and CD8+ cells in CD3+cdTCR+ cells was observed after infection, comparing with CD3 cdTCR+ cells (Figure 5). It indicated that the phenotypes of two subsets were different and may modulate cell growth and function. Herein, we have found that the lower expression of activation markers, including CD25 and CD69 on the CD3 cdTCR+ cells, compared with CD3+cdTCR+ cells (Figure 6). Our results showed that the expressions of CD25 and CD69 were significantly higher on the CD3+cdTCR+ cells than CD3 cdTCR+ cells, indicating that CD3+cdTCR+ cells are more activated in this infectious process. We reasoned that as CD69 is one of the earliest expressed markers in T-cell activation, its higher level may mainly reflect CD3+cdTCR+ cells activation status early upon infection. The different activation markers expression of these two subsets might be explained on the basis of surface CD3 expression, suggesting that their activated function was in close contact with CD3 expression. To further explore the potent mechanisms and role of different hepatic cd cells subsets contributing to disease progression, we investigated the cytokine profiles of these cells after infection. Analyses of intracellular cytokines of these cells demonstrated that these infected hepatic CD3+cdTCR+cells revealed a classical inflammatory cytokine profile, with high production of IL-4 and IL-17 (15) after stimulation with PMA and Ionomycin, compared with normal mice (Figure 7). Conversely, no differences of IFN-c, IL-4, IL-9 and IL-17 in hepatic CD3 cdTCR+cells were noted between normal control and infected mice. These results indicated that CD3+cdTCR+cells instead of CD3 cdTCR+cells might play a role during infection. Consistently, previous studies have indicated that cdT cells were capable of producing Th2-type cytokines (39). Particularly, cdT cells were found to be the major source of IL-17 in naive mice, and IL-17 production by cdT cells can be further modulated in various pathological condition (40, 41). Moreover, IL-17 has been shown to be a
crucial regulator of the migration and activation of neutrophils and also secreted by the cdT cells (42, 43). Overall, our findings suggested that CD3+cdTCR+ cells, but not CD3 cdTCR+ cells played important roles in S. japonicum infection. NKG2D, like many other activating receptors, associates with an adaptor molecule to initiate signal transduction and cellular activation (44). NKG2D recognizes different families of ligand, which are distantly related to MHC, including the MHC class I chain related molecule A and B (MICA and MICB), or act as receptors for the UL16 protein (the UL16 binding proteins, ULBPs) (45, 46). NKG2D receptor engagement on T cells functions may as a co-stimulatory signal by amplifying T-cell activation through the TCR (44). Thus, CD3+cdTCR+cells may continue exerting the activity, due to the higher expression of NKG2D as shown in Figure 8 (P < 005). Moreover, it suggested that higher expression of NKG2D might be the mechanistic response to the activation ofCD3+cdTCR+ cells compared with CD3 cdTCR+ cells in this study, but this requires further investigation. In summary, our data suggest that S. japonicum infection could induce cdT cells in the liver of C57BL/6 mice, though the content of CD3 cdTCR+ cells increased significantly, the CD3+cdTCR+ cells, which expressed higher level of CD69, NKG2D, IL-4 and IL-17 seemed to play a more important role during S. japonicum.
ACKNOWLEDGEMENTS This study was supported by a grant from the Natural Science Foundation of China (30901353), Science and Technology Planning Project of Guangzhou City (2011J22007), and College Scientific Research Project in Guangzhou City (2012C117).
DISCLOSURES None.
