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The Journal of Immunology, 2003, 170: 5625-5629.
Copyright © 2003 by The American Association of Immunologists

Dendritic Cell-Based Immunotherapy Combined with Antimony-Based Chemotherapy Cures Established Murine Visceral Leishmaniasis1

Monidipa Ghosh*, Chiranjib Pal2,*, Mitali Ray*, Sudipta Maitra*, Labanya Mandal* and Santu Bandyopadhyay3,*

* Division of Immunology, Indian Institute of Chemical Biology, Jadavpur, Kolkata India


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Dendritic cells (DCs) have been proposed to play a critical role as adjuvants in vaccination and immunotherapy. In this study we evaluated the combined effect of soluble Leishmania donovani Ag (SLDA)-pulsed syngeneic bone marrow-derived DC-based immunotherapy and antimony-based chemotherapy for the treatment of established murine visceral leishmaniasis. Three weekly injections of SLDA-pulsed DCs into L. donovani-infected mice reduced liver and splenic parasite burden significantly, but could not clear parasite load from these organs completely. Strikingly, the conventional antileishmanial chemotherapy (sodium antimony gluconate) along with injections of SLDA-pulsed DCs resulted in complete clearance of parasites from both these organs. Repetitive in vitro stimulation of splenocytes from uninfected or L. donovani-infected mice with SLDA-pulsed DCs led to the emergence of CD4+ T cells with characteristics of Th1 cells. Our data indicate that DC-based immunotherapy enhances the in vivo antileishmanial potential of antimony or vice versa.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The visceral form of leishmaniasis, commonly known as kala-azar, is caused by the parasite Leishmania donovani (L. donovani). Approximately 350 million people in eight countries are estimated to be threatened by the disease (1). The World Health Organization estimated that there are 12 million cases of all forms of leishmaniasis worldwide, with >500,000 new cases of visceral disease occurring each year (1). Although recent reports suggested the efficacy of amphotericin B (AmB)4-lipid complex (2) or miltefosine, an orally active phosphocholine analog (3), for the treatment of visceral leishmaniasis, sodium antimony gluconate (Sb) remains the mainstay of treatment. Sb, AmB, and miltefosine are directly leishmanicidal toward intracellular L. donovani amastigotes when applied to parasitized macrophages (M{phi}) in vitro (4, 5, 6). However, in contrast to AmB and miltefosine, the in vivo efficacy of Sb was shown to depend on IFN-{gamma} (7). Due to the nonexistence of an effective vaccine, improved chemotherapy for visceral leishmaniasis remains desirable.

After the discovery of dendritic cells (DCs) by Steinman and Cohn in 1973 (8), emerging evidence suggested that DCs may be used as adjuvants to treat a variety of diseases. Several studies in mouse models (9) and humans (10) suggested the potential of tumor Ag-pulsed DCs to induce antitumor immunity. However, the reports on DC-based therapy in an infectious disease setting, especially against parasitic diseases, are scanty (11, 12). To our knowledge, only one report suggested the immunoprophylactic and immunotherapeutic efficacy of DC-based treatment against murine visceral leishmaniasis, although this strategy could not cure established infection (13). Sb-based antileishmanial chemotherapy depends in part on the Th1-type response (7), which could be induced by DC-based treatment (14). In the present report we evaluated whether a combination of DC-based immunotherapy and Sb-based chemotherapy cures established murine visceral leishmaniasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Reagents

The following mAbs were used: CD1d, CD4, CD11b, CD11c, CD86, I-A, IFN-{gamma}, IL-4, and IL-10 (BD PharMingen, San Diego, CA). FACS permeabilizing solution was obtained from BD Biosciences (Mountain View, CA). Brefeldin A was obtained from Sigma-Aldrich (St. Louis, MO). RNA extraction reagent, TRIzol, and Superscript one-step RT-PCR kit were purchased from Life Technologies (New Delhi, India). Recombinant mouse (rm) cytokines rmGM-CSF, rmIL-4, rmIL-2, and rmTNF-{alpha} were purchased from R&D Systems (Minneapolis, MN). Complete RPMI medium consisted of RPMI 1640, 1% L-glutamine, 1% penicillin/streptomycin, 50 µM 2-ME, 1% essential amino acids, and heat-inactivated 10% FCS (all from Life Technologies).

