TLR7 Modulated T Cell Response in the Mesenteric Lymph Node of Schistosoma japonicum-Infected C57BL/6 Mice

Toll-like receptors (TLRs) play an important role in regulating immune responses during pathogen infection. However, roles of TLRs on T cells reside in the mesenteric lymph node (MLN) were not be fully elucidated in the course of S. japonicum infection. In this study, T lymphocytes from the mesenteric lymph node (MLN) of S. japonicum-infected mice were isolated and the expression and roles of TLR2, TLR3, TLR4, and TLR7 on both CD4+ and CD8+ T cells were compared. We found that the expression of TLR7 was increased in the MLN cells of S. japonicum-infected mice, particularly in CD4+ and CD8+ T cells (P < 0.05). R848, a TLR7 agonist, could enhance the production of IFN-γ from MLN T cells of infected mice (P < 0.05), especially in CD8+ T cells (P < 0.01). In TLR7 gene knockedout (KO) mice, the S. japonicum infection caused a significant decrease (P < 0.05) of the expression of CD25 and CD69, as well as the production of IFN-γ and IL-4 inducted by PMA plus ionomycin on both CD4+ and CD8+ T cells. Furthermore, the decreased level of IFN-γ and IL-4 in the supernatants of SEA- or SWA-stimulated mesenteric lymphocytes was detected (P < 0.05). Our results indicated that S. japonicum infection could induce the TLR7 expression on T cells in the MLN of C57BL/6 mice, and TLR7 mediates T cell response in the early phase of infection.


Introduction
Schistosomiasis is a chronic, parasitic disease caused by blood flukes with significant morbidity and mortality, especially in vertebrates, including humans [1]. Immunopathological studies have shown that schistosomiasis results predominantly from the evoked host immune response to schistosome eggs and the granulomatous reaction [2]. After infection, schistosomula and its eggs migrate through a variety of tissues, such as the skin, lung [3], liver [4,5], and intestinal and vesical mucosa [6]. Schistosoma eggs must migrate from the mesenteric vessels, across the intestinal wall and into the feces. A vast proportion of eggs fail to leave their definite host, instead becoming lodged within intestinal or hepatic tissue, where they could evoke potentially lifethreatening pathology [7].
The mesenteric lymph node (MLN) is the main draining lymph node in mouse enterocoelia which contains many types of immune cells [8]. MLN has been associated with initiation of immunological responses to bacterial translocation and inflammatory bowel diseases (IBDs) [9]. Moreover, it was reported that MLN CD4 + T lymphocytes could migrate to liver and contribute to nonalcoholic fatty liver disease [10]. Our previous study have found that S. japonicum infection could stimulate the responses of multiple immune cells, including Th cells, NK cells, NKT cells, and γδT cells in the B6 mouse MLN [11,12].
CD4 + Th cells could modulate the immune response by secreting many kinds of cytokines. According to the different cytokine production profiles, divide into different subtypes, such as Th1, Th2, Th9, and Th17 [13]. It was reported that CD4 + Th2 cell is the main effector T cell response to S. japonicum infection by producing produce IL-4, IL-5, and IL-13 [14]. IL-17-secreting Th17 cell was reported playing an important roles in S. japonicum infection inducing liver granuloma damage [4,5]. Th9 cells could influence the progress of S. japonicum infection-induced liver damage, too [15]. IFN-γ and IL-4 were classic Th1 and Th2 cytokines, respectively. IFN-γ could mediate cellular immune response, including the activity of CD8 + cytotoxin T cell and macrophages. On the contrary, IL-4 is the most important cytokine in induced B cell activation and antibody production. IFN-γ and IL-4 were the most important cytokines secreting by Th cell which influence the progress S. japonicum infectioninduced disease [16].
TLRs are the best characterized class of pattern recognition receptors (PRRs) that prevent pathogen invasion by recognizing pathogen-associated molecular patterns (PAMPs), which are highly conserved components derived from bacteria, viruses, fungi, and parasites [17]. Studies show that TLRs are the most important sensors to parasite components during Schistosoma mansoni infection [18,19]. Although TLRs are predominantly expressed in innate immune cells, such as dendritic cells, macrophages, and natural killer (NK) cells [20]. TLRs have also been detected in T cells [21] and were found to be able to modulate the function of T lymphocytes [22]. For example, the report by Lee et al. indicated that TLR2 was constitutively expressed on Listeria-specific memory CD8 + T cells [23]. In addition, Caron et al. reported that effector memory T cells exhibit an enhanced response to TLR activation and are more sensitive to TLR-mediated activation than naive CD4 + T cells [24].
Among all the TLRs identified, TLR7 is an intracellular member of the innate immune receptor that recognizes intracellular single-stranded and double-stranded RNA [25]. It was reported that TLR7 was involved in the progress of autoimmune disease [26], graft-versus-host disease [27], and infectious diseases [28]. For example, TLR7 could be detected on different CD4 + and CD8 + T cell subpopulations from blood of hepatitis C virus infected patients by flow cytometry [29]. Resiquimod (R848), a TLR7 and TLR8 agonist, could not only induce immune response as an adjuvant [30], but also blocks virus replication by inducing the antiviral protein viperin [31]. To date, however, the exact role of the TLR7 in S. japonicum infection remains elusive. In this study, we utilized both in vivo TLR7 gene knockedout (KO) mice and in vitro schistosome worm (SWA)-and egg (SEA)-stimulated mesenteric lymphocytes to investigate the roles of TLR7 on T cells residue in the mesenteric lymph node (MLN) in the course of S. japonicum infection.

