Leishmania donovani: impairment of the cellular immune response against recombinant ornithine decarboxylase protein as a possible evasion strategy of Leishmania in visceral leishmaniasis.

International Journal for Parasitology xxx (2014) xxx–xxx

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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara

Leishmania donovani: impairment of the cellular immune response against recombinant ornithine decarboxylase protein as a possible evasion strategy of Leishmania in visceral leishmaniasis Anupam Yadav a,1, Ajay Amit a,1, Rajesh Chaudhary a, Arvind Singh Chandel b, Vijay Mahantesh b, Shashi Shekhar Suman c, Subhankar Kumar Singh d, Manas Ranjan Dikhit a,e, Vahab Ali c, Vidyanand Rabidas f, Krishna Pandey f, Anil Kumar g, Pradeep Das h, Sanjiva Bimal a,⇑ a

Division of Immunology, Rajendra Memorial Research Institute of Medical Sciences, Patna 800007, India Dept. of Biotechnology, National Institute of Pharmaceutical Education and Research, Hajipur 844102, India c Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna 800007, India d Division of Microbiology, Rajendra Memorial Research Institute of Medical Sciences, Patna 800007, India e Dept. of Biomedical Informatics, Rajendra Memorial Research Institute of Medical Sciences, Patna 800007, India f Dept. of Clinical Medicine, Rajendra Memorial Research Institute of Medical Sciences, Patna 800007, India g Animal House, Rajendra Memorial Research Institute of Medical Sciences, Patna 800007, India h Dept. of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Patna 800007, India b

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Article history: Received 12 May 2014 Received in revised form 5 August 2014 Accepted 7 August 2014 Available online xxxx Keywords: Visceral leishmaniasis Leishmania donovani ODC IL-10 IFN-c ROS

a b s t r a c t Ornithine decarboxylase, the rate limiting enzyme of the polyamine biosynthesis pathway, is significant in the synthesis of trypanothione, T(SH)2, the major reduced thiol which is responsible for the modulation of the immune response and pathogenesis in visceral leishmaniasis. Data on the relationship between ornithine decarboxylase and the cellular immune response in VL patients are limited. Therefore, we purified a recombinant ornithine decarboxylase from Leishmania donovani (r-LdODC) of approximately 77 kDa and examined its effects on the immunological responses in peripheral blood mononuclear cells of human visceral leishmaniasis cases. For these studies, a-difluoromethylornithine was tested as an inhibitor and was used in parallel in all experiments. The r-LdODC was identified as having a direct correlation with parasite growth and significantly increased the number of promastigotes as well as axenic amastigotes after 96 h of culture. The stimulation of peripheral blood mononuclear cells with r-LdODC up-regulated IL-10 production but not IFN-c production from CD4+ T cells in active as well as cured visceral leishmaniasis cases, indicating a pivotal role for r-LdODC in causing strong immune suppression in a susceptible host. In addition, severe hindrance of the immune response and anti-leishmanial macrophage function due to r-LdODC, as revealed by decreased IL-12 and nitric oxide production, and down-regulation in mean fluorescence intensities of reactive oxygen species, occurred in visceral leishmaniasis patients. There was little impact of r-LdODC in the killing of L. donovani amastigotes in macrophages of visceral leishmaniasis patients. In contrast, when cultures of promastigotes and amastigotes, and patients’ blood samples, were directed against a-difluoromethylornithine, parasite numbers significantly reduced in culture, whereas the levels of IFN-c and IL-12, and the production of reactive oxygen species and nitric oxide, were significantly elevated. Therefore, we demonstrated cross-talk with the use of adifluoromethylornithine which can reduce the activity of ornithine decarboxylase of L. donovani, eliminating the parasite-induced immune suppression and inducing collateral host protective responses in visceral leishmaniasis. Ó 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Division of Immunology, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna 800007, India. Tel.: +91 7677435674; fax: +91 612 2634379. E-mail address: [email protected] (S. Bimal). 1 These authors contributed equally to this work.

1. Introduction Leishmania, a protozoan pathogen, is the causative agent of various forms of leishmaniasis such as cutaneous (CL), mucocutaneous, and visceral leishmaniasis (VL), the latter of which can be

http://dx.doi.org/10.1016/j.ijpara.2014.08.013 0020-7519/Ó 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Yadav, A., et al. Leishmania donovani: impairment of the cellular immune response against recombinant ornithine decarboxylase protein as a possible evasion strategy of Leishmania in visceral leishmaniasis. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/ j.ijpara.2014.08.013

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A. Yadav et al. / International Journal for Parasitology xxx (2014) xxx–xxx

