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CpG oligodeoxynucleotides with crude parasite antigens reduce worm recovery in Opisthorchis viverrini infected hamsters
Acta Tropica 164 (2016) 395–401
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Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
CpG oligodeoxynucleotides with crude parasite antigens reduce worm recovery in Opisthorchis viverrini infected hamsters Chamraj Kaewraemruaen a,c , Rasana W. Sermswan b,c , Surasakdi Wongratanacheewin a,c,∗ a
Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand c Melioidosis Research Center, Khon Kaen University, Khon Kaen, 40002, Thailand b
a r t i c l e
i n f o
Article history: Received 25 April 2016 Received in revised form 4 October 2016 Accepted 9 October 2016 Available online 11 October 2016 Keywords: CpG ODN Opisthorchis viverrini Protection Hamster T cells IFN-␥
a b s t r a c t Opisthorchis viverrini, a human liver fluke, is still an endemic parasitic infection in Thailand and nearly all countries in Southeast Asia. O. viverrini induces a chronic stage of infection in hamsters. During the first 2 weeks of infection, Th1 inducing cytokine, IL-12, increased but was down regulated in chronic infection. In this study it was found that unmethylated-CpG ODN (oligodeoxynucleotides) 1826 increased hamster mononuclear cell proliferation and stimulated IFN-␥ production in vitro. The IFN-␥ levels in hamster sera were significantly increased in hamsters injected with CpG ODN 1826 alone or plus crude somatic antigens (CSAg). Further investigation using the flow cytometer found that CD4+ T cells and IFN-␥+ CD4+ T cells (Th1-like cells) in the hamster blood were significantly increased. The role of these cells in the protective responses in hamsters was evaluated by challenging with 25 metacercaria and observation for 3 months. The number of worms recovered was significantly reduced in the hamsters injected with CpG ODN 1826 with CSAg, but not in CpG ODN 1826 alone groups when compared to PBS control. The percent of reduction in hamsters against this parasite were 32.95% and 21.49% in the CpG ODN 1826 with CSAg and CpG ODN 1826 alone. This study indicates that CpG ODN 1826 plus parasite antigens elicit a Th1-like response that leads to the enhancement of worm reduction. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Opisthorchis viverrini is a helminth parasite that causes a public health problem in the Southeastern Asia region. Opisthorchiasis is endemic in the northeast (Thaewnongiew et al., 2014) and north (Wongsawad et al., 2012) of Thailand, and other neighboring countries i.e. Laos, Cambodia, and Vietnam (Sithithaworn et al., 2012; Sripa et al., 2011). O. viverrini infection causes chronic infection and can be a risk factor for cholangiocarcinoma (CCA) in patients (Srivatanakul et al., 1991a, 1991b). Humans or animals, for example dogs, cats, pigs, minks, weasels, civets, and house rats are the hosts of this parasite, and the hamster is the suitable animal model for O. viverrini infection (Boonmars et al., 2009). In the chronic stage of O. viverrini infection in hamsters, IL-10 and TGF- were the regulatory cytokines that were found significantly increased. In contrast, the IL-12 responses (Th1 induced cytokine) were highly
∗ Corresponding author at: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand. E-mail address: sura [email protected] (S. Wongratanacheewin). http://dx.doi.org/10.1016/j.actatropica.2016.10.005 0001-706X/© 2016 Elsevier B.V. All rights reserved.
expressed during the first two weeks of infection but were significantly lower in the chronic stage. This indicated that IL-12 responses could be found during the stage of juvenile parasite development (Jittimanee et al., 2007). Similarly, Th1- IFN-␥ levels were found increased in rats infected with a trematode, F. hepatica, on day 7 post infection (Tliba et al., 2002). Moreover, Th2 cytokine (IL-4) and Treg cytokine responses such as IL-10 and TGF-, were found significantly expressed more than Th1 responses in spleens and mesenteric lymph nodes (MLN) in the chronic stage (Jittimanee et al., 2007). It was then hypothesized that since the Th1 cytokine responses were upregulated during the early stage of infection, this cytokine might play a role in the protection against this infection. Moreover, the parasites from infected hamsters have been shown to induce significantly high levels of regulatory cytokines (IL-10 and TGF-) (Jittimanee et al., 2007) and such TGF- levels were stimulated by CSAg. It was found that when hamsters were immunized to protect against O. viverrini infection, such protection was correlated with the reduction of TGF- and IL-10, but not IFN-␥ expression. (Jittimanee et al., 2012). To determine whether the stimulation of Th1 responses could provide increased protection against O. viverrini infection by reduction of worm revovery, the Th1 inducing
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adjuvant, unmethylated-CpG oligodeoxynucleotide (CpG ODN) has been used in this study. The CpG ODN stimulates the immune cells via TLR9 (Toll-like receptor 9) (Hemmi et al., 2000), and induces a strong inflammatory response to activate the immune system. CpG ODN was used as the adjuvant of immune activation in the vaccination of bacterial infections (Liu et al., 2011), viral infections (Davis et al., 1998), and parasitic infections (Aldridge et al., 2010; Cervi et al., 2004; Gupta et al., 2011; Pinzon-Charry et al., 2010; Ribeiro et al., 2009; Sanchez et al., 2011; Sane et al., 2010). BALB/c mice treated with CpG ODN reduced Taenia crassiceps burden (Aldridge et al., 2010). Moreover, CpG ODN was used as an adjuvant to enhance the partial reduction of Schistosoma mansoni (Teixeira de Melo et al., 2013). In this study, the effect of CpG ODN on Th1-like cytokine activation in hamsters was determined. It was demonstrated that CpG ODN could stimulate significant levels of CD4+ T cells producing IFN-␥ in hamster blood. It was demonstrated herein that these cells could increase the resistance to O. viverrini infection in hamsters.
2. Materials and methods 2.1. Metacercarial preparation O. viverrini metacercaria were prepared as previously described (Sirisinha et al., 1984). Briefly, infected cyprinid fish, obtained from a fresh water lake in the endemic area of the northeast Thailand, were homogenized and placed in a digesting solution (0.25% Pepsin, Sigma, MO), normal saline and 15% hydrochloric acid (BDH, Poole England) at a ratio of 1:3 and then incubated at 37 ◦ C for one hour. The digested solution was sieved through 1000, 300, and 106 m meshes. The debris obtained by filtering on 106 m mesh was washed repeatedly many times with normal saline until clear. Cleared sediments were investigated for metacercarial isolation under a dissecting microscope. These metacercaria were stored in normal saline at 4 ◦ C until infection.
2.2. Antigen preparations Crude somatic antigens (CSAg) were prepared from adult O. viverrini worms as described by Wongratanacheewin and colleagues (Wongratanacheewin et al., 1988). The worms, aged approximately 3 months, were collected from bile ducts of O. viverrini infected hamsters and washed with normal saline 3 times. The worms then were transferred into a grinder tube containing complete protease inhibitor cocktails (Roche Diagnostic GmbH, Mannheim Germany) and homogenized at 4 ◦ C. The homogenate was sonicated in an ultrasonic disintegrator set to operate at 1 min intervals for 10 min at 4 ◦ C. The sonicated homogenate was stored overnight at 4 ◦ C and centrifuged at 10,000g for 30 min at 4 ◦ C. The supernatant was transferred into new tubes, and sterilized with filter membranes with a pore size of 0.2 m (Whatman, Buckinghamshire England) and kept at −20 ◦ C. The protein concentration was determined in the supernatant by Bradford’s reagent (Bio-Rad, CA), and protein patterns (Supplement Fig. 1) were checked by the silver staining method.
2.3. Oligodeoxynucleotides Oligodeoxynucleotides (ODNs) were used with the following sequences: CpG ODN 1826, 5 TCCATGACG TTCCTGACGTT3 and non-CpG ODN 2138, 5 TCCATGAGCTTCCTG AGCTT3 . All ODNs were purchased from Invivogen (InvivoGen, CA).
