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Cellular Immunology 285 (2013) 149–157
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Roles of Th17 cells in pulmonary granulomas induced by Schistosoma japonicum in C57BL/6 mice Dianhui Chen a,1, Hongyan Xie b,1, Xueping Luo a, Xiuxue Yu a, Xiaoying Fu c,d, Haigang Gu e, Changyou Wu c,d, Xiaoping Tang f, Jun Huang a,g,⇑ a
Department of Pathogenic Biology and Immunology, Guangzhou Medical University, 510182 Guangzhou, China Functional Experiment Centre, Guangzhou Medical University, 510182 Guangzhou, China Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, 510080 Guangzhou, China d Key Laboratory of Tropical Disease Control Research of Ministry of Education, Sun Yat-sen University, 510080 Guangzhou, China e Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA f Department of Infectious Diseases, Affiliated No. 8 Guangzhou People’s Hospital, Guangzhou Medical University, 510060 Guangzhou, China g State Key Laboratory of Respiratory Disease, First Affiliated Hospital of Guangzhou Medical College, 151 Yanjiang Rd., 510120 Guangzhou, China b c
a r t i c l e
i n f o
Article history: Received 16 August 2013 Accepted 30 September 2013 Available online 14 October 2013 Keywords: Th17 Schistosoma japonicum Lung Granulomas
a b s t r a c t In schistosomiasis, limited information is available about the role of interleukin-17 (IL-17) in lung, despite the fact that this cytokine plays a crucial role during pro-inflammatory immune responses. In our study, we observed CD4+T cells changed after the infection. Furthermore, ELISA and FACS results revealed that Schistosoma japonicum infection could induce a large amount of IL-17 in mouse pulmonary lymphocytes. IL-17-producing cells, including Th17 cells, CD8+T (Tc) cells, cdT cells and natural killer T cells, was also associated with the development of lung inflammatory diseases. FACS results indicated that Th17 cell was the main source of IL-17 in the infected pulmonary lymphocytes after phorbol-12myristate-13-acetate (PMA) and Ionomycin stimulation. Moreover, FACS results revealed that the percentage of Th17 cells continued to increase as over the course of S. japonicum infection. Additionally, cytokines co-expression results demonstrated that Th17 cells could express more IL-4 and IL-5 than IFNc. Reducing IL-17 activity by using anti-IL-17 ameliorated the damage and decreased infiltration of inflammatory cells in infected C57BL/6 mouse lungs. Collectively, these results suggest Th17 cells is the major IL-17-producing cells population and IL-17 contributes to pulmonary granulomatous inflammatory during the S. japonicum infection. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Schistosomiasis is a worldwide, chronic, parasitic disease caused by blood flukes and causes significant morbidity and mortality especially in developing countries [1]. The main human species are Schistosoma mansoni, S. japonicum and S. haematobium [2] and the S. japonicum is endemic in Asian developing countries, including China [3]. Schistosomula and its eggs migrate through a variety of tissue including skin, lung, liver, intestinal and vesical mucosa [4], which may induce pathological changes and immune ⇑ Corresponding author at: Department of Pathogenic Biology and Immunology, Guangzhou Medical University, 510182 Guangzhou, China. E-mail addresses: [email protected] (D. Chen), [email protected] (H. Xie), [email protected] (X. Luo), [email protected] (X. Yu), xiaoying_ [email protected] (X. Fu), [email protected] (H. Gu), [email protected] (C. Wu), [email protected] (X. Tang), [email protected], [email protected] (J. Huang). 1 These authors share equal first authorship. 0008-8749/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2013.09.008
response. Moreover, granulomatous inflammation against parasite eggs is the pathological hallmarks of schistosome infection [5]. Immune cells, such as T cells, B cells, macrophages and DCs, are involved in the process of granulomatous inflammation, with eosinophils the most frequent cells [6]. Infection of S. japonicum, a multi-cellular parasite which has an extremely diverse repertoire of antigens, induces the production of bulk cytokines that play important roles in the immune response to infection [7]. Interleukin-17 (IL-17) is a major proinflammatory mediator that works through several mechanisms, including the production of chemokines, cytokines, and growth factors [8]. It has been reported to participate in host defense against various types of pathogens [9,10] and thought to be an important cytokine in the immune response against S. japonicum infection [7,11]. In addition to Th17 cells, CD8+T (Tc) cells [12], cdT cells and natural killer (NK) T cells have also been shown to produce IL-17 in the lung [13]. Multiple studies have demonstrated that IL-17 is largely expressed by Th17 cells [8,14], whereas some reports showed that
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IL-17 production was dominated by cdT cells rather than CD4+T cells during some infections, such as Mycobacterium tuberculosis [15] and Escherichia coli [16] infection. However, there is limited information to know which population is the main source of producing IL-17 cells during the S. japonicum infected lung. Therefore, the aim of current study was to observe lymphocyte subpopulations that produce IL-17 in the pathogenic processes of the S. japonicum infected lung and the characteristics of main IL-17-producing cells.
2. Materials and methods 2.1. Mice Female C57BL/6 mice, 6–8 weeks old, were purchased from Zhongshan University Animal Center (Guangzhou, China) and maintained in an animal care facility under pathogen-free conditions. Animal experiments were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (1988.11.1). All protocols for animal use were approved to be appropriate and humane by institutional animal care and use committee of Guangzhou Medical University (2011–44).
2.2. Parasite infection S. japonicum cercariae were shed from naturally infected Oncomelania hupensis snails, which were purchased from Jiangsu Institute of Parasitic Disease (Wuxi, China). Twenty mice were infected percutaneously with 40 ± 5 cercariae. At 4, 5, 6 and 7 weeks post-infection, five mice were randomly chosen and sacrificed. Five pathogen-free mice constituted the control group.
2.3. Antibodies APC-cy7-conjugated anti-mouse CD3 (145-2C11), FITC-conjugated anti-mouse CD3 (17A2), FITC conjugated anti-mouse CD8 (53–6.7), PerCP-cy5.5-conjugated anti-mouse CD4 (RM4-5), FITC-conjugated anti-mouse cdTCR (17A2), PE-cy7-conjugated anti-mouse NK1.1 (PK136), PE-conjugated anti-mouse IL-17 (TC11-18H10), PE-conjugated anti-mouse IL-4 (11B11), APC-conjugated anti-mouse IL-5 (TRFK5), APC-conjugated anti-mouse IL-9 (D9302C12), APC-conjugated anti-mouse IL-10 (JES5-16E3), APCconjugated anti-mouse IFN-c (XMG1.2) and isotype-matched control monoclonal antibodies (X39, G155-178) were purchased from BD/Pharmingen (San Diego, CA). The neutralizing rat anti-mouse IL-17A mAb (TC11-18H10.1) and an isotype-matched rat IgG2a mAb (RTK2758) were purchased from BioLegend.
2.4. Lymphocytes isolation Mice were narcotized and fixed on weeks 0, 4, 5, 6, 7 after infection. The excised lung was cut to small pieces and incubated in 5 ml of digestion buffer (collagenase IV/DNase I mix, Invitrogen Corporation) for 30 min at 37 °C. The digested lung tissue was pressed through 200-gauge stainless-steel mesh, and then was suspended in Hank’s balanced salt solution (HBSS). Lymphocytes were isolated by Ficoll-Hypaque (DAKEWE) density gradient centrifugation. Isolated cells were washed twice in HBSS and resuspended at 2 106 cells/ml in complete RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 lg/ml streptomycin, 2 mM glutamine, and 50 lM 2-mercaptoethanol.