Ethics Statement.
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 the Institutional Animal Care and Use Committee of Guangzhou Medical University (2012-11).
2.2. Mice, Parasites, and Infection. Sixty female C57BL/6 mice, 6 to 8 weeks old, weighted 20-25 g, were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China), and TLR7 KO mice were purchased from the Jackson Laboratory (B6.129S1-Tlr7 tm1Fl v/J, strains: 008380). All mice were maintained in a specific pathogenfree microenvironment (SPF) at the Laboratory Animal Centre, Guangzhou Medical University. Mice were fed with standard diet, allowed ad libitum access to food and water and taken care of on a 12 h light-dark cycle. S. japonicum cercariae were shed from naturally infected Oncomelania hupensis snails, which were purchased from Jiangsu Institute of Parasitic Disease (Wuxi, China). There are 3 groups of mice in this study. 40 C57BL/6 mice were divided into normal and infected group randomly, twenty mice per group. 20 C57BL/6 mice in the infected group and 10 TLR7 KO mice (TLR7 KO group) were infected percutaneously with 40 ± 5 cercariae and sacrificed at 6 weeks after infection; 10 uninfec-tedTLR7 KO mice were served as control, too. The animal experiments were performed in strict accordance with the regulations for the Administration of Affairs Concerning Experimental Animals, and all efforts were made to minimize suffering. The bodies of the mice were frozen in -20°C and sent to the Laboratory Animal Centre of Guangzhou Medical University after the experiment. 2.4. SEA and SWA Preparation. SEA and SWA were obtained from the Jiangsu Institute of Parasitic Diseases as previously described [32]. In brief, SEA and SWA were sterile filtered and the endotoxin was removed with polymyxin B agarose beads (Sigma-Aldrich). A Limulus amebocyte lysate assay kit (Lonza, Basel, Switzerland) was used to confirm the removal of the endotoxin from SEA and SWA.
2.5. Isolation of Lymphocytes. At 6 weeks after infection, the mice were sacrificed by cervical dislocation in laboratory, and the mesenteric lymph nodes (MLN) were harvested [11,12]. A 100 μm cell strainer (BD, CA, USA) was used for preparing the single cell suspensions. The isolated cells were washed twice in Hanks' balanced salt solution, stained by 0.4% trypan blue (Guangzhou chemical reagent factory), and counted under a microscope (the rate of ling cell >98%). The cells were resuspended and adjusted to 2 × 10 6 cells/ml in complete RPMI-1640 medium supplemented with 10% heatinactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine, and 50 μm 2-mercaptoethanol.

ELISA Detection of Cytokines.
Single cell suspensions from the normal, infected, and TLR7 KO group were prepared, respectively. Cells were plated in 96-well plates at 4 × 10 5 cells/200 μl medium per well and cultured for 72 h at 37°C with 5% CO 2 in the presence or absence of anti-CD3 mAb (1 μg/ml) plus PAMPs (PGN, 10 μg/ml, Poly I:C 25 μg/ml, LPS 1 μg/ml, or R848 2 μg/ml) or not. The supernatants were collected 72 h later and the released cytokines were measured using mouse ELISA kits for IFN-γ (R&D Systems Inc., Minneapolis, MN, USA) and IL-4 (BD Pharmingen, Franklin Lakes, NJ, USA). ELISAs were performed in accordance with the manufacturer's instructions. The optical density of each well was read at 450 nm using a microplate reader (Model ELX-800; BioTek Instruments Inc., Winooski, VT, USA).

Cell Surface and Intracellular Cytokine Staining (ICS).
For cell surface staining, single cell suspensions from the MLN of the normal group, infected group, and TLR KO group were washed twice in PBS contained 0.5% BSA and then stained for 30 min at 4°C in the dark with conjugated antibodies specific for the cell surface antigens CD3, CD4, CD8 CD25, CD69, TLR2, and TLR4. Cells were washed twice in PBS, fixed with 4% paraformaldehyde, and permeabilized overnight at 4°C in PBS buffer containing 0.1% saponin (Sigma), 0.1% BSA, and 0.05% NaN 3 . The cells were then stained for 30 min at 4°C in the dark with conjugated antibodies specific for TLR3 and TLR7. Stained cells were washed twice and detected by using flow cytometry (Cytoflex, Beckman Coulter, USA) and data were analyzed by the program CytExpert 1.1 (Beckman Coulter, USA).
For intracellular cytokine staining, single cell suspensions from the MLN of control mice and mice infected with S. japonicum were stimulated with TLR ligands (PGN 10 μg/ml, Poly I:C 25 μg/ml, LPS 1 μg/ml, or R848 2 μg/ml) plus 1 μg/ml anti-CD3 for 5 h at 37°C under a 5% CO 2 atmosphere. Brefeldin A (1 μg/ml, Sigma) was added during the last 4 h of incubation. The cells were washed twice in PBS and stained for 30 min at 4°C in the dark with conjugated antibodies specific for the cell surface antigens CD3, CD4, and CD8. The cells were washed twice in PBS, fixed with 4% paraformaldehyde, and permeabilized overnight at 4°C in PBS buffer containing 0.1% saponin (Sigma), 0.1% BSA, and 0.05% NaN 3 . The cells were then stained for 30 min at 4°C in the dark with conjugated antibodies specific for the intracellular IFN-γ and IL-4. Stained cells were washed twice and detected by using flow cytometry (Cytoflex, Beckman Coulter, USA), and data were analyzed by the program CytExpert 1.1 (Beckman Coulter, USA).
2.9. Statistics. Data from each group were analysed using SPSS (v11.0). Statistical evaluation of the difference between means was performed by unpaired, two-tailed Student's t-tests; P < 0:05 was considered to be significant.

Accumulation of CD3 + T Cells in Infected Mesenteric
Lymph Nodes. Six weeks after infection, the mice were sacrificed, and MLNs were harvested. Compared to the normal group, the infected MLN had significantly increased in size (Figure 1(a)). Single mononuclear cell solutions were prepared and stained by trypan blue; the living cells were counted. The average number of cells in nontreated MLN was ð12:88 ± 3:26Þ × 10 6 . This number significantly increased to ð22:54 ± 5:90Þ × 10 6 after 6 weeks of infection (Figure 1(b), P < 0:01).
To investigate whether MLN T cells were involved in the host response to S. japonicum infection, mononuclear cells from normal or infected mouse MLN were stained by fluorescence-labeled anti-CD3 antibody and were detected by FACS (Figure 1(c)). As shown in Figure 1 percentage of CD3 + T cells in infected mouse MLN was 46:76 ± 8:43%, which was lower compared to normal mice (70:9 ± 6:31%, P < 0:01). However, because of the number of MLN mononuclear cells in response to infection dramatically increased, the absolute number of MLN CD3 + T cells was obviously increased after infection (P < 0:05, Figure 1(e)). CD25 and CD69 were classic markers for T cell activation [33]. To detect the degree of activation, the expression of CD25 and CD69 on CD3 + T cells was detected by the cell surface staining. As shown in Figure 1(f), the expression of CD69 on the CD3 + cells after infection (31:87 ± 9:58%) was significantly higher than normal mice (12:26 ± 2:54%, P < 0:05, Figure 1(g)). However, no significant change was found in the population of CD3 + CD25 + T cells (P > 0:05, Figure 1(g)).
3.2. Expression of TLRs in S. Japonicum-Infected Mouse MLN. To explore the expression changes of TLRs in the S. japonicum-infected mouse MLN, we isolated MLN from both normal and infected mice and performed the qRT-PCR. The expression of TLR2, TLR3, TLR4, TLR7, and β-actin genes was detected as described in Materials and Methods. As shown in Figures 2(a) and 2(b), the amount of TLR7 mRNA in infected mice (2:040 ± 0:2062) was higher than that in nontreated mice (1:327 ± 0:1436, P < 0:05). Although there were also changes in the expression of TLR2, TLR3, and TLR4, the difference was not statistically significant (P > 0:05).
Moreover, lymphocytes from MLN were cultured with stimulation of SEA, SWA, or CD3, respectively, with CD28 for 72 hours. The concentration of IFN-γ (Figure 4(e)) and IL-4 (Figure 4