2.4. Animal infections Male Syrian golden hamsters, 6–8 weeks old were orally infected with either 25 (for challenging) or 50 (for CSAg preparation) O. viverrini metacercaria (Mc). They were housed in the animal care unit at Faculty of Medicine, Khon Kaen University on a 12 h light/dark cycle and fed a commercial feed. Uninfected hamsters were used as controls. These O. viverrini infected hamsters were housed and taken care of for 3 months. Animal experiments were approved by the Animal Ethics Committee of Khon Kaen University (AEKKU 7/2558).
2.5. Experimental design for study of CpG ODN stimulation in hamsters Hamsters were divided into five groups (5/group). The animals were injected either with PBS (Group 1), 100 g CpG ODN 1826 (Group 2), 100 g CSAg (Group 3), 100 g non-CpG-ODN 2138 (Group 4) or 100 g CpG ODN 1826 plus 100 g CSAg (Group 5). Blood was collected via lateral saphenous vein from hamsters 2 days before and 2 days after CpG ODN injections. The injections were administrated via the intraperitoneal (i.p.) route. Each hamster’s blood sample was prepared as serum for cytokine detection while whole blood was prepared for cell staining for investigation of T cell populations by flow cytometry.
2.6. CpG ODN stimulation 2.6.1. Cell proliferation in vitro Normal hamster spleens collected from 4 to 6 week naïve hamsters were used for the in vitro study of CpG ODN stimulation. Mononuclear cells were prepared and isolated by ficoll-paque solution (GE Healthcare, Uppsala Sweden) then suspended in RPMI medium supplemented with 10% inactivated fetal bovine serum (FBS) and 100 U/100 g/ml penicillin/streptomycin (Gibco, Life Technologies, Grand Island, NY). The 2 × 106 cells/ml in 96 well plates (Corning Costar, Corning, NY) were cultured either with CpG ODN 1826 at 10 g/ml, 10 g/ml non-CpG ODN 2138 or 10 g/ml CSAg. The 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Enzo, Lausen Switzerland) with 1 g/ml ionomycin (Enzo, Lausen Switzerland) was used as a positive control. The cell suspensions were incubated at 37 ◦ C, 5% CO2 for 72 h. The XTT (sodium 3 -[1-(phenyl-aminocarbonyl)-3, 4-tetrazolium]-bis (4-methoxy6-nitro) benzene sulfonic acid hydrate) (Sigma, MO), that is a tetrazolium salt reagent, was used to measure mitochondrial dehydrogenase activity of viable cells. The XTT solution was added to the final concentration of 20% for at least 4–6 h before harvesting. Finally, cell culture plates were gently shaken and measured using a spectrophotometer at 450 nm.
2.6.2. IFN- production assay Mononuclear cells were prepared from naïve hamster spleens and adjusted to be 2 × 106 cells/ml. These cells were added into 24 well plates (Corning Costar, Corning, NY) containing 1 ml complete medium. Cell suspensions (2 × 106 cells/ml) were cultured either with medium alone, 10 g/ml CpG ODN 1826, 10 g/ml non-CpG ODN 2138, 10 g/ml CSAg or 50 ng/ml PMA plus 1 g/ml ionomycin, and incubated at 37 ◦ C, 5%CO2 . After 72 h incubation, supernatants were harvested for detection of IFN-␥ production levels. Culture supernatants or sera from hamsters were collected for determination of IFN-␥ levels by Enzyme-Linked Immunosorbent Assay (ELISA) (BioLegend, CA). The protocols were performed according to the manufacturer’s recommended protocols.
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2.7. Flow cytometry analysis The 100 l hamster blood samples collected before and 2 days after injection of either with PBS, 100 g CpG ODN 1826, 100 g non-CpG ODN 2138, 100 g CpG ODN 1826 plus 100 g CSAg, or only 100 g CSAg were stained with PE/Cy7-conjugated antimouse CD4 (BioLegend, CA) and incubated for 20 min in the dark at room temperature. The red blood cell lysis buffer (BioLegend, CA) was then added to the stained blood for 10 min at room temperature and washed with cell staining buffer and centrifuged at 300g for 5 min at 20 ◦ C. The stained cells were resuspended in a fixative buffer (BioLegend, CA) and incubated for 20 min in the dark at room temperature and then washed with cell staining buffer. The cells were then washed and resuspended with perm buffer (BioLegend, CA). The PerCP/Cy5.5-conjugated anti-mouse IFN-␥ solution (BioLegend, CA) was added into cell suspension tubes and incubated for 40 min in the dark at room temperature. This antibody was found to be cross-reactive with hamster IFN␥. Cells were washed twice with perm buffer. Finally stained cell pellets were resuspended in 0.5 ml cell staining buffer. Cells were analyzed on a BD FACS Canto II flow cytometer (BD Biosciences, CA) and BD FACSDiva software (BD Bioscience, CA). Isotype control, PerCP/Cy5.5-conjugated rat IgG1, a RTK2071 clone that does not cross-react with hamsters, was included in each intracellular staining protocol.
2.8. Immunization and challenge studies To study the protective effect of CpG ODN 1826 either with or without parasite antigens, hamsters were divided into 3 groups (10/group). The animals were injected either with PBS as control (Group 1), 100 g CpG ODN 1826 (Group 2) or 100 g CpG ODN 1826 plus 100 g CSAg (Group 3). Each injection was administrated intraperitoneally (i.p.) before the infection. On day 2 after injection, all hamsters were then infected orally with Mc (25 Mc/animal). Immunizations were repeated on day 14 post-infections in order to enhance the immunity during the early stage of infections as the parasites need 1 month to develop into adult worms. At 3 months after infection all hamsters were sacrificed and their livers were removed to investigate the number of adult O. viverrini worms recovered.
Fig. 1. CpG ODN induced spleen cell proliferation and IFN-␥ production in vitro. The 2 × 106 cells/ml cells of naïve hamster mononuclear cells obtained from spleens and stimulated either with 10 g/ml CpG ODN 1826, 10 g/ml non-CpG ODN 2138, 10 g/ml CSAg or medium alone. The XTT reagent was added at 4–6 h before determination at 72 h of incubation. The cell proliferation values were determined by OD at 450 nm (A). Cells stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) plus 1 g/ml ionomycin (IO) were used as a positive control. Cell supernatants were harvested and the IFN-␥ levels were detected by ELISA (B). Data represent the means from duplicate cultures, and each bar represents the mean ± SD. All results from non-CpG groups are not significantly different from medium controls.
3. Results 2.9. Calculation of worm recovery Percent worm recovery was calculated from the number of adult worms recovered from livers divided by number of metacercaria that were given at the beginning and multiplied by 100. The percent worm reduction (protection) in each group was calculated from the equation below: %Worm reduction (%Protection) = ((Wn − Wi)/Wn) × 100 Wn = Worm recovery in control group Wi = Worm recovery in injected group
2.10. Statistical analysis The statistical analysis was performed using SPSS 17.0 software. The Student’s paired t-test was used in the comparisons between before and after injections, but one-way ANOVA was used in the comparisons between each group. Data were considered statistically significant if the p value was less than 0.05.
3.1. CpG ODN 1826 induces hamster spleen cell proliferations and IFN- production To determine the effect of CpG ODN 1826 on hamster cell division, mononuclear cells obtained from the spleens of naïve hamsters were used. It was found that CpG ODN 1826 could significantly stimulate mononuclear cell proliferation (p = 0.0001) 72 h after incubation compared to media alone or non-CpG ODN 2138 as the negative control (Fig. 1A). In addition, CSAg also induced cell proliferation but its level was significantly lower (p = 0.001) than those of CpG ODN 1826 (Fig. 1A). To investigate whether CpG ODN 1826 could induce IFN-␥ production in hamsters, the experiments were done using CpG ODN 1826 stimulated naïve mononuclear cells in vitro. High IFN-␥ levels could be significantly detected (p = 0.0001) at 72 h after incubation (Fig. 1B). CpG ODN 1826 could also induce IFN-␥ in vivo. Significant IFN-␥ production in sera either from CpG ODN 1826 (p = 0.0001), CpG ODN 1826 with CSAg (p = 0.0001) or CSAg alone groups (p = 0.0001) was shown when compared with control naïve hamsters (Fig. 2). Although CSAg could stimulate the increased IFN-␥ level, its level was significantly lower than those of CpG ODN 1826 alone (p = 0.001) or CpG ODN 1826 with CSAg (p = 0.0001) (Fig. 2). The CSAg did not
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Fig. 2. CpG ODN induced IFN-␥ levels in hamsters. Blood was collected from all hamsters before, and after injection either with PBS, 100 g CpG ODN 1826, 100 g non-CpG ODN 2138, 100 g CpG ODN 1826 plus 100 g CSAg, or 100 g CSAg only. The IFN-␥ levels were detected in hamster sera by ELISA and calculated for statistical analysis. Data represent the means from duplicate experiments, and each bar represents the mean ± SD. The IFN-␥ levels in CpG ODN 1826, CpG ODN 1826 plus CSAg, and CSAg injected hamsters were significantly different from PBS and non-CpG ODN 2138 injected groups. The IFN-␥ levels in the non-CpG group were not significantly different from PBS controls as shown.
Fig. 4. Quantification of CD4+ IFN-␥ producing T cells from hamster blood. Blood was collected from hamsters similar to those in Fig. 3. Each hamster’s blood was stained with anti-mouse PE/Cy7 conjugated CD4 and anti-mouse PerCP/Cy5.5 conjugated IFN-␥. The percent of IFN-␥+ CD4+ T cells was investigated by flow cytometer. The profiles of IFN-␥+ CD4+ T cells were detected in hamster blood on day 2 after the injection. The percent of IFN-␥+ CD4+ T cells in hamster blood were compared before as shown in bar graph. Data represent the means from duplicate experiments, and each bar represents the mean ± SD. The percent of IFN-␥+ CD4+ T cells in hamster blood injected either with CpG ODN 1826, CpG ODN 1826 plus CSAg or CSAg alone were significantly (p < 0.05) different from PBS or non-CpG ODN 2138 injected groups. The IFN-␥+ CD4+ T cells in hamster blood from non-CpG groups are not significantly different from PBS control.
duction and CD4+ T cells, the numbers of IFN-␥+ CD4+ T cells in CpG ODN with CSAg were not significantly (p = 0.799) different from CpG ODN alone (Fig. 4 and Supplement Fig. 3). 3.3. CpG ODN with CSAg significantly stimulates worm reduction
Fig. 3. Quantification of CD4+ T cells from hamster blood. Blood was collected from hamsters after injection either with PBS, 100 g CpG ODN 1826, 100 g non-CpG ODN 2138, 100 g CpG ODN 1826 plus 100 g CSAg, or only 100 g CSAg. Each hamster blood sample was stained with anti-mouse PE/Cy7 conjugated CD4 and the percent of CD4+ T cells were investigated by flow cytometer. The profiles of CD4+ T cells in hamster blood on day 2 after injection were compared as shown in the bar graph. Data represent the mean from duplicate experiments, and each bar represents the mean ± SD. The percent of CD4+ T cells in hamster blood injected either with CpG ODN 1826, CpG ODN 1826 plus CSAg or CSAg alone were significantly (p < 0.05) different from PBS or non-CpG ODN 2138 injected groups. The percent of CD4+ T cells in hamster blood from non-CpG group were not significantly different from PBS control as shown.
significantly (p = 0.879) enhance the IFN-␥ stimulation when used with CpG ODN 1826 when compared to CpG ODN alone (Fig. 2). 3.2. CpG ODN 1826 stimulates IFN- + CD4+ T cell in hamsters The CD4+ T cell populations were significantly increased in hamster blood either from CpG ODN 1826 (p = 0.0001), CpG ODN 1826 with CSAg (p = 0.0001) or CSAg alone (p = 0.0001) groups when compared to non-CpG ODN or PBS controls (Fig. 3 and Supplement Fig. 2). There were no significant differences (p = 0.817) of CD4+ T cells between CpG ODN 1826 alone and CpG ODN 1826 with CSAg in the injected hamster blood groups (Fig. 3 and Supplement Fig. 2). IFN-␥+ CD4+ T cells were found significantly increased (p = 0.0001) in hamster blood (Fig. 4 and Supplement Fig. 3) similar to those found in CD4+ T cells (Fig. 3) or IFN-␥ levels (Fig. 2). Although CpG ODN 1826 with or without CSAg was able to activate the IFN-␥ pro-
The number of worms recovered were significantly reduced (p = 0.0001) only in the hamsters injected with CpG ODN 1826 with CSAg, but not in CpG ODN 1826 alone compared to the PBS group (Fig. 5A). The percents of reduction in hamsters against this parasite were 32.95% and 21.49% in the CpG ODN 1826 with CSAg and CpG ODN 1826 alone groups (Fig. 5B). Although, CpG ODN 1826 alone did not give a significant (p = 0.154) worm reduction from control, the reduction of worm numbers was significantly (p = 0.006) found in CpG ODN 1826 with the CSAg group. In addition, there were no significant differences (p = 0.203) between CpG ODN 1826 alone and CpG ODN 1826 plus CSAg (Figs. 5B). 4. Discussion Several attempts have been made in the past to describe the protective immunity against O. viverrini infection. The protective responses to O. viverrini infection in previous reports were not successful enough to clear this worm from infected hamsters (Jittimanee et al., 2012; Papatpremsiri et al., 2016; Sirisinha et al., 1983a,b; Sirisinha and Wongratanacheewin, 1986). In an early stage of this parasitic infection, Th1 inducing cytokine responses were found but decreased during the progression of chronic infection. The chronic stage of O. viverrini infection showed the high expression of IL-4 and regulatory cytokines (IL-10 and TGF-) (Jittimanee et al., 2007). This means that the adult stage development of this parasite may modulate the immune responses via down regulation of the Th1 cytokine responses. The parasitic molecules with such functions, however, have never been identified. If Th1 responses were elicited in this parasitic infection, they might enhance activation of immune responses to clear or reduce worm numbers. In addition, CpG ODN was used as a protective adjuvant for treatment of other parasites (Sane et al., 2010). Herein,
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Fig. 5. Worm recoveries from O. viverrini infected hamsters with or without CpG ODN 1826 pretreatment. All hamsters were injected either with PBS, 100 g CpG ODN 1826, 100 g CpG ODN 1826 plus 100 g CSAg 2 days before infection and on 14 days post-infection intraperitoneally (i.p). Each hamster group (10/group) was then orally infected with 25 metacercaria per hamster, and worm recoveries were determined at 3 months post-infection. The percentages of worm recovery were calculated from O. viverrini infected hamsters (A), and the percentages of worm reduction in hamsters (B). Each bar represents the mean ± SD.