2.5. ELISA detection of cytokines Microtiter plates were coated with anti-CD3 (1) and anti-CD28 (1 lg/ml) and incubated overnight at 4 °C. Single-cell suspensions were cultured in 96-well microtiter plates at 4 105 cells/200 ll medium per well and supernatants were collected 72 h later. Levels of the released cytokines in supernatants were determined by using mouse cytokine multiplex assay kits for IFN-c (BD Pharmingen), IL-4 (BD Biosciences) and IL-17 (R&D Systems). ELISAs were performed in accordance with the manufacturer’s instructions. The optical density of each well was read at 450 nm by using a microplate reader (Model ELX-800, BioTek). 2.6. Detection of cell surface markers and intracellular cytokine expression Single cell suspensions from the lungs of control mice and mice infected with S. japonicum were incubated with 20 ng/ml phorbol12-myristate-13-acetate (PMA) plus 1 lg/ml ionomycin at 37 °C under a 5% CO2 atmosphere. One hour later, cells were treated with brefeldin A (10 mg/ml, Sigma) and incubated for an additional 4 h. Cells were stained with conjugated antibodies specific for the cell surface antigens CD3, CD4, CD8, NK1.1, and cdTCR, respectively. After washing in PBS, cells were fixed with 4% paraformaldehyde, and permeabilized overnight at 4 °C in PBS buffer containing 0.1% saponin (Sigma), 0.1% BSA, and 0.05% NaN3, then stained with conjugated antibodies specific for the cytokines, including IFN-c, IL-4, IL-5, IL-9, IL-10 and IL-17. Antibody-labeled lymphocytes (200,000–300,000 cells per run) were acquired on flow cytometry (BD Calibur and Aria II) and data were analyzed by using CellQuest software (BD Biosciences). Isotype-matched controls for cytokines were included in each staining protocol. 2.7. Neutralizing anti-mouse IL-17A mAb administration and S. japonicum infection 10 mice were randomly assigned in two groups (five mice per group). Each mouse was challenged with 40 cercariae of S. japonicum as described above. Neutralizing rat anti-mouse IL-17A mAb or an isotype-matched rat IgG2a mAb was first administered intraperitoneally 3 weeks after S. japonicum infection (62.5 lg per mouse) at the same dose every third day until 2 days before sacrifice. 2.8. Histology studies Lungs were removed from the mice, perfused three times with 0.01 M phosphate-buffered saline (pH = 7.4), fixed in 10% formalin, embedded in paraffin, and sectioned. Paraffin tissue sections (5 lm) of mice in different groups were stained with hematoxylin and eosin. Briefly, lung tissue sections were immersed in xylene to remove paraffin, then in consecutive ethanol concentrations from 100% to 80% (v/v). The tissue sections were immersed in hematoxylin and eosin. Subsequently, sections were dehydrated in ethanol and immersed in xylene. The sections were then examined by light microscopy under 100 and 400 magnification after standard hematoxylin-eosin (H&E) staining for visualization of cellular changes. 2.9. Statistics Statistical evaluation of differences between means was performed by unpaired two-tailed t tests. P < 0.05 was considered significant.
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3. Results 3.1. The accumulated Th cells in infected lung Firstly, the absolute numbers of mononuclear cell in the total lung was counted under microscopy. The absolute numbers of mononuclear cells (MNC) in the lung showed that there was an apparently increase after infection, from 1.91 ± 0.23 106 to 4.15 ± 0.99 106, which increased up to 2-fold after infection (Fig. 1A, P < 0.05). To investigate whether pulmonary T cells and especially CD4+T cells are involved in host response to S. japonicum infection, the percentages of the CD3+ cells and CD4+T cells in the pulmonary mononuclear cells of normal and infected mice were examined by FACS. Of the normal pulmonary mononuclear cells, CD3+cells and CD4+T cells comprised 24.86 ± 6.71%, 9.39 ± 2.40%, respectively. After infection, the percentage was significantly increased (CD3+: 33.32 ± 8.10%, CD4+T: 12.56 ± 2.70%; P < 0.05, Fig. 1B and C). In addition, considering the dramatically increased number of pulmonary mononuclear cells in response to infection, absolute numbers of pulmonary CD3+ cells and CD4+T cells were significant increased after infection (CD3+: 1.31 ± 0.45 106 vs. 0.50 ± 0.14 106 , P < 0.05; CD4+T: 0.49 ± 0.16 106 vs. 0.19 ± 0.05 106 , P < 0.01, Fig. 1D). 3.2. ELISA detection of cytokines in the supernatant of cultured pulmonary lymphocytes To explore the production of IFN-c, IL-4, IL-17 induced by infection, single mononuclear lung cell suspensions of normal and infected mice were cultured in the presence of anti-CD3 plus anti-CD28. 72 h later, the culture supernatants were collected, and cytokines levels were detected by ELISA. As shown in Fig. 2, we noted that the levels of these cytokines in the supernatant were considerably low in cultures of un-stimulated lymphocytes from normal and infected lungs. However, release of IFN-c, IL-4 and IL-17 from pulmonary MNC was significantly induced in normal and S. japonicum infected group by anti-CD3/anti-CD28 stimulation. Results (Fig. 2) indicated that concentration of IFNc, IL-4, IL-17 in the supernatant of anti-CD3/anti-CD28 stimulated
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lungs from infected mice could reach 4.38 ± 1.69, 2.19 ± 0.73, and 7.47 ± 1.27 ng/ml, respectively, which was obviously higher than that from normal mice (0.83 ± 0.40, 0.04 ± 0.03, and 3.62 ± 2.33 ng/ml, respectively) (⁄P < 0.05, ⁄⁄P < 0.01). The results suggest that the production of INF-c, IL-4 and IL-17 were induced after infection. 3.3. FACS analysis of the proportion of IL-17+ cells in CD3+ cells To further explore the IFN-c, IL-4 and IL-17 expression in CD3+ cells, lymphocytes were isolated from normal and infected C57BL/ 6 mice lungs, and stimulated by PMA and Ionomycin. ICS (intracellular cytokines staining) was done and cells were detected by FACS. The proportions of IFN-c+ CD3+ cells , IL-4+ CD3+ cells and IL-17+ CD3+ in the infected cell population were significantly higher than uninfected cells (IFN-c: 3.84 ± 2.37% vs. 1.35 ± 0.54%, P < 0.01; IL-4: 0.58 ± 0.16% vs. 0.21 ± 0.13%, P < 0.05; IL-17: 1.35 ± 0.53% vs. 0.89 ± 0.42%, P < 0.05, Fig. 3). These results were consistent with the release of cytokines in the supernatant of anti-CD3/anti-CD28 stimulated lung (Fig. 2), suggesting that infection induced the production of IFN-c, IL-4 and IL-17. In accordance with the result of CD3+ cells, the generation of cytokines (IFN-c, IL-4, IL-17) in the CD3 cells from the infected lung (1.57 ± 1.13%, 0.35 ± 0.16%, 0.31 ± 0.23%, respectively) were increased in comparison to the normal lung (0.79 ± 0.42%, 0.21 ± 0.15%, 0.18 ± 0.16%, respectively, Fig. 3B). However, when compared with CD3+ cells, the proportions of IFN-c+CD3 , IL-4+CD3- and IL-17+ CD3 cells were lower in both normal control and infected group (P < 0.05, Fig. 3B). Furthermore, since there was a dramatical raise of lung cells in response to S. japonicum infection, absolute numbers of IFN-c+CD3+, IL-4+CD3+ and IL-17+ CD3+ cells were significantly increased after the infection (P < 0.01, Fig. 3C). 3.4. IL-17 expression in different T cell subsets IL-17-producing T cells, including a population of cdT cells, natural killer T cells, CD8+Tc cells, and CD4+ helper T cells, have also been associated with the development of granulomatous disease [17–19]. Therefore, FACS was performed to investigate whether
Fig. 1. The increased Th cells in infected lung from C57BL/6 mice. Female C57BL/6 mice were infected with 40 ± 5 S. japonicum cercariae per mouse. Six weeks after the infection, the mice were sacrificed. (A) The pulmonary cells were stained with anti-CD3 and anti-CD4 mAbs. CD3+ cells and CD4+CD3+ cells in the lungs of normal and infected mice were analyzed by FCM. (B) Percentage of the CD3+ cells and CD4+CD3+ cells in the pulmonary mononuclear cells were calculated from twelve independent experiments with similar results. (C) Absolute numbers of mononuclear cells (MNC) in normal and infected lungs were counted under a microscope. (D) The absolute number of CD3+ cells and CD4+CD3+ cells in the normal and infected lungs were shown.(⁄P < 0.05, ⁄⁄P < 0.01, the error bars indicate SD).
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Fig. 2. IL-17 was increased in the supernatant of cultured infected lung cells. Single lung cell suspensions of normal and infected mice were prepared and then cultured in the presence of anti-CD3 plus anti-CD28. The culture supernatants were collected after 72 h of incubation for detection of INF-c (A), IL-4 (B) and IL-17 (C) by ELISA. The data are representative of four experiments, each with three or four replicates per group. The asterisks indicate significant differences compared with infected mice: ⁄P < 0.05. The error bars indicate SD.