Discussion
In S. japonicum infection, T cells, stimulated mainly by the soluble adult worm antigens and soluble egg antigens of S. japonicum, were believed to play an important role in the infection-induced pathogenic immune response [11,34]. In this study, the bigger size of MLN, which contained a greater number of CD3 + T cells, was found in the S. japonicum-infected B6 mouse. These results suggested that S. japonicum infection could induce strong immune response in intestinal tract. CD25 and CD69 were classic markers for T cell activation [33], though CD4 + CD25 + foxp3 + T cells were served as nature regulatory T cells (Treg) [35], and CD69 was seem to be a marker for tissue resident memory T cells (TRM) [36]. Higher percentages of CD25 and CD69-expressing CD3 + T were found in the infected mouse MLN. It further indicated that CD3 + T lymphocytes might be a component of the immune response during S. japonicum infection, as our previous study reported [12]. TLRs are the most well-described PRRs, which promote both innate defense mechanisms and adaptive immune responses to invasive pathogen infection [37]. Previous studies showed that TLR4 might be involved in the protection against S. japonicum infection [19], and the absence of TLR7 could influence the immune response against S. japonicum infection [19]. In this study, higher expression levels of TLR7 mRNA were found in infected MLN lymphocytes (P < 0:05), which suggested that TLR7 might involved in the infected-induced immune response in the lymph nodes. Recently, Applequist and Mac Leod et al. reported the detection of TLRs expression on T cells [21] and the functional roles of TLRs on the modulation of both CD4 + and CD8 + T lymphocytes [22]. Our FACS results showed that the expressions of TLR2 in CD4 + T cells and TLR7 in CD4 + Three independent experiments (5-6 mice per group) were performed, and one representative result is shown. * P < 0:05, * * P < 0:01, ns P > 0:05. and CD8 + T cells from infected mouse were higher than that in normal T cells (P < 0:05), especially the expression of TLR7 (P < 0:05). This finding suggested that TLR7 might have important effects directly on T cells in response to S. japonicum infection. The binding of TLRs with their specific ligands could initiate a signaling cascade that results in the secretion of cytokines, which subsequently drives an inflammatory response and activates the adaptive immune system [38]. As shown in Figure 3, significant higher levels of IFN-γ and IL-4 could be induced by R848-stimulated lymphocytes from infected mice. It further confirmed that TLR7 played an important role in S. japonicum infection-induced immune response. It implied that many kinds of TLR7-expressing innate immune cells played an important role in this progress. Moreover, the ELISA and FACS results showed that TLR7 could help anti-CD3 antibody inducing IFN-γ releasing and promoting the percentage of IFN-γ + CD8 + T cells during S. japonicum infection. It implied that S. japonicum infection could induce a Th1 immune response and CTL activity through TLR7. Consistent with our results, the levels of Th1 cytokines, TNF-α, and INF-γ in the supernatant of cultured spleen cells from TLR7 -/infected mice were found lower than those of WT mice [19]. Similarly, TLR7 was confirmed to promote Th1 polarization and may thus contribute to the pathogenesis of immune thrombocytopenia [39].

IFN-
In the same time, we found that in the infected mice, the percentage of IFN-γ and IL-4 producing both CD4 + and CD8 + T cells induced by CD3 plus R848 was similar to that induced by CD3 plus CD28. However, significantly higher level of IFN-γ and lower level of IL-4 were induced by CD3 plus R848 in the supernatant of cultured cells from infected mice (P < 0:05). It meant that S. japonicum infection induce TLR7-expressing innate cells in the mesenteric lymph nodes apt to induce Th1 response.
Previous study, however, showed S. japonicum infection could induce a Th2-dominant immune response in the body [40]. To further evaluate the role of TLR7 in S. japonicum infection in the induction of T cell response in MLN, we performed further phenotypic and functional characterization of CD4 + and CD8 + T cells from both TLR7 KO mice and cultured lymphocytes. As showed in Figure 4, results indicated that decreased CD25, CD69, IFN-γ, and IL-4 expressed on CD4 + and CD8 + T cells from MLN of S. japonicum-infected TLR7 KO mice (P < 0:05). In the same time, ELISA results showed that both SEA-and SWA-specific IFN-γ and IL-4 decreased significantly in culture lymphocytes from MLN of S. japonicum-infected TLR7 KO mice (P < 0:05). Together, these findings imply that the effect of TLR7 might only play an early or limited effect on T cell responses in the course of S. japonicum infection.
In conclusion, this study indicated that S. japonicum infection could induce TLR7 expression in both CD4 + and CD8 + T cells of the MLN in C57BL/6 mice, and importantly, the alteration of TLR7 mediates T cell response in the early phase of infection. Further clinic investigations are warranted to define the roles of TLR7 in human host infection of S. japonicum.

Data Availability
The datasets used in the current study are available from the corresponding authors on reasonable request.

Ethical Approval
All protocols for animal use were approved to be appropriate and humane by the institutional animal care and use committee of Guangzhou Medical University (2012-11). Every effort was made to minimize suffering.

Disclosure
We declare that the partial funder did not participate in the design of the study, data collection, analysis, interpretation, and in the manuscript preparation.