Parasites and infection of mice

The L. donovani virulent strain AG83 was originally obtained from an Indian kala-azar patient (15) and was maintained in golden hamsters. Promastigotes were obtained from transforming amastigotes derived from the spleens of infected animals. Soluble parasite Ag (SLDA) was prepared from stationary phase promastigotes of L. donovani following published procedure (16). BALB/c mice (5–6 wk old) were infected i.v. (through the tail vein) with freshly purified amastigotes of L. donovani (1 x 107/mouse). Splenic and liver parasite burdens were determined from impression smears after Giemsa staining and are reported as the total parasite load per organ using the formula: organ weight (milligrams) x number of amastigotes per cell nucleus x (2 x 105) (17). To further evaluate whether these organs contained live parasites, in selected experiments homogenates of these organs were cultured for 2 wk after serial dilution (18).

Preparation of APCs

Bone marrow-derived DCs were generated by culturing BALB/c bone marrow cells in the presence of GM-CSF and IL-4 as previously reported (19) with some modifications. Briefly, bone marrow was collected from tibias and femurs of BALB/c mice, passed through a nylon mesh to remove small pieces of muscles and debris, resuspended in complete RPMI medium, and cultured in tissue culture dishes for 2 h. Nonadherent cells were collected, and aliquots of 1 x 106 cells were placed in 24-well plates containing 1 ml of complete medium with GM-CSF (150 U/ml) and IL-4 (75 U/ml). Two-thirds of the medium was replaced on day 3. On days 5 and 7 of culture, nonadherent cells were transferred into six-well plates in complete medium with cytokines (2.5 x 106 cells/2 ml/well) and maintained for 3 additional days in culture. On day 10 of culture, most nonadherent cells had acquired typical dendritic morphology. Phenotypic characteristics of these cells were evaluated by flow cytometry. These cells were used as the source of DC in subsequent experiments. M{phi} were purified from splenocytes by plastic adherence and were at least 92% pure as judged by surface staining with anti-CD11b mAb (16).

Immunotherapy

For APC-based therapy and vaccination, two types of APCs were used: purified splenic macrophages and bone marrow-derived DCs. Each type of APC was divided into two parts. One part was pulsed with SLDA in the presence of rmTNF-{alpha}, and the other part was cultured with rmTNF-{alpha} alone as an unpulsed control. Pulsing with SLDA for 18 h was performed as described previously (13) with the following modifications. After 4-h incubation with SLDA (25 µg/ml), rmTNF-{alpha} (20 ng/ml) was included for the last 14 h to allow maturation of pulsed DCs. SLDA-pulsed or unpulsed APCs were washed with PBS and injected i.v. (105 cells/mouse in 100 µl of PBS) into groups of 1 mo postinfected mice (four mice per group) through the tail vein once a week for 3 wk. An additional group of infected mice received Sb (50 mg/kg body weight i.m.) once a week for 3 wk. Infected mice in other groups received SLDA-pulsed/unpulsed DCs or M{phi} i.v. and Sb i.m. simultaneously. Infected mice in the control group received 100 µl of PBS through the tail vein. Mice in all groups were sacrificed 3 wk after receiving the last treatment (10 wk postinfection) for the determination of parasite burden. The animal ethics committee of Indian Institute of Chemical Biology (Kolkata, India) approved these studies.