Fig. 3. Proportions of IL-17+CD3+T cells in infected mice. Female C57BL/6 mice were infected with 40 ± 5 S. japonicum cercariae per mouse. Six weeks after the infection, the mice were sacrificed. Single cell suspensions of lung cells were stimulated with PMA, ionomycin and BFA. Cells were stained with different fluorophore-conjugated antibodies for CD3, IFN-c, IL-4, IL-17 and isotype IgG control antibody for FACS analysis. (A) The percentages of IFN-c, IL-4 and IL-17 expression by CD3-negative population and CD3positive population cells are shown. Flow cytometric analysis from one representative experiment. (B and C) Average percentages and absolute numbers of IFN-c+ CD3+ cells or IFN-c+ CD3 cells populations(upper panels), IL-4+ CD3+ cells or IL-4+ CD3 cells (middle panels), IL-17+ CD3+ cells and IL-17+ CD3 cells populations (lower panels) in normal and infected mice were calculated from FACS data. The asterisks indicate significant differences between normal and infected mices: ⁄P < 0.05.
CD4+ Th, CD8+ Tc, cd T cells, and natural killer T cells are the producing-IL-17 cells during S. japonicum infected lung and which is the main producing-IL-17 cells population. Moreover, the production of IFN-c and IL-4 by these T subsets was also analyzed by intracellular staining. The lated by PMA and Ionomycin. ICS was done and cells were detected by Fpulmonary lymphocytes were isolated from infected C57BL/6 mice and stimuACS. Firstly, cells were gated on the CD3+population, and then the proportion of IFN-c+, IL-4+ and IL-17+ cells were detected in CD4, CD8, NK1.1 and cdTCR positive cells, respectively. As shown in Fig. 4, CD4+T cells, CD8+T cells, NKT cells and cd T cells could secrete IL-17 after non-specific stimulation during S. japonicum infection. Meanwhile, the percentages of IL-17+CD4+T after infection detected by FACS analysis was 2.52%, which was higher than that in CD8+Tc cells (1.23%), NK1.1+NKT cells (1.65%), and cd TCR+ cd T cells (0.87%). Moreover, in parallel with the result of IL-17+ cells, the largest proportion of IFN-c+-producing cells and IL-4+-producing cells were in CD4+T cells, with 15.08% and 9.36% respectively. Thus, we concluded that CD4+Th cells, CD8+Tc cells, NKT cells and cd T cells in infected lung were sources of IL-17 after non-specific stimulation and CD4+ Th cells represented the largest population producing IL-17.
3.5. The kinetics of Th17 cells during S. japonicum infection To observe the change of Th17 cells during the course of S. japonicum infection, lymphocytes were isolated from lungs at 0, 4, 5, 6, 7 weeks after infection and stimulated by PMA and Ionomycin. Cells were gated on CD3+CD4+ cells and the expression of IFNc, IL-4, and IL-17 were detected (Fig. 5A). The results showed that IFN-c+Th1 cells and IL-4+Th2 cells increased on week 4. After that, the percentages slightly decreased on week 5 ,followed by a second increase on week 6, reaching 22.39 ± 3.38%, 22.51 ± 2.23%, respectively, by week 7 (Fig. 5B and C). In contrast to the aforementioned cell subgoups, the percentage of IL-17+cells among CD4+T cells continued to increase, reaching 6.63 ± 1.42% by week 7 (Fig. 5D). These results indicated that infection induced a significant increase of Th17 cells. 3.6. Co-expression of IFN-c, IL-4, IL-5, IL-9, IL-10 in Th17 cells To explore the correlation of Th17 cells to IFN-c, IL-4, IL-5, IL-9 and IL-10 secreted Th cells. The cytokines of CD4+ cells in the lungs of S. Japonicum infected mice were examined by flow cytometry. FACS analysis revealed that in CD4+T lymphocytes the percentage
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Fig. 4. IFN-c, IL-4 and IL-17 expression of different T cell subsets in infected lungs. Female C57BL/6 mice were infected with 40 ± 5 S. japonicum cercariae per mouse weeks after the infection, the mice were sacrificed. Single cell suspensions of lung cells were stimulated with PMA and ionomycin. Cells were stained with different fluorophoreconjugated antibodies for CD3, CD4, CD8, cdTCR, NK1.1, IFN-c, IL-4 and IL-17 for FACS analysis. Numbers in quadrants are percentages of cells in each expression phenotype (n = 3–5 mice per group). A representative of two independent experiments is shown.
Fig. 5. Kinetics of Th1, Th2 and Th17 cells in S. japonicum infection. 20 Female C57BL/6 mice were infected with 40 ± 5 S. japonicum cercariae per mouse. Four mice were randomly chosen and sacrificed at 0 (before infection), 4, 5, 6 or 7 weeks post-infection. Single cell suspensions of lung cells were stimulated with PMA, ionomycin and BFA. Cells were stained with anti-CD3-FITC, anti-CD4-Percp5.5 and then intracellularly stained with PE-conjugated antibodies against IL-17 for FACS analysis. Intracellular IFN-c, IL-4 and IL-17 expression by gated populations of CD3+CD4+ T cells during the course of infection. (A) Representative flow cytometry dot-plots were shown. (B–D) The kinetics of the percentages of Th1, Th2 and Th17 cells from eight independent experiments with similar results. The asterisks indicate significant differences: ⁄P < 0.05; ⁄⁄P < 0.01. The error bars indicate SD.
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Fig. 6. Proportions of IL-17+ Th17 cell expressing IFN-c, IL-4, IL-5, IL-9 and IL-10 in lungs of infected C57BL/6 mice. C57BL/6 mice were infected with 40 ± 5 S. japonicum cercariae per mouse. 6–8 weeks after the infection, the mice were sacrificed. Single cell suspensions of lung cells were stimulated with PMA and ionomycin. Expressing IFN-c, IL-4 IL-5, IL-9 and IL-10 were detected in IL-17+ Th17 cell by FACS analysis. Numbers in quadrants are percentages of cells in each expression phenotype (n = 3–5 mice per group). A representative of two independent experiments is shown.
of IL-4+ IL-17+ (0.45%) and IL-5+ IL-17+ (0.29%) were higher than IFN-c+ IL-17+ cells (0.13%). It indicated that the Th17 cells after S. japonicum infection could secrete high proportion IL-4 and IL-5 compared with other cytokines. Additionally, the proportions of IL-9+ IL-17+ cells (0.01%) and IL-10+Th17 cells (0.06%) were very low, indicating the Th17 cell hardly secret IL-9 and IL-10 (Fig. 6). 3.7. Reduction of pulmonary granulomatous inflammation by anti-IL17 in vivo Different groups of normal, infected, infected/anti-IL-17-treated mice were sacrificed and the differences in lungs among them were observed. Compared to the normal lung, the infected lung dwindled in size, indicating the infected lung become pyknotic and the infection changed the structure of the mouse lung. In contrast, lungs from infected/anti-IL-17-treated mice resembled the control normal lungs, with a smooth surface, indicating antiIL-17 mAb reduced inflammation and granuloma response (Fig. 7A). Since granulomatous inflammation against parasite eggs was the pathological hallmarks of schistosome infection. The sections were stained with haematoxylin and eosin to observe the effects of infection on lung microstructure. As control, uninfected mice showed normal lung parenchyma. After the infection, the pathologic damage and the infiltration of large amounts of inflammatory cells, including eosinophils, macrophages and lymphocytes, were observed in lung (Fig. 7B). Meanwhile, we also observed that the extent of pulmonary granulomatous inflammation around schistosome eggs in the infected/anti-IL-17-treated lungs was smaller than infected lungs, indicating anti-IL-17A neutralizing mAb markedly reduced inflammatory cell number in infected lung. All together, these results suggested that generation of IL-17 during S. japonicum infection may enhance pulmonary granulomatous inflammation, implicating that IL-17 promoted the severity of S. japonicum infected lung pathogenesis.