fatal to humans if left untreated (Desjeux, 1992; Croft et al., 2006; Mookerjee et al., 2008; Sunder, 2011). Leishmaniasis is a complex disease that affects approximately 12 million people worldwide and 500,000 new cases are reported annually (McCall et al., 2013). The drugs that are currently used for chemotherapy of leishmaniasis, such as antimonials, miltefosine, paromomycin and amphotericin B, are very toxic and resistance to these drugs occurs in endemic areas (Sundar et al., 1997; Abdo et al., 2003; Das et al., 2005; Jha, 2006). Thus, the major thrust of research in this area is to either inhibit essential transporters or block vital metabolic pathways in the parasite (Guha et al., 2013) as a rationale for new drug development (Barrett et al., 1999). Gene amplification in Leishmania strains is a frequent mechanism through which the parasite resists the action of cytotoxic drugs (Mukherjee et al., 2007). The first amplified gene in Leishmania promastigotes or in axenic amastigotes of Leishmania infantum for Sb(III) resistance was the ATP-binding cassette (ABC) transporter gene (Haimeur et al., 2000; El Fadili et al., 2005), which is believed to efflux antimonials. There are reports that ABC transporters confer resistance to drugs by the sequestration of trypanothione, T(SH)2, which is the major reduced thiol and is found exclusively in Leishmania in an intracellular organelle located close to the flagellar pocket (Legare et al., 2001) or extruded outside the cell by an efflux pump (Dey et al., 1996). Therefore, regulation through appropriate interference in the T(SH)2 pathway can provide clues for rational drug design and vaccine development against VL. T(SH)2 is a peptide amine conjugate that is synthesised due to the overproduction of ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthetic pathway (Haimeur et al., 2000), and c-glutamylcysteine synthetase (cGCS), the rate-limiting enzyme of glutathione (GSH) biosynthesis (Haimeur et al., 2000). Studies related to polyamine synthesis in cloned proteins of Leishmania genes encoding arginase (ARG), ODC, S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase (SPDSYN) have demonstrated that the ARGknockout Leishmania mexicana promastigotes can survive only in the presence of exogenously supplemented ornithine, putrescine or spermidine, whereas ODC-knockout Leishmania donovani promastigotes require putrescine or spermidine supplementation (Jiang et al., 1999). Such details of polyamine synthesis are relatively less available in Leishmania amastigotes. As previously suggested, L. mexicana amastigotes may survive in the absence of an intact polyamine pathway because they can derive sufficient amounts of ornithine/putrescine/spermidine from the phagolysosome of macrophages (MUs). Leishmania is a severely harmful parasite globally, second only to malaria. Any effort to block its metabolic pathway can be beneficial in interrupting its survival strategies, thereby enabling the host’s immune response to control the disease. Because ODC is essential for T(SH)2 biosynthesis, polyamines of parasitic protozoa may be potential targets for the development of new drugs with activity against L. donovani. The inhibitor of ODC, namely L-a-difluoromethylornithine (DFMO), was previously used to treat African sleeping sickness caused by Trypanosoma brucei gambiense, a protozoan parasite that is phylogenetically linked to Leishmania (Reguera et al., 1995; Burri and Brun, 2003). Reports showing that DFMO inhibits short-term L. donovani infections in experimental animals are also available (Keithly and Fairlamb, 1987; Gradoni et al., 1989; Mukhopadhyay and Madhubala, 1993). Previous studies have shown that the suppression of the specific Th1-type immune response, as shown by decreasing production of IFN-c and IL-12 in patients, promotes disease susceptibility and this suppression is critically regulated by IL-10, a pleiotropic cytokine secreted from different cell types including MUs (Carvalho et al., 1994). Recombinant IL-10 was reported to have an inhibitory effect on the nitric oxide (NO)-mediated killing of Leishmania in

human MUs (Vouldoukis et al., 1997). Thus, one goal of drug development is to evaluate the potential of Leishmania antigen for evoking a Th1 response against Leishmania infection. The precise mechanism, particularly in relation to ODC and its effects on the immunological response in human VL cases, is not completely understood. The present study addresses the cloning, expression and purification, as well as molecular characterisation, of L. donovani ODC (LdODC) during Leishmania infection. Because a cellular immune response involving T cells is important for controlling infection with an intracellular pathogen, it was also of interest to determine the host protective role of an anti-ODC CD4+ T cell response and anti-leishmanial MU function in VL patients.

2. Materials and methods 2.1. Isolation and purification of ODC protein from L. donovani Soluble L. donovani promastigote antigen (SLA) was prepared from a L. donovani clinical strain procured from a patient admitted to the Rajendra Medical Research Institute of Medical Science, Patna, India as described by Gupta et al. (2007). The protein content of the supernatant was determined and SLA was stored at 70 °C. Subsequently, to obtain recombinant LdODC (r-LdODC), L. donovani genomic DNA was isolated from 108 promastigotes (DNA Isolation Kit, Qiagen, Germany) and was subjected to RNase (100 lg/ml) treatment. The LdODC gene was amplified using a Taq polymerase lacking 30 50 exonuclease activity (Qiagen). PCR was performed using Ld-ODC-specific primers (EcoRI and XhoI restriction sites shown are in bold): forward 50 -GAATTCATGGGTGATCATGACGTCG-30 and reverse 50 -CTCGAGTTATCACTCGCTCACA0 CACCTCA-3 in a Thermo cycler (Applied Biosystems, Singapore) using one cycle of 95 °C for 2 min and 30 cycles of 95 °C for 30 s, 58 °C for 50 s and 72 °C for 2 min 5 s, and one cycle of 72 °C for 10 min. The PCR products were separated in a 1% agarose gel and DNA from the specific ODC gene (approximately 2.214 kb) was eluted from the gel by Gel Elute columns (Qiagen). The eluted product was ligated in a pET28a cloning vector (Novagen, Germany) and transformed into competent Escherichia coli DH5a cells. The expression of r-LdODC protein was confirmed by transforming the pET-LdODC construct into E. coli BL21 competent cells. For purification, the recombinant plasmid was grown in 200 mL of Luria–Bertani (LB) medium containing kanamycin (30 lg/ml) overnight at 37 °C and 200 rpm in a shaker incubator. The recombinant protein was subsequently induced by isopropyl b-D-1-thiogalactopyranoside (IPTG, 1 mM) overnight in a shaker incubator at 37 °C and 200 rpm. The bacterial cells were harvested, pelleted (4332 g) and lysed in cell lysing solution (150 mM NaCl, 10 mM Tris–HCl, 2% SDS; pH 8.0) at a ratio of 1:4 for 30 min at 4 °C and then ultra-sonicated at 85% amplitude with 0.5s pulses for 5 min. After centrifugation (17,968g at 4 °C), the recombinant protein was purified on a nickel–nitrilotriacetic acid (Ni–NTA) super flow column (Qiagen) according to the manufacturer’s instructions. In brief, the supernatant was added to 1.5 ml of Ni–NTA slurry and incubated for 3 h at 4 °C with gentle shaking. The resin was divided into three 10 ml disposable columns and washed with five to eight column volumes of lysis buffer containing 250 mM imidazole. The purity of the r-LdODC protein was checked by 12% SDS–PAGE analysis and Coomassie Brilliant Blue R-250 staining. The eluted fractions were combined and dialysed twice against a 300-fold volume of 50 mM Tris–HCl (pH 8.0) and 150 mM NaCl supplemented with 10% glycerol overnight at 4 °C. The concentration of dialysed protein was spectroscopically determined by the Bradford method using BSA as the standard. The protein was stored in 10% glycerol at 20 °C in 1.5 ml aliquots until used.