Repetitive in vitro stimulation of splenocytes with SLDA-pulsed DCs

Plastic-nonadherent splenocytes of uninfected and L. donovani-infected BALB/c mice (1 mo postinfection) were cultured in complete RPMI medium containing 0.5% autologous uninfected mouse serum and 50 U/ml rmIL-2 in the presence of SLDA-pulsed or unpulsed DCs (responder/stimulator ratio, 20/1). The cultures were restimulated every 7 days with SLDA-pulsed or unpulsed DCs. When unpulsed DCs were used as stimulators, lymphocytes did not increase in number, and cell death began after 7 days of culture. In contrast, after repetitive stimulation with SLDA-pulsed DCs, increased lymphocyte numbers were detected. Phenotypic and intracellular cytokine analyses were performed on ex vivo expanded lymphocytes of uninfected and L. donovani-infected mice that were stimulated with SLDA-pulsed DCs at least three times.

Flow cytometry

Flow cytometry was performed to define the phenotypic characteristics of DCs generated in vitro from bone marrow progenitors and to analyze intracellular cytokine production by ex vivo expanded T cells. Two-color flow cytometry was performed for intracellular analysis of IFN-{gamma}, IL-4, and IL-10 by ex vivo expanded CD4+ T lymphocytes at the single-cell level. Ex vivo expanded T cells (after three in vitro stimulations with SLDA-pulsed DCs) were restimulated with SLDA-pulsed DCs for 6 h. Brefeldin A (10 µg/ml) was added to the culture 2 h before harvest. The cells were washed and stained with PE-conjugated anti-CD4 mAb, permeabilized by treatment with FACS, and then stained with FITC-conjugated anti-mouse cytokine mAbs (IFN-{gamma}, IL-4, IL-10) or isotype-matched control mAb and analyzed on a flow cytometer (FACSCalibur; BD Biosciences, Mountain View, CA) using the CellQuest program. Staining with FITC-conjugated anti-cytokine mAbs before permeabilization always resulted in <0.2% cytokine-positive cells.

Semiquantitative RT-PCR

Inguinal and popliteal lymph nodes were isolated from L. donovani-infected mice receiving PBS, unpulsed DCs, Sb alone, SLDA-pulsed DCs, or SLDA-pulsed DCs plus Sb 3 days after receiving the last injection, and total RNA was extracted from single-cell suspensions of lymph nodes with the RNA extraction reagent, TRIzol. RT of RNA into DNA and PCR were performed from 2 µg of total RNA using the Superscript One-Step RT-PCR kit. PCR amplification was performed with a thermal cycler (PE Applied Biosystems, Foster City, CA) for 35 cycles as previously reported (20). A 10-µl portion of each PCR product was electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. Sequences of the oligonucleotide primers used were as follows: hypoxanthine phosphoribosyltransferase: sense, 5'-GTTGGATACAGGCCAAGACTTTGTTG-3'; antisense, 5'-GATTCAACTTGCGCTCATCTTAGGC-3'; and IFN-{gamma}: sense, 5'-AACGCTACACACTGCATCT-3'; antisense, 5'-TGCTCATTGTAATGCTTGG-3'.

Ag presentation assay

Inguinal and popliteal lymph node cells (105/well) from L. donovani-infected mice receiving the indicated therapies were cultured in complete RPMI medium containing 0.5% autologous normal mouse serum in the presence or the absence of SLDA (25 µg/ml) for 96 h. Cells were pulsed for 16 h with 0.5 µCi of [3H]thymidine/well (NEN, Boston, MA). In parallel experiments cell-free supernatants were harvested after 24- and 72-h culture for quantitation of IL-12 and IFN-{gamma}, respectively, using commercial ELISA kits (R&D Systems). The sensitivity of both assays was 5 pg/ml.