4. Discussion T cell subsets, especially, CD4 T helper (Th) lymphocytes are essential regulators of immune responses and in inflammatory diseases [20,21]. CD4+T lymphocytes have also been found to be necessary for the development of a parasitic protozoan. Houpt et al.
reported that the depletion of CD4+cells significantly diminished both parasite burden and inflammation in the mouse model of amebic colitis [22]. Previous studies showed CD4+T cells were the critical immune element for normal S. japonicum development, and the absence of CD4+T cells impaired the growth of this parasite in mice [23]. It has been supposed that the main adaptive immune response against schistosomes is mediated by MHC class II-restricted CD4+ T cells [7]. Consistent with previous report, we observed that the percentage and absolute number of CD4+T cells after infection were significantly increased, indicating that CD4+T lymphocytes might be a component of the immune response during S. japonicum infection. It is well known that an effective T-cell response is critical for the development of the granulomatous response and host survival [24]. Moreover, besides Th1 cells and Th2 cells, other reports and researches demonstrated Th17 cells, preferentially secreting cytokine IL-17, might play an important role in mediating inflammation of Schistosomiasis [7,25,26]. IFN-c, IL-4 and IL-17 were the classic cytokines secreted by Th1, Th2, Th17 subset T cell, respectively [25,27,28]. To investigate whether pulmonary IL-17 are involved in host response to S. japonicum infection, we tested IFN-c, IL-4, IL-17 production of mononuclear cells isolated from lungs of normal mice and infected mice by ELISA and FACS. Our results showed that anti-CD3 plus anti-CD28 stimulation could induce high level of IL-17 in the supernatant of cultured cells from S. japonicum infected mouse lungs, along with IFN-c and IL-4 (Fig. 2). Moreover, FACS analysis showed the proportion of IL-17 was significantly increased after infection in both CD3+ cells and CD3 cells compared with the control group. However, when comparing the CD3+ cells with CD3 cells, the data showed that the infection preferentially induced higher levels of IL-17+CD3+ cells. Moreover, similar characteristic was observed in the proportions and absolute numbers of IFN-c- and IL-4-producing cells in the lung following S. japonicum challenge. Our results indicated that IL-17 was induced in infected lung T cells. IL-17 inself and IL-17-producing cells, including Th17 cells, CD8+ Tc cells, cdT cells and natural killer T cells, have also been associated with the development of lung inflammatory diseases [13,29]. IL-17+ CD3+ cell comprised approximately 80% of IL-17+ cells in the infected lung and were significantly higher than CD3 cells (Fig. 3). Therefore, we could conclude that the prominent producer of IL-17 after the infection was CD3+ cells, not CD3 cells.
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Fig. 7. Effect of anti-IL-17 mAb on the histopathological changes in infected C57BL/6 mice lung. 30 female C57BL/6 mice were divided into three groups, normal group, infected group,anti-mouse IL-17 group. The infected group and anti-mouse IL-17 group were infected with 40 ± 5 cercariae of S. japonicum per mouse. For infected and antiIL-17 group, 62.5 lg of control IgG mAb or anti-IL-17 mAb per mouse were administered intraperitoneally every third days, total four times, respectively. Six weeks after the infection, the mice were sacrificed. (A) The gross appearance of the normal lungs,infected lungs anti-mouse IL-17 group lungs. (B) Lungs were flushed with 0.01 M phosphatebuffered saline three times, fixed in 10% formalin, embedded in paraffin, and sectioned. Sections of the lung of normal mice (left panels), infected mice (middle panels) and anti-mouse IL-17 mAb mice (right panels) were examined by H&E staining (original magnification 100 for upper panels and 400 for lower panels). The multicellular granuloma could be observed in the infected group.
Flow cytometry data confirmed that CD4+ Th cells, CD8+ Tc cells, NKT cells and cdT cells were all the source of IL-17 in the infected lung. Of note, Th17 cells were shown to be the initial source of IL17 during pulmonary infections with S. japonicum, which made up about 40% of all IL-17 positive cells (Fig. 4). In accordance with our results, Th17 cells have also been implicated in the pathogenesis of inflammatory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis [30–32], mainly through the recruitment and activation of neutrophils and macrophages [13]. Consistent with the IL-17 expression in CD4+ Th cells, the percentage of IFN-c and IL-4 in CD4+T was the highest among the four different T cell subsets. Hence CD4+ Th cells were the dominant population not only producing IL-17, but also producing IFN-c and IL-4 during the phase of S. japonicum infection. Some studies found that a subset of Tc cells was observed to produce IL-17 (Tc17 cells) in the lung and digestive mucosa [33,34]. Our results were consistent with their results. Findings have demonstrated that the development of Tc17 cells was similar to Th17 cells [34], which might occur during the infection. Besides, NKT and cdT cells
provided a rapid response (4–8 h) to pathogens and promoted a more potent adaptive immune response [33]. The IL-17-producing cells analysis in the infected lung revealed the important role of Th17, which differed with a previous study [7] in which the difference between the percentage of IL-17+ CD4+ T cells and IL-17+cdT + cells in the infected liver was not obvious. One potential reason for this discrepancy is tissue specificity. There may be more APCs in the lung, which can process and present antigens to activate T cells. Therefore, Th17 cells may be easier to act than cdT cells. Further studies should be taken to provide more suggestive evidence. Furthermore, we investigated the dynamic change of IL-17 producing Th17 cells from different stages of S. japonicum infection in mice (Fig. 5). The result demonstrated that the proportion of Th17 cells in the lung increased continuously as over the course of infection indicating that the infection induced the simultaneous generation of Th17 cells, an observation that was consistent with many previous studies [19,20,35]. Whereas the percentages of other cytokines producing cells, including Th1 and Th2 cell, showed a slight decrease at week 5 post-infection, which was different when
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compared to Th17 cells. It might relate to the life cycle of schistosoma [36,37]. In an immune response, CD4+ cells further differentiate into various helper subsets characterized by distinct cytokine profiles. As mentioned before, IFN-c, IL-4 and IL-17 was classical cytokine for Th1, Th2 and Th17 cells, respectively. IL-5 was another Th2 cytokine which was involved in a number of immune responses such as helminth infection and allergy [38]. IL-9 was a typical Th9 cytokines [39,40]. IL-10 was an anti-inflammatory cytokine, which produced by both Th9 and classical Th2 cells [41]. Studies revealed that subsets of cells produced both IFN-c and IL-17 in the inflamed CNS as well as in the eye [42]. And IL-17 was produced by some pro-inflammatory Th1/Th0 cells, not by Th2 cells, during rheumatoid arthritis [43]. In this study, co-expression of IL-17 with IFN-c, IL-4, IL-5, IL-9 and IL-10 were detected by FACS in S. japonicum infected mouse lung CD4+ T cells after PMA plus Ionomycin stimulation. As shown in Fig. 6, not only IFN-c+ IL-17+ cells (0.13%), but also IL-4+ IL-17+ (0.45%) and IL-5+ IL-17+ (0.29%) were detected. It suggested that Th17 cells could secrete Th2 cytokines, or Th2 could produce IL-17. Therefore, we believed that Th17 cells were tightly associated with Th2 cells in the S. japonicum infection. However, there seems little existence of IL-17 +IL-9+ or IL-17 +IL-10+ cells in the CD4+T cells from the S. japonicum infected lung. S. japonicum infection is initiated by cercariae, which burrow into the skin, transform into schistosomula, and then enter the vasculature and migrate to the portal system, where they mature to be adult worms [25]. During the migration, the passage of schistosomula, adult schistosomes and eggs across the lung may cause damage to this organ. In agreement with previous reports [21], the immune response and pathological changes were seen in the lungs of infected S. japonicum compared to uninfected controls in this study (Fig. 7). It is known that IL-17 plays a significant role in the development of large granulomas, possibly released by inflammatory cells such as neutrophils, macrophages, and eosinophils in the granulomas [28,44,45]. We administered anti-IL-17A mAb to infected mice to evaluate the role of IL-17 in the host protective responses against S. japonicum infection. The anti-IL-17 mAb reduced pulmonary granulomatous inflammation induced by infection, indicating that IL-17 level was positively related to the severity of pulmonary pathogenesis during S. japonicum infection (Fig. 7B). Previous studies revealed that a large amount of eggs were carried by the blood flow into liver in S. japonicum infection, after which the liver became fibrotic [25]. However, the sinusoids of liver are too small for the eggs to traverse and most eggs eventually die within the liver. Only a small amount of eggs enter capillaries and lymphatic vessels en route to the lungs. Therefore, we observed that pulmonary fibrosis was not obvious in the S. japonicum infection (data are not shown). In conclusion, our findings demonstrated that Th17 cells was the primary source of IL-17 in lungs after S. japonicum infection and IL-17 level was positively related to the severity of pulmonary pathogenesis during the infection. Moreover, there was a close relationship betweenTh17 and Th2 cells. Disclosures The authors have no financial conflicts of interest. Acknowledgments This work 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).
