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Infectionand Clears Organ Parasites in Experimental

Leishmania donovaniAntimony-Resistant Macrophages Infected withTo Increase the IL-12/IL-10 Ratio in Imipramine Exploits Histone Deacetylase 11

Jean-Claude Dujardin and Syamal RoyMukhopadhyay, Kshudiram Naskar, Shyam Sundar, Sandip Mukherjee, Budhaditya Mukherjee, Rupkatha

http://www.jimmunol.org/content/193/8/4083doi: 10.4049/jimmunol.1400710September 2014;

2014; 193:4083-4094; Prepublished online 12J Immunol 

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The Journal of Immunology

Imipramine Exploits Histone Deacetylase 11 To Increasethe IL-12/IL-10 Ratio in Macrophages Infected withAntimony-Resistant Leishmania donovani and ClearsOrgan Parasites in Experimental Infection

Sandip Mukherjee,*,1 Budhaditya Mukherjee,*,1 Rupkatha Mukhopadhyay,*

Kshudiram Naskar,* Shyam Sundar,† Jean-Claude Dujardin,‡ and Syamal Roy*

The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kala-

azar patients infected with antimony-resistant Leishmania donovani (SbRLD). Previously we showed that MDR-1 upregulation in

SbRLD infection is IL-10–dependent. Imipramine, a drug in use for the treatment of depression and nocturnal enuresis in children,

inhibits IL-10 production from SbRLD-infected macrophages (SbRLD-Mfs) and favors accumulation of surrogates of antimonials.

It inhibits IL-10–driven nuclear translocation of c-Fos/c-Jun, critical for enhanced MDR-1 expression. The drug upregulates

histone deacetylase 11, which inhibits acetylation of IL-10 promoter, leading to a decrease in IL-10 production from SbRLD-

Mfs. It abrogates SbRLD-mediated p50/c-Rel binding to IL-10 promoter and preferentially recruits p65/RelB to IL-12 p35 and

p40 promoters, causing a decrease in IL-10 and overproduction of IL-12 in SbRLD-Mfs. Histone deacetylase 11 per se does

not influence IL-12 promoter activity. Instead, a imipramine-mediated decreased IL-10 level allows optimal IL-12 production in

SbRLD-Mfs. Furthermore, exogenous rIL-12 inhibits intracellular SbRLD replication, which can be mimicked by the presence of Ab

to IL-10. This observation indicated that reciprocity exists between IL-10 and IL-12 and that imipramine tips the balance toward an

increased IL-12/IL-10 ratio in SbRLD-Mfs. Oral treatment of infected BALB/c mice with imipramine in combination with sodium

stibogluconate cleared organ SbRLD parasites and caused an expansion of the antileishmanial T cell repertoire where sodium

stibogluconate alone had no effect. Our study deciphers a detailed molecular mechanism of imipramine-mediated regulation of

IL-10/IL-12 reciprocity and its impact on SbRLD clearance from infected hosts. The Journal of Immunology, 2014, 193: 4083–4094.

Resistance to sodium stibogluconate (SSG) or pentavalentantimonials in the treatment of visceral leishmaniasis(VL) or kala-azar is a major problem in the Indian sub-

continent (1). In Bihar, the epicenter of Indian kala-azar, tradi-tional pentavalent antimonial treatment was abandoned notbecause of toxicity but owing to high resistance (2). Pentavalentantimonials remain the treatment of choice in Africa, SouthAmerica, Bangladesh, Nepal, and India (except north Bihar) (3).

The resistance is due to upregulation of the multidrug resistanceprotein (MDR)-1 pump, which causes efflux of antimonials (4).

New drugs such as miltefosine and amphotericin B are in use for

the treatment of antimony-resistant (SbR) kala-azar cases, but re-

lapse cases are also increasing (5, 6). Because antimonials and

miltefosine use the same efflux pump (7), this raises serious

concern about the efficacy of miltefosine, either alone or in

combination, in view of the rampant antimony resistance in the

field. Recent studies from our group showed that ∼78% of the

recent clinical isolates are resistant to antimonials although

the drug has not been in use for more than a decade in Bihar (8).

Moreover, there is an increasing number of reports of post–kala-

azar dermal leishmaniasis after successful treatment with milte-

fosine (9, 10). In 2010, the World Health Organization’s expert

committee on leishmaniasis “strongly recommended not to use

miltefosine monotherapy” owing to a serious form of leishmani-

asis relapse in Nepal after a year when one in five people treated

developed the disease (11). The question is how the pre-existing

rampant antimony resistance in the field would influence the

efficacy of future drugs. Considering the health problem in the

Indian subcontinent and Sudan, one would expect that anti-

leishmanial drugs must ideally have the following criteria: they

must be orally active, inexpensive, and less toxic and show

immunostimulatory properties. Previously we showed that most

of the antileishmanial drugs possess immunostimulatory ability

(12). The emergence of drug resistance in leishmania is however

hampering the control program.One of the strategies to discover new drugs is to reposition or

reemploy existing drugs that will reduce the cost and time for drug

*Department of Infectious Diseases and Immunology, Council of Scientific andIndustrial Research–Indian Institute of Chemical Biology, Kolkata 700032, India;†Institute of Medical Sciences, Benaras Hindu University, Varanasi 221005, India;and ‡Institute of Tropical Medicine, 2000 Antwerp, Belgium

1S.M. and B.M. contributed equally to this work.

Received for publication March 18, 2014. Accepted for publication August 8, 2014.

This work was supported by Council of Scientific and Industrial Research (NewDelhi) Network Projects BSC 0114 and BSC 0120, as well as by European Com-mission–funded Kaladrug-R Project Grant Health-F3-2008-222895. S.M., B.M., andR.M. are recipients of a fellowship from the Council of Scientific and IndustrialResearch (New Delhi).

Address correspondence and reprint requests to Dr. Syamal Roy, Department ofInfectious Diseases and Immunology, Council of Scientific and Industrial Re-search–Indian Institute of Chemical Biology, Kolkata 700032, India. E-mail address:sroy@iicb.res.in

The online version of this article contains supplemental material.

Abbreviations used in this article: ChIP, chromatin immunoprecipitation; HDAC,histone deacetylase; Mf, macrophage; MDR, multidrug resistance protein; PEC,peritoneal exudate cell; RAW-Mf, RAW264.7 cell line Mf; Rh123, rhodamine123; SbR, antimony-resistant; SbRLD, antimony-resistant Leishmania donovani; SbS,antimony-sensitive; SbSLD, antimony-sensitive L. donovani; SLA, soluble leishmanialAg; SSG, sodium stibogluconate; VL, visceral leishmaniasis.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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development (13). Fleximidazole, a nitroimidazole recently testedin a phase I clinical trial for treating African trypanosomiasis (14),could potentially be used to treat VL (15). Thioridazole, belongingto the class of antipsychotic drugs, shows promising activitiesagainst multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis in a mouse model (16). Most importantly,it shows unique activity against nonreplicating Mycobacteriumtuberculosis culture grown under hypoxic conditions (17). Similarto phenothiazines, imipramine, another antipsychotic drug usedfor treating depression (18) and pediatric nocturnal enuresis (19),alters the proton motive force in Leishmania donovani promasti-gotes (20) and kills antimony-sensitive (SbS) and SbR intracellularamastigotes without affecting the host cell viability. Additionