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Recognition of Schistosoma mansoni egg-expressed ovalbumin by T cell receptor transgenic mice
Experimental Parasitology 206 (2019) 107767
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Recognition of Schistosoma mansoni egg-expressed ovalbumin by T cell receptor transgenic mice
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Mebrahtu G. Tedla1,∗, Musammat F. Nahar1, Jana Hagen2, Alison L. Every3, Jean-Pierre Y. Scheerlinck Centre for Animal Biotechnology, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: OT II T cells Ovalbumin Schistosoma mansoni Transgenesis
Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.
1. Introduction Schistosomiasis, a parasitic disease caused by trematodes or digenetic blood flukes, is one of the most devastating tropical diseases. It is endemic in 74 countries and affects more than 200 million people worldwide, with 85% of patients living in Africa [Chitsulo et al., 2017]. Regular and periodic chemotherapy of at-risk groups with praziquantel, with potency against the Schistosoma adult parasite, is the only available intervention strategy [WHO, 2015]. As a result, the global and regional occurrence of the diseases has declined in the past few decades. However, elimination of the disease remains a global challenge [WHO, 2015]. While an effective vaccine is not yet available, the recent discovery of specific vaccine antigens has shown promise. Drug treatment targeting the parasite and control of the arthropod vector combined with vaccines might offer a long-term solution (Moreno-Cid et al., 2013; Sinden, 2017), despite genetic variation among parasitic strains, which could affect vaccine coverage (Sheerin et al., 2017). However, induction of effective protective immunity remains out of reach at least
in part because of the ability of the parasite to modulate immune response. Therefore, understanding how the parasite persists in the face of robust stimulation of the host immune system is one of the most important aspects in the study of host-parasite interactions [Hagen et al., 2015]. Indeed, S. mansoni, is known to have specific tactics to manipulate the host immune response in order to ensure its survival in the host [Bergquist, 1995]. Modulating the immune system at a molecular level via, in particular, soluble egg antigens (SEAs) is one of the key strategies for S. mansoni to compromise host immunity [Kane et al., 2008]. However, the mechanism of this specific immunomodulation remains elusive, particularly in vivo [Fallon and Antonio, 2006]. The immunomodulatory potential of S. mansoni eggs at the onset of chronic infection, is well documented and characterized by a Th2 polarized immune response [Pearce et al., 2004]. This Th2 response is associated with down-regulation of the initial Th1 or pro-inflammatory responses to migrating cercaria and leads to granuloma formation [Brunet et al., 1997; Fallon et al., 2000]. Many SEAs have been identified in S.
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Corresponding author. E-mail address: [email protected] (M.G. Tedla). 1 Both authors contributed equally. 2 Current address: Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK. 3 Current address: College of Science, Health and Engineering, La Trobe University, Victoria, 3086. https://doi.org/10.1016/j.exppara.2019.107767 Received 6 May 2019; Received in revised form 7 September 2019; Accepted 8 September 2019 Available online 11 September 2019 0014-4894/ © 2019 Elsevier Inc. All rights reserved.
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protocol described previously [Mann et al., 2011]. Briefly, livers were extracted from mice, diced and digested overnight in shaking incubator at 120 rpm, 37 °C with 25 mg collagenase IV (Sigma Aldrich) in 40 mL of PBS and 10 mL of DMEM (Gibco, Life Technologies) containing 200 μg/mL gentamicin (Sigma Aldrich), 100 U/mL of penicillin (Gibco, Life Technologies), and 100 μg/mL of streptomycin (Gibco, Life Technologies). Subsequently, liver homogenates were washed three times with PBS containing Penicillin-Streptomycin (10,000U/mL) and resuspended in 5 mL of PBS. Eggs were purified over a percoll-sucrose gradient (Sigma Aldrich) consisting of 8 mL percoll and 32 mL of 250 mM sucrose by centrifuging at 800×g for 12 min. Finally, eggs were washed twice and resuspended in DMEM media (Sigma Aldrich) supplemented with 4.5 g of glucose, 1 mM sodium pyruvate, 2 mM Lglutamine, 1 mM HEPES, 100 U/mL penicillin, 100 μg/mL of streptomycin and 10% v/v of FBS and cultured at 37 °C and 5% CO2. Freshly isolated eggs of S. mansoni were transduced as described previously [Hagen et al., 2014]. Briefly, eggs were exposed to 100 μL of virus particles at a titter of 25 × 103 pfu per mL of OVA-encoding lentiviral particles or left untreated in complete DMEM medium (10 mM HEPES, 100U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 8 μg/mL polybrene) and incubated at 37 °C and 5% CO2. Further controls included a virus encoding mCherry or virus lacking a transgene sequence (empty vector). After 24 h, eggs were washed three times with PBS and cultured for an additional four days in complete DMEM medium. Afterwards, eggs were washed in PBS and resuspended in RPMI medium for subsequent T cell assays. Throughout all the experiments, fresh eggs were used and maintained in complete DMEM media. Expression of functional OVA protein confirmed egg viability.
mansoni, including omega-1 now understood to drive Th2-polarized responses, through interaction with dendritic cells (DCs). This protein plays an important role in switching the Th1 response into a Th2 response through inhibition of IL-12 production [Everts et al., 2009; Steinfelder et al., 2001]. Moreover, other SEAs, such as, IPSE/α-1, trigger Ig-E-driven IL-4 secretion by basophils and induces alternatively activated macrophages and, thus, Th2-polarized responses [Donnelly et al., 2008]. Kappa-5 is another immunomodulatory SEA present in S. mansoni. In the present study, we utilised S. mansoni-expressed chicken ovalbumin (OVA), to study the immune response of parasite-associated antigens in inducing biased Th responses in naïve T cells. OVA being a model antigen avoids the induction of biased immune response and is ideal for such investigations as it is difficult to exclude that parasitederived antigens have intrinsic biases selected as a result of prolonged evolutionary pressures. The expression of OVA in the parasite therefore helps to elucidate the immunomodulatory properties of the soluble egg antigens of the parasite egg and, using the expressed protein as a neutral antigen, we are able to further characterise functions of specific SEAs. As a first step, demonstrating that T cell receptor transgenic mice (OT-II mice) known for having higher frequency of CD4+ T cell receptors that recognise OVA, as a source of naïve CD4+ T cells that can recognise S. mansoni expressed OVA, is essential. Here we show that, OT-II cells respond to this antigen by proliferating and producing key cytokines in vitro. 2. Material and methods 2.1. Experimental mice and maintenance of the S. mansoni life cycle Six- to eight-week-old female BALB/c and OT-II mice were purchased from the Walter and Eliza Hall Institute of Medical Research (WEHI) and were used in the present study to maintain the life cycle of S. mansoni and as a source of naïve OT-II T cells, respectively. All the experiments related with mice were conducted after getting an approval from the Animal Ethical Committee of the University of Melbourne (Ethics ID: 1312952). Snails (Biomphlaria glabrata, NMRI strain), an intermediate host of the parasite were purchased from NIHNI-AID, Schistosomiasis resource centre, USA.
2.4. RNA extraction, RT-PCR To detect and analyse mRNA synthesis, parasite eggs were extracted and homogenized four days after transduction with lentivirus, based on the protocol modified from TRI Reagent® Protocol (Sigma Aldrich). Briefly, eggs were homogenized and further ultra-sonicated to break the egg shells and the homogenates were resuspended in 1 mL of Tri reagents (Sigma Aldrich) and incubated for 5 min at room temperature. Chloroform (0.2 mL) was added to the suspension and the mixture was further incubated at room temperature for 15 min. After incubation, the mixture was centrifuged at 12,000×g for 10 min at 4 °C and the clear aqueous layer was collected to purify RNA and 0.5 mL of isopropanol was added to precipitate the RNA. The precipitated RNA was further incubated at room temperature for 10 min and centrifuged at 12,000×g for 8 min. The RNA pellet was washed twice with 1 mL of 75% ethanol and centrifuged at 7500×g for 5 min at 4 °C. Finally, the RNA pellets were air dried for 5 min and resuspended in 50 μL of nuclease free water. The RNA pellets were solubilized by passing through a pipet tip and further incubated for 10 min at 55 °C. Average purity of RNA was confirmed by a ratio of absorbance at A260/A230 of 1.93. Finally, the solubilized RNA was stored at −20 °C.
2.2. Generation of constructs and production of lentiviral particles For expression and secretion in S. mansoni the OVA sequence (GenBank access no: V00438) was modified by N-terminal replacing the first 47 amino acids with the signal peptide of S. mansoni omega-1 (GenBank access no: DQ013207) and a 6-Histidine (his)-tag to the Cterminus. The modified OVA sequence was optimized for schistosome codon usage and synthesized by Gen Script (NJ, USA) and cloned into pGIPZ vector [Hagen et al., 2014], via SpeI and NotI restriction sites, replacing mCherry (Fig. 1). Plasmids were maintained in Stbl3 E. coli and clones were verified by Sanger sequencing. Replication-incompetent lentiviral particles were produced in HEK293T cells after cotransfection of the lentiviral expression plasmid and TransLenti™ packaging mix (Open Biosystems, Dharmacon™) according to the manufactures instructions. Functional virus titres were estimated following a modified protocol of the commercially available retro-viral detection kit given by Takara Bio Inc, the virus titter was determined by semi quantitative real time PCR reaction using MX3000P real-time PCR cycler (Stratagene, La Jolla, CA, USA). Forward (5ʹ-TGGACAGGGGCT CGGCTGTT-3ʹ) and Reverse (5ʹ-CCGCTGGATTGAGGGCCGA-3ʹ) primers were used to amplify the cDNA. The samples were run in triplicate and average Ct value and copy number was taken.
2.5. Synthesis of cDNA from viral genomic RNA For the reverse transcription of viral genomic RNA into complementary DNA (cDNA), virus supernatants collected from cell culture were used as a source of template. The thermal profile of the reverse transcription process was adjusted in each step using the thermocycler (MJ mini personal thermal cycler, Bio-Rad Laboratories, San Francisco, CA, USA), following the manufacturers recommendation. Sequence of the primers used for RT-PCR for OVA transcript in the RNA recovered from the schistosome eggs were F-5′-ATGAAGATACACAAGCAATG CCA-‘3; R-5’-TCATAGTTCCAGAAGCGAATGGT′-3’.
2.3. Isolation and lentiviral transduction of S. mansoni eggs S. mansoni eggs were isolated from mouse livers following the 2
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Fig. 1. (A) OVA, mCherry and pGIPZ vector without transgene constructs: LTR, long terminal repeat; ψ, packaging signal; cPPT, poly-purinetract; ZeoR, zeomycin resistant gene; CMV, cytomegalovirus promoter; mCherry, mCherry encoding transgene; WPRE, woodchuck hepatitis virus post-translational regulatory element; SIN, self-inactivating LTR; OVA-CMV, Optimized OVA with CMV promoter. (B) Screening of clones by colony PCR yielded Opt-OVA positive clones. DNA region amplified from the upstream of the promoter to the middle of the Opt-OVA insert. (C) Generation of 163 bp RT-PCR product using agarose gel electrophoresis. (D) PCR amplification product of the cDNA synthesized from virus supernatants containing the mCherry construct. The numerical labelling 1, 2, 3, 4 are the 5-fold serial dilutions of the viruses. Generation of the same PCR products and the dilutions are also similar with the exception in which the reference of the viral construct is the optimized OVA. The pGIPZ empty vector was used as a negative control.
2.6. Preparation of soluble egg antigen and Western blot
2.7. T cell proliferation assay
According to the protocol modified from Boros and Warren (1970), SEAs were prepared five days after virus exposure. Approximately 6000 transduced and un-transduced eggs were suspended in cold PBS and homogenized using a T10 basic ultra-turrax® until the shells of the eggs were completely crushed, which was confirmed using light microscopy. The egg homogenates were further exposed to ultrasonic pulses using a microson™ ultrasonic cell disruptor at 30 s bursts until complete breakdown was achieved. After centrifugation at 8765×g, the supernatants were passed through a 0.2 μm filter and analysed by SDS-PAGE and Western blot. Immunoblotting was carried out after transfer to a nitrocellulose membrane with 1:1000 anti-rabbit IgG fraction to chicken egg ovalbumin (MP Bio medicals) and 1:2000 anti-HRP-conjugated swine anti-rabbit immunoglobulins (DAKO). Proteins were visualised by chemiluminescence (ECL™ western blot detection reagents, GE Health care life science, Australia).
Spleen, maxillary, axillary and inguinal lymph nodes of OT-II mice were collected and pooled for cell extraction using complete RPMI1640 medium (Invitrogen, Life Technologies). Single cell suspensions were obtained by passing the tissue through a 70 μm nylon cell strainer. Red blood cells were lysed by water and lysis was stopped using PBS. Cells were then resuspended in complete RPMI-1640 medium. For proliferation assays, cells were stained with carboxyfluorescein-diacetate-succinimidyl-ester (CFSE; Bio Legend, San Diago, California, USA) according to manufacturer's instruction. A total of 2 × 105 labelled cells per well were co-incubated with 25, 50, 100, or 200 transduced or untransduced S. mansoni eggs for 4–5 days in 96 well plates at 37 °C, 5% CO2. After four days culturing, OT-II cells were collected and washed with FACS buffer (10% FCS in 1× PBS). For proliferation analysis, cells were stained with PE-anti-mouse CD4 (Bio Legend) following an FcγRsurface block (antibody, manufacturer). To exclude dead cells, 7-aminoactinomycin D (1 μg/mL; Sigma Aldrich, Steinheim, Germany) was added to cell suspension prior data acquisition using The BD
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FACSVerse™ flow cytometer (BD Bioscience, New Jersey, USA). 2.8. Cytokine assay Cell culture supernatants from T cell proliferation assays were analysed for cytokine production by mouse Th1, Th2, and Th17 cytometric bead array (BD™). According to the manufacturer, the detection limits for each cytokine were: IL-2 (0.1 pg/mL), IL-4 (0.03 pg/mL), IL-6 (1.4 pg/mL), IFN-γ (0.5 pg/mL), TNF-α (0.9 pg/mL), IL-17A (0.8 pg/ mL) and IL-10 (16.8 pg/mL). 2.9. Statistical analysis Data was computed and analysed using the single cell analysis software: flowjo version 10 (Ashland, Orgeon, USA). All statistical analyses were performed using one-way ANOVA followed by t-test using Graph Pad Prism version 7.00 for Windows (GraphPad Software, La Jolla, California, USA). In all the analyses, the confidence level was held at 95% and p < 0.05 was required for significance. 3. Results 3.1. Optimisation of OVA for expression and secretion in S. mansoni eggs following lentiviral transduction The first 47-amino acid signal peptide sequence of original OVA was replaced by the 23-amino acid of S. mansoni ω-1 signal sequence. And 6histidine sequence was added at the C-terminal end of the optimized sequence. Two restriction sites, SpeI at the N-terminal end and NotI at the C-terminal end were included to the sequence. After the sequence was synthesized commercially by GenScript (NJ, USA), the gene was cloned in to the pGIPZ_CMV_mCherry via SpeI and NotI replacing mCherry to produce pGIPZ Opt-OVA (Fig. 1A). In order to generate a promoter-less expression vector (OVA-PL), Opt-OVA was cloned into pGIPZ-SmACT vector construct via SpeI and NotI replacing the SmACT_mCherry region and a colony PCR screening confirmed successful transformation (Data not shown). Due to the similarities in the total number of amino acids and following expression of both the optimized OVA and normal OVA by the parasite egg, we used both names interchangeably. Integration of Opt-OVA into the vector was confirmed by amplifying the segment upstream of the promoter to the middle of the Opt-OVA insert, using colony PCR (Fig. 1B). The presence of the OVA (Fig. 1C) and mCherry (Fig. 1D) in the virus was confirmed by reverse transcription PCR, followed by agarose gel electrophoresis.
Fig. 2. Transcription of the Opt-OVA encoding gene and expression of OVA by transduced S. mansoni eggs. Reverse transcription PCR of OVA-transduced (6000 and 5000 eggs) and control eggs (6000 eggs) showing the presence of an OVA-specific mRNA band of 149 bp in transduced eggs that is absent in the untransduced eggs (A). Additional controls (B), showing the presence of parasite genetic material in each lane through the expression of IPSE irrespective of whether the parasites were transduced with the OVA gene or not. Panel B also shows that OVA mRNA is increased when OVA was optimized for expression in S. mansoni (OVA vs Opt-OVA). PCR product sizes: Opt-OVA; 149bp and IPSE; 263bp. M, molecular weight marker; UnTd, untransduced; Opt-OVA-PL, optimized OVA promoter-less; Opt-OVA, optimized OVA. SDS-PAGE of OVAtransduced (6,000, 5000 and < 2400) and control S. mansoni eggs (6,000) (C) and the same gel following probing with specific primary antibodies to OVA followed by enzyme-labelled secondary antibody (D).
the same number of un-transduced eggs were used as a negative control; in these samples, there was no strongly stained band at this molecular weight. To further confirm the expression, proteins were transferred electrophoretically onto a nitrocellulose membrane and a specific band of 45 kDa was stained with antibodies to OVA, confirming the expression of this protein (Fig. 2C and D). To demonstrate that the OVA protein was produced by the transduced eggs and was not transferred together with the virus as a contaminant in the HEK-293T cell supernatant, we performed the transduction protocol with both heat inactivated (90 °C for 10 min) and Opt-OVA-promoter-less virus constructs. As expected no OVA was detected unless a viable and functional OVA-expressing virus was used in the transduction protocol (Data not shown). To determine if the OVA protein was secreted into the supernatant following viral transduction of the eggs, we subjected the supernatant and egg lysate to a capture ELISA and could only detect the presence of OVA in the lysate. Using purified OVA as a control the detection limit of the capture ELISA was estimated to be 5 ng/mL.
3.2. Detection of Opt-OVA in transduced S. mansoni eggs The expression of OVA was analysed by SDS-PAGE using 6,000, 5,000, < 2400 transduced eggs per lane. The control contained 6000 un-transduced eggs per lane. First the expression of the OVA gene was confirmed using a reverse transcription PCR after performing cDNA synthesis from mRNA of transfected eggs. To achieve this, pools of transduced or un-transduced eggs were used for the extraction of total RNA. After synthesis of cDNA, the OVA backbone was amplified using OVA-specific forward and reverse primers and revealed a 163 bp amplicon and RNA from un-transduced eggs was also extracted and reverse transcribed, but it showed no amplification. The Opt-OVA- and S. mansoni IPSE gene were also amplified by standard PCR followed by cDNA synthesized from mRNA by reverse transcriptase and revealed a PCR product sizes of 149 bp of Opt-OVA- and 263bp of S. mansoni IPSE (Fig. 2A and B). SEA was separated by SDS-PAGE and Coomassie blue staining was performed to analyse total protein in each sample. The lysate from eggs transduced with Opt-OVA virus, contained an additional band corresponding to a molecular weight of OVA (45 kDa). SEA extracted from
3.3. Activation of OT-II CD4+ T cells in vitro For evaluating the proliferation of the OT-II T cells in vitro, purified cells (2 × 105 cells/well) from spleen and lymph nodes of OT-II transgenic mice were labelled with CFSE and incubated with different 4
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Fig. 3. Proliferation of OT-II cells in response to Opt-OVA-expressing S. mansoni eggs. The proliferated cells were gated using the forward and side scatter analysis of the cells. The first lane represents, anti-mouse CD4-PE positive (both proliferated and un-proliferated cells) up on stimulation with OVA peptide 0.1 μg/mL, 10 μg/mL and without stimuli. The next lane represents proliferated cells where cells exposed with un-transduced eggs, cells exposed with empty vector transduced eggs, and cells exposed with eggs transduced with mCherry containing viral constructs. In the third and last lower lane, from left to right represents the transduced and untransduced eggs from high dose to lower dose. The % of proliferating cells is written in the top left corner each graph. The reproducibility of the experiment was confirmed by conducting the whole experiment twice using two biological replicates for each experimental group.
4. Discussion
numbers of transduced/un-transduced eggs or controls. As shown in Fig. 3, CD4+ T cells proliferated in the presence of OVA peptide (10.9% at 0.1 μg/mL and 51.2% at 10 μg/mL). Non-specific proliferation was marginal when OT-II cells were incubated with 25 un-transduced eggs per well (1.96%) or cells only (2.63%). No proliferation was observed when OT-II cells were stimulated with 25 eggs per well, transduced with either empty vector contain no transgene or mCherry encoding vector. In contrast, the proportion of CD4+ T cells that proliferated was 8.99%, 9.82%, 9.79%, and 9.67% when exposed to 200, 100, 50, and 25 transduced eggs, respectively. Un-transduced eggs induced a lower percentage of proliferation in line with background proliferation observed in the negative controls (1.87%–2.49%).
As a result of the complex biology of the schistosome parasite, generation of a transgenic parasite eggs has been challenging. However, recently, introduction of foreign genes was achieved using lentiviruses as a vector [Hagen et al., 2014]. Lentiviral vectors, which are modified from the type 1 human immunodeficiency virus (HIV-1), have become the main tool for gene-delivery to mammalian cells and are known for their important features of mediating strong transduction and stable expression both in vitro and in vivo. Here we demonstrated that eggs can be transduced by a lentivirus and that these transduced eggs not only produce the OVA, but most importantly that this antigen can be recognised by OT-II T cells expressing a well-defined Vα2 and Vβ5 TCR recognising an MHC-restricted antigenic OVA323-339 peptide [Kouskoff et al., 1995]. The OVA protein sequence used was optimized by replacing the first 47 amino acids by the signal sequence of omega-1, containing 23 amino acids and adding 6-histidine sequence at 3ʹ end and for cloning in to the vector, restriction sites of Spel and NotI were also added at the 5ʹ and 3ʹ end of the sequence respectively. Unexpectedly, we did not observe higher levels of OT-II cell proliferation, when we used more OVA-transduced eggs. Although we have shown, by co-incubation of eggs in the presence of a mitogen, that T cells can be activated in the presence of 200 eggs, we can't exclude that eggs might reduce proliferation, especially when low amounts of OVA expressed in the eggs. Thus, at low levels of OVA-transduced eggs there might be insufficient OVA to induce full proliferation, while at high egg-concentrations the eggs might inhibit proliferation to some degree. This is supported by the observation that proliferation was much higher when
3.4. Secretion of cytokines by OT-II CD4+ T cells The level of cytokine expression in the culture supernatants of CD4+ OT-II T cells incubated with either transduced or un-transduced eggs was calculated (Fig. 4). The OT-II cells stimulated with transduced eggs produce significantly more TNF-α, IFN-γ, IL-6 and IL-17, than cells stimulated with un-transduced eggs at all egg concentration except 25 eggs/well. The expression of IL-10 and IL-4 was only marginally higher than the detection limit of the assay for certain conditions only (16.8 pg/mL for IL-10 and 0.03 pg/mL respectively) and no statistically significant differences between groups were detected. Interestingly, and in contrast to the other cytokines, the levels of IL2 detected by the assay was higher with lower concentrations of transduced eggs and as a result the largest differences were detected only at the lowest concentrations of eggs (50 and 25 eggs/well). 5
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Fig. 4. A bar graph showing the amounts of cytokine expressed by OT-II cells following stimulation with transduced (T) and un-transduced (U), S. mansoni eggs. The number of eggs used to stimulate the OT-II cells, in each well follows each condition (either T or U). Horizontal bar line with ns showed non-significant and a symbol with * showed level of significant difference (*: p < 0.05; **: p < 0.01; ***: p < 0.001). A horizontal line in each graph indicates the detection limit for each cytokine. The reproducibility of the experiment was confirmed by conducting the whole experiment twice using two biological replicates for each experimental group.
we used 10 μg/mL of OVA ppt (51.20%) compared to 0.1 μg/mL of OVA (10.90%). During schistosome infections, cytokines are important in determining the type of the immune response induced including Th1, Th2 or Th17 cells. However, the precise function of cytokines in the regulation of granulomatous response is not fully understood. Most of the existing knowledge about the granuloma formation suggests that it is associated with increasing production of Th2 cytokines such as IL-4, IL5 and IL-13 [Chensue et al., 1994; Chiaramonte et al., 1999] and this is down- modulated by IL-10 and egg antigen specific antibodies [Mola et al., 1999]. Here we established that naïve antigen-specific T cells stimulated with S. mansoni eggs induce the Th1 cytokines TNF-α and IFN-γ as well as low levels of IL-17A. The production of IL-6, a predominantly Th2 cytokine may appear out of place in this context. However, macrophages as well as DCs, and B cells also produce IL-6. This cytokine regulates both Th1 and Th2 differentiation under two mechanisms; (1) by inhibiting Th1 induction in the absence of IL-4 and nuclear factor of activated T cells or NFAT, (2) by up-regulating Th2 differentiation in the presence of IL-4 [Diehl and Mercedes, 2002]. The apparent greater concentration of IL-2 in the wells stimulated with lower number of transduced eggs could be explained by the fact
that T cell activation increases the expression of the high affinity IL-2R on T cells. Thus, if more T cells are activated in the presence of larger number of transduced eggs, as suggested by the proliferation assay (Fig. 4), one could envisage that more of the produced IL-2 could be consumed by these activated T cells, leaving less for detection in the supernatant [Boymann and Jonathan. 2012]. The low amount of IL-10 detected, suggest that this cytokine does not play a critical role in our model system. Evidences from human patients also showed that the amount of IL-10 was low during S. mansoni infection but nevertheless this IL-10 could contribute to the development of fibrosis [Booth et al., 2004; Oliviera et al., 2006]. Taken together, our results suggest that a predominant Th1 response is induced in vitro following exposure to S. mansoni eggs. This result appears in contradiction with the observation that S. mansoni eggs induce a Th2 response during in vivo infection. One possible explanation for this discrepancy is that not all cells that implicated in the induction of the expected Th2 response in vivo are present in our in vitro. For example, it is possible that some of the critical DC populations might not be present in sufficient numbers in our OT-II-based system. Another possibility is that OT-II cells are intrinsically biased towards Th1 responses. This has been suggested previously [Leung et al., 2013], at
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least when the OT-II cells are stimulated with B cells as APCs. In our case however, the OT-II cells can be stimulated not only by B cells but also by a range of other APCs present in our cell cultures. The results presented here suggest that the expression of OVA in the parasite can be recognised by CD4+ T cells and that this will be useful for the analysis of immune responses in vivo particularly following adoptive transfer of in vitro differentiated or naïve OT-II cells. Hence these experiments would shed light on the cellular and molecular mechanisms utilised by the parasite to modulate the host's immunity.
alternatively activated macrophages. FASEB J. 22 (11), 4022–4032. https://doi.org/ 10.1096/fj.08-106278. Everts, B., Perona-Wright, G., Smits, H.H., Hokke, C.H., Van Der Ham, A.J., Fitzsimmons, C.M., Doenhoff, M.J., et al., 2009. Omega-1 , a glycoprotein secreted by Schistosoma mansoni Eggs, drives Th2 responses. J. Exp. Med. 206 (8), 1673. https://doi.org/10. 1084/jem.20082460. Fallon, P.G., Antonio, A., 2006. Pathogen-derived immunomodulatory molecules: future immunotherapeutics? Trends Immunol. 27 (10), 470–476. https://doi.org/10.1016/ j.it.2006.08.002. Fallon, P.G., Richardson, E.J., McKenzie, G.J., McKenzie, A.N.J., 2000. Schistosome infection of transgenic mice defines distinct and contrasting pathogenic roles for IL-4 and IL-13: IL-13 is a profibrotic agent. J. Immunol. 164 (5), 2585–2591. https://doi. org/10.4049/jimmunol.164.5.2585. Hagen, J., Young, N.D., Every, A.L., Pagel, C.N., Schnoeller, C., Scheerlinck, J.Y., Gasser, R.B., et al., 2014. Omega-1 knockdown in Schistosoma mansoni eggs by lentivirus transduction reduces granuloma size in vivo”. Nat. Commun. 5, 1–9. https://doi.org/ 10.1038/ncomms6375. Hagen, J., Scheerlinck, J.Y., Young, N.D., Gasser, R.B., Kalinna, B.H., 2015. Prospects for vector-based gene silencing to explore immunobiological features of Schistosoma mansoni. Adv. Parasitol. 88, 85–122. https://doi.org/10.1016/bs.apar.2015.02.002. Kane, C.M., Jung, E., Pearce, E.J., 2008. Schistosoma mansoni egg antigen-mediated modulation of toll-like receptor (TLR) -induced activation accurs andependently. Infect. Immun. 76 (12), 5754–5759. https://doi.org/10.1128/IAI.00497-08. Kouskoff, V., Signorelli, K., Benoist, C., Mathis, D., 1995. Cassette vectors directing expression of T cell receptor genes in transgenic mice. J. Immunol. Methods 180 (95), 273–280. https://doi.org/10.1016/0022-1759(95)00002-R. Leung, S., Smith, D., Myc, A., Morry, J., Baker Jr., J.R., 2013. OT-II TCR transgenic mice fail to produce anti-ovalbumin antibodies upon vaccination. Cell. Immunol. 282 (2), 79–84. Mann, V.H., Maria, E.M., Gabriel, R., Paul, J., 2011. Culture for genetic manipulation of developmental stages of Schistosoma mansoni. Parasitology 137 (3), 451–462. https:// doi.org/10.1017/S0031182009991211. Mola, P.W., Farah, I.O., Kariuki, T.M., Nyindo, M., Blanton, R.E., King, C.L., 1999. Cytokine control of the granulomatous response in Schistosoma mansoni- infected baboons: role of exposure and treatment. Infect. Immun. 67 (12), 6565–6571. Moreno-Cid, J.A., de La Lastra, J.M., Villar, M., Jiménez, M., Pinal, R., Estrada-Peña, A., et al., 2013. Control of multiple arthropod vector infestations with subolesin/akirin vaccines. Vaccine 31, 1187–1196. https://doi.org/10.1016/j.vaccine.2012.12.073. Oliveira, L.F., Alves, E.C., Moreno, G.G., Silveira, A.M.S., Gazzinelli, A., Loverde, P., Martins, L.P., 2006. Cytokine production associated with periportal fibrosis during chronic Schistosomiasis mansoni in humans. Infect. Immun. 74 (2), 1215–1221. https://doi.org/10.1128/IAI.74.2.1215. Pearce, E.J., Kane, C., Sun, J.J., Taylor, J., McKee, A.S., Cervi, L., 2004. Th2 response polarization during infection with the helminth parasite Schistosoma mansoni. Immunol. Rev. 201, 117–126. Sheerin, D., Openshaw, P.J., Pollard, A.J., 2017. Issues in vaccinology: present challenges and future directions. Eur. J. Immunol. 47, 2017–2025. https://doi.org/10.1002/eji. 201746942. Sinden, R.E., 2017. Developing transmission-blocking strategies for malaria control. PLoS Pathol. 13, e1006336. https://doi.org/10.1371/journal.ppat.1006336. Steinfelder, Svenja, Andersen, John F., Cannons, Jennifer L., Feng, Carl G., Joshi, Manju, Dwyer, Dennis, Caspar, Pat, Schwartzberg, Pamela L., Sher, Alan, Jankovic, Dragana, 2001. The major component in schistosome eggs responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease (Omega-1). J. Exp. Med. 206 (8), 1681–1690. https://doi.org/10.1084/jem.20082462. World Health Organization, 2015. Fact Sheet No 115 (May). (Geneva, Switzerland).
Acknowledgements We are grateful to Melbourne International Research Scholarship for research training sponsorship. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.exppara.2019.107767. References Bergquist, N.R., 1995. Controlling schistosomiasis by vaccination : a realistic option ? Trends Parasitol. 1 (5), 3–6. https://doi.org/10.1016/0169-4758(95)80157-X. Booth, M., Mwatha, J.K., Joseph, S., Jones, F.M., Kadzo, H., Ireri, E., Kazibwe, F., et al., 2004. Periportal fibrosis in human Schistosoma mansoni infection is associated with low IL-10, low IFN-γ , high TNF-α , or low RANTES, depending on age and gender. J. Immunol. 172 (2), 1295–1303. https://doi.org/10.4049/jimmunol.172.2.1295. Boros, D.L., Warren, K.S., 1970. Delayed hypersensitivity granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. Exp. Med. 132, 488–507. https://doi.org/10.1084/jem.132.3.488. Boyman, O., Jonathan, S., 2012. The role of interleukin-2 during homeostasis and activation of the immune system. Nat. Rev. Immunol. 12 (3), 180–190. https://doi.org/ 10.1038/nri3156. Brunet, L.R., Finkelman, F.D., Cheever, A.W., Kopf, M.A., Pearce, E.J., 1997. IL-4 protects against TNF-α-mediated cachexia and death during acute schistosomiasis. J. Immunol. 159 (2), 777–785. Chensue, S.W., Warmington, K.S., Ruth, J., Lincoln, P.M., Kunkel, S.L., 1994. Cross-regulatory role of interferon-gamma (IFN-γ), IL-4 and IL-10 in schistosome egg granuloma formation: in vivo regulation of Th activity and inflammation. Clin. Exp. Immunol. 98, 395–400. Chiaramonte, M.G., Schopf, L.R., Neben, T.Y., Cheever, A.W., Donaldson, D.D., Wynn, T.A., 1999. IL-13 is a key regulatory cytokine for Th2 cell-mediated mulmonary granuloma formation and IgE responses induced by Schistosoma mansoni Eggs. J. Immunol. 162 (2), 920–930. https://doi.org/10.4049/jimmunol.167.11.6533. Chitsulo, L., Engels, D., Montresor, A., Savioli, L., 2017. Europe PMC funders group. The global status of schistosomiasis and its control. Acta Trop. 77 (1), 41–51. Diehl, S., Mercedes, R., 2002. The two faces of IL-6 on Th1/Th2 differentiation. Mol. Immunol. 39 (9), 531–536. https://doi.org/10.1016/S0161-5890(02)00210-9. Donnelly, S., Stack, C.M., O'Neill, S.M., Sayed, A.A., Williams, D.L., Dalton, J.P., 2008. Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Recognition of Schistosoma mansoni egg-expressed ovalbumin by T cell receptor transgenic mice
T
Mebrahtu G. Tedla1,∗, Musammat F. Nahar1, Jana Hagen2, Alison L. Every3, Jean-Pierre Y. Scheerlinck Centre for Animal Biotechnology, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: OT II T cells Ovalbumin Schistosoma mansoni Transgenesis
Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.
1. Introduction Schistosomiasis, a parasitic disease caused by trematodes or digenetic blood flukes, is one of the most devastating tropical diseases. It is endemic in 74 countries and affects more than 200 million people worldwide, with 85% of patients living in Africa [Chitsulo et al., 2017]. Regular and periodic chemotherapy of at-risk groups with praziquantel, with potency against the Schistosoma adult parasite, is the only available intervention strategy [WHO, 2015]. As a result, the global and regional occurrence of the diseases has declined in the past few decades. However, elimination of the disease remains a global challenge [WHO, 2015]. While an effective vaccine is not yet available, the recent discovery of specific vaccine antigens has shown promise. Drug treatment targeting the parasite and control of the arthropod vector combined with vaccines might offer a long-term solution (Moreno-Cid et al., 2013; Sinden, 2017), despite genetic variation among parasitic strains, which could affect vaccine coverage (Sheerin et al., 2017). However, induction of effective protective immunity remains out of reach at least
in part because of the ability of the parasite to modulate immune response. Therefore, understanding how the parasite persists in the face of robust stimulation of the host immune system is one of the most important aspects in the study of host-parasite interactions [Hagen et al., 2015]. Indeed, S. mansoni, is known to have specific tactics to manipulate the host immune response in order to ensure its survival in the host [Bergquist, 1995]. Modulating the immune system at a molecular level via, in particular, soluble egg antigens (SEAs) is one of the key strategies for S. mansoni to compromise host immunity [Kane et al., 2008]. However, the mechanism of this specific immunomodulation remains elusive, particularly in vivo [Fallon and Antonio, 2006]. The immunomodulatory potential of S. mansoni eggs at the onset of chronic infection, is well documented and characterized by a Th2 polarized immune response [Pearce et al., 2004]. This Th2 response is associated with down-regulation of the initial Th1 or pro-inflammatory responses to migrating cercaria and leads to granuloma formation [Brunet et al., 1997; Fallon et al., 2000]. Many SEAs have been identified in S.
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Corresponding author. E-mail address: [email protected] (M.G. Tedla). 1 Both authors contributed equally. 2 Current address: Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK. 3 Current address: College of Science, Health and Engineering, La Trobe University, Victoria, 3086. https://doi.org/10.1016/j.exppara.2019.107767 Received 6 May 2019; Received in revised form 7 September 2019; Accepted 8 September 2019 Available online 11 September 2019 0014-4894/ © 2019 Elsevier Inc. All rights reserved.