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Roles of Th17 cells in pulmonary granulomas induced by Schistosoma japonicum in C57BL/6 mice Dianhui Chen a,1, Hongyan Xie b,1, Xueping Luo a, Xiuxue Yu a, Xiaoying Fu c,d, Haigang Gu e, Changyou Wu c,d, Xiaoping Tang f, Jun Huang a,g,⇑ a
Department of Pathogenic Biology and Immunology, Guangzhou Medical University, 510182 Guangzhou, China Functional Experiment Centre, Guangzhou Medical University, 510182 Guangzhou, China Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, 510080 Guangzhou, China d Key Laboratory of Tropical Disease Control Research of Ministry of Education, Sun Yat-sen University, 510080 Guangzhou, China e Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA f Department of Infectious Diseases, Affiliated No. 8 Guangzhou People’s Hospital, Guangzhou Medical University, 510060 Guangzhou, China g State Key Laboratory of Respiratory Disease, First Affiliated Hospital of Guangzhou Medical College, 151 Yanjiang Rd., 510120 Guangzhou, China b c
a r t i c l e
i n f o
Article history: Received 16 August 2013 Accepted 30 September 2013 Available online 14 October 2013 Keywords: Th17 Schistosoma japonicum Lung Granulomas
a b s t r a c t In schistosomiasis, limited information is available about the role of interleukin-17 (IL-17) in lung, despite the fact that this cytokine plays a crucial role during pro-inflammatory immune responses. In our study, we observed CD4+T cells changed after the infection. Furthermore, ELISA and FACS results revealed that Schistosoma japonicum infection could induce a large amount of IL-17 in mouse pulmonary lymphocytes. IL-17-producing cells, including Th17 cells, CD8+T (Tc) cells, cdT cells and natural killer T cells, was also associated with the development of lung inflammatory diseases. FACS results indicated that Th17 cell was the main source of IL-17 in the infected pulmonary lymphocytes after phorbol-12myristate-13-acetate (PMA) and Ionomycin stimulation. Moreover, FACS results revealed that the percentage of Th17 cells continued to increase as over the course of S. japonicum infection. Additionally, cytokines co-expression results demonstrated that Th17 cells could express more IL-4 and IL-5 than IFNc. Reducing IL-17 activity by using anti-IL-17 ameliorated the damage and decreased infiltration of inflammatory cells in infected C57BL/6 mouse lungs. Collectively, these results suggest Th17 cells is the major IL-17-producing cells population and IL-17 contributes to pulmonary granulomatous inflammatory during the S. japonicum infection. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Schistosomiasis is a worldwide, chronic, parasitic disease caused by blood flukes and causes significant morbidity and mortality especially in developing countries [1]. The main human species are Schistosoma mansoni, S. japonicum and S. haematobium [2] and the S. japonicum is endemic in Asian developing countries, including China [3]. Schistosomula and its eggs migrate through a variety of tissue including skin, lung, liver, intestinal and vesical mucosa [4], which may induce pathological changes and immune ⇑ Corresponding author at: Department of Pathogenic Biology and Immunology, Guangzhou Medical University, 510182 Guangzhou, China. E-mail addresses: [email protected] (D. Chen), [email protected] (H. Xie), [email protected] (X. Luo), [email protected] (X. Yu), xiaoying_ [email protected] (X. Fu), [email protected] (H. Gu), [email protected] (C. Wu), [email protected] (X. Tang), [email protected], [email protected] (J. Huang). 1 These authors share equal first authorship. 0008-8749/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2013.09.008
response. Moreover, granulomatous inflammation against parasite eggs is the pathological hallmarks of schistosome infection [5]. Immune cells, such as T cells, B cells, macrophages and DCs, are involved in the process of granulomatous inflammation, with eosinophils the most frequent cells [6]. Infection of S. japonicum, a multi-cellular parasite which has an extremely diverse repertoire of antigens, induces the production of bulk cytokines that play important roles in the immune response to infection [7]. Interleukin-17 (IL-17) is a major proinflammatory mediator that works through several mechanisms, including the production of chemokines, cytokines, and growth factors [8]. It has been reported to participate in host defense against various types of pathogens [9,10] and thought to be an important cytokine in the immune response against S. japonicum infection [7,11]. In addition to Th17 cells, CD8+T (Tc) cells [12], cdT cells and natural killer (NK) T cells have also been shown to produce IL-17 in the lung [13]. Multiple studies have demonstrated that IL-17 is largely expressed by Th17 cells [8,14], whereas some reports showed that
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IL-17 production was dominated by cdT cells rather than CD4+T cells during some infections, such as Mycobacterium tuberculosis [15] and Escherichia coli [16] infection. However, there is limited information to know which population is the main source of producing IL-17 cells during the S. japonicum infected lung. Therefore, the aim of current study was to observe lymphocyte subpopulations that produce IL-17 in the pathogenic processes of the S. japonicum infected lung and the characteristics of main IL-17-producing cells.
2. Materials and methods 2.1. Mice Female C57BL/6 mice, 6–8 weeks old, were purchased from Zhongshan University Animal Center (Guangzhou, China) and maintained in an animal care facility under pathogen-free conditions. Animal experiments were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (1988.11.1). All protocols for animal use were approved to be appropriate and humane by institutional animal care and use committee of Guangzhou Medical University (2011–44).
2.2. Parasite infection S. japonicum cercariae were shed from naturally infected Oncomelania hupensis snails, which were purchased from Jiangsu Institute of Parasitic Disease (Wuxi, China). Twenty mice were infected percutaneously with 40 ± 5 cercariae. At 4, 5, 6 and 7 weeks post-infection, five mice were randomly chosen and sacrificed. Five pathogen-free mice constituted the control group.
2.3. Antibodies APC-cy7-conjugated anti-mouse CD3 (145-2C11), FITC-conjugated anti-mouse CD3 (17A2), FITC conjugated anti-mouse CD8 (53–6.7), PerCP-cy5.5-conjugated anti-mouse CD4 (RM4-5), FITC-conjugated anti-mouse cdTCR (17A2), PE-cy7-conjugated anti-mouse NK1.1 (PK136), PE-conjugated anti-mouse IL-17 (TC11-18H10), PE-conjugated anti-mouse IL-4 (11B11), APC-conjugated anti-mouse IL-5 (TRFK5), APC-conjugated anti-mouse IL-9 (D9302C12), APC-conjugated anti-mouse IL-10 (JES5-16E3), APCconjugated anti-mouse IFN-c (XMG1.2) and isotype-matched control monoclonal antibodies (X39, G155-178) were purchased from BD/Pharmingen (San Diego, CA). The neutralizing rat anti-mouse IL-17A mAb (TC11-18H10.1) and an isotype-matched rat IgG2a mAb (RTK2758) were purchased from BioLegend.
2.4. Lymphocytes isolation Mice were narcotized and fixed on weeks 0, 4, 5, 6, 7 after infection. The excised lung was cut to small pieces and incubated in 5 ml of digestion buffer (collagenase IV/DNase I mix, Invitrogen Corporation) for 30 min at 37 °C. The digested lung tissue was pressed through 200-gauge stainless-steel mesh, and then was suspended in Hank’s balanced salt solution (HBSS). Lymphocytes were isolated by Ficoll-Hypaque (DAKEWE) density gradient centrifugation. Isolated cells were washed twice in HBSS and resuspended at 2 106 cells/ml in complete RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 lg/ml streptomycin, 2 mM glutamine, and 50 lM 2-mercaptoethanol.