Statistical analysis

The statistical significance of variants between groups and within each group of experimental animals was evaluated by one-way ANOVA.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Combination therapy with SLDA-pulsed DCs and Sb cures established murine visceral leishmaniasis

DCs were generated in vitro by culturing bone marrow cells in the presence of GM-CSF and IL-4 for 10 days. In agreement with other reports (21), the DC expressed high levels of CD11c, CD86, CD1d, and MHC class II (Fig. 1). GM-CSF and IL-4 differentiated bone marrow DCs pulsed with SLDA in the presence of TNF-{alpha} produced significant amounts of IL-12 (276 ± 40 vs 76 ± 22 pg/ml by unpulsed DCs), and their administration in infected mice significantly reduced splenic and hepatic parasite burden (96.6 ± 3.5 and 90.9 ± 4.2%, respectively, compared with PBS-treated infected controls; p < 0.0005 for each comparison; Fig. 2A). The parasite burden in both organs also decreased significantly following administration of unpulsed DCs (p < 0.05). SLDA-pulsed or unpulsed M{phi} reduced parasite burden in both organs only marginally, and the differences were not statistically significant (Fig. 2, A and B). Of interest, the level of parasite burden in the spleens of mice was significantly less compared with that in liver after receiving SLDA-pulsed DC-based therapy. This difference may be attributed to the DC subtypes and/or CCR7-mediated migration of DCs, which is dependent on the expression of CCL21 and CCL19 chemokines by stromal cells within the T cell areas of secondary lymphoid organs (22).



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  		FIGURE 1. Immunophenotyping of bone marrow-derived DCs. BALB/c mice bone marrow cells were cultured in the presence of GM-CSF and IL-4 for 10 days. The data show histograms of cell number against fluorescence intensity and are representative of three experiments. Dotted lines indicate staining with isotype-matched control mAbs; solid lines show staining with the indicated mAbs.

 


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  		FIGURE 2. Combined therapy with APCs and antimony against murine visceral leishmaniasis. One month postinfected BALB/c mice received the indicated treatment once a week for 3 wk; the i.v. route was used for APC-based treatment, and the i.m. route was used for Sb (50 mg/kg body weight). DCs (A) and M{phi} (B) were used as APCs. Three weeks after the last treatment (10 wk postinfection) animals were sacrificed for the determination of parasite burden. Data represent the mean ± SD for four animals per group. Results are representative of four experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.0005 (vs PBS). *****, p < 0.05 (vs Sb).

 
Recent evidence that the in vivo antileishmanial potential of Sb depends on endogenous IFN-{gamma} (7) and the possibility that Sb augments IFN-{gamma} signaling in macrophages via inhibiting the Src homology protein tyrosine phosphatase 1 (23) prompted us to examine whether immunotherapy with SLDA-pulsed DCs could enhance the antileishmanial therapeutic potential of Sb. Immunotherapy with SLDA-pulsed/unpulsed M{phi} or unpulsed DC was also combined with Sb-based chemotherapy as a control. In the mouse model of visceral leishmaniasis, the optimum antileishmanial effect by Sb is achieved by a high dose of Sb (~500 mg/kg body weight), and only a marginal effect is seen at 50 mg/kg body weight (24). A dose of Sb (50 mg/kg body weight) was chosen that could only partially clear the parasite load from the organs tested (Fig. 2A). This dose of Sb did not induce detectable liver toxicity as evident from the assays of serum glutamate oxaloacetate transaminase and serum glutamate pyruvate transaminase (results not shown). Treatment with Sb alone reduced parasite burden by 39.6 ± 13.5% (p < 0.05) and 51.7 ± 13.3% (p < 0.01) in spleen and liver, respectively. Strikingly, complete clearance of parasite burden from spleen and liver was achieved when infected mice received both SLDA-pulsed DCs and Sb. To further evaluate whether these organs had live parasites, homogenates of these organs were cultured for 2 wk after serial dilution as previously described (18). No transformed promastigotes were detected in this group of animals, although promastigotes were readily detectable in animals receiving either treatment alone (Table I). In contrast, combination therapy with Sb plus unpulsed DCs, SLDA-pulsed M{phi}, or unpulsed M{phi} was ineffective (Fig. 2, A and B).