2.2. Production of L. donovani ODC antibodies and immunoblot analysis Polyclonal antiserum against r-LdODC was raised in a rabbit through four repeated s.c. injections after obtaining approval from the animal ethical committee of Rajendra Memorial Research Institute of Medical Sciences. Pre-immune serum was collected prior to immunisation, and the first dose of 30 lg of r-LdODC in FCA was followed by three booster doses of 25 lg of r-LdODC, each in incomplete Freund’s adjuvant (FIA), after 15 days at 2 week intervals. For serum samples, blood was collected 8 days after the last immunisation to determine the anti-ODC titre by ELISA. Finally, the rabbit was sacrificed to collect serum, which was stored at 20 °C in 1.5 ml aliquots. For the immune-blotting experiment, the total cell lysate from stationary phase promastigotes of L. donovani (1 108 cells/ml) was solubilised in PBS (pH 7.2) in the presence of 1 protease inhibitors and was subjected to 10% SDS PAGE prior to its immobilisation on a nitrocellulose membrane. The membrane was probed with polyclonal anti-ODC serum (1:5000 dilution) for 120 min at room temperature (RT). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Bangalore Genei, India) was used as a secondary antibody and the blot was developed with diaminobenzidine, imidazole and H2O2 (Sigma, USA). 2.3. Effect of r-LdODC on parasite growth The effects of r-LdODC on the growth of promastigotes and axenic amastigotes of L. donovani were evaluated. For these tests, a total of 2 106 early-stationary-phase L. donovani promastigotes per ml of RPMI-1640 complete media with 20% FBS and pH 7.2– 7.4 were dispersed into a 24-well culture plate. The culture was supplemented with purified r-LdODC or crude SLA in duplicate series at final concentrations of 5, 10 and 20 lg/ml. The culture was further incubated at 24 ± 1 °C and subjected to microscopic analysis after 24, 48 and 96 h (Fig. 1C) using a 0.1 mm Neubauer chamber (Jaffe et al., 1984). Axenic amastigotes were generated from the promastigote forms at 37 °C in a CO2 incubator using the media described above at pH 5.5 (Chattopadhyay et al., 1996). The culture stimulation and analysis used were the same as those described above. 2.4. Samples from VL patients and controls A total of 40 human subjects (aged between 17 and 40 years) of both sexes were studied. These included 10 pre-treatment and recently diagnosed patients with VL, 10 who had failed to respond to anti-leishmanial therapy, 10 successfully treated and cured subjects and 10 apparently healthy individuals. The study was conducted between March 2012 and February 2014. Each control subject was sero-negative for antibody to Leishmania (Bimal et al., 2005), and none had a history of Leishmania infection. The diagnosis of VL was based on typical symptoms and signs and the finding of characteristic amastigotes in splenic aspirate smears (Sundar et al., 1995). This study was approved by the ethics committee at Rajendra Memorial Research Institute of Medical Sciences. Informed consent was obtained from the patients and volunteers. 2.5. Cytokine studies 2.5.1. Intracellular cytokines produced from CD4+ and CD8+ T cells We initially examined the capacity of CD4+ T lymphocytes to generate cytokines (IL-10 and IFN-c) after stimulation with rLdODC protein. In brief, to obtain an initial antigen response, peripheral blood mononuclear cells (PBMCs) were stimulated with 2% formaldehyde-fixed L. donovani promastigote antigen in a

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responder antigen presenting cells (APCs), to stimulator (fixed L. donovani antigen) ratio of 1:50 for 2 h at 37 °C (Bimal et al., 2008). A subset of 1 106 cells were cultured in 96-well roundbottomed plates in the presence of r-LdODC protein (10 lg/ml). Control cultures were set up in medium alone or medium containing 10 lg/ml of phytohaemagglutinin (PHA). The intracytoplasmic cytokine levels were detected using a FACS-Calibur flow cytometer as previously described. The cells were cultured for 14 h and then incubated for 4 h with 1 lg/ml of brefeldin-A, a protein transport inhibitor. The harvested cells were consecutively co-incubated with phycoerythrin (PE)-conjugated anti-CD4 – PE antibodies, Cytofix/Cytoperm solution, and Fluorescein isothiocyanate (FITC) conjugated IL-10/IFN-c (BD Pharmingen, USA) before each sample was resuspended in 500 ll of stain buffer. The samples were analysed using a FACS-Calibur flow cytometer. 2.5.2. Assessment of cytokine levels (IFN-c and IL-10) against r-LdODC in VL patients under different clinical manifestations and responses to anti-Leishmania treatment Heparinised venous blood (10 ml each) was collected from all of the study subjects, and PBMCs were isolated from the blood by Ficoll-Hypaque™ density gradient centrifugation (Sigma, USA). A final suspension of 1 106 cells/ml was prepared in complete RPMI 1640 medium after determining the cell viability by the trypan blue staining method. PBMCs (1 106/ml) from VL patients and uninfected healthy controls were cultured in 96-well culture plates, and r-LdODC or crude SLA was added in triplicate wells at a concentration of 10 lg/ml. Control panels with un stimulated culture (ex vivo) or with PHA were run in parallel for all of the experiments. The quantitative yield of IFN-c and IL-10 in the supernatant after 5 days of incubation was determined by ELISA (Thermo Scientific, USA). The detection limit for IFN-c was < 2 pg/ml, and that for IL-10 was 92% MUs and 95% of the cells were viable during incubation. The purified MUs were cultured on a 24-well plate (106 cells/ well) in complete RPMI-1640, infected at a MU-to-parasites ratio of 1:10 for 22 h and subjected to extracellular promastigote removal. The infected MUs (106 cells/well) were treated with SAG (20 lg/ml) and DFMO (10 lg/ml). In parallel, experiments were also set up in the presence of r-LdODC. After 2 days of drug exposure, the plates containing adherent MUs were washed and used for further studies. 2.6.2. Cytokine ELISA To understand the nature of the immune responses generated by r-LdODC and its inhibitor DFMO in the infected MUs, we compared the levels of IL-12 and IL-10 in MUs after in vitro infection