it was shown that CpG ODN 1826 stimulated IFN-␥ production in hamsters. Although the numbers of CD4+ T cells and IFN-␥ levels were the same in animals that received CpG ODN alone and animals that received CpG ODN + CSAg; however, IFN-␥+ CD4+ T cells in the CpG ODN + antigen group were higher (Fig. 4). This might lead to a greater worm reduction in these animals (Fig. 5). The IFN-␥ found in IFN-␥+ CD4+ T cell (Th1-like) responses therefore conferred the protective immune responses by reduction of worms in challenged animals. The hamster models are the most common animal model and a suitable model for O. viverrini infection as they give similar pathologies and chronic inflammation in bile ducts, around the liver area and can develop to be cholangiocarcinoma similar to humans (Tansurat, 1971; Bhamarapravati et al., 1978; Koompirochana et al., 1978; Harinasuta et al., 1984; Riganti et al., 1989; Sripa and Kaewkes, 2002; Sripa et al., 2003). The size of hamster liver is not a reason for the numbers of worm recovery because the liver in the rat is larger but there are no adult worn parasites found in this species (Boonmars et al., 2009). The reason for the hamster as the best model of O. viverrini infection may be the presence of the gall bladder which is not found in rat (Boonmars et al., 2009). The immune responses against O. viverrini infections were therefore tested in the hamster as the animal model for antibody, cell mediated immune responses and protective approaches (Flavell et al., 1980; Flavell, 1982; Sirisinha et al., 1983a,b; Wongratanacheewin et al., 1987; Jittimanee et al., 2007, 2012). The strategies for protection against O. viverrini infection in hamsters were designed and
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tested (Jittimanee et al., 2012). The Th1 stimulator adjuvant, CFA, a complete Freund’s adjuvant agent, increased partial protection to O. viverrini infection of hamsters (Jittimanee et al., 2012). CFA enhanced the resistance to O. viverrini infection, and could activate IL-12, a pro-inflammatory cytokine, and increased Th1 responses (Shibaki and Katz, 2002). Alternatively, CpG ODN 1826 as a Th1 stimulator was used to treat the hamster model against parasitic infections. The CpG ODN 1826 was used as a murine immune stimulation agent. It was found in this study that it could activate immune stimulation in the hamster similar to that found in mouse (Sane et al., 2010). Results in the present study demonstrated that CpG ODN 1826 could stimulate significant mononuclear cell proliferation in vitro (Fig. 1A). This agent could also increase the phagocytic index in hamsters when compared to untreated controls (Sane et al., 2010). In addition, rat spleen cells were also activated by this CpG ODN (Cervi et al., 2004). This indicated that murine CpG ODN could be used in these current hamster experiments. The use of hamsters in various studies in infectious diseases is limited by available monoclonal antibodies (mAb) for this model. Recently, some anti-mouse mAbs were reported to cross react with hamsters such as anti-mouse CD4 mAb (GK 1.5 clone) (Dondji et al., 2010; Hammerbeck and Hooper, 2011). This mAb clone was used in a T cell study in the hamster model of hanta virus diseases (Hammerbeck and Hooper, 2011). Moreover, this specific murine CD4 mAb was documented in hamsters infected with hook worms (Dondji et al., 2010). Other anti-mouse mAbs from the R&D systems company, such as TGF- and IL-10 were reported to cross react with hamsters (Zivcec et al., 2011). The present study compared the IFN-␥ amino acid sequences between mouse and hamster and found that they gave 56% identity. It was then tested in the preliminary study with anti-mouse IFN-␥ from BioLegend. Results showed this mAb could cross-react with hamster IFN-␥ in detection by both ELISA (Fig. 2) and flow cytometry (Figs. 4). Those data and the results herein could then be used to design the present hamster experiments. The mechanism of CpG ODN activates immune cells via internalization. This agent interacts with TLR9 that is located in the endocytic vesicle. The results within cellular activation involve a signaling cascade process through the myeloid differentiation primary gene 88 (MyD88) (Klinman, 2004). B cells and dendritic cells express TLR9 and responded to CpG ODN (Davis et al., 1998; Hemmi et al., 2000; Klinman, 2004). The indirect activation by CpG ODN can provide an immune stimulatory cascade to stimulate monocyte/macrophage cells or NK or T cells (Klinman et al., 1996; Stacey et al., 1996; Sun et al., 1998). These effects give the results of Th1biased cytokine secreting such as TNF-␣, IFN-␥ and IL-12 (Ballas et al., 1996; Klinman et al., 1996). In this study, it was found that the TLR9 protein sequence similarity between hamster and mouse was 85%, and the murine TLR9 activator or CpG ODN to activate hamster immune response was used. It might therefore activate hamster immune cells like mouse immune cells to stimulate Th1 cytokine responses. The current results found this agent could elicit CpG ODN in hamster mononuclear cells to produce significant IFN␥ levels in 72 h (Fig. 1B) similar to the IFN-␥ production that was found in the experiment of rat spleen cells treated with CpG ODN for 72 h (Cervi et al., 2004). This result showed that the Th1 cytokine response was activated by CpG ODN administration and also found significant IFN-␥ production was activated on hamster mononuclear cells by CSAg (Figs. 1B). The CpG ODN activation was also done in vivo and it was found that IFN-␥ levels were induced (Fig. 2). Although CSAg could significantly induce IFN-␥ production in vivo, it was lower than CpG ODN 1826 stimulation (Fig. 2). Further investigations in the present study found that CD4+ T cells (Fig. 3) and IFN-␥+ CD4+ T cells (Th1-like) in the blood of hamsters were stimulated by CpG ODN 1826 or CpG ODN 1826 with CSAg or only CSAg injections (Fig. 4). These results were related to the increased IFN-
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␥ levels in CpG ODN 1826 or CSAg treated-hamster sera (Fig. 2). This indicated that the CpG ODN mechanism in vivo could induce a significant increase in IFN-␥ levels from Th1-like cells in hamster blood. IFN-␥ and Th1-like cells in hamsters were increased by CpG ODN 1826 stimulation both in vitro and in vivo. It was then hypothesized that this CpG ODN might stimulate Th1 activation and help to eliminate the worms in hamsters. Although CpG ODN 1826 alone treated hamsters could significantly stimulate Th1-like responses in hamster blood (Fig. 4), this agent alone did not provide significant reduction of worm recovery (Fig. 5). In contrast, CpG ODN 1826 with CSAg could significantly protect hamsters against infection by increasing worm reductions (Fig. 5B). This might be due to the enhancement of CSAg on CpG ODN to increase IFN-␥ levels (Fig. 2) from IFN-␥+ CD4+ Th1-like cells (Fig. 4). The antigens are important to stimulate adaptive immunity as CpG ODN only stimulates innate immunity. The combination of CSAg and CpG ODN may shift the adaptive immunity to be more Th1-like using CpG ODN as an adjuvant leading to more protection. The CSAg alone, however, was shown to stimulate high levels of TGF- and inhibition of cell proliferation (Jittimanee et al., 2012) thus making fewer IFN-␥+ CD4+ cells. These findings were similar to the significant reduction of parasite burden by CpG ODN that could be found in mice infected with T. crassiceps (Aldridge et al., 2010) and in rats infected with F. hepatica (Cervi et al., 2004). Th1 cytokines were shown to be involved in the reduction of worm recovery and egg production when the CpG ODN formula vaccine was used against S. mansoni in mice (Ricciardi et al., 2015). Recently, mice immunized with the CpG ODN-Cathepsin B recombinant protein induced high IgG2c titers and reduced S. mansoni worm numbers (Ricciardi et al., 2015). Also, mice immunized with the tegument-CpG ODN combination gave high IgG2c titers (Teixeira de Melo et al., 2013). It was shown that antibody titers might be due to neutralization of the worms via antibody mediation by an antibody dependent cell mediated cytotoxicity (ADCC) mechanism. NK cells might modulate and play a role via the ADCC mechanism to eliminate worms in schistosomiasis. In a previous report, this mechanism was a key mediator providing protection against schistosomiasis (Comin et al., 2008). In this study it was found that IFN-␥ and Th1-like cells were induced by CpG ODN and might have activated other immune cells that are involved in parasite elimination. In conclusion, the CpG ODN plus this parasite antigen could elicit the protective Th1-like responses and Th1 cytokine responses in hamsters leading to increased partial protection. CpG ODN might be an alternative prophylactic agent or adjuvant in the protection of this parasitic infection.
Conflict of interests None declared
Ethical approval Animal experiments were approved by the Animal Ethics Committee of Khon Kaen University, Thailand (AEKKU 7/2558).