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protocol described previously [Mann et al., 2011]. Briefly, livers were extracted from mice, diced and digested overnight in shaking incubator at 120 rpm, 37 °C with 25 mg collagenase IV (Sigma Aldrich) in 40 mL of PBS and 10 mL of DMEM (Gibco, Life Technologies) containing 200 μg/mL gentamicin (Sigma Aldrich), 100 U/mL of penicillin (Gibco, Life Technologies), and 100 μg/mL of streptomycin (Gibco, Life Technologies). Subsequently, liver homogenates were washed three times with PBS containing Penicillin-Streptomycin (10,000U/mL) and resuspended in 5 mL of PBS. Eggs were purified over a percoll-sucrose gradient (Sigma Aldrich) consisting of 8 mL percoll and 32 mL of 250 mM sucrose by centrifuging at 800×g for 12 min. Finally, eggs were washed twice and resuspended in DMEM media (Sigma Aldrich) supplemented with 4.5 g of glucose, 1 mM sodium pyruvate, 2 mM Lglutamine, 1 mM HEPES, 100 U/mL penicillin, 100 μg/mL of streptomycin and 10% v/v of FBS and cultured at 37 °C and 5% CO2. Freshly isolated eggs of S. mansoni were transduced as described previously [Hagen et al., 2014]. Briefly, eggs were exposed to 100 μL of virus particles at a titter of 25 × 103 pfu per mL of OVA-encoding lentiviral particles or left untreated in complete DMEM medium (10 mM HEPES, 100U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 8 μg/mL polybrene) and incubated at 37 °C and 5% CO2. Further controls included a virus encoding mCherry or virus lacking a transgene sequence (empty vector). After 24 h, eggs were washed three times with PBS and cultured for an additional four days in complete DMEM medium. Afterwards, eggs were washed in PBS and resuspended in RPMI medium for subsequent T cell assays. Throughout all the experiments, fresh eggs were used and maintained in complete DMEM media. Expression of functional OVA protein confirmed egg viability.
mansoni, including omega-1 now understood to drive Th2-polarized responses, through interaction with dendritic cells (DCs). This protein plays an important role in switching the Th1 response into a Th2 response through inhibition of IL-12 production [Everts et al., 2009; Steinfelder et al., 2001]. Moreover, other SEAs, such as, IPSE/α-1, trigger Ig-E-driven IL-4 secretion by basophils and induces alternatively activated macrophages and, thus, Th2-polarized responses [Donnelly et al., 2008]. Kappa-5 is another immunomodulatory SEA present in S. mansoni. In the present study, we utilised S. mansoni-expressed chicken ovalbumin (OVA), to study the immune response of parasite-associated antigens in inducing biased Th responses in naïve T cells. OVA being a model antigen avoids the induction of biased immune response and is ideal for such investigations as it is difficult to exclude that parasitederived antigens have intrinsic biases selected as a result of prolonged evolutionary pressures. The expression of OVA in the parasite therefore helps to elucidate the immunomodulatory properties of the soluble egg antigens of the parasite egg and, using the expressed protein as a neutral antigen, we are able to further characterise functions of specific SEAs. As a first step, demonstrating that T cell receptor transgenic mice (OT-II mice) known for having higher frequency of CD4+ T cell receptors that recognise OVA, as a source of naïve CD4+ T cells that can recognise S. mansoni expressed OVA, is essential. Here we show that, OT-II cells respond to this antigen by proliferating and producing key cytokines in vitro. 2. Material and methods 2.1. Experimental mice and maintenance of the S. mansoni life cycle Six- to eight-week-old female BALB/c and OT-II mice were purchased from the Walter and Eliza Hall Institute of Medical Research (WEHI) and were used in the present study to maintain the life cycle of S. mansoni and as a source of naïve OT-II T cells, respectively. All the experiments related with mice were conducted after getting an approval from the Animal Ethical Committee of the University of Melbourne (Ethics ID: 1312952). Snails (Biomphlaria glabrata, NMRI strain), an intermediate host of the parasite were purchased from NIHNI-AID, Schistosomiasis resource centre, USA.
2.4. RNA extraction, RT-PCR To detect and analyse mRNA synthesis, parasite eggs were extracted and homogenized four days after transduction with lentivirus, based on the protocol modified from TRI Reagent® Protocol (Sigma Aldrich). Briefly, eggs were homogenized and further ultra-sonicated to break the egg shells and the homogenates were resuspended in 1 mL of Tri reagents (Sigma Aldrich) and incubated for 5 min at room temperature. Chloroform (0.2 mL) was added to the suspension and the mixture was further incubated at room temperature for 15 min. After incubation, the mixture was centrifuged at 12,000×g for 10 min at 4 °C and the clear aqueous layer was collected to purify RNA and 0.5 mL of isopropanol was added to precipitate the RNA. The precipitated RNA was further incubated at room temperature for 10 min and centrifuged at 12,000×g for 8 min. The RNA pellet was washed twice with 1 mL of 75% ethanol and centrifuged at 7500×g for 5 min at 4 °C. Finally, the RNA pellets were air dried for 5 min and resuspended in 50 μL of nuclease free water. The RNA pellets were solubilized by passing through a pipet tip and further incubated for 10 min at 55 °C. Average purity of RNA was confirmed by a ratio of absorbance at A260/A230 of 1.93. Finally, the solubilized RNA was stored at −20 °C.
2.2. Generation of constructs and production of lentiviral particles For expression and secretion in S. mansoni the OVA sequence (GenBank access no: V00438) was modified by N-terminal replacing the first 47 amino acids with the signal peptide of S. mansoni omega-1 (GenBank access no: DQ013207) and a 6-Histidine (his)-tag to the Cterminus. The modified OVA sequence was optimized for schistosome codon usage and synthesized by Gen Script (NJ, USA) and cloned into pGIPZ vector [Hagen et al., 2014], via SpeI and NotI restriction sites, replacing mCherry (Fig. 1). Plasmids were maintained in Stbl3 E. coli and clones were verified by Sanger sequencing. Replication-incompetent lentiviral particles were produced in HEK293T cells after cotransfection of the lentiviral expression plasmid and TransLenti™ packaging mix (Open Biosystems, Dharmacon™) according to the manufactures instructions. Functional virus titres were estimated following a modified protocol of the commercially available retro-viral detection kit given by Takara Bio Inc, the virus titter was determined by semi quantitative real time PCR reaction using MX3000P real-time PCR cycler (Stratagene, La Jolla, CA, USA). Forward (5ʹ-TGGACAGGGGCT CGGCTGTT-3ʹ) and Reverse (5ʹ-CCGCTGGATTGAGGGCCGA-3ʹ) primers were used to amplify the cDNA. The samples were run in triplicate and average Ct value and copy number was taken.
2.5. Synthesis of cDNA from viral genomic RNA For the reverse transcription of viral genomic RNA into complementary DNA (cDNA), virus supernatants collected from cell culture were used as a source of template. The thermal profile of the reverse transcription process was adjusted in each step using the thermocycler (MJ mini personal thermal cycler, Bio-Rad Laboratories, San Francisco, CA, USA), following the manufacturers recommendation. Sequence of the primers used for RT-PCR for OVA transcript in the RNA recovered from the schistosome eggs were F-5′-ATGAAGATACACAAGCAATG CCA-‘3; R-5’-TCATAGTTCCAGAAGCGAATGGT′-3’.
2.3. Isolation and lentiviral transduction of S. mansoni eggs S. mansoni eggs were isolated from mouse livers following the 2
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Fig. 1. (A) OVA, mCherry and pGIPZ vector without transgene constructs: LTR, long terminal repeat; ψ, packaging signal; cPPT, poly-purinetract; ZeoR, zeomycin resistant gene; CMV, cytomegalovirus promoter; mCherry, mCherry encoding transgene; WPRE, woodchuck hepatitis virus post-translational regulatory element; SIN, self-inactivating LTR; OVA-CMV, Optimized OVA with CMV promoter. (B) Screening of clones by colony PCR yielded Opt-OVA positive clones. DNA region amplified from the upstream of the promoter to the middle of the Opt-OVA insert. (C) Generation of 163 bp RT-PCR product using agarose gel electrophoresis. (D) PCR amplification product of the cDNA synthesized from virus supernatants containing the mCherry construct. The numerical labelling 1, 2, 3, 4 are the 5-fold serial dilutions of the viruses. Generation of the same PCR products and the dilutions are also similar with the exception in which the reference of the viral construct is the optimized OVA. The pGIPZ empty vector was used as a negative control.