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Fig. 1. Effect of recombinant Leishmania donovani ornithine decarboxylase (r-LdODC) on the growth of L. donovani culture. (A) Leishmania donovani promastigotes (2 106) in the early stationary phase were cultured with or without a-difluoromethylornithine. Growth levels were analysed at 48 and 96 h using a Neubauer chamber and the numbers of parasite were counted. (B) The mean values of axenic amastigote numbers after 48 and 96 h of culture with soluble Leishmania antigen (10 lg/ml) r-LdODC (10 lg/ml) alone or with DFMO. Unstimulated (ex vivo) culture is used as a control. (C) Evaluation of the minimum effective concentrations of r-LdODC (a), a-difluoromethylornithine (b) and soluble Leishmania antigen (c).

and treatment in the presence or absence of the indicated proteins. In brief, after polyclonal stimulation with SLA, we further cultured MUs (1 106 cells/ml) from patients and controls for 72 h. The cytokine levels (IL-12 and IL-10) were measured in culture supernatants using a sandwich ELISA kit (BD Pharmingen).

2.6.3. Measurement of reactive oxygen species (ROS) Measurements of ROS activity were accomplished through flow cytometry, as described previously (O’Donnell et al., 1993). Briefly, 100 ll of cultured and washed MUs were triggered by SLA (10 lg/ ml) and lipopolysaccharide (LPS, 100 lg/ml) plus formylmethionyl-leucyl-phenylalanine (fMLP, 5 mg/ml) at 37 °C in a water bath (10 min). This process was followed by further incubation with 20 mL of 10 mM dihydrorhodamine 123 at 37 °C in a water bath (15 min) to allow the internalisation of the latter into the cell, its conversion into a green fluorescent compound and its subsequent binding to oxidative bursts produced by the stimulated cells. Following incubation, MUs were lysed at RT with 2 ml of FACS lysing reagent (Becton–Dickinson), washed (1 PBS, 268g, 5 min) and resuspended in 450 ll of PBS containing 1% paraformaldehyde. The ROS produced by the stimulated cells were measured based

on the mean fluorescence intensity (MFI), as detected by flow cytometry. 2.6.4. Measurement of NO Alterations in NO generated in stimulated and unstimulated MUs were monitored using a Griess reagent, as described previously (Mookerjee et al., 2006). This method is based on the colorimetric detection of nitrite (oxidation product of NO) as an azo dye product of the Griess reaction. Briefly, 100 ll of the culture supernatants from a MU culture were dispensed in each well of a 96-well microtiter plate and then incubated with 100 ll of Griess reagent in each well at RT for 20 min. The O.D. of the coloured product formed was measured using an ELISA reader (BIO-RAD, Japan) at 540 nm. The amount of NO formed in molar/million (M/106) cells was calculated by comparing the standard sodium nitrite concentration curve. The lower limit of the sensitivity of the nitrite assay was 0.08 lM/ml. 2.6.5. Leishmanicidal activity against intracellular amastigotes The cultured MUs from L. donovani-pulsed wells that were either left untreated or treated earlier with SAG (20 lg/ml), DFMO



(10 lg/ml) or r-LdODC (10 lg/ml) were further tested to compare their leishmanicidal activities against intracellular amastigotes. The wells were washed with PBS and stained with 0.1 lg/ml of propidium iodide (PI, BD Pharmingen) for 10 min in the dark (Martin et al., 1995). The stained cells (30,000) were subjected to FL2 detector/Side Scattered Cells (SSC) plot analysis using a FACS Calibur sorter and CellQuestPro software (Beckton Dickinson). The cell population appearing in the lower right quadrant of each plot was analysed to compare the leishmanicidal effects exerted on amastigotes. An additional sample tube of MU cells supplemented with PBS only was prepared at the same time as the other experimental tubes and used as a negative control. 2.7. Statistical analysis All of the data are expressed as the means ± S.E.M. The statistical analyses were performed using the GraphPad Prism5 (USA) software through one-way ANOVA with a post-test, which was only performed if P < 0.05 (Tukey’s test was used to compare all pairs of columns). A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Characterisation of r-LdODC The ODC gene of L. donovani was amplified and cloned in a pET28a vector (Supplementary Fig. S1A). The recombinant pET28a-LdODC construct was further transformed into E. coli BL21 (Supplementary Fig. S1B), purified and eluted with 250 mM imidazole. The size of the eluted r-LdODC was approximately 77 kDa (Supplementary Fig. S1C). A western blot analysis of L. donovani promastigote lysates was performed with the polyclonal anti-r-LdODC antibody, which detected one dominant protein of approximately 77 kDa (Supplementary Fig. S1D). 3.2. r-LdODC is a virulent factor and its inhibitor, DFMO, inhibits parasite growth There was significantly higher growth of L. donovani parasites in the presence of r-LdODC compared with the parasite culture in the absence of any stimulation of both promastigotes (P < 0.05; Fig. 1A) and amastigotes after 72 h (P < 0.05; Fig. 1B), although it was higher in amastigotes (P < 0.05; Fig. 1B). It is evident from Fig. 1 that increases in L. donovani (both promastigote and amastigote) growth was primarily due to the addition of r-LdODC because DFMO (an established inhibitor of ODC), when used in ODC-supplemented culture, resulted in a decrease in the cell viability (P < 0.001). The dose finding analysis revealed (Fig. 1C) that a threshold concentration of 10 lg/ml of r-LdODC was necessary to enhance the growth of L. donovani (both promastigotes and axenic amastigotes) after 48 h. Furthermore, the addition of SLA to the culture media was also shown to enhance the parasite replication (both promastigotes and amastigotes), as shown in Fig. 1A and B, which was even greater than that obtained with r-LdODC stimulation. The inhibitory effect of DFMO on the growth of parasites was also observed to be optimal at the concentration of 10 lg/ml relative to the concentrations of 5, 15 and 20 lg/ml. 3.3. Functional profile of r-LdODC-induced CD4+ and CD8+ T cell responses The cellular immune response involving CD4+ and CD8+ T cells is important for controlling infection by intracellular pathogens (Guha et al., 2013). To understand the nature of the T cell immune