Acknowledgements We thank the grant of the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Health Cluster (ShePGMS), Khon Kaen University for supporting the scholarship during Ph.D study. This work is partially supported by Faculty of Medicine, Khon Kaen University and Melioidosis Research Center, Khon Kaen
University. We would like to acknowledge Prof. James A. Will for editing the MS via Publication Clinic KKU, Thailand. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actatropica.2016. 10.005. References Aldridge, J.R., Johnson, E.C., Kuhn, R.E., 2010. CpG stimulates protective immunity in Balb/Cj mice infected with larval Taenia crassiceps. J. Parasitol. 96, 920–928. Ballas, Z.K., Rasmussen, W.L., Krieg, A.M., 1996. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J. Immunol. 157, 1840–1845. Bhamarapravati, N., Thammavit, W., Vajrasthira, S., 1978. Liver changes in hamsters infected with a liver fluke of man, Opisthorchis viverrini. Am. J. Trop. Med. Hyg. 27, 787–794. Boonmars, T., Boonjaraspinyo, S., Kaewsamut, B., 2009. Animal models for Opisthorchis viverrini infection. Parasitol. Res. 104, 701–703. 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Opisthorchiasis: a clinicopathologic study of 154 autopsy cases. Southeast Asian J. Trop. Med. Public Health 9, 60–64. Liu, Y., Luo, X., Yang, C., Yu, S., Xu, H., 2011. Three CpG oligodeoxynucleotide classesdifferentially enhance antigen-specific humoral and cellular immune responses in mice. Vaccine 29, 5778–5784. Papatpremsiri, A., Junpue, P., Loukas, A., Brindley, P.J., Bethony, J.M., Sripa, B., Laha, T., 2016. Immunization and challenge shown by hamsters infected with Opisthorchisviverrini following exposure to gamma-irradiated metacercariae of this carcinogenic liver fluke. J. Helminthol. 90, 39–47. Pinzon-Charry, A., McPhun, V., Kienzle, V., Hirunpetcharat, C., Engwerda, C., McCarthy, J., Good, M.F., 2010. Low doses of killed parasite in CpG elicit vigorous CD4+ T cellresponses against blood-stage malaria in mice. J. Clin. Invest. 120, 2967–2978. Ribeiro, D.P., Freitas, M.M., Cardoso, M.R., Pajuaba, A.C., Silva, N.M., Mineo, T.W., Silva, J.S., Mineo, J.R., Silva, D.A., 2009. 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CpG oligodeoxynucleotides with crude parasite antigens reduce worm recovery in Opisthorchis viverrini infected hamsters Chamraj Kaewraemruaen a,c , Rasana W. Sermswan b,c , Surasakdi Wongratanacheewin a,c,∗ a
Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand c Melioidosis Research Center, Khon Kaen University, Khon Kaen, 40002, Thailand b
a r t i c l e
i n f o
Article history: Received 25 April 2016 Received in revised form 4 October 2016 Accepted 9 October 2016 Available online 11 October 2016 Keywords: CpG ODN Opisthorchis viverrini Protection Hamster T cells IFN-␥
a b s t r a c t Opisthorchis viverrini, a human liver fluke, is still an endemic parasitic infection in Thailand and nearly all countries in Southeast Asia. O. viverrini induces a chronic stage of infection in hamsters. During the first 2 weeks of infection, Th1 inducing cytokine, IL-12, increased but was down regulated in chronic infection. In this study it was found that unmethylated-CpG ODN (oligodeoxynucleotides) 1826 increased hamster mononuclear cell proliferation and stimulated IFN-␥ production in vitro. The IFN-␥ levels in hamster sera were significantly increased in hamsters injected with CpG ODN 1826 alone or plus crude somatic antigens (CSAg). Further investigation using the flow cytometer found that CD4+ T cells and IFN-␥+ CD4+ T cells (Th1-like cells) in the hamster blood were significantly increased. The role of these cells in the protective responses in hamsters was evaluated by challenging with 25 metacercaria and observation for 3 months. The number of worms recovered was significantly reduced in the hamsters injected with CpG ODN 1826 with CSAg, but not in CpG ODN 1826 alone groups when compared to PBS control. The percent of reduction in hamsters against this parasite were 32.95% and 21.49% in the CpG ODN 1826 with CSAg and CpG ODN 1826 alone. This study indicates that CpG ODN 1826 plus parasite antigens elicit a Th1-like response that leads to the enhancement of worm reduction. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Opisthorchis viverrini is a helminth parasite that causes a public health problem in the Southeastern Asia region. Opisthorchiasis is endemic in the northeast (Thaewnongiew et al., 2014) and north (Wongsawad et al., 2012) of Thailand, and other neighboring countries i.e. Laos, Cambodia, and Vietnam (Sithithaworn et al., 2012; Sripa et al., 2011). O. viverrini infection causes chronic infection and can be a risk factor for cholangiocarcinoma (CCA) in patients (Srivatanakul et al., 1991a, 1991b). Humans or animals, for example dogs, cats, pigs, minks, weasels, civets, and house rats are the hosts of this parasite, and the hamster is the suitable animal model for O. viverrini infection (Boonmars et al., 2009). In the chronic stage of O. viverrini infection in hamsters, IL-10 and TGF- were the regulatory cytokines that were found significantly increased. In contrast, the IL-12 responses (Th1 induced cytokine) were highly
∗ Corresponding author at: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand. E-mail address: sura [email protected] (S. Wongratanacheewin). http://dx.doi.org/10.1016/j.actatropica.2016.10.005 0001-706X/© 2016 Elsevier B.V. All rights reserved.
expressed during the first two weeks of infection but were significantly lower in the chronic stage. This indicated that IL-12 responses could be found during the stage of juvenile parasite development (Jittimanee et al., 2007). Similarly, Th1- IFN-␥ levels were found increased in rats infected with a trematode, F. hepatica, on day 7 post infection (Tliba et al., 2002). Moreover, Th2 cytokine (IL-4) and Treg cytokine responses such as IL-10 and TGF-, were found significantly expressed more than Th1 responses in spleens and mesenteric lymph nodes (MLN) in the chronic stage (Jittimanee et al., 2007). It was then hypothesized that since the Th1 cytokine responses were upregulated during the early stage of infection, this cytokine might play a role in the protection against this infection. Moreover, the parasites from infected hamsters have been shown to induce significantly high levels of regulatory cytokines (IL-10 and TGF-) (Jittimanee et al., 2007) and such TGF- levels were stimulated by CSAg. It was found that when hamsters were immunized to protect against O. viverrini infection, such protection was correlated with the reduction of TGF- and IL-10, but not IFN-␥ expression. (Jittimanee et al., 2012). To determine whether the stimulation of Th1 responses could provide increased protection against O. viverrini infection by reduction of worm revovery, the Th1 inducing
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adjuvant, unmethylated-CpG oligodeoxynucleotide (CpG ODN) has been used in this study. The CpG ODN stimulates the immune cells via TLR9 (Toll-like receptor 9) (Hemmi et al., 2000), and induces a strong inflammatory response to activate the immune system. CpG ODN was used as the adjuvant of immune activation in the vaccination of bacterial infections (Liu et al., 2011), viral infections (Davis et al., 1998), and parasitic infections (Aldridge et al., 2010; Cervi et al., 2004; Gupta et al., 2011; Pinzon-Charry et al., 2010; Ribeiro et al., 2009; Sanchez et al., 2011; Sane et al., 2010). BALB/c mice treated with CpG ODN reduced Taenia crassiceps burden (Aldridge et al., 2010). Moreover, CpG ODN was used as an adjuvant to enhance the partial reduction of Schistosoma mansoni (Teixeira de Melo et al., 2013). In this study, the effect of CpG ODN on Th1-like cytokine activation in hamsters was determined. It was demonstrated that CpG ODN could stimulate significant levels of CD4+ T cells producing IFN-␥ in hamster blood. It was demonstrated herein that these cells could increase the resistance to O. viverrini infection in hamsters.
2. Materials and methods 2.1. Metacercarial preparation O. viverrini metacercaria were prepared as previously described (Sirisinha et al., 1984). Briefly, infected cyprinid fish, obtained from a fresh water lake in the endemic area of the northeast Thailand, were homogenized and placed in a digesting solution (0.25% Pepsin, Sigma, MO), normal saline and 15% hydrochloric acid (BDH, Poole England) at a ratio of 1:3 and then incubated at 37 ◦ C for one hour. The digested solution was sieved through 1000, 300, and 106 m meshes. The debris obtained by filtering on 106 m mesh was washed repeatedly many times with normal saline until clear. Cleared sediments were investigated for metacercarial isolation under a dissecting microscope. These metacercaria were stored in normal saline at 4 ◦ C until infection.
2.2. Antigen preparations Crude somatic antigens (CSAg) were prepared from adult O. viverrini worms as described by Wongratanacheewin and colleagues (Wongratanacheewin et al., 1988). The worms, aged approximately 3 months, were collected from bile ducts of O. viverrini infected hamsters and washed with normal saline 3 times. The worms then were transferred into a grinder tube containing complete protease inhibitor cocktails (Roche Diagnostic GmbH, Mannheim Germany) and homogenized at 4 ◦ C. The homogenate was sonicated in an ultrasonic disintegrator set to operate at 1 min intervals for 10 min at 4 ◦ C. The sonicated homogenate was stored overnight at 4 ◦ C and centrifuged at 10,000g for 30 min at 4 ◦ C. The supernatant was transferred into new tubes, and sterilized with filter membranes with a pore size of 0.2 m (Whatman, Buckinghamshire England) and kept at −20 ◦ C. The protein concentration was determined in the supernatant by Bradford’s reagent (Bio-Rad, CA), and protein patterns (Supplement Fig. 1) were checked by the silver staining method.