2.6. Preparation of soluble egg antigen and Western blot
2.7. T cell proliferation assay
According to the protocol modified from Boros and Warren (1970), SEAs were prepared five days after virus exposure. Approximately 6000 transduced and un-transduced eggs were suspended in cold PBS and homogenized using a T10 basic ultra-turrax® until the shells of the eggs were completely crushed, which was confirmed using light microscopy. The egg homogenates were further exposed to ultrasonic pulses using a microson™ ultrasonic cell disruptor at 30 s bursts until complete breakdown was achieved. After centrifugation at 8765×g, the supernatants were passed through a 0.2 μm filter and analysed by SDS-PAGE and Western blot. Immunoblotting was carried out after transfer to a nitrocellulose membrane with 1:1000 anti-rabbit IgG fraction to chicken egg ovalbumin (MP Bio medicals) and 1:2000 anti-HRP-conjugated swine anti-rabbit immunoglobulins (DAKO). Proteins were visualised by chemiluminescence (ECL™ western blot detection reagents, GE Health care life science, Australia).
Spleen, maxillary, axillary and inguinal lymph nodes of OT-II mice were collected and pooled for cell extraction using complete RPMI1640 medium (Invitrogen, Life Technologies). Single cell suspensions were obtained by passing the tissue through a 70 μm nylon cell strainer. Red blood cells were lysed by water and lysis was stopped using PBS. Cells were then resuspended in complete RPMI-1640 medium. For proliferation assays, cells were stained with carboxyfluorescein-diacetate-succinimidyl-ester (CFSE; Bio Legend, San Diago, California, USA) according to manufacturer's instruction. A total of 2 × 105 labelled cells per well were co-incubated with 25, 50, 100, or 200 transduced or untransduced S. mansoni eggs for 4–5 days in 96 well plates at 37 °C, 5% CO2. After four days culturing, OT-II cells were collected and washed with FACS buffer (10% FCS in 1× PBS). For proliferation analysis, cells were stained with PE-anti-mouse CD4 (Bio Legend) following an FcγRsurface block (antibody, manufacturer). To exclude dead cells, 7-aminoactinomycin D (1 μg/mL; Sigma Aldrich, Steinheim, Germany) was added to cell suspension prior data acquisition using The BD
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FACSVerse™ flow cytometer (BD Bioscience, New Jersey, USA). 2.8. Cytokine assay Cell culture supernatants from T cell proliferation assays were analysed for cytokine production by mouse Th1, Th2, and Th17 cytometric bead array (BD™). According to the manufacturer, the detection limits for each cytokine were: IL-2 (0.1 pg/mL), IL-4 (0.03 pg/mL), IL-6 (1.4 pg/mL), IFN-γ (0.5 pg/mL), TNF-α (0.9 pg/mL), IL-17A (0.8 pg/ mL) and IL-10 (16.8 pg/mL). 2.9. Statistical analysis Data was computed and analysed using the single cell analysis software: flowjo version 10 (Ashland, Orgeon, USA). All statistical analyses were performed using one-way ANOVA followed by t-test using Graph Pad Prism version 7.00 for Windows (GraphPad Software, La Jolla, California, USA). In all the analyses, the confidence level was held at 95% and p < 0.05 was required for significance. 3. Results 3.1. Optimisation of OVA for expression and secretion in S. mansoni eggs following lentiviral transduction The first 47-amino acid signal peptide sequence of original OVA was replaced by the 23-amino acid of S. mansoni ω-1 signal sequence. And 6histidine sequence was added at the C-terminal end of the optimized sequence. Two restriction sites, SpeI at the N-terminal end and NotI at the C-terminal end were included to the sequence. After the sequence was synthesized commercially by GenScript (NJ, USA), the gene was cloned in to the pGIPZ_CMV_mCherry via SpeI and NotI replacing mCherry to produce pGIPZ Opt-OVA (Fig. 1A). In order to generate a promoter-less expression vector (OVA-PL), Opt-OVA was cloned into pGIPZ-SmACT vector construct via SpeI and NotI replacing the SmACT_mCherry region and a colony PCR screening confirmed successful transformation (Data not shown). Due to the similarities in the total number of amino acids and following expression of both the optimized OVA and normal OVA by the parasite egg, we used both names interchangeably. Integration of Opt-OVA into the vector was confirmed by amplifying the segment upstream of the promoter to the middle of the Opt-OVA insert, using colony PCR (Fig. 1B). The presence of the OVA (Fig. 1C) and mCherry (Fig. 1D) in the virus was confirmed by reverse transcription PCR, followed by agarose gel electrophoresis.
Fig. 2. Transcription of the Opt-OVA encoding gene and expression of OVA by transduced S. mansoni eggs. Reverse transcription PCR of OVA-transduced (6000 and 5000 eggs) and control eggs (6000 eggs) showing the presence of an OVA-specific mRNA band of 149 bp in transduced eggs that is absent in the untransduced eggs (A). Additional controls (B), showing the presence of parasite genetic material in each lane through the expression of IPSE irrespective of whether the parasites were transduced with the OVA gene or not. Panel B also shows that OVA mRNA is increased when OVA was optimized for expression in S. mansoni (OVA vs Opt-OVA). PCR product sizes: Opt-OVA; 149bp and IPSE; 263bp. M, molecular weight marker; UnTd, untransduced; Opt-OVA-PL, optimized OVA promoter-less; Opt-OVA, optimized OVA. SDS-PAGE of OVAtransduced (6,000, 5000 and < 2400) and control S. mansoni eggs (6,000) (C) and the same gel following probing with specific primary antibodies to OVA followed by enzyme-labelled secondary antibody (D).
the same number of un-transduced eggs were used as a negative control; in these samples, there was no strongly stained band at this molecular weight. To further confirm the expression, proteins were transferred electrophoretically onto a nitrocellulose membrane and a specific band of 45 kDa was stained with antibodies to OVA, confirming the expression of this protein (Fig. 2C and D). To demonstrate that the OVA protein was produced by the transduced eggs and was not transferred together with the virus as a contaminant in the HEK-293T cell supernatant, we performed the transduction protocol with both heat inactivated (90 °C for 10 min) and Opt-OVA-promoter-less virus constructs. As expected no OVA was detected unless a viable and functional OVA-expressing virus was used in the transduction protocol (Data not shown). To determine if the OVA protein was secreted into the supernatant following viral transduction of the eggs, we subjected the supernatant and egg lysate to a capture ELISA and could only detect the presence of OVA in the lysate. Using purified OVA as a control the detection limit of the capture ELISA was estimated to be 5 ng/mL.
3.2. Detection of Opt-OVA in transduced S. mansoni eggs The expression of OVA was analysed by SDS-PAGE using 6,000, 5,000, < 2400 transduced eggs per lane. The control contained 6000 un-transduced eggs per lane. First the expression of the OVA gene was confirmed using a reverse transcription PCR after performing cDNA synthesis from mRNA of transfected eggs. To achieve this, pools of transduced or un-transduced eggs were used for the extraction of total RNA. After synthesis of cDNA, the OVA backbone was amplified using OVA-specific forward and reverse primers and revealed a 163 bp amplicon and RNA from un-transduced eggs was also extracted and reverse transcribed, but it showed no amplification. The Opt-OVA- and S. mansoni IPSE gene were also amplified by standard PCR followed by cDNA synthesized from mRNA by reverse transcriptase and revealed a PCR product sizes of 149 bp of Opt-OVA- and 263bp of S. mansoni IPSE (Fig. 2A and B). SEA was separated by SDS-PAGE and Coomassie blue staining was performed to analyse total protein in each sample. The lysate from eggs transduced with Opt-OVA virus, contained an additional band corresponding to a molecular weight of OVA (45 kDa). SEA extracted from
3.3. Activation of OT-II CD4+ T cells in vitro For evaluating the proliferation of the OT-II T cells in vitro, purified cells (2 × 105 cells/well) from spleen and lymph nodes of OT-II transgenic mice were labelled with CFSE and incubated with different 4
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Fig. 3. Proliferation of OT-II cells in response to Opt-OVA-expressing S. mansoni eggs. The proliferated cells were gated using the forward and side scatter analysis of the cells. The first lane represents, anti-mouse CD4-PE positive (both proliferated and un-proliferated cells) up on stimulation with OVA peptide 0.1 μg/mL, 10 μg/mL and without stimuli. The next lane represents proliferated cells where cells exposed with un-transduced eggs, cells exposed with empty vector transduced eggs, and cells exposed with eggs transduced with mCherry containing viral constructs. In the third and last lower lane, from left to right represents the transduced and untransduced eggs from high dose to lower dose. The % of proliferating cells is written in the top left corner each graph. The reproducibility of the experiment was confirmed by conducting the whole experiment twice using two biological replicates for each experimental group.