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DOI: 10.1111/pim.12135
Immune response of cdT cells in Schistosome japonicum–infected C57BL/6 mouse liver H. XIE,1,† D. CHEN,2,† L. LI,3,† X. YU,3 C. WU,4,5 H. GU,6 X. TANG,7 A. PENG8 & J. HUANG3 Functional Experiment Centre, Guangzhou Medical University, Guangzhou, China, 2Department of Laboratory Medicine, Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, China, 3Department of Pathogenic Biology and Immunology, institute of immunology, Guangzhou Medical University, Guangzhou, China, 4Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China, 5Key Laboratory of Tropical Disease Control Research of Ministry of Education, Sun Yat-sen University, Guangzhou, China, 6Department of Pharmacology, Vanderbilt University, Nashville, TN, USA, 7Department of Infectious Diseases, Affiliated No.8 Guangzhou Peoples Hospital, Guangzhou Medical University, Guangzhou, China, 8Clinical laboratory, Traditional Chinese Medicine Hospital of Guangdong province, Guangzhou, China 1
SUMMARY Systematic evaluation of the role of cdT cells during the Schistosoma japonicum infection has not been reported, despite the fact that cdT cells contribute to many infectious diseases in innate immunity. Therefore, the aim of this study was to observe the properties of cdT cells in the liver of C57BL/6 mice infected by S. japonicum. In this report, using immuno-fluorescent histological analysis, cdT cells were found around hepatic granulomatous. Moreover, the flow cytometry results revealed that the percentage of hepatic cdT cells increased significantly after S. japonicum infection. More interestingly, a subset of CD3 cdTCR+ cells were found and markedly increased after infection. Furthermore, expression of activation markers (CD25 and CD69) and cytokine profiles were detected in these hepatic CD3+cdTCR+and CD3 cdTCR+ cells. The significantly higher level of CD69, IL-4 and IL-17 were observed in CD3+cdTCR+ cells after infection, suggesting that CD3+cdTCR+ cells instead of CD3 cdTCR+ cells might play a predominant role during the infection. Finally, our results indicated that the expression of NKG2D on CD3+cdTCR+ cells was higher than that on CD3 cdTCR+ cells. Collectively, cdT cells could play an important role in the liver of C57BL/6 mouse during japonicum infection.
Correspondence: Jun Huang, Department of Pathogenic Biology and Immunology, Guangzhou Medical College, 510182 Guangzhou, China (e-mail: [email protected]) and Anping Peng, Clinical laboratory, Traditional Chinese Medicine Hospital of Guangdong province, 510120 Guangzhou, China (e-mail: penganping @21cn.com). † These authors share equal first authorship. Received: 8 April 2014 Accepted for publication: 29 July 2014
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Keywords cytokines, NKG2D, Schistosoma japonicum, cdT cell
INTRODUCTION Schistosomiasis is a chronic parasitic disease caused by extracellular parasite Schistosoma, such as Schistosoma japonicum, and widespread in vertebrates including humans (1). A key feature of the immune response in S. japonicum-infected mice is triggered by parasite eggs that are gradually deposited in host tissues, particularly in the liver (2, 3). Infection of S. japonicum, a multicellular parasite which has an extremely diverse repertoire of antigens, induces the production of bulk cytokines to induce immune cells that play important roles in the immune response to infection (1). The main adaptive immune response against schistosomes is mediated by MHC class II-restricted CD4+T cells (4). Moreover, cdT cells, CD1restricted NKT cells and CD8+T-cell responses are also induced, although their effects are less clear (5). T cells are subdivided into two large populations distinguished by their surface expression of ab and cd T-cell receptors (TCR) (6). T cells undergo extensive DNA rearrangements at the a, b, c and d TCR loci aiming to express functional TCR chains and make a selection between two developmental programs during the DN3 stage. Thus, two distinct characteristics and functions of T-cell subsets are generated (7, 8). Cells with the ab TCR generally express CD4 or CD8 lineage markers and mostly fall into helper or effector/cytotoxic subsets. Whereas cd cells in humans usually do not express these lineage markers. They usually do not require conventional antigen presentation in the context of MHC (8). In fact, the ab and cdT-cell populations recognize different types of antigens. © 2014 John Wiley & Sons Ltd
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abT cells recognize nonself-peptide fragments restricted by MHC molecules. However, cdT cells recognize unconventional antigens including stress molecules and nonpeptidic metabolites of isoprenoid biosynthesis, and so on (9). Although the cdT cells comprise a very small percentage of hepatic immune cells, they seem to be the first line of immune response against invading pathogens (10). cdT cells display a number of effective functions leading to cells proliferation, release of pro-inflammatory cytokines (e.g. IFN-c) and anti-inflammatory cytokines (e.g. IL-4 and IL-10) (11, 12). They also express a high level of cytolytic molecules, such as granzyme A, granzyme B and Fas ligand and may help defense against pathogens via targeted killing (13). Recently, data have been obtained suggesting that cdT cells acted as early responders that promoted inflammation through the production of interleukin-17 (IL-17) (14). In some cases, cdT cells but not Th17 cells have been shown to be the primary source of IL-17A production, such as in the early phase of Mycobacterium tuberculosis (15) and Escherichia coli infections (16). cdT cells could also act as antigen-presenting cells (APC) to initiate immunity, hence acting as a bridge between innate and adaptive immunity (17). It also has an important role in regulating the initial immune response by influencing the migration and activity of neutrophils, macrophages, T and NK cells (18–20). It has been reported that the liver was probably an important organ for activation and expansion of cdT cells in humans and mice (21). In previous study, we found that the largest proportion of cdT cells produced IL-17 among three cell types (CD4+ T cells, NKT cells and cdT cells) in the S. japonicum-infected mouse liver (22). However, there is limited information about the immune response of cdT cells in Schistosomiasis. The purpose of this study is to identify the potential role of cdT cells during S. japonicum infection in C57BL/6 mice liver.