2.3. Oligodeoxynucleotides Oligodeoxynucleotides (ODNs) were used with the following sequences: CpG ODN 1826, 5 TCCATGACG TTCCTGACGTT3 and non-CpG ODN 2138, 5 TCCATGAGCTTCCTG AGCTT3 . All ODNs were purchased from Invivogen (InvivoGen, CA).
2.4. Animal infections Male Syrian golden hamsters, 6–8 weeks old were orally infected with either 25 (for challenging) or 50 (for CSAg preparation) O. viverrini metacercaria (Mc). They were housed in the animal care unit at Faculty of Medicine, Khon Kaen University on a 12 h light/dark cycle and fed a commercial feed. Uninfected hamsters were used as controls. These O. viverrini infected hamsters were housed and taken care of for 3 months. Animal experiments were approved by the Animal Ethics Committee of Khon Kaen University (AEKKU 7/2558).
2.5. Experimental design for study of CpG ODN stimulation in hamsters Hamsters were divided into five groups (5/group). The animals were injected either with PBS (Group 1), 100 g CpG ODN 1826 (Group 2), 100 g CSAg (Group 3), 100 g non-CpG-ODN 2138 (Group 4) or 100 g CpG ODN 1826 plus 100 g CSAg (Group 5). Blood was collected via lateral saphenous vein from hamsters 2 days before and 2 days after CpG ODN injections. The injections were administrated via the intraperitoneal (i.p.) route. Each hamster’s blood sample was prepared as serum for cytokine detection while whole blood was prepared for cell staining for investigation of T cell populations by flow cytometry.
2.6. CpG ODN stimulation 2.6.1. Cell proliferation in vitro Normal hamster spleens collected from 4 to 6 week naïve hamsters were used for the in vitro study of CpG ODN stimulation. Mononuclear cells were prepared and isolated by ficoll-paque solution (GE Healthcare, Uppsala Sweden) then suspended in RPMI medium supplemented with 10% inactivated fetal bovine serum (FBS) and 100 U/100 g/ml penicillin/streptomycin (Gibco, Life Technologies, Grand Island, NY). The 2 × 106 cells/ml in 96 well plates (Corning Costar, Corning, NY) were cultured either with CpG ODN 1826 at 10 g/ml, 10 g/ml non-CpG ODN 2138 or 10 g/ml CSAg. The 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Enzo, Lausen Switzerland) with 1 g/ml ionomycin (Enzo, Lausen Switzerland) was used as a positive control. The cell suspensions were incubated at 37 ◦ C, 5% CO2 for 72 h. The XTT (sodium 3 -[1-(phenyl-aminocarbonyl)-3, 4-tetrazolium]-bis (4-methoxy6-nitro) benzene sulfonic acid hydrate) (Sigma, MO), that is a tetrazolium salt reagent, was used to measure mitochondrial dehydrogenase activity of viable cells. The XTT solution was added to the final concentration of 20% for at least 4–6 h before harvesting. Finally, cell culture plates were gently shaken and measured using a spectrophotometer at 450 nm.
2.6.2. IFN- production assay Mononuclear cells were prepared from naïve hamster spleens and adjusted to be 2 × 106 cells/ml. These cells were added into 24 well plates (Corning Costar, Corning, NY) containing 1 ml complete medium. Cell suspensions (2 × 106 cells/ml) were cultured either with medium alone, 10 g/ml CpG ODN 1826, 10 g/ml non-CpG ODN 2138, 10 g/ml CSAg or 50 ng/ml PMA plus 1 g/ml ionomycin, and incubated at 37 ◦ C, 5%CO2 . After 72 h incubation, supernatants were harvested for detection of IFN-␥ production levels. Culture supernatants or sera from hamsters were collected for determination of IFN-␥ levels by Enzyme-Linked Immunosorbent Assay (ELISA) (BioLegend, CA). The protocols were performed according to the manufacturer’s recommended protocols.
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2.7. Flow cytometry analysis The 100 l hamster blood samples collected before and 2 days after injection of either with PBS, 100 g CpG ODN 1826, 100 g non-CpG ODN 2138, 100 g CpG ODN 1826 plus 100 g CSAg, or only 100 g CSAg were stained with PE/Cy7-conjugated antimouse CD4 (BioLegend, CA) and incubated for 20 min in the dark at room temperature. The red blood cell lysis buffer (BioLegend, CA) was then added to the stained blood for 10 min at room temperature and washed with cell staining buffer and centrifuged at 300g for 5 min at 20 ◦ C. The stained cells were resuspended in a fixative buffer (BioLegend, CA) and incubated for 20 min in the dark at room temperature and then washed with cell staining buffer. The cells were then washed and resuspended with perm buffer (BioLegend, CA). The PerCP/Cy5.5-conjugated anti-mouse IFN-␥ solution (BioLegend, CA) was added into cell suspension tubes and incubated for 40 min in the dark at room temperature. This antibody was found to be cross-reactive with hamster IFN␥. Cells were washed twice with perm buffer. Finally stained cell pellets were resuspended in 0.5 ml cell staining buffer. Cells were analyzed on a BD FACS Canto II flow cytometer (BD Biosciences, CA) and BD FACSDiva software (BD Bioscience, CA). Isotype control, PerCP/Cy5.5-conjugated rat IgG1, a RTK2071 clone that does not cross-react with hamsters, was included in each intracellular staining protocol.
2.8. Immunization and challenge studies To study the protective effect of CpG ODN 1826 either with or without parasite antigens, hamsters were divided into 3 groups (10/group). The animals were injected either with PBS as control (Group 1), 100 g CpG ODN 1826 (Group 2) or 100 g CpG ODN 1826 plus 100 g CSAg (Group 3). Each injection was administrated intraperitoneally (i.p.) before the infection. On day 2 after injection, all hamsters were then infected orally with Mc (25 Mc/animal). Immunizations were repeated on day 14 post-infections in order to enhance the immunity during the early stage of infections as the parasites need 1 month to develop into adult worms. At 3 months after infection all hamsters were sacrificed and their livers were removed to investigate the number of adult O. viverrini worms recovered.
Fig. 1. CpG ODN induced spleen cell proliferation and IFN-␥ production in vitro. The 2 × 106 cells/ml cells of naïve hamster mononuclear cells obtained from spleens and stimulated either with 10 g/ml CpG ODN 1826, 10 g/ml non-CpG ODN 2138, 10 g/ml CSAg or medium alone. The XTT reagent was added at 4–6 h before determination at 72 h of incubation. The cell proliferation values were determined by OD at 450 nm (A). Cells stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) plus 1 g/ml ionomycin (IO) were used as a positive control. Cell supernatants were harvested and the IFN-␥ levels were detected by ELISA (B). Data represent the means from duplicate cultures, and each bar represents the mean ± SD. All results from non-CpG groups are not significantly different from medium controls.
3. Results 2.9. Calculation of worm recovery Percent worm recovery was calculated from the number of adult worms recovered from livers divided by number of metacercaria that were given at the beginning and multiplied by 100. The percent worm reduction (protection) in each group was calculated from the equation below: %Worm reduction (%Protection) = ((Wn − Wi)/Wn) × 100 Wn = Worm recovery in control group Wi = Worm recovery in injected group
2.10. Statistical analysis The statistical analysis was performed using SPSS 17.0 software. The Student’s paired t-test was used in the comparisons between before and after injections, but one-way ANOVA was used in the comparisons between each group. Data were considered statistically significant if the p value was less than 0.05.