4. Discussion
numbers of transduced/un-transduced eggs or controls. As shown in Fig. 3, CD4+ T cells proliferated in the presence of OVA peptide (10.9% at 0.1 μg/mL and 51.2% at 10 μg/mL). Non-specific proliferation was marginal when OT-II cells were incubated with 25 un-transduced eggs per well (1.96%) or cells only (2.63%). No proliferation was observed when OT-II cells were stimulated with 25 eggs per well, transduced with either empty vector contain no transgene or mCherry encoding vector. In contrast, the proportion of CD4+ T cells that proliferated was 8.99%, 9.82%, 9.79%, and 9.67% when exposed to 200, 100, 50, and 25 transduced eggs, respectively. Un-transduced eggs induced a lower percentage of proliferation in line with background proliferation observed in the negative controls (1.87%–2.49%).
As a result of the complex biology of the schistosome parasite, generation of a transgenic parasite eggs has been challenging. However, recently, introduction of foreign genes was achieved using lentiviruses as a vector [Hagen et al., 2014]. Lentiviral vectors, which are modified from the type 1 human immunodeficiency virus (HIV-1), have become the main tool for gene-delivery to mammalian cells and are known for their important features of mediating strong transduction and stable expression both in vitro and in vivo. Here we demonstrated that eggs can be transduced by a lentivirus and that these transduced eggs not only produce the OVA, but most importantly that this antigen can be recognised by OT-II T cells expressing a well-defined Vα2 and Vβ5 TCR recognising an MHC-restricted antigenic OVA323-339 peptide [Kouskoff et al., 1995]. The OVA protein sequence used was optimized by replacing the first 47 amino acids by the signal sequence of omega-1, containing 23 amino acids and adding 6-histidine sequence at 3ʹ end and for cloning in to the vector, restriction sites of Spel and NotI were also added at the 5ʹ and 3ʹ end of the sequence respectively. Unexpectedly, we did not observe higher levels of OT-II cell proliferation, when we used more OVA-transduced eggs. Although we have shown, by co-incubation of eggs in the presence of a mitogen, that T cells can be activated in the presence of 200 eggs, we can't exclude that eggs might reduce proliferation, especially when low amounts of OVA expressed in the eggs. Thus, at low levels of OVA-transduced eggs there might be insufficient OVA to induce full proliferation, while at high egg-concentrations the eggs might inhibit proliferation to some degree. This is supported by the observation that proliferation was much higher when
3.4. Secretion of cytokines by OT-II CD4+ T cells The level of cytokine expression in the culture supernatants of CD4+ OT-II T cells incubated with either transduced or un-transduced eggs was calculated (Fig. 4). The OT-II cells stimulated with transduced eggs produce significantly more TNF-α, IFN-γ, IL-6 and IL-17, than cells stimulated with un-transduced eggs at all egg concentration except 25 eggs/well. The expression of IL-10 and IL-4 was only marginally higher than the detection limit of the assay for certain conditions only (16.8 pg/mL for IL-10 and 0.03 pg/mL respectively) and no statistically significant differences between groups were detected. Interestingly, and in contrast to the other cytokines, the levels of IL2 detected by the assay was higher with lower concentrations of transduced eggs and as a result the largest differences were detected only at the lowest concentrations of eggs (50 and 25 eggs/well). 5
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Fig. 4. A bar graph showing the amounts of cytokine expressed by OT-II cells following stimulation with transduced (T) and un-transduced (U), S. mansoni eggs. The number of eggs used to stimulate the OT-II cells, in each well follows each condition (either T or U). Horizontal bar line with ns showed non-significant and a symbol with * showed level of significant difference (*: p < 0.05; **: p < 0.01; ***: p < 0.001). A horizontal line in each graph indicates the detection limit for each cytokine. The reproducibility of the experiment was confirmed by conducting the whole experiment twice using two biological replicates for each experimental group.
we used 10 μg/mL of OVA ppt (51.20%) compared to 0.1 μg/mL of OVA (10.90%). During schistosome infections, cytokines are important in determining the type of the immune response induced including Th1, Th2 or Th17 cells. However, the precise function of cytokines in the regulation of granulomatous response is not fully understood. Most of the existing knowledge about the granuloma formation suggests that it is associated with increasing production of Th2 cytokines such as IL-4, IL5 and IL-13 [Chensue et al., 1994; Chiaramonte et al., 1999] and this is down- modulated by IL-10 and egg antigen specific antibodies [Mola et al., 1999]. Here we established that naïve antigen-specific T cells stimulated with S. mansoni eggs induce the Th1 cytokines TNF-α and IFN-γ as well as low levels of IL-17A. The production of IL-6, a predominantly Th2 cytokine may appear out of place in this context. However, macrophages as well as DCs, and B cells also produce IL-6. This cytokine regulates both Th1 and Th2 differentiation under two mechanisms; (1) by inhibiting Th1 induction in the absence of IL-4 and nuclear factor of activated T cells or NFAT, (2) by up-regulating Th2 differentiation in the presence of IL-4 [Diehl and Mercedes, 2002]. The apparent greater concentration of IL-2 in the wells stimulated with lower number of transduced eggs could be explained by the fact
that T cell activation increases the expression of the high affinity IL-2R on T cells. Thus, if more T cells are activated in the presence of larger number of transduced eggs, as suggested by the proliferation assay (Fig. 4), one could envisage that more of the produced IL-2 could be consumed by these activated T cells, leaving less for detection in the supernatant [Boymann and Jonathan. 2012]. The low amount of IL-10 detected, suggest that this cytokine does not play a critical role in our model system. Evidences from human patients also showed that the amount of IL-10 was low during S. mansoni infection but nevertheless this IL-10 could contribute to the development of fibrosis [Booth et al., 2004; Oliviera et al., 2006]. Taken together, our results suggest that a predominant Th1 response is induced in vitro following exposure to S. mansoni eggs. This result appears in contradiction with the observation that S. mansoni eggs induce a Th2 response during in vivo infection. One possible explanation for this discrepancy is that not all cells that implicated in the induction of the expected Th2 response in vivo are present in our in vitro. For example, it is possible that some of the critical DC populations might not be present in sufficient numbers in our OT-II-based system. Another possibility is that OT-II cells are intrinsically biased towards Th1 responses. This has been suggested previously [Leung et al., 2013], at
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least when the OT-II cells are stimulated with B cells as APCs. In our case however, the OT-II cells can be stimulated not only by B cells but also by a range of other APCs present in our cell cultures. The results presented here suggest that the expression of OVA in the parasite can be recognised by CD4+ T cells and that this will be useful for the analysis of immune responses in vivo particularly following adoptive transfer of in vitro differentiated or naïve OT-II cells. Hence these experiments would shed light on the cellular and molecular mechanisms utilised by the parasite to modulate the host's immunity.
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