TLR3 Modulates the Response of NK Cells against Schistosoma japonicum

Natural killer (NK) cells are classic innate immune cells that play roles in many types of infectious diseases. NK cells possess many kinds of TLRs that allow them to sense and respond to invading pathogens. Our previous study found that NK cells could modulate the immune response induced by Schistosoma japonicum (S. japonicum) in C57BL/6 mice. In the present study, the role of TLRs in the progress of S. japonicum infection was investigated. Results showed that the expression of TLR3 on NK cells increased significantly after S. japonicum infection by using RT-PCR and FACS (P < 0.05). TLR3 agonist (Poly I:C) increased IFN-γ and IL-4 levels in the supernatant of cultured splenocytes and induced a higher percentage of IFN-γ- and IL-4-secreting NK cells from infected mouse splenocytes (P < 0.05). Not only the percentages of MHC II-, CD69-, and NKG2A/C/E-expressing cells but also the percentages of IL-4-, IL-5-, and IL-17-producing cells in TLR3+ NK cells increased significantly after infection (P < 0.05). Moreover, the expression of NKG2A/C/E, NKG2D, MHC II, and CD69 on the surface of splenic NK cells was changed in S. japonicum-infected TLR3−/− (TLR3 KO mice, P < 0.05); the abilities of NK cells in IL-4, IL-5, and IL-17 secretion were decreased too (P < 0.05). These results indicate that TLR3 is the primary molecule which modulates the activation and function of NK cells during the course of S. japonicum infection in C57BL/6 mice.


Introduction
Schistosomiasis japonica is a chronic helminth infection of humans caused by S. japonicum [1,2]. The eggs of S. japonicum are deposited in the liver, lung, and intestinal wall and induce granulomatous inflammation and progressive fibrosis, which are the primary clinical pathological changes. There are many types of cells involved in the fight against invading S. japonicum and its eggs, including Th cells, natural killer (NK) cells, NKT cells, myeloid-derived suppressor cells (MDSCs), and macrophages [3][4][5][6]. Thus, obvious changes could be detected in the immune organs, such as the spleen and local lymph nodes [7,8].
NK cells are innate lymphocytes that respond rapidly to invading pathogens by exerting a direct cytotoxic effect or secreting various cytokines, particularly interferon-gamma (IFN-γ) [7] Recent studies have reported that NK cells are able to survive long enough to take part in the adaptive immune response [9], and NK cells could play an important role in the immune response of host against pathogen and tumor [10]. In parasite infection, both activated and inhibitory receptors such as CD16, CD69, NKG2D, and Ly49a on NK cells were downregulated after a 16-day post-Angiostrongylus cantonensis infection in mice [11]. The decrease of circulating frequency of CD56 + CD161 + NK cells in human visceral leishmaniasis [12] and the downmodulation of effector functions in NK cells upon Toxoplasma gondii infection were both found too [13]. The negative regulatory role of NK cells in S. japonicum egg-induced liver fibrosis was found [14]. Our previous research has found that Th2-like response was induced in the splenic NK cells of S. japonicum-infected mice [7].
Toll-like receptors (TLRs) are evolutionarily conserved molecules that were originally identified in vertebrates on the basis of their homology with Toll [15] mammalian TLRs are a family of at least 12 membrane proteins that trigger innate immune responses. The TLR family members are pattern recognition receptors (PRRs) that recognize lipid, carbohydrate, peptide, and nucleic acid structures collectively, which are expressed widely by different groups of microorganisms [16]. TLR2, TLR3, and TLR4 could response to helminth antigens and modulate the activation of dendritic cells during S. japonicum infection [17,18]. TLR3 was reported to modulate immunopathology during Schistosoma mansoni egg-driven Th2 responses in the lung [19].
NK cells possess many kinds of TLRs that allow them to sense and respond to invading pathogens. It was reported that in healthy controls, TLR2 and TLR4 of NK cells are mainly intracellular expressed which is similar to TLR9 [20]. TLRs could mediate activation of NK cells in bacterial/viral immune responses in mammals [21]. TLR3 and TLR7 activation in uterine NK cells might play important roles in nonobese diabetic (NOD) mice [22]. Immune response modifiers (IRMs) could modulate NK cell function both in vitro and in vivo, and human NK cell activation was controlled in distinct indirect pathways by TLR7 and TLR8 agonists [23]. In this study, the roles of TLRs on NK cells from the S. japonicum-infected mouse spleen were investigated.