3.1. CpG ODN 1826 induces hamster spleen cell proliferations and IFN- production To determine the effect of CpG ODN 1826 on hamster cell division, mononuclear cells obtained from the spleens of naïve hamsters were used. It was found that CpG ODN 1826 could significantly stimulate mononuclear cell proliferation (p = 0.0001) 72 h after incubation compared to media alone or non-CpG ODN 2138 as the negative control (Fig. 1A). In addition, CSAg also induced cell proliferation but its level was significantly lower (p = 0.001) than those of CpG ODN 1826 (Fig. 1A). To investigate whether CpG ODN 1826 could induce IFN-␥ production in hamsters, the experiments were done using CpG ODN 1826 stimulated naïve mononuclear cells in vitro. High IFN-␥ levels could be significantly detected (p = 0.0001) at 72 h after incubation (Fig. 1B). CpG ODN 1826 could also induce IFN-␥ in vivo. Significant IFN-␥ production in sera either from CpG ODN 1826 (p = 0.0001), CpG ODN 1826 with CSAg (p = 0.0001) or CSAg alone groups (p = 0.0001) was shown when compared with control naïve hamsters (Fig. 2). Although CSAg could stimulate the increased IFN-␥ level, its level was significantly lower than those of CpG ODN 1826 alone (p = 0.001) or CpG ODN 1826 with CSAg (p = 0.0001) (Fig. 2). The CSAg did not
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Fig. 2. CpG ODN induced IFN-␥ levels in hamsters. Blood was collected from all hamsters before, and after injection either with PBS, 100 g CpG ODN 1826, 100 g non-CpG ODN 2138, 100 g CpG ODN 1826 plus 100 g CSAg, or 100 g CSAg only. The IFN-␥ levels were detected in hamster sera by ELISA and calculated for statistical analysis. Data represent the means from duplicate experiments, and each bar represents the mean ± SD. The IFN-␥ levels in CpG ODN 1826, CpG ODN 1826 plus CSAg, and CSAg injected hamsters were significantly different from PBS and non-CpG ODN 2138 injected groups. The IFN-␥ levels in the non-CpG group were not significantly different from PBS controls as shown.
Fig. 4. Quantification of CD4+ IFN-␥ producing T cells from hamster blood. Blood was collected from hamsters similar to those in Fig. 3. Each hamster’s blood was stained with anti-mouse PE/Cy7 conjugated CD4 and anti-mouse PerCP/Cy5.5 conjugated IFN-␥. The percent of IFN-␥+ CD4+ T cells was investigated by flow cytometer. The profiles of IFN-␥+ CD4+ T cells were detected in hamster blood on day 2 after the injection. The percent of IFN-␥+ CD4+ T cells in hamster blood were compared before as shown in bar graph. Data represent the means from duplicate experiments, and each bar represents the mean ± SD. The percent of IFN-␥+ CD4+ T cells in hamster blood injected either with CpG ODN 1826, CpG ODN 1826 plus CSAg or CSAg alone were significantly (p < 0.05) different from PBS or non-CpG ODN 2138 injected groups. The IFN-␥+ CD4+ T cells in hamster blood from non-CpG groups are not significantly different from PBS control.
duction and CD4+ T cells, the numbers of IFN-␥+ CD4+ T cells in CpG ODN with CSAg were not significantly (p = 0.799) different from CpG ODN alone (Fig. 4 and Supplement Fig. 3). 3.3. CpG ODN with CSAg significantly stimulates worm reduction
Fig. 3. Quantification of CD4+ T cells from hamster blood. Blood was collected from hamsters after injection either with PBS, 100 g CpG ODN 1826, 100 g non-CpG ODN 2138, 100 g CpG ODN 1826 plus 100 g CSAg, or only 100 g CSAg. Each hamster blood sample was stained with anti-mouse PE/Cy7 conjugated CD4 and the percent of CD4+ T cells were investigated by flow cytometer. The profiles of CD4+ T cells in hamster blood on day 2 after injection were compared as shown in the bar graph. Data represent the mean from duplicate experiments, and each bar represents the mean ± SD. The percent of CD4+ T cells in hamster blood injected either with CpG ODN 1826, CpG ODN 1826 plus CSAg or CSAg alone were significantly (p < 0.05) different from PBS or non-CpG ODN 2138 injected groups. The percent of CD4+ T cells in hamster blood from non-CpG group were not significantly different from PBS control as shown.
significantly (p = 0.879) enhance the IFN-␥ stimulation when used with CpG ODN 1826 when compared to CpG ODN alone (Fig. 2). 3.2. CpG ODN 1826 stimulates IFN- + CD4+ T cell in hamsters The CD4+ T cell populations were significantly increased in hamster blood either from CpG ODN 1826 (p = 0.0001), CpG ODN 1826 with CSAg (p = 0.0001) or CSAg alone (p = 0.0001) groups when compared to non-CpG ODN or PBS controls (Fig. 3 and Supplement Fig. 2). There were no significant differences (p = 0.817) of CD4+ T cells between CpG ODN 1826 alone and CpG ODN 1826 with CSAg in the injected hamster blood groups (Fig. 3 and Supplement Fig. 2). IFN-␥+ CD4+ T cells were found significantly increased (p = 0.0001) in hamster blood (Fig. 4 and Supplement Fig. 3) similar to those found in CD4+ T cells (Fig. 3) or IFN-␥ levels (Fig. 2). Although CpG ODN 1826 with or without CSAg was able to activate the IFN-␥ pro-
The number of worms recovered were significantly reduced (p = 0.0001) only in the hamsters injected with CpG ODN 1826 with CSAg, but not in CpG ODN 1826 alone compared to the PBS group (Fig. 5A). The percents of reduction in hamsters against this parasite were 32.95% and 21.49% in the CpG ODN 1826 with CSAg and CpG ODN 1826 alone groups (Fig. 5B). Although, CpG ODN 1826 alone did not give a significant (p = 0.154) worm reduction from control, the reduction of worm numbers was significantly (p = 0.006) found in CpG ODN 1826 with the CSAg group. In addition, there were no significant differences (p = 0.203) between CpG ODN 1826 alone and CpG ODN 1826 plus CSAg (Figs. 5B). 4. Discussion Several attempts have been made in the past to describe the protective immunity against O. viverrini infection. The protective responses to O. viverrini infection in previous reports were not successful enough to clear this worm from infected hamsters (Jittimanee et al., 2012; Papatpremsiri et al., 2016; Sirisinha et al., 1983a,b; Sirisinha and Wongratanacheewin, 1986). In an early stage of this parasitic infection, Th1 inducing cytokine responses were found but decreased during the progression of chronic infection. The chronic stage of O. viverrini infection showed the high expression of IL-4 and regulatory cytokines (IL-10 and TGF-) (Jittimanee et al., 2007). This means that the adult stage development of this parasite may modulate the immune responses via down regulation of the Th1 cytokine responses. The parasitic molecules with such functions, however, have never been identified. If Th1 responses were elicited in this parasitic infection, they might enhance activation of immune responses to clear or reduce worm numbers. In addition, CpG ODN was used as a protective adjuvant for treatment of other parasites (Sane et al., 2010). Herein,
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Fig. 5. Worm recoveries from O. viverrini infected hamsters with or without CpG ODN 1826 pretreatment. All hamsters were injected either with PBS, 100 g CpG ODN 1826, 100 g CpG ODN 1826 plus 100 g CSAg 2 days before infection and on 14 days post-infection intraperitoneally (i.p). Each hamster group (10/group) was then orally infected with 25 metacercaria per hamster, and worm recoveries were determined at 3 months post-infection. The percentages of worm recovery were calculated from O. viverrini infected hamsters (A), and the percentages of worm reduction in hamsters (B). Each bar represents the mean ± SD.