MATERIALS AND METHODS Mice, parasites and infection Sixty female C57BL/6 mice, 6–8 weeks old, were purchased from Zhongshan University Animal Center (Guangzhou, China) and maintained in a specific-pathogen-free facility at the Guangzhou Medical University. Schistosoma japonicum cercariae were shed from naturally infected Oncomelania hupensis snails, which were purchased from Jiangsu Institute of Parasitic Disease (Wuxi, China). Mice were infected percutaneously with 40 5 cercariae, and they were sacrificed at different times after infection. Animal experiments were performed in strict accordance with the regulations for the Administration of © 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 658–667
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Affairs Concerning Experimental Animals, and all efforts were made to minimize suffering.
Antibodies FITC-conjugated anti-mouse cdTCR (17A2), PE-conjugated anti-mouse CD8 (53-6.7), PerCP-cy5.5-conjugated anti-mouse CD4 (RM4-5), PE-conjugated anti-mouse CD25 (3C7), APC-conjugated anti-mouse CD69 (H1.2F3), APC-cy7-conjugated anti-mouse CD3 (145-2C11), PE-conjugated anti-mouse NKG2D(CX5), APC-conjugated antimouse IFN-c (XMG1.2), PE-conjugated anti-mouse IL-4 (11B11), APC-conjugated anti-mouse IL-9 (D9302C12), PE-conjugated anti-mouse IL-17 (TC11-18H10) and isotype-matched rat IgG2a mAb were purchased from BD/ Pharmingen (San Diego, CA, USA).
Immuno-fluorescence histology To detect cdT cells, fixed livers were embedded in OCT compound (Sakura Finetec Japan, Tokyo, Japan), frozen in liquid nitrogen and stored at 70°C. Frozen tissues were cut (7 lm), mounted on slides, fixed in ice-cold acetone for 20 min, and permeabilized with 01% Triton/PBS. Slides were then treated with the blocking buffer (10% foetal calf serum in PBS) for 1 h. Slides were incubated with FITC conjugated anti-mouse cdTCR (BD, Inc., San Diego, CA, USA) in a 1 : 20 dilution overnight at 4°C. Finally, samples were washed with PBS three times. Fluorescent staining patterns were detected and acquired by serial imaging on a CARL ZEISS Axio Imager confocal microscope.
Isolation lymphocytes The precava was cut and sterile normal saline was injected to remove blood from the liver through the ventriculus sinister. The livers were then harvested. Single cell suspensions were prepared by digesting tissue in Hanks balanced salt solution (HBSS). Digested tissue was passed through both gauze and glass wool filters to remove clumped cells. Lymphocytes were isolated by Ficoll-Hypaque (DAKEWE) density-gradient centrifugation. Isolated cells were washed twice in HBSS and resuspended at 2 9 106 cells/ mL in complete RPMI 1640 medium supplemented with 10% heat inactivated foetal calf serum (FCS), 100 U/mL penicillin, 100 lg/mL streptomycin, 2 mM glutamine and 50 lM 2-mercaptoethanol.
Cell surface and intracellular cytokine staining (ICS) Single cell suspensions from liver of control mice and mice infected with S. japonicum were stimulated with 20 ng/mL
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Figure 1 Detection of cd T cells in livers of C57BL/6 mice by immuno-fluorescence and flow cytometry. Female C57BL/6 mice were infected with 40 5 Schistosome japonicum cercariae per mouse. Six weeks after the infection, the mice were sacrificed. (a) cdTCR is identified by green fluorescence (FITC), whereas nuclei are visualized using DAPI staining (blue).cd T cells are found around granulomatous (white arrows). (b) Lymphocytes isolated either from livers of normal controls or infected C57BL/6 mice were stained with anti-cdTCR-FITC mAbs and analyzed by flow cytometry. The cells were gated on cd T lymphocytes via their forward and side scatter properties and their cdTCR expression profile. Representative flow cytometry dot plots were shown. (c) Results represent mean percentages (SD) of cd T cells within hepatic lymphocytes isolated either from liver of normal controls or infected mice. The result represents for four independent experiments. **, P < 001.