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response generated by ODC stimulation, VL patients and healthy controls were studied to determine the in vitro intracellular cytokine production in CD4+ and CD8+ lymphocytes after stimulation in the presence or absence of r-LdODC, SLA, DFMO and PHA. As shown in the representative dot plot of the FACS results (Fig. 2A– D and Supplementary Figs. S2 and S3) and histogram analysis, the majority of the IL-10-producing T cells in VL patients were CD4+ cells: 18.35% and 7.43% after stimulation, respectively, with r-LdODC and SLA. It was also observed that r-LdODC induced a 3.23-fold decrease in the CD4+ T cell response to IFN-c compared with the Ex vivo results (P < 0.001), and SLA induced a 2.63-fold decrease (P < 0.001). In contrast, the CD4+ T cell-secreting cytokines following r-LdODC stimulation were mostly IL-10, and its inhibitor, DFMO, induced 4.32-fold more IFN-c in CD4+ T cells (P < 0.001) and decreased IL-10 production by 3.59-fold compared with r-LdODC (P < 0.001). However, the CD8+ T cells activated following r-LdODC stimulation triggered a higher frequency of IFN-c, which was 5-fold greater compared with that observed with the CD4+ T cells (P < 0.001). In general, although the T cells following r-LdODC stimulation triggered increased IL-10 production among CD4+ T cells, the majority of the CD8+ T cells were those in which IFN-c production was least affected. The analysis of the CD8+ T cells showed a significant response only in terms of IL-10 and IFN-c production: IL-10 production was reduced 1.32-fold, whereas IFN-c production was increased 1.14-fold compared with r-LdODC. Comparatively, there was a marked impact of DFMO on CD4+ T cells; IL-10 production was reduced 3.59-fold and IFN-c production was increased 3.98fold compared with r-LdODC (P < 0.001). Primarily, the IFN-c produced by CD4+ T cells mediates pathogen killing by activating the microbicidal properties of MUs by inducing NO production. Thus, a suppressed CD4+ Th1 cell-associated mechanism exists during VL infection, and more CD4+ T cells show decreased sensitivity for IFN-c production after r-LdODC stimulation.

3.4. Activation of the endogenous polyamine biosynthetic pathway by r-LdODC suppresses protective immunity in VL patients We simultaneously measured the secreted levels of cytokines (IFN-c and IL-10) to assess the overall abilities of the r-LdODCinduced IFN-c and IL-10 responses in different groups (active, cured, and non-cured infections) of VL patients (Supplementary Fig. S4A, B). The addition of SLA as a source of antigen stimulation in the PBMCs of cured VL cases showed enhanced IFN-c production (771.5 ± 65.10 pg/ml) in comparison with the controls (232.26 ± 95.36 pg/ml, P < 0.001; Supplementary Fig. S4A). Similarly, our results showed that the IFN-c production was arrested 2.66-fold by the addition of r-LdODC (289.08 ± 110.3 pg/ml) in comparison with the SLA (771.5 ± 65.10 pg/ml; P < 0.001), or 3.53-fold by the addition of PHA (P < 0.001; 1023.31 ± 232.2 pg/ ml) in the cultured supernatant of cured VL cases (Supplementary Fig. S4A). We also found that the addition of PHA to cultured supernatant of active VL cases triggered IFN-c production (427.63 ± 74.65 pg/ml), whereas little effect was observed in the modulation of IFN-c production by the addition of r-LdODC (271.6 ± 117.6 pg/ml, P < 0.05) or SLA (292.46 pg/ml; P > 0.05). Because pre-treatment with DFMO resulted in the inhibition of Leishmania growth in culture, it was tested for its abilities to revert IFN-c unresponsiveness in VL cases upon r-LdODC treatment. We found that IFN-c production was markedly increased against DFMO in all categories of VL patients. These results indicate that the activation of an endogenous polyamine biosynthetic pathway by r-LdODC suppresses protective immunity in VL patients and that the inhibition of r-LdODC can direct the active IFN-c dominant immune responses necessary for parasite clearance in VL patients.


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Fig. 2. Recombinant Leishmania donovani ornithine decarboxylase (r-LdODC)-inducedCD4+ and CD8+ T cell immune responses in visceral leishmaniasis. Production of IFN-c and IL-10 by CD4+ and CD8+ T cells in the peripheral blood mononuclear cell culture were determined. Peripheral blood mononuclear cells were stained for different cytokines and surface markers. Cells were analyzed by multiparameter flow cytometry using CellQuest-Pro software to determine the frequency of IFN-c and IL-10 positive T cells. Histogram plot showing frequency of intracellular IFN-c produced in response to different stimulants: soluble Leishmania antigen, r-LdODC, a-difluoromethylornithine, phytohemagglutinin and unstimulated (ex vivo) conditions in cells from (A) visceral leishmaniasis patients and (B) health controls. Flow cytometry analysis of IL-10 produced in response to the same stimulants in (C) visceral leishmaniasis patients and (D) healthy controls.