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  		Table I. SLDA-pulsed DC-based immunotherapy combined with Sb-based chemotherapy clears established parasite load from liver and spleen

 
The immunoprophylactic and immunotherapeutic efficacy of SLDA-pulsed DCs against murine visceral leishmaniasis has been reported (13). SLDA-pulsed DCs could not induce sterile protection or cure established infection. However, vaccination with DCs engineered to constitutively produce IL-12 (IL-12-transduced) and pulsed with SLDA resulted in sterile immunity against murine visceral leishmaniasis (13).

IL-12-transduced SLDA-pulsed DCs had an enhanced protective effect compared with SLDA-pulsed DCs. Whether IL-12-transduced SLDA-pulsed DCs have enhanced therapeutic efficacy against established L. donovani infection has yet to be determined. Here we report that simply combining DC-based immunotherapy with Sb-based chemotherapy results in the cure of established L. donovani infection in mice. Complete clearance of parasite burden from liver and spleen was confirmed by Giemsa staining of organ smears and limiting dilution culture assay.

Treatment with SLDA-pulsed DCs induces Th1-type response even after establishment of L. donovani infection

SLDA-pulsed DC vaccination in uninfected mice induced a protective Th1-type response in cutaneous (14) and visceral (13) leishmaniasis. Here we show that treatment of L. donovani-infected mice with SLDA-pulsed DCs induced IFN-{gamma} mRNA accumulation in lymph node cells (Fig. 3A). The expression of IFN-{gamma} mRNA was not further enhanced when infected mice received combined therapy with SLDA-pulsed DCs plus Sb (Fig. 3A). Treatment with PBS, unpulsed DCs, or Sb alone did not induce the expression of IFN-{gamma} in lymph node cells of infected mice. Similarly, Ag-specific T cell proliferation and production of IL-12 and IFN-{gamma} were significantly greater in infected mice receiving SLDA-pulsed DCs than in infected control mice receiving PBS, SLDA-pulsed M{phi}s, unpulsed DCs, or Sb alone (Fig. 3, B and C). In accordance with the data from RT-PCR, neither Ag-specific T cell proliferation nor induction of IL-12/IFN-{gamma} proteins was further enhanced by combined therapy over those in the SLDA-pulsed DC-treated group (Fig. 3, B and C). Recent reports indicated that Sb inhibits Src homology protein tyrosine phosphatase 1, leading to augmentation of Janus kinase 2 (JAK2) and Stat5 phosphorylation (23). JAK/Stat pathways are known to be involved in IFN-{gamma} signaling (25), and augmentation of JAK2 and Stat5 phosphorylation leads to enhanced IFN-{gamma} signaling and the expression of inducible NO synthase (26). Sb can potentiate the production of reactive oxygen species in murine visceral leishmaniasis (27) and induces programmed cell death in Leishmania (28). Our data on combined therapy may have the following explanations. Sb may have potentiated the antileishmanial activity of DC-based therapy-induced IFN-{gamma} by enhancing IFN-{gamma} signaling. Alternatively, Sb, by its ability to potentiate the production of reactive oxygen species and to induce programmed cell death in Leishmania, may provide additional antileishmanial activity over DC-based therapy-induced IFN-{gamma}.



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  		FIGURE 3. Treatment with SLDA-pulsed DCs induces a Th1-type response in L. donovani-infected mice. A, RT-PCR of IFN-{gamma} mRNA in the lymph nodes of L. donovani-infected mice after treatment with PBS (lane 1), unpulsed DCs (lane 2), Sb alone (lane 3), SLDA-pulsed DCs (lane 4), and SLDA-pulsed DCs plus Sb (lane 5). One month postinfected mice received the indicated treatment once a week for 3 wk. Three days after receiving the last treatment, lymph node cells were pooled from four mice from each group, and RT-PCR was performed. The housekeeping gene hypoxanthine phosphoribosyltransferase was used as an internal control. B, Ag-specific T cell proliferation by lymph node cells of L. donovani-infected mice after receiving indicated therapies. Proliferation was measured by [3H]thymidine incorporation (counts per minute). Data represent the mean ± SD of triplicate wells. C, Ag-specific induction of IL-12 and IFN-{gamma} by lymph node cells of L. donovani-infected mice (mean of duplicate wells). The experimental protocols for B and C are the same as that for A, except that lymph node cells were cultured in complete RPMI medium containing 0.5% autologous normal mouse serum in the presence or the absence of SLDA. Results are representative of three experiments.