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Figure 2 Expression of CD4 and CD8 on hepatic cd T cells. Six weeks after Schistosome Japonicum infection, mice were sacrificed. Cells were stained with anti-cdTCR-FITC, anti-CD4-Percp5.5 and anti-CD8-PE. cd T cells were gated in the FACS analysis (a) The dot plots show staining with PerCP-cy5.5-conjugated anti-CD4 mAbs and PE-conjugated anti-CD8 mAbs of cd T cells. The representative dot plots were shown. (b) Results represent mean percentages (SD) of CD4+cdT cells, CD8+cdT cells, CD4 CD8 cdT cells from normal and infected liver. The results represent for four independent experiments.
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phorbol 12-myristate 13-acetate (PMA) plus 1 lg/mL Ionomycin for 5 h at 37°C under a 5% CO2 atmosphere. Brefeldin A (10 g/mL, Sigma Shanghai, China) was added during the last 4 h of incubation. Cells were washed twice in PBS, fixed with 4% paraformaldehyde, and permeabilized overnight at 4°C in PBS buffer containing 01% saponin (Sigma), 01% BSA and 005% NaN3. Cells were then stained for 30 min at 4°C in the dark with conjugated antibodies specific for the cell surface antigens CD3, cdTCR, CD4, CD8, CD25, CD69 and NKG2D, the cytokines IFN-c, IL-4, IL-9 and IL-17. The expression phenotypes of antibody-labelled lymphocytes were analysed by flow cytometry (BD Calibur and Aria II) and results analyzed with the program FlowJo version 6.0 (Tree Star, Inc. Ashland, OR USA). Isotype-matched controls for cytokines were included in each staining protocol.
Statistics Statistical evaluation of difference between means was performed by unpaired, two-tailed t-tests. P value of 005). CD69 is one of the earliest expressed markers on T cell. Its expression pattern may reflect T-cell activation status early upon stimulation (31). Further, increasing evidence suggests the activation antigen CD69 may be involved in the development or regulation of important T-cell subsets, and may exert an activating function to trigger pro-inflammatory responses (32, 33). CD25, the IL-2 receptor a-chain, is proposed to be a phenotypical marker of regulatory T cells (34) and expressed on both activated T cells and some regulatory T cells (35). Therefore, the upregulation we observed for cdT cells in infected mice (Figure 3) might be either due to activation or increased receptor expression based on the regulatory background of cdT cells. These results indicated that cdT cells were actively involved in immunity respond to infection and the enhanced activation of cdT cells again suggested that these cells were more primed for action at baseline or resting. TCR assembly starts in the endoplasmic reticulum (ER), where cTCR and dTCR subunits are synthesized and CD3ce and TCRff dimers are formed (36). The CD3 dimer then pair with individual clonotypic TCR chains (37). In this study, we found that the liver of normal mice contained a subset of CD3 cdTCR+cells (125 029%), which markedly increased to 847 047% after infection (Figure 4). The invariant CD3 play a role not only in TCR signal transduction but also in TCR assembly and surface expression (38). The role of CD3 cdTCR+ cells may be predictably blocked owing to lack of surface CD3 expression, compared with CD3+cdTCR+ cells. These
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Figure 7 Cytokine profile of hepatic cd T cells. Six weeks after Schistosome japonicum infection, the mice were sacrificed. Single cell suspensions of liver cells were stimulated with PMA, Ionomycinand BFA. Cells were stained with anti-CD3-APC-cy7, anti-cdTCR-FITC, and then intracellular stained with anti-IFN-c-APC, anti-IL-4-PE, anti-IL-9-APC and anti-IL-17-PE for FACS analysis. (a)Intracellular IFN-c, IL-4,IL-9 and IL-17 expression by gated populations of CD3+cdTCR+ cells, CD3 cdTCR+ and CD3+T cells isolated from normal and infected mice, respectively. Flow cytometric analysis from one representative experiment. (b) Statistical analysis of the expression of IFN-c, IL-4, IL-9 and IL-17 by CD3+cdTCR+ cells, CD3 cdTCR+ and CD3+T cells. The results represent for four independent experiments. The error bars indicate SD.*, P < 005; **, P < 001.