3.5. Increased tendency of infected MU to convert to IL-10 effector cells following stimulation with r-LdODC in VL patients Subsequent experiments were performed for the analysis of IL10 production in active, cured and unresponsive VL patients (Supplementary Fig. S4B). Patients with active VL infection induced a significantly higher concentration of IL-10 (260.54 ± 102.4 pg/ml) against r-LdODC antigen (P < 0.001) compared with the controls (26.04 ± 35.72 pg/ml). This increase in IL-10 against r-LdODC antigen assumes significance by the finding that there was a 6.63-fold increase in the levels of IL-10 cytokines in active VL compared with cured cases (39.28 ± 25.03 pg/ml) (P < 0.01). In contrast, the IL-10 produced in VL cases with treatment failure also remained high (164 ± 54.25 pg/ml), whereas compared with the controls, no significant difference was observed and IL-10 production was reduced in cured VL cases (39.28 pg/ml, P > 0.05). Similarly, our results showed that the effects on IL-10 production by r-LdODC and SLA were inhibited 2.52-fold and 3.36-fold by the addition of DFMO (10 lg/ml) in active VL cases. These results suggested that r-LdODC up-regulated Th2-promoting cytokines in infected MUs. 3.6. r-LdODC suppresses MU, and its inhibitor, DFMO, overcomes defects in cytokine production in MUs following L. donovani infection To understand the nature of immune responses generated in the infected MUs by r-LdODC stimulation, we compared by ELISA the levels of IL-12, a cytokine that is required for host protection and IL-10, which is known to induce strong immune suppression following L. donovani infection (Fig. 3A and B). MUs with active VL, following stimulation with r-LdODC, increased the production of IL-10 (405.5 ± 33.62) by 3.112-fold (Fig. 3B) and reduced the production of IL-12 (61.26 ± 6.851) by 4.164-fold compared with the controls (255.1 ± 44.37). Similarly, MUs from active VL cases stimulated with SLA (633.5 ± 75.24) showed higher amounts of IL-10

(P < 0.001) and down-regulation of IL-12 production (77.69 ± 9.075) compared with the controls (P < 0.01) and r-LdODC stimulation. In contrast, in response to DFMO, MUs showed approximately 1.825-fold more IL-12, whereas stimulation with DFMO inhibited the production of IL-10 (206.9 ± 12.77) by 1.959-fold compared with r-LdODC (405.5 ± 33.62, P < 0.05).

3.7. Alteration in r-LdODC activity stimulates macrophages for enhanced ROS release and NO production To determine whether r-LdODC stimulation can accelerate the impaired anti-leishmanial function of MUs observed in VL, we performed a ROS assay in cultured MUs in the presence of r-LdODC, DFMO and SAG. MUs stimulated with r-LdODC showed decreased ROS production upon antigenic re-stimulation compared with the infected controls (Fig. 3C and D). The MUs of control individuals exhibited higher ROS activity (1050 MFI) compared with those of the VL participants (80.13 MFI) against r-LdODC, and this difference was highly significant (P < 0.01). Similarly, when the MUs were tested against parasite SLA, the ROS activity of MUs (136.1 MFI) was significantly reduced in the VL participants compared with the healthy control group (951.7 MFI, P < 0.01; Fig. 3C). Moreover, the induced ROS production in the stimulated MUs of VL patients was complemented by increased production of ROS by MUs after DFMO stimulation in comparison to r-LdODC (P < 0.05). We also observed lower levels of NO with r-LdODC (P < 0.05) compared with infected controls, whereas stimulation with DFMO induced significant nitrite production (Fig. 3E and F). These results indicate the requirement of IL-12 for anti-leishmanial function of MUs and confirm that the inhibition of ODC activity is also necessary to activate the MUs and promote anti-leishmanial function during VL.



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Fig. 3. Secreted levels of IL-12 and IL-10 in macrophages (MU) and assessment of anti-leishmanial MU function after recombinant Leishmania donovani ornithine decarboxylase (r-LdODC) stimulation. Comparison of IL-12 (A) and IL-10 (B) released by MU after co-stimulation by r-LdODC, a-difluoromethylornithine, soluble Leishmania antigen and phytohaemagglutinin. Unstimulated (ex vivo) culture is used as a control. Levels of cytokines were measured by ELISA (as described in Section 2.6) after 48 h. (C) The decrease in mean fluorescence intensity of MU for the release of reactive oxygen species. Culture MU in the presence of di-hydrorhodamine and N-formyl-methionylleucyl-phenylalanine acquired using a FL-1 detector on a FACS Calibur. The ROS produced were measured via mean fluorescence intensity. (D) Flow diagram of cultured MU of a visceral leishmaniasis patient showing a decrease in mean fluorescence intensity for the release of reactive oxygen species using a FACS Calibur after activation with rLdODC. Results for cultured MU stimulated with soluble Leishmania antigen, a-difluoromethylornithine and phytohaemagglutinin are also shown. Unstimulated (ex vivo) culture is used as a control. (E) Retardation in anti-leishmanial MU function for the production of nitric oxide by r-LdODC in visceral leishmaniasis patients. (F) Antileishmanial MU function for the production of nitric oxide by r-LdODC in healthy controls. Nitric oxide generation in response to r-LdODC, a-difluoromethylornithine and phytohaemagglutinin were measured using a Griess reagent. Ex vivo culture is used as a control. Nitric oxide in culture supernatant was measured by colorimetric detection of nitrite as an azo dye product of the Griess reaction.