it was shown that CpG ODN 1826 stimulated IFN-␥ production in hamsters. Although the numbers of CD4+ T cells and IFN-␥ levels were the same in animals that received CpG ODN alone and animals that received CpG ODN + CSAg; however, IFN-␥+ CD4+ T cells in the CpG ODN + antigen group were higher (Fig. 4). This might lead to a greater worm reduction in these animals (Fig. 5). The IFN-␥ found in IFN-␥+ CD4+ T cell (Th1-like) responses therefore conferred the protective immune responses by reduction of worms in challenged animals. The hamster models are the most common animal model and a suitable model for O. viverrini infection as they give similar pathologies and chronic inflammation in bile ducts, around the liver area and can develop to be cholangiocarcinoma similar to humans (Tansurat, 1971; Bhamarapravati et al., 1978; Koompirochana et al., 1978; Harinasuta et al., 1984; Riganti et al., 1989; Sripa and Kaewkes, 2002; Sripa et al., 2003). The size of hamster liver is not a reason for the numbers of worm recovery because the liver in the rat is larger but there are no adult worn parasites found in this species (Boonmars et al., 2009). The reason for the hamster as the best model of O. viverrini infection may be the presence of the gall bladder which is not found in rat (Boonmars et al., 2009). The immune responses against O. viverrini infections were therefore tested in the hamster as the animal model for antibody, cell mediated immune responses and protective approaches (Flavell et al., 1980; Flavell, 1982; Sirisinha et al., 1983a,b; Wongratanacheewin et al., 1987; Jittimanee et al., 2007, 2012). The strategies for protection against O. viverrini infection in hamsters were designed and
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tested (Jittimanee et al., 2012). The Th1 stimulator adjuvant, CFA, a complete Freund’s adjuvant agent, increased partial protection to O. viverrini infection of hamsters (Jittimanee et al., 2012). CFA enhanced the resistance to O. viverrini infection, and could activate IL-12, a pro-inflammatory cytokine, and increased Th1 responses (Shibaki and Katz, 2002). Alternatively, CpG ODN 1826 as a Th1 stimulator was used to treat the hamster model against parasitic infections. The CpG ODN 1826 was used as a murine immune stimulation agent. It was found in this study that it could activate immune stimulation in the hamster similar to that found in mouse (Sane et al., 2010). Results in the present study demonstrated that CpG ODN 1826 could stimulate significant mononuclear cell proliferation in vitro (Fig. 1A). This agent could also increase the phagocytic index in hamsters when compared to untreated controls (Sane et al., 2010). In addition, rat spleen cells were also activated by this CpG ODN (Cervi et al., 2004). This indicated that murine CpG ODN could be used in these current hamster experiments. The use of hamsters in various studies in infectious diseases is limited by available monoclonal antibodies (mAb) for this model. Recently, some anti-mouse mAbs were reported to cross react with hamsters such as anti-mouse CD4 mAb (GK 1.5 clone) (Dondji et al., 2010; Hammerbeck and Hooper, 2011). This mAb clone was used in a T cell study in the hamster model of hanta virus diseases (Hammerbeck and Hooper, 2011). Moreover, this specific murine CD4 mAb was documented in hamsters infected with hook worms (Dondji et al., 2010). Other anti-mouse mAbs from the R&D systems company, such as TGF- and IL-10 were reported to cross react with hamsters (Zivcec et al., 2011). The present study compared the IFN-␥ amino acid sequences between mouse and hamster and found that they gave 56% identity. It was then tested in the preliminary study with anti-mouse IFN-␥ from BioLegend. Results showed this mAb could cross-react with hamster IFN-␥ in detection by both ELISA (Fig. 2) and flow cytometry (Figs. 4). Those data and the results herein could then be used to design the present hamster experiments. The mechanism of CpG ODN activates immune cells via internalization. This agent interacts with TLR9 that is located in the endocytic vesicle. The results within cellular activation involve a signaling cascade process through the myeloid differentiation primary gene 88 (MyD88) (Klinman, 2004). B cells and dendritic cells express TLR9 and responded to CpG ODN (Davis et al., 1998; Hemmi et al., 2000; Klinman, 2004). The indirect activation by CpG ODN can provide an immune stimulatory cascade to stimulate monocyte/macrophage cells or NK or T cells (Klinman et al., 1996; Stacey et al., 1996; Sun et al., 1998). These effects give the results of Th1biased cytokine secreting such as TNF-␣, IFN-␥ and IL-12 (Ballas et al., 1996; Klinman et al., 1996). In this study, it was found that the TLR9 protein sequence similarity between hamster and mouse was 85%, and the murine TLR9 activator or CpG ODN to activate hamster immune response was used. It might therefore activate hamster immune cells like mouse immune cells to stimulate Th1 cytokine responses. The current results found this agent could elicit CpG ODN in hamster mononuclear cells to produce significant IFN␥ levels in 72 h (Fig. 1B) similar to the IFN-␥ production that was found in the experiment of rat spleen cells treated with CpG ODN for 72 h (Cervi et al., 2004). This result showed that the Th1 cytokine response was activated by CpG ODN administration and also found significant IFN-␥ production was activated on hamster mononuclear cells by CSAg (Figs. 1B). The CpG ODN activation was also done in vivo and it was found that IFN-␥ levels were induced (Fig. 2). Although CSAg could significantly induce IFN-␥ production in vivo, it was lower than CpG ODN 1826 stimulation (Fig. 2). Further investigations in the present study found that CD4+ T cells (Fig. 3) and IFN-␥+ CD4+ T cells (Th1-like) in the blood of hamsters were stimulated by CpG ODN 1826 or CpG ODN 1826 with CSAg or only CSAg injections (Fig. 4). These results were related to the increased IFN-
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␥ levels in CpG ODN 1826 or CSAg treated-hamster sera (Fig. 2). This indicated that the CpG ODN mechanism in vivo could induce a significant increase in IFN-␥ levels from Th1-like cells in hamster blood. IFN-␥ and Th1-like cells in hamsters were increased by CpG ODN 1826 stimulation both in vitro and in vivo. It was then hypothesized that this CpG ODN might stimulate Th1 activation and help to eliminate the worms in hamsters. Although CpG ODN 1826 alone treated hamsters could significantly stimulate Th1-like responses in hamster blood (Fig. 4), this agent alone did not provide significant reduction of worm recovery (Fig. 5). In contrast, CpG ODN 1826 with CSAg could significantly protect hamsters against infection by increasing worm reductions (Fig. 5B). This might be due to the enhancement of CSAg on CpG ODN to increase IFN-␥ levels (Fig. 2) from IFN-␥+ CD4+ Th1-like cells (Fig. 4). The antigens are important to stimulate adaptive immunity as CpG ODN only stimulates innate immunity. The combination of CSAg and CpG ODN may shift the adaptive immunity to be more Th1-like using CpG ODN as an adjuvant leading to more protection. The CSAg alone, however, was shown to stimulate high levels of TGF- and inhibition of cell proliferation (Jittimanee et al., 2012) thus making fewer IFN-␥+ CD4+ cells. These findings were similar to the significant reduction of parasite burden by CpG ODN that could be found in mice infected with T. crassiceps (Aldridge et al., 2010) and in rats infected with F. hepatica (Cervi et al., 2004). Th1 cytokines were shown to be involved in the reduction of worm recovery and egg production when the CpG ODN formula vaccine was used against S. mansoni in mice (Ricciardi et al., 2015). Recently, mice immunized with the CpG ODN-Cathepsin B recombinant protein induced high IgG2c titers and reduced S. mansoni worm numbers (Ricciardi et al., 2015). Also, mice immunized with the tegument-CpG ODN combination gave high IgG2c titers (Teixeira de Melo et al., 2013). It was shown that antibody titers might be due to neutralization of the worms via antibody mediation by an antibody dependent cell mediated cytotoxicity (ADCC) mechanism. NK cells might modulate and play a role via the ADCC mechanism to eliminate worms in schistosomiasis. In a previous report, this mechanism was a key mediator providing protection against schistosomiasis (Comin et al., 2008). In this study it was found that IFN-␥ and Th1-like cells were induced by CpG ODN and might have activated other immune cells that are involved in parasite elimination. In conclusion, the CpG ODN plus this parasite antigen could elicit the protective Th1-like responses and Th1 cytokine responses in hamsters leading to increased partial protection. CpG ODN might be an alternative prophylactic agent or adjuvant in the protection of this parasitic infection.
Conflict of interests None declared
Ethical approval Animal experiments were approved by the Animal Ethics Committee of Khon Kaen University, Thailand (AEKKU 7/2558).
Acknowledgements We thank the grant of the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Health Cluster (ShePGMS), Khon Kaen University for supporting the scholarship during Ph.D study. This work is partially supported by Faculty of Medicine, Khon Kaen University and Melioidosis Research Center, Khon Kaen
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