2.5. ELISA detection of cytokines Microtiter plates were coated with anti-CD3 (1) and anti-CD28 (1 lg/ml) and incubated overnight at 4 °C. Single-cell suspensions were cultured in 96-well microtiter plates at 4 105 cells/200 ll medium per well and supernatants were collected 72 h later. Levels of the released cytokines in supernatants were determined by using mouse cytokine multiplex assay kits for IFN-c (BD Pharmingen), IL-4 (BD Biosciences) and IL-17 (R&D Systems). ELISAs were performed in accordance with the manufacturer’s instructions. The optical density of each well was read at 450 nm by using a microplate reader (Model ELX-800, BioTek). 2.6. Detection of cell surface markers and intracellular cytokine expression Single cell suspensions from the lungs of control mice and mice infected with S. japonicum were incubated with 20 ng/ml phorbol12-myristate-13-acetate (PMA) plus 1 lg/ml ionomycin at 37 °C under a 5% CO2 atmosphere. One hour later, cells were treated with brefeldin A (10 mg/ml, Sigma) and incubated for an additional 4 h. Cells were stained with conjugated antibodies specific for the cell surface antigens CD3, CD4, CD8, NK1.1, and cdTCR, respectively. After washing in PBS, cells were fixed with 4% paraformaldehyde, and permeabilized overnight at 4 °C in PBS buffer containing 0.1% saponin (Sigma), 0.1% BSA, and 0.05% NaN3, then stained with conjugated antibodies specific for the cytokines, including IFN-c, IL-4, IL-5, IL-9, IL-10 and IL-17. Antibody-labeled lymphocytes (200,000–300,000 cells per run) were acquired on flow cytometry (BD Calibur and Aria II) and data were analyzed by using CellQuest software (BD Biosciences). Isotype-matched controls for cytokines were included in each staining protocol. 2.7. Neutralizing anti-mouse IL-17A mAb administration and S. japonicum infection 10 mice were randomly assigned in two groups (five mice per group). Each mouse was challenged with 40 cercariae of S. japonicum as described above. Neutralizing rat anti-mouse IL-17A mAb or an isotype-matched rat IgG2a mAb was first administered intraperitoneally 3 weeks after S. japonicum infection (62.5 lg per mouse) at the same dose every third day until 2 days before sacrifice. 2.8. Histology studies Lungs were removed from the mice, perfused three times with 0.01 M phosphate-buffered saline (pH = 7.4), fixed in 10% formalin, embedded in paraffin, and sectioned. Paraffin tissue sections (5 lm) of mice in different groups were stained with hematoxylin and eosin. Briefly, lung tissue sections were immersed in xylene to remove paraffin, then in consecutive ethanol concentrations from 100% to 80% (v/v). The tissue sections were immersed in hematoxylin and eosin. Subsequently, sections were dehydrated in ethanol and immersed in xylene. The sections were then examined by light microscopy under 100 and 400 magnification after standard hematoxylin-eosin (H&E) staining for visualization of cellular changes. 2.9. Statistics Statistical evaluation of differences between means was performed by unpaired two-tailed t tests. P < 0.05 was considered significant.
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3. Results 3.1. The accumulated Th cells in infected lung Firstly, the absolute numbers of mononuclear cell in the total lung was counted under microscopy. The absolute numbers of mononuclear cells (MNC) in the lung showed that there was an apparently increase after infection, from 1.91 ± 0.23 106 to 4.15 ± 0.99 106, which increased up to 2-fold after infection (Fig. 1A, P < 0.05). To investigate whether pulmonary T cells and especially CD4+T cells are involved in host response to S. japonicum infection, the percentages of the CD3+ cells and CD4+T cells in the pulmonary mononuclear cells of normal and infected mice were examined by FACS. Of the normal pulmonary mononuclear cells, CD3+cells and CD4+T cells comprised 24.86 ± 6.71%, 9.39 ± 2.40%, respectively. After infection, the percentage was significantly increased (CD3+: 33.32 ± 8.10%, CD4+T: 12.56 ± 2.70%; P < 0.05, Fig. 1B and C). In addition, considering the dramatically increased number of pulmonary mononuclear cells in response to infection, absolute numbers of pulmonary CD3+ cells and CD4+T cells were significant increased after infection (CD3+: 1.31 ± 0.45 106 vs. 0.50 ± 0.14 106 , P < 0.05; CD4+T: 0.49 ± 0.16 106 vs. 0.19 ± 0.05 106 , P < 0.01, Fig. 1D). 3.2. ELISA detection of cytokines in the supernatant of cultured pulmonary lymphocytes To explore the production of IFN-c, IL-4, IL-17 induced by infection, single mononuclear lung cell suspensions of normal and infected mice were cultured in the presence of anti-CD3 plus anti-CD28. 72 h later, the culture supernatants were collected, and cytokines levels were detected by ELISA. As shown in Fig. 2, we noted that the levels of these cytokines in the supernatant were considerably low in cultures of un-stimulated lymphocytes from normal and infected lungs. However, release of IFN-c, IL-4 and IL-17 from pulmonary MNC was significantly induced in normal and S. japonicum infected group by anti-CD3/anti-CD28 stimulation. Results (Fig. 2) indicated that concentration of IFNc, IL-4, IL-17 in the supernatant of anti-CD3/anti-CD28 stimulated
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lungs from infected mice could reach 4.38 ± 1.69, 2.19 ± 0.73, and 7.47 ± 1.27 ng/ml, respectively, which was obviously higher than that from normal mice (0.83 ± 0.40, 0.04 ± 0.03, and 3.62 ± 2.33 ng/ml, respectively) (⁄P < 0.05, ⁄⁄P < 0.01). The results suggest that the production of INF-c, IL-4 and IL-17 were induced after infection. 3.3. FACS analysis of the proportion of IL-17+ cells in CD3+ cells To further explore the IFN-c, IL-4 and IL-17 expression in CD3+ cells, lymphocytes were isolated from normal and infected C57BL/ 6 mice lungs, and stimulated by PMA and Ionomycin. ICS (intracellular cytokines staining) was done and cells were detected by FACS. The proportions of IFN-c+ CD3+ cells , IL-4+ CD3+ cells and IL-17+ CD3+ in the infected cell population were significantly higher than uninfected cells (IFN-c: 3.84 ± 2.37% vs. 1.35 ± 0.54%, P < 0.01; IL-4: 0.58 ± 0.16% vs. 0.21 ± 0.13%, P < 0.05; IL-17: 1.35 ± 0.53% vs. 0.89 ± 0.42%, P < 0.05, Fig. 3). These results were consistent with the release of cytokines in the supernatant of anti-CD3/anti-CD28 stimulated lung (Fig. 2), suggesting that infection induced the production of IFN-c, IL-4 and IL-17. In accordance with the result of CD3+ cells, the generation of cytokines (IFN-c, IL-4, IL-17) in the CD3 cells from the infected lung (1.57 ± 1.13%, 0.35 ± 0.16%, 0.31 ± 0.23%, respectively) were increased in comparison to the normal lung (0.79 ± 0.42%, 0.21 ± 0.15%, 0.18 ± 0.16%, respectively, Fig. 3B). However, when compared with CD3+ cells, the proportions of IFN-c+CD3 , IL-4+CD3- and IL-17+ CD3 cells were lower in both normal control and infected group (P < 0.05, Fig. 3B). Furthermore, since there was a dramatical raise of lung cells in response to S. japonicum infection, absolute numbers of IFN-c+CD3+, IL-4+CD3+ and IL-17+ CD3+ cells were significantly increased after the infection (P < 0.01, Fig. 3C). 3.4. IL-17 expression in different T cell subsets IL-17-producing T cells, including a population of cdT cells, natural killer T cells, CD8+Tc cells, and CD4+ helper T cells, have also been associated with the development of granulomatous disease [17–19]. Therefore, FACS was performed to investigate whether
Fig. 1. The increased Th cells in infected lung from C57BL/6 mice. Female C57BL/6 mice were infected with 40 ± 5 S. japonicum cercariae per mouse. Six weeks after the infection, the mice were sacrificed. (A) The pulmonary cells were stained with anti-CD3 and anti-CD4 mAbs. CD3+ cells and CD4+CD3+ cells in the lungs of normal and infected mice were analyzed by FCM. (B) Percentage of the CD3+ cells and CD4+CD3+ cells in the pulmonary mononuclear cells were calculated from twelve independent experiments with similar results. (C) Absolute numbers of mononuclear cells (MNC) in normal and infected lungs were counted under a microscope. (D) The absolute number of CD3+ cells and CD4+CD3+ cells in the normal and infected lungs were shown.(⁄P < 0.05, ⁄⁄P < 0.01, the error bars indicate SD).
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Fig. 2. IL-17 was increased in the supernatant of cultured infected lung cells. Single lung cell suspensions of normal and infected mice were prepared and then cultured in the presence of anti-CD3 plus anti-CD28. The culture supernatants were collected after 72 h of incubation for detection of INF-c (A), IL-4 (B) and IL-17 (C) by ELISA. The data are representative of four experiments, each with three or four replicates per group. The asterisks indicate significant differences compared with infected mice: ⁄P < 0.05. The error bars indicate SD.