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Figure 8 Expression of NKG2D on the CD3+cdTCR+ cells and CD3 cdTCR+ cells. Six weeks after Schistosome japonicum infection, the mice were sacrificed. (a) Single liver cells were stained with anti-CD3 and anti-cdTCR mAbs. Then, cells were gated on CD3+cdTCR+ cells and CD3 cdTCR+ cells population for the analysis of NKG2D in the livers of infected mice by FCM. (b) The percentages of NKG2D on CD3+cdTCR+ cells and CD3 cdTCR+ cells in the hepatic mononuclear cells were calculated from eight independent experiments with similar results. *, P < 005. © 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 658–667
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results indicated that the infection might influence cd cells differentiation and development. A higher level of CD4+cells and CD8+ cells in CD3+cdTCR+ cells was observed after infection, comparing with CD3 cdTCR+ cells (Figure 5). It indicated that the phenotypes of two subsets were different and may modulate cell growth and function. Herein, we have found that the lower expression of activation markers, including CD25 and CD69 on the CD3 cdTCR+ cells, compared with CD3+cdTCR+ cells (Figure 6). Our results showed that the expressions of CD25 and CD69 were significantly higher on the CD3+cdTCR+ cells than CD3 cdTCR+ cells, indicating that CD3+cdTCR+ cells are more activated in this infectious process. We reasoned that as CD69 is one of the earliest expressed markers in T-cell activation, its higher level may mainly reflect CD3+cdTCR+ cells activation status early upon infection. The different activation markers expression of these two subsets might be explained on the basis of surface CD3 expression, suggesting that their activated function was in close contact with CD3 expression. To further explore the potent mechanisms and role of different hepatic cd cells subsets contributing to disease progression, we investigated the cytokine profiles of these cells after infection. Analyses of intracellular cytokines of these cells demonstrated that these infected hepatic CD3+cdTCR+cells revealed a classical inflammatory cytokine profile, with high production of IL-4 and IL-17 (15) after stimulation with PMA and Ionomycin, compared with normal mice (Figure 7). Conversely, no differences of IFN-c, IL-4, IL-9 and IL-17 in hepatic CD3 cdTCR+cells were noted between normal control and infected mice. These results indicated that CD3+cdTCR+cells instead of CD3 cdTCR+cells might play a role during infection. Consistently, previous studies have indicated that cdT cells were capable of producing Th2-type cytokines (39). Particularly, cdT cells were found to be the major source of IL-17 in naive mice, and IL-17 production by cdT cells can be further modulated in various pathological condition (40, 41). Moreover, IL-17 has been shown to be a
crucial regulator of the migration and activation of neutrophils and also secreted by the cdT cells (42, 43). Overall, our findings suggested that CD3+cdTCR+ cells, but not CD3 cdTCR+ cells played important roles in S. japonicum infection. NKG2D, like many other activating receptors, associates with an adaptor molecule to initiate signal transduction and cellular activation (44). NKG2D recognizes different families of ligand, which are distantly related to MHC, including the MHC class I chain related molecule A and B (MICA and MICB), or act as receptors for the UL16 protein (the UL16 binding proteins, ULBPs) (45, 46). NKG2D receptor engagement on T cells functions may as a co-stimulatory signal by amplifying T-cell activation through the TCR (44). Thus, CD3+cdTCR+cells may continue exerting the activity, due to the higher expression of NKG2D as shown in Figure 8 (P < 005). Moreover, it suggested that higher expression of NKG2D might be the mechanistic response to the activation ofCD3+cdTCR+ cells compared with CD3 cdTCR+ cells in this study, but this requires further investigation. In summary, our data suggest that S. japonicum infection could induce cdT cells in the liver of C57BL/6 mice, though the content of CD3 cdTCR+ cells increased significantly, the CD3+cdTCR+ cells, which expressed higher level of CD69, NKG2D, IL-4 and IL-17 seemed to play a more important role during S. japonicum.
ACKNOWLEDGEMENTS This study was supported by a grant from the Natural Science Foundation of China (30901353), Science and Technology Planning Project of Guangzhou City (2011J22007), and College Scientific Research Project in Guangzhou City (2012C117).
DISCLOSURES None.
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