 
Splenocytes from L. donovani-infected mice repeatedly stimulated (three times) in vitro with SLDA-pulsed DCs in the presence of IL-2 resulted in the emergence of CD4+ IFN-{gamma}+ T cells with the characteristic Th1 phenotype (Fig. 4A). To consider the possibility of in vitro priming as intracellular cytokines were analyzed after three rounds of stimulation, splenocytes from uninfected mice were analyzed under the same experimental conditions. Intracellular IFN-{gamma} in normal CD4+ T cells was also detected (Fig. 4B). The percentage of CD4+ IFN-{gamma}+ T cells was, however, higher in splenocytes from infected mice (16.4%) compared with those from normal mice (9.9%), possibly because of the existence of primed T cells in infected mice.



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  		FIGURE 4. Emergence of CD4+ T cells with Th1 characteristics after repetitive in vitro stimulation of splenocytes from L. donovani-infected (A) and uninfected (B) mice with SLDA-pulsed DCs. Twenty-five-day-old cultures of splenocytes (after three in vitro stimulations) were studied for CD4 surface molecules (y-axis) and intracellular IFN-{gamma}, IL-4, and IL-10 (x-axis). Values in the quadrants represent the percentage of positive cells (representative of two experiments).

 
Our data suggest that SLDA-pulsed DC may induce a Th1 response in uninfected as well as L. donovani-infected mice. Neither DC-based immunotherapy nor Sb-based chemotherapy alone was sufficient to cure established infection in mice. This was achieved only by combining these two therapeutic strategies. A similar strategy of chemoimmunotherapy may be beneficial for other intracellular infections.


   Acknowledgments
 
We thank Anirban Manna for preparation of the manuscript.


   Footnotes
 
1 This work was supported by grants from the Council of Scientific and Industrial Research, the Department of Science and Technology, the Department of Biotechnology, and the Indian Council of Medical Research, Government of India. Back

2 Current address: Department of Zoology, Brahmananda Keshab Chandra College, Kolkata 700 035, India. Back

3 Address correspondence and reprint requests to Dr. Santu Bandyopadhyay, Division of Immunology, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India. E-mail address: santu2{at}iicb.res.in Back

4 Abbreviations used in this paper: AmB, amphotericin B; DC, dendritic cell; JAK, Janus kinase 2; M{phi}, macrophage; rm, recombinant mouse; Sb, sodium antimony gluconate; SLDA, soluble L. donovani Ag. Back

Received for publication July 29, 2002. Accepted for publication March 21, 2003.


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 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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H. W. Murray, K. C. Flanders, D. D. Donaldson, J. P. Sypek, P. J. Gotwals, J. Liu, and X. Ma
Antagonizing Deactivating Cytokines To Enhance Host Defense and Chemotherapy in Experimental Visceral Leishmaniasis
Infect. Immun., July 1, 2005; 73(7): 3903 - 3911.
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C. De Trez, M. Brait, O. Leo, T. Aebischer, F. A. Torrentera, Y. Carlier, and E. Muraille
Myd88-Dependent In Vivo Maturation of Splenic Dendritic Cells Induced by Leishmania donovani and Other Leishmania Species
Infect. Immun., February 1, 2004; 72(2): 824 - 832.
[Abstract] [Full Text] [PDF]

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