Ethics Statement.
Six-to eight-week-old female C57BL/6 mice (wild-type, Laboratory Animal Centre of Sun Yat-sen University, China) were used for the experiments. Experiments were also performed by using TLR3 −/− mice (B6; 129S1-Tlr3 tm1Fl v/J) purchased from Model Animal Resource Information Platform (Nanjing, China; strains: J005217). All mice were maintained in a specific pathogen-free microenvironment at the Laboratory Animal Centre, Guangzhou Medical University. Animal experiments were performed in strict accordance with the regulations and guidelines of the institutional animal care and use committee of Guangzhou Medical University. 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.
2.2. Mice, Parasites, and Infection. The Schistosoma japonicum cercariae used in experiments were obtained from Oncomelania hupensis-infected snails (Jiangsu Institute of Parasitic Disease, China). 30 C57BL/6 mice and 10 TLR3 −/− mice were percutaneously infected with 40 ± 5 cercariae, and an equal number of uninfected normal mice were used as control. All mice were sacrificed 6 weeks after S. japonicum infection as reported before [5].

Preparation of Splenocytes and NK Cells.
Mice were sacrificed after infection for 6 weeks. The spleens were mechanically dissociated and processed through a 100 μm cell strainer (BD Falcon). After erythrocyte was removed by RBC lysis buffer (NH 4 Cl 8.29 g, KHCO 3 1 g, and Na 2 EDTA 37.2 mg per liter), the cells were washed twice in Hanks' balanced salt solution and resuspended in complete RPMI-1640 medium, which contained 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 50 μM 2-mercaptoethanol. Splenocytes were counted under microscope and then diluted to a final concentration of 2 × 10 6 cells/ml for cell culture. For cell staining, splenocytes were stained with fluorescent-labeled anti-mouse CD3 and NK1.1 antibodies for 30 min, followed by washing twice and resuspending in sterile PBS with 0.5 Bull Serum Albumin (BSA). Then, CD3 − NK1.1 + NK cells were sorted by using flow cytometry (MoFlo XDP, Beckman, USA). The purity of NK cells was above 90%, which was used immediately after sorting.
2.7. ELISA Detection. Cells were cultured with different stimulations in 96-well plates at 37°C under a 5% CO 2 atmosphere for 72 h. Supernatant was collected, and the levels of IFN-γ and IL-4 were detected by using ELISA according to the manufacturer's instructions (IFN-γ: 551866, BD; IL-4: 555232, BD). The lower detection limit for IFN-γ is 3.126 pg/ml and 7.8 pg/ml for IL-4. Samples were read at 450 nm by using a microplate reader (Moder ELX-800, BioTek).

Statistical
Analysis. Data were analyzed with SPSS 11.0 software (SPSS Inc., Chicago, IL, USA). Differences in mean values between groups were assessed by using Mann-Whitney U test. One-way ANOVA was used to analyze data of TLR3 KO mouse in Figure 1. P < 0 05 was considered statistically significant.

Increasing TLR3 Expression on Splenic NK Cells in S.
japonicum-Infected Mice. To explore the role of TLRs on NK cells in the progress of S. japonicum infection, the expression of TLRs on NK cells from S. japonicum-infected mice was detected firstly. As shown in Figure 2, the spleens from normal and infected mice were isolated, and single cell suspensions were prepared, and different fluorescent-labeled anti-mouse CD3 and NK1.1 antibodies were used to sort CD3 − NK1.1 + NK cells by FACS. The purity of sorted NK cells was above 95%. Moreover, mRNA was extracted from both splenocytes and splenic NK cells, respectively. cDNAs were synthesized, and qPCR was performed. The results demonstrated that the expressions of TLR2, TLR3, TLR4, and TLR7 mRNA in splenic NK cells significantly increased after S. japonicum infection (P < 0 05). TLR3 mRNA expression increased almost fivefold when compared to normal mice (P < 0 01). Moreover, splenocytes were stained and the expression of TLR2, TLR3, and TLR4 on CD3 − NK1.1 + NK cells was detected (Figure 2(c)). Results (Figure 2(d)) showed that the percentages of TLR2 and TLR3 in splenic NK cells in the infected group were higher than the normal group (TLR2: 12.80 ± 2.442 versus 21.23 ± 2.409, P < 0 05; TLR3: 13.75 ± 1.623% versus 1.558 ± 0.266%, P < 0 01). However, there were no significant differences in the expressions of TLR4 and TLR7 on NK cells between normal and infected mice (P > 0 05).

Poly I:C Promotes IL-4 Secretion from Infected Mouse
Splenic NK Cells. To further explore the roles of TLRs on NK cells, splenocytes from normal and infected mice were cultured with PGN, poly I:C, or LPS, respectively. Supernatants were collected 72 h later, and IFN-γ and IL-4 levels were detected by using ELISA. As shown in Figure 3(a), the IL-4 level in the supernatants of infected mice was significantly higher than normal mice (P < 0 05). After poly I:C stimulation, IL-4 level was significantly higher than the nonstimulated controls (38.76 ± 4.45% versus 23.88 ± 2.33%, P < 0 05).