3.8. Effect of r-LdODC on the killing of amastigotes in L. donovaniinfected MUs MUs were infected with virulent L. donovani and stimulated with r-LdODC or DFMO. PBS-supplemented MUs were used as controls. Both treated and untreated samples were stained with PI, and the percentage of the cell populations appearing in the lower right quadrant of each plot (representing PI-stained dead cells) was analysed to compare the leishmanicidal effects exerted on intracellular amastigotes in MUs. Our results showed that the percentage of PIstained dead cells in the population of r-LdODC-stimulated cells was 26.87% and that the live cells were inhibited only by 2.66% compared with the infected untreated controls (Supplementary Figs. S5A and S6). However, significantly greater inhibition of the parasite by DFMO-stimulated MUs was observed compared with the infected untreated controls (P < 0.001). 4. Discussion Frequent resistance occurs against several drugs that are currently used for chemotherapy of leishmaniasis. As such, the major thrust of research on new drug development against leishmaniasis has become the identification of antigens as virulent factors and efforts to block some vital metabolic pathways linked to those

antigens in the parasite. Several studies have demonstrated a correlation between Leishmania resistance to antimonial drugs and increased T(SH)2 levels (Mukhopadhyay et al., 1996; Wyllie et al., 2004), and T(SH)2 is also crucial for facilitation of infection with the Leishmania parasite. An appropriate interference in the T(SH)2 pathway could provide clues for rational drug design and vaccine development against VL. Increased resistance to antimonial drugs has been found in cells that overproduce T(SH)2 as a result of increased expression of ODC (Haimeur et al., 1999; Mukherjee et al., 2007), the rate-limiting enzyme of the polyamine biosynthetic pathway. The ODC enzyme is an obligate homodimer with two symmetry-related active sites located at the dimer interface. As such, the pharmacological inhibition of ODC is a promising strategy for the treatment of visceral and perhaps other forms of leishmaniasis; however, the influence of ODC on host factors and its relation to disease pathogenesis have remained open questions. Cellular immune responses involving CD4+ cells are important for controlling infection in patients by an intracellular pathogen (Amit et al., 2014; Chaudhary et al., 2014). Because ODC is linked to T(SH)2 synthesis, the goal of the present study was to evaluate the potential of r-LdODC to influence immunopathogenicity during L. donovani infection in immunosuppressed VL patients. To evolve the function of LdODC, the LdODC gene was amplified, cloned in the bacterial expression vector pET28a, purified and


8


eluted in imidazole. A western blot analysis of L. donovani promastigote lysates was performed with the polyclonal anti-r-LdODC antibody, which detected one dominant protein of 77 kDa. The results reported here show that ODC may play a role in the virulence of L. donovani. These results are in agreement with a previous report that showed that LdODC is indispensable for parasite survival in the mammalian host (Boitz et al., 2009). We agree with a previous suggestion that variations in the ornithine and putrescine pools exist within the phagolysosomes of MUs in the skin (Langerhans cells, skin dendritic cells and histiocytes), liver (Kupffer cells, parenchymal cells and non-resident MUs) and spleen (splenic MUs), where L. donovani resides, and these differences could account for its virulence (Boitz et al., 2009). Many research groups may not support this hypothesis due to the existence of salvageable polyamines in the host phagolysosome to which the parasites would have access (Basselin et al., 2000). The results reported here also show that SLA extracted from promastigotes of L. donovani, when used as a supplement in the leishmanial culture, can enhance parasite growth. Our results are in agreement with previous reports on the Leishmania propagating abilities of this SLA, which shows that the increase in parasite growth may be due to activation of multiple antigens present in the SLA construct (Amit et al., 2014). Because it was found that r-LdODC is required to promote infection, a pharmacological inhibition of this enzyme can be a strategy to restrict Leishmania growth. Recent studies have shown that DFMO (an irreversible inhibitor of ODC) induces the inhibition of ODC, thereby providing better control over Leishmania growth (Boitz et al., 2009). Therefore, we compared the pattern of growth kinetics from the r-LdODC versus DFMO-supplemented Leishmania culture. Our results showed that Leishmania promastigote and axenic amastigote culture in the presence of DFMO inhibited the growth of L. donovani (Kaur et al., 1986). DFMO, a substrate analogue, makes a stable covalent bond with the conserved Cys residue and other residues in the active site of the ODC enzyme, and inhibits its catalytic reaction (Preeti et al., 2013). In addition to DFMO, other inhibitors of ODC, such as 3-aminooxy-1 aminopropane (APA), have been reported to have an ability to block the proliferation of parasites and tumour cells (Robin et al., 2005; Singh et al., 2007). The effects of APA were not measured in this study. Because ODC is linked to T(SH)2 synthesis and relatively less is known of its impact on immune responses in patients, we further examined the potential of r-LdODC during L. donovani infection in PBMC samples of immunosuppressed VL patients (Thakur et al., 2003; Kushwaha et al., 2012; Guha et al., 2013; Amit et al., 2014). Methods other than the isolation and culture of PBMCs, such as whole blood assays, are also available to investigate the immune response in simple, live, whole, unmanipulated blood and have also been employed in research related to leishmaniasis (Vikash et al., 2014). Our concern in the present study was to determine which cytokines (and their cellular sources) are involved in the disease process such that they may be distinguished from those that may merely be correlated with immune activation. Therefore, we preferred PBMC analyses over whole blood assays for anticipating the presence of many cell types that, during a particular culture condition provided to the whole blood after antigenic stimulation, may produce a particular cytokine, such as IFN-c. For instance, similar to a T cell, polymorphonuclear neutrophils (PMNs) also produce IFN-c (Vikash et al., 2014). Most importantly, our aim in this study was to both quantitatively and qualitatively examine the immune response generated exclusively by a specific type of immune cell. Cellular immune responses involving CD4+ and CD8+ T cells are important for controlling infection by intracellular pathogens (Guha et al., 2013). The present investigation showed that stimulation with r-LdODC induced significantly higher production of IL-10 in VL cases, and our FACS data revealed that the majority