Fig. 3. Proportions of IL-17+CD3+T cells in infected mice. Female C57BL/6 mice were infected with 40 ± 5 S. japonicum cercariae per mouse. Six weeks after the infection, the mice were sacrificed. Single cell suspensions of lung cells were stimulated with PMA, ionomycin and BFA. Cells were stained with different fluorophore-conjugated antibodies for CD3, IFN-c, IL-4, IL-17 and isotype IgG control antibody for FACS analysis. (A) The percentages of IFN-c, IL-4 and IL-17 expression by CD3-negative population and CD3positive population cells are shown. Flow cytometric analysis from one representative experiment. (B and C) Average percentages and absolute numbers of IFN-c+ CD3+ cells or IFN-c+ CD3 cells populations(upper panels), IL-4+ CD3+ cells or IL-4+ CD3 cells (middle panels), IL-17+ CD3+ cells and IL-17+ CD3 cells populations (lower panels) in normal and infected mice were calculated from FACS data. The asterisks indicate significant differences between normal and infected mices: ⁄P < 0.05.
CD4+ Th, CD8+ Tc, cd T cells, and natural killer T cells are the producing-IL-17 cells during S. japonicum infected lung and which is the main producing-IL-17 cells population. Moreover, the production of IFN-c and IL-4 by these T subsets was also analyzed by intracellular staining. The lated by PMA and Ionomycin. ICS was done and cells were detected by Fpulmonary lymphocytes were isolated from infected C57BL/6 mice and stimuACS. Firstly, cells were gated on the CD3+population, and then the proportion of IFN-c+, IL-4+ and IL-17+ cells were detected in CD4, CD8, NK1.1 and cdTCR positive cells, respectively. As shown in Fig. 4, CD4+T cells, CD8+T cells, NKT cells and cd T cells could secrete IL-17 after non-specific stimulation during S. japonicum infection. Meanwhile, the percentages of IL-17+CD4+T after infection detected by FACS analysis was 2.52%, which was higher than that in CD8+Tc cells (1.23%), NK1.1+NKT cells (1.65%), and cd TCR+ cd T cells (0.87%). Moreover, in parallel with the result of IL-17+ cells, the largest proportion of IFN-c+-producing cells and IL-4+-producing cells were in CD4+T cells, with 15.08% and 9.36% respectively. Thus, we concluded that CD4+Th cells, CD8+Tc cells, NKT cells and cd T cells in infected lung were sources of IL-17 after non-specific stimulation and CD4+ Th cells represented the largest population producing IL-17.
3.5. The kinetics of Th17 cells during S. japonicum infection To observe the change of Th17 cells during the course of S. japonicum infection, lymphocytes were isolated from lungs at 0, 4, 5, 6, 7 weeks after infection and stimulated by PMA and Ionomycin. Cells were gated on CD3+CD4+ cells and the expression of IFNc, IL-4, and IL-17 were detected (Fig. 5A). The results showed that IFN-c+Th1 cells and IL-4+Th2 cells increased on week 4. After that, the percentages slightly decreased on week 5 ,followed by a second increase on week 6, reaching 22.39 ± 3.38%, 22.51 ± 2.23%, respectively, by week 7 (Fig. 5B and C). In contrast to the aforementioned cell subgoups, the percentage of IL-17+cells among CD4+T cells continued to increase, reaching 6.63 ± 1.42% by week 7 (Fig. 5D). These results indicated that infection induced a significant increase of Th17 cells. 3.6. Co-expression of IFN-c, IL-4, IL-5, IL-9, IL-10 in Th17 cells To explore the correlation of Th17 cells to IFN-c, IL-4, IL-5, IL-9 and IL-10 secreted Th cells. The cytokines of CD4+ cells in the lungs of S. Japonicum infected mice were examined by flow cytometry. FACS analysis revealed that in CD4+T lymphocytes the percentage
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Fig. 4. IFN-c, IL-4 and IL-17 expression of different T cell subsets in infected lungs. Female C57BL/6 mice were infected with 40 ± 5 S. japonicum cercariae per mouse weeks after the infection, the mice were sacrificed. Single cell suspensions of lung cells were stimulated with PMA and ionomycin. Cells were stained with different fluorophoreconjugated antibodies for CD3, CD4, CD8, cdTCR, NK1.1, IFN-c, IL-4 and IL-17 for FACS analysis. Numbers in quadrants are percentages of cells in each expression phenotype (n = 3–5 mice per group). A representative of two independent experiments is shown.
Fig. 5. Kinetics of Th1, Th2 and Th17 cells in S. japonicum infection. 20 Female C57BL/6 mice were infected with 40 ± 5 S. japonicum cercariae per mouse. Four mice were randomly chosen and sacrificed at 0 (before infection), 4, 5, 6 or 7 weeks post-infection. Single cell suspensions of lung cells were stimulated with PMA, ionomycin and BFA. Cells were stained with anti-CD3-FITC, anti-CD4-Percp5.5 and then intracellularly stained with PE-conjugated antibodies against IL-17 for FACS analysis. Intracellular IFN-c, IL-4 and IL-17 expression by gated populations of CD3+CD4+ T cells during the course of infection. (A) Representative flow cytometry dot-plots were shown. (B–D) The kinetics of the percentages of Th1, Th2 and Th17 cells from eight independent experiments with similar results. The asterisks indicate significant differences: ⁄P < 0.05; ⁄⁄P < 0.01. The error bars indicate SD.
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Fig. 6. Proportions of IL-17+ Th17 cell expressing IFN-c, IL-4, IL-5, IL-9 and IL-10 in lungs of infected C57BL/6 mice. C57BL/6 mice were infected with 40 ± 5 S. japonicum cercariae per mouse. 6–8 weeks after the infection, the mice were sacrificed. Single cell suspensions of lung cells were stimulated with PMA and ionomycin. Expressing IFN-c, IL-4 IL-5, IL-9 and IL-10 were detected in IL-17+ Th17 cell by FACS analysis. Numbers in quadrants are percentages of cells in each expression phenotype (n = 3–5 mice per group). A representative of two independent experiments is shown.
of IL-4+ IL-17+ (0.45%) and IL-5+ IL-17+ (0.29%) were higher than IFN-c+ IL-17+ cells (0.13%). It indicated that the Th17 cells after S. japonicum infection could secrete high proportion IL-4 and IL-5 compared with other cytokines. Additionally, the proportions of IL-9+ IL-17+ cells (0.01%) and IL-10+Th17 cells (0.06%) were very low, indicating the Th17 cell hardly secret IL-9 and IL-10 (Fig. 6). 3.7. Reduction of pulmonary granulomatous inflammation by anti-IL17 in vivo Different groups of normal, infected, infected/anti-IL-17-treated mice were sacrificed and the differences in lungs among them were observed. Compared to the normal lung, the infected lung dwindled in size, indicating the infected lung become pyknotic and the infection changed the structure of the mouse lung. In contrast, lungs from infected/anti-IL-17-treated mice resembled the control normal lungs, with a smooth surface, indicating antiIL-17 mAb reduced inflammation and granuloma response (Fig. 7A). Since granulomatous inflammation against parasite eggs was the pathological hallmarks of schistosome infection. The sections were stained with haematoxylin and eosin to observe the effects of infection on lung microstructure. As control, uninfected mice showed normal lung parenchyma. After the infection, the pathologic damage and the infiltration of large amounts of inflammatory cells, including eosinophils, macrophages and lymphocytes, were observed in lung (Fig. 7B). Meanwhile, we also observed that the extent of pulmonary granulomatous inflammation around schistosome eggs in the infected/anti-IL-17-treated lungs was smaller than infected lungs, indicating anti-IL-17A neutralizing mAb markedly reduced inflammatory cell number in infected lung. All together, these results suggested that generation of IL-17 during S. japonicum infection may enhance pulmonary granulomatous inflammation, implicating that IL-17 promoted the severity of S. japonicum infected lung pathogenesis.