Discussion
It was reported that NK cells could express many kinds of TLRs, which could play an important role in the progress of NK cell activation in response to bacterial and viral infection [21] and tumors [24]. Here, roles of TLRs on NK cells in the progress of S. japonicum infection were investigated in the spleen of C57BL/6 mice after infection for 6 weeks, which is the acute phase of infection in previous report [6][7][8]. As shown in Figure 2, splenic NK cells expressed a higher level of TLR3 than TLR2, TLR4, TLR7, and TLR9 from S. japonicum-infected mouse (P < 0 05). It suggested that TLR3 might be involved in modulating the activation of S. japonicum infection-induced NK cell.
As we know that S. japonicum infection could induce a Th2-dominant immune response in the body [25]. Our previous research has found that the ability of splenic NK cells in secreting IFN-γ from S. japonicum-infected mouse is decreased while the IL-4 secretion is increased [7]. Here, our results demonstrated that Poly I:C could induce a higher level of IL-4 from infected mouse splenocytes (Figure 3(a), P < 0 05) and the percentage of IL-4 + NK cells in poly I:C cocultured infected splenocytes was extremely higher (P < 0 05). It suggested that S. japonicum infection-induced TLR3 on NK cells was involved in modulating the function of NK cells. However, it was reported that poly I:C is not only a TLR3 ligand but also can interact with RIG-I [26,27], which might influence the expression of IL-4 [28].
On the other hand, it was reported that TLR3 could modulate immunopathology during Schistosoma mansoni egg-driven Th2 responses in the lung [19]. Many types of immune cells express TLR3, such as DC cells, macrophages, NK cells, T cells, and B cells [29][30][31][32]. In this study, the expression of TLR3 was examined on different types of splenocytes from normal and S. japonicum-infected mice. Results showed that TLR3 expression only increased obviously in NK cell from infected mice (P < 0 01, Figure 4(a)). It suggested that NK cells might be the main target cells which could respond to materials through TLR3 in the spleen in the course of S. japonicum infection. Although the engagement of both TLR2 and TLR3 by schistosome eggs is important for the production of inflammatory cytokines and interferon-stimulated genes, such as some chemokines, by DCs [18], no significant differences of TLR3 expressions were observed in pDCs, cDCs, neutrophils, and macrophages (P > 0 05, Figure 4(b)). It might relate to the fact that myeloid cells mainly play function on the site of local inflammation [33].
MHC II, CD69, NKG2A/C/E, and NKG2D are activation-and function-associated molecules which were expressed on the surface of NK cells [34][35][36][37]. Our results demonstrated that the percentages of MHC II-, CD69-, and NKG2A/C/E-expressing cells in TLR3 + NK cells increased significantly after infection (P < 0 05, Figures 5(a) and 5(b)). It indicated that S. japonicum infection could induce the activation of TLR3 + NK cell in the spleen. Moreover, it was reported that NK lineage may develop into cytokine-producing/APCs which affect the priming and progress of systemic autoimmune disease [37]. And many cytokines, such as IFN-γ, IL-4, IL-5, and IL-17, were reported to be secreted by NK cells [7,38]. Our results showed a significant increase in the percentage of IL-4-, IL-5-, and IL-17-producing cells in TLR3 + NK cells population from infected mice (P < 0 05, Figures 5(c) and 5(d)). It indicated that TLR3 might mediate the Th2-like immune response of splenic NK cells in the progress of S. japonicum infection.
Additionally, TLR3 −/− mice were infected with S. japonicum to further evaluate the role of TLR3 on NK cells during infection. Results showed that the expression of NKG2A/C/E, NKG2D, MHC II, and CD69 on NK cells was decreased significantly in S. japonicum-infected TLR3 −/− mice (Figures 1(c) and 1(d)). These results suggested that TLR3 mediated the activation of NK cells during S. japonicum infection. Meanwhile, our results showed the significant decrease of IFN-γ-producing NK cells and increase of IL-4-, IL-5-, and IL-17-producing NK cells in S. japonicum-infected TLR3 −/− mice (P < 0 05, Figures 1(e) and 1(f)). It confirmed that TLR3 could modulate the function of NK cells in the course of S. japonicum infection.
In summary, our study demonstrated that splenic NK cells from S. japonicum-infected C57BL/6 mice expressed a higher level of TLR3, and TLR3-expressing NK cells were more sensitive, both in vitro and in vivo. Our results suggested that TLR3 might be involved in modulating the immune response of NK cells in the course of S. japonicum infection in C57BL/6 mice.

Data Availability
All data used to support the findings of this study are available from the corresponding author upon 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.