of the IL-10 that was produced during VL originates from CD4+ cells, in which r-LdODC induces a sharp decrease in IFN-c production. In contrast, the CD8+ T cells activated following r-LdODC stimulation triggered a higher frequency of IFN-c than CD4+ T-cells. Surprisingly, the ODC inhibitor DFMO induced 4.32-fold more IFN-c in CD4+ T cells and decreased IL-10 production by 3.59-fold compared with r-LdODC. Because IFN-c mediates Leishmania killing by activating MUs to release free radicals such as ROS and NO (Murray and Nathan, 1999; Thakur et al., 2003; Guha et al., 2013), and the effect is decreased by IL-10, leading to severe disseminated forms of the disease (Uzonna et al., 2001), these findings are worth pursuing to demonstrate the role of ODC in influencing the CD4+ T cell function during VL. Our experiments examining the anti-leishmanial activity of MUs indicated a down-regulated IL-12 pro-inflammatory response triggered by r-LdODC, and this made a marked impact on the free radical (super-oxide and NO) generation in MUs during VL infection. Pro-inflammatory cytokines such as IL-12 and TNF- a play a critical role in the induction of NO and ROS, thus ensuring an intact anti-leishmanial function of phagocytic cells during VL (Adhikari et al., 2012). Given that we earlier observed a defective IFN-c response with markedly elevated IL-10 secretion against r-LdODC, and correlating these results with the impairments observed in infected MUs, it is suggested that r-LdODC leads to the indispensability of IL-12, which may interfere in the induction of protective immunity and anti-Leishmania defence during VL. The study further reflected that the obstruction of the activities of ODC by inhibitors such as DFMO may lead to the generation of adequate amounts of IL-12, induce NO and ROS production in MUs, and restore the Th1 response required for parasite clearance. Previous results have shown a link between polyamine and antimonial resistance in trypanosomatids. In Leishmania, glutathione is replaced by T(SH)2, which is a conjugate between the polyamine spermidine and glutathione. A correlation between the polyamine to antimonial drugs and increased T(SH)2 levels appears to exist in Leishmania (Mukhopadhyay et al., 1996; Wyllie et al., 2004). A role of ODC is supported by our finding that immune suppression in VL patients becomes more predominant against r-LdODC. We have also demonstrated that DFMO is a potent stimulator of a protective immune response and that its immune stimulatory effects are due to the inhibition of ODC. It was also demonstrated that the inhibitory effect of DFMO on ODC activity is correlated with its effect on parasite growth. Through this study we show that, in addition to its role in growth and proliferation of the parasite, r-LdODC directly influences the host immune response in VL patients. These findings may help researchers better design future drug targets for the control of VL. Acknowledgements This work was supported by the Indian Council of Medical Research (ICMR), Ministry of Health and Family Welfare, Government of India. We are indebted to the Council of Scientific and Industrial Research (CSIR) – University Grant Commission (UGC) and ICMR for the fellowship awarded to Mr. Ajay Amit and Mr. Rajesh Chaudhary, respectively, to pursue this study. Technical assistance provided by Mr. Manish Kumar Ranjan, Mr. Santosh Sinha, Mr. Ajay Kumar and Mr Santosh Singh is highly acknowledged and appreciated. 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.ijpara.2014. 08.013.



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Evaluation of Leishmania donovani disulfide isomerase as a potential target of cellular immunity against visceral leishmaniasis.

Recombinant NAD-dependent SIR-2 protein of Leishmania donovani: immunobiochemical characterization as a potential vaccine against visceral leishmaniasis.

Leishmania donovani: dynamics of L. donovani evasion of innate immune cell attack due to malnutrition in visceral leishmaniasis.

Immunoblot analysis of the humoral immune response to Leishmania donovani infantum polypeptides in human visceral leishmaniasis.

Leishmania donovani phosphoproteins pp41 and pp29 re-establishes host protective immune response in visceral leishmaniasis.

Comparative Analysis of Cellular Immune Responses in Treated Leishmania Patients and Hamsters against Recombinant Th1 Stimulatory Proteins of Leishmania donovani.

Live Attenuated Leishmania donovani Centrin Knock Out Parasites Generate Non-inferior Protective Immune Response in Aged Mice against Visceral Leishmaniasis.

Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis.

Role of CD8(+) T cells in protection against Leishmania donovani infection in healed Visceral Leishmaniasis individuals.

Immunogenicity in dogs and protection against visceral leishmaniasis induced by a 14kDa Leishmania infantum recombinant polypeptide.

Mapping of a visceral leishmaniasis-specific immunodominant B-cell epitope of Leishmania donovani Hsp70.

The activity of hydroxynaphthoquinones against Leishmania donovani.

Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity.

Use of a recombinant cysteine proteinase from Leishmania (Leishmania) infantum chagasi for the Immunotherapy of canine visceral Leishmaniasis.

A Leishmania-specific hypothetical protein expressed in both promastigote and amastigote stages of Leishmania infantum employed for the serodiagnosis of, and as a vaccine candidate against, visceral leishmaniasis.

Risk factors for visceral leishmaniasis and asymptomatic Leishmania donovani infection in India and Nepal.

c Mice.

Leishmania donovani Nucleoside Hydrolase (NH36) Domains Induce T-Cell Cytokine Responses in Human Visceral Leishmaniasis.

Efficacy of Leishmania donovani trypanothione reductase, identified as a potent Th1 stimulatory protein, for its immunogenicity and prophylactic potential against experimental visceral leishmaniasis.

An imported case of cutaneous leishmaniasis caused by Leishmania (Leishmania) donovani in Japan.

Leishmania donovani influenced cytokines and Toll-like receptors expression among Sudanese visceral leishmaniasis patients.

Melittin induces in vitro death of Leishmania (Leishmania) infantum by triggering the cellular innate immune response.

HIV-Leishmania co-infections in Italy. Isoenzyme characterization of Leishmania causing visceral leishmaniasis in HIV patients.

Novel Agents against Miltefosine-Unresponsive Leishmania donovani.