4. Discussion T cell subsets, especially, CD4 T helper (Th) lymphocytes are essential regulators of immune responses and in inflammatory diseases [20,21]. CD4+T lymphocytes have also been found to be necessary for the development of a parasitic protozoan. Houpt et al.
reported that the depletion of CD4+cells significantly diminished both parasite burden and inflammation in the mouse model of amebic colitis [22]. Previous studies showed CD4+T cells were the critical immune element for normal S. japonicum development, and the absence of CD4+T cells impaired the growth of this parasite in mice [23]. It has been supposed that the main adaptive immune response against schistosomes is mediated by MHC class II-restricted CD4+ T cells [7]. Consistent with previous report, we observed that the percentage and absolute number of CD4+T cells after infection were significantly increased, indicating that CD4+T lymphocytes might be a component of the immune response during S. japonicum infection. It is well known that an effective T-cell response is critical for the development of the granulomatous response and host survival [24]. Moreover, besides Th1 cells and Th2 cells, other reports and researches demonstrated Th17 cells, preferentially secreting cytokine IL-17, might play an important role in mediating inflammation of Schistosomiasis [7,25,26]. IFN-c, IL-4 and IL-17 were the classic cytokines secreted by Th1, Th2, Th17 subset T cell, respectively [25,27,28]. To investigate whether pulmonary IL-17 are involved in host response to S. japonicum infection, we tested IFN-c, IL-4, IL-17 production of mononuclear cells isolated from lungs of normal mice and infected mice by ELISA and FACS. Our results showed that anti-CD3 plus anti-CD28 stimulation could induce high level of IL-17 in the supernatant of cultured cells from S. japonicum infected mouse lungs, along with IFN-c and IL-4 (Fig. 2). Moreover, FACS analysis showed the proportion of IL-17 was significantly increased after infection in both CD3+ cells and CD3 cells compared with the control group. However, when comparing the CD3+ cells with CD3 cells, the data showed that the infection preferentially induced higher levels of IL-17+CD3+ cells. Moreover, similar characteristic was observed in the proportions and absolute numbers of IFN-c- and IL-4-producing cells in the lung following S. japonicum challenge. Our results indicated that IL-17 was induced in infected lung T cells. IL-17 inself and IL-17-producing cells, including Th17 cells, CD8+ Tc cells, cdT cells and natural killer T cells, have also been associated with the development of lung inflammatory diseases [13,29]. IL-17+ CD3+ cell comprised approximately 80% of IL-17+ cells in the infected lung and were significantly higher than CD3 cells (Fig. 3). Therefore, we could conclude that the prominent producer of IL-17 after the infection was CD3+ cells, not CD3 cells.
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Fig. 7. Effect of anti-IL-17 mAb on the histopathological changes in infected C57BL/6 mice lung. 30 female C57BL/6 mice were divided into three groups, normal group, infected group,anti-mouse IL-17 group. The infected group and anti-mouse IL-17 group were infected with 40 ± 5 cercariae of S. japonicum per mouse. For infected and antiIL-17 group, 62.5 lg of control IgG mAb or anti-IL-17 mAb per mouse were administered intraperitoneally every third days, total four times, respectively. Six weeks after the infection, the mice were sacrificed. (A) The gross appearance of the normal lungs,infected lungs anti-mouse IL-17 group lungs. (B) Lungs were flushed with 0.01 M phosphatebuffered saline three times, fixed in 10% formalin, embedded in paraffin, and sectioned. Sections of the lung of normal mice (left panels), infected mice (middle panels) and anti-mouse IL-17 mAb mice (right panels) were examined by H&E staining (original magnification 100 for upper panels and 400 for lower panels). The multicellular granuloma could be observed in the infected group.
Flow cytometry data confirmed that CD4+ Th cells, CD8+ Tc cells, NKT cells and cdT cells were all the source of IL-17 in the infected lung. Of note, Th17 cells were shown to be the initial source of IL17 during pulmonary infections with S. japonicum, which made up about 40% of all IL-17 positive cells (Fig. 4). In accordance with our results, Th17 cells have also been implicated in the pathogenesis of inflammatory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis [30–32], mainly through the recruitment and activation of neutrophils and macrophages [13]. Consistent with the IL-17 expression in CD4+ Th cells, the percentage of IFN-c and IL-4 in CD4+T was the highest among the four different T cell subsets. Hence CD4+ Th cells were the dominant population not only producing IL-17, but also producing IFN-c and IL-4 during the phase of S. japonicum infection. Some studies found that a subset of Tc cells was observed to produce IL-17 (Tc17 cells) in the lung and digestive mucosa [33,34]. Our results were consistent with their results. Findings have demonstrated that the development of Tc17 cells was similar to Th17 cells [34], which might occur during the infection. Besides, NKT and cdT cells
provided a rapid response (4–8 h) to pathogens and promoted a more potent adaptive immune response [33]. The IL-17-producing cells analysis in the infected lung revealed the important role of Th17, which differed with a previous study [7] in which the difference between the percentage of IL-17+ CD4+ T cells and IL-17+cdT + cells in the infected liver was not obvious. One potential reason for this discrepancy is tissue specificity. There may be more APCs in the lung, which can process and present antigens to activate T cells. Therefore, Th17 cells may be easier to act than cdT cells. Further studies should be taken to provide more suggestive evidence. Furthermore, we investigated the dynamic change of IL-17 producing Th17 cells from different stages of S. japonicum infection in mice (Fig. 5). The result demonstrated that the proportion of Th17 cells in the lung increased continuously as over the course of infection indicating that the infection induced the simultaneous generation of Th17 cells, an observation that was consistent with many previous studies [19,20,35]. Whereas the percentages of other cytokines producing cells, including Th1 and Th2 cell, showed a slight decrease at week 5 post-infection, which was different when
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compared to Th17 cells. It might relate to the life cycle of schistosoma [36,37]. In an immune response, CD4+ cells further differentiate into various helper subsets characterized by distinct cytokine profiles. As mentioned before, IFN-c, IL-4 and IL-17 was classical cytokine for Th1, Th2 and Th17 cells, respectively. IL-5 was another Th2 cytokine which was involved in a number of immune responses such as helminth infection and allergy [38]. IL-9 was a typical Th9 cytokines [39,40]. IL-10 was an anti-inflammatory cytokine, which produced by both Th9 and classical Th2 cells [41]. Studies revealed that subsets of cells produced both IFN-c and IL-17 in the inflamed CNS as well as in the eye [42]. And IL-17 was produced by some pro-inflammatory Th1/Th0 cells, not by Th2 cells, during rheumatoid arthritis [43]. In this study, co-expression of IL-17 with IFN-c, IL-4, IL-5, IL-9 and IL-10 were detected by FACS in S. japonicum infected mouse lung CD4+ T cells after PMA plus Ionomycin stimulation. As shown in Fig. 6, not only IFN-c+ IL-17+ cells (0.13%), but also IL-4+ IL-17+ (0.45%) and IL-5+ IL-17+ (0.29%) were detected. It suggested that Th17 cells could secrete Th2 cytokines, or Th2 could produce IL-17. Therefore, we believed that Th17 cells were tightly associated with Th2 cells in the S. japonicum infection. However, there seems little existence of IL-17 +IL-9+ or IL-17 +IL-10+ cells in the CD4+T cells from the S. japonicum infected lung. S. japonicum infection is initiated by cercariae, which burrow into the skin, transform into schistosomula, and then enter the vasculature and migrate to the portal system, where they mature to be adult worms [25]. During the migration, the passage of schistosomula, adult schistosomes and eggs across the lung may cause damage to this organ. In agreement with previous reports [21], the immune response and pathological changes were seen in the lungs of infected S. japonicum compared to uninfected controls in this study (Fig. 7). It is known that IL-17 plays a significant role in the development of large granulomas, possibly released by inflammatory cells such as neutrophils, macrophages, and eosinophils in the granulomas [28,44,45]. We administered anti-IL-17A mAb to infected mice to evaluate the role of IL-17 in the host protective responses against S. japonicum infection. The anti-IL-17 mAb reduced pulmonary granulomatous inflammation induced by infection, indicating that IL-17 level was positively related to the severity of pulmonary pathogenesis during S. japonicum infection (Fig. 7B). Previous studies revealed that a large amount of eggs were carried by the blood flow into liver in S. japonicum infection, after which the liver became fibrotic [25]. However, the sinusoids of liver are too small for the eggs to traverse and most eggs eventually die within the liver. Only a small amount of eggs enter capillaries and lymphatic vessels en route to the lungs. Therefore, we observed that pulmonary fibrosis was not obvious in the S. japonicum infection (data are not shown). In conclusion, our findings demonstrated that Th17 cells was the primary source of IL-17 in lungs after S. japonicum infection and IL-17 level was positively related to the severity of pulmonary pathogenesis during the infection. Moreover, there was a close relationship betweenTh17 and Th2 cells. Disclosures The authors have no financial conflicts of interest. Acknowledgments This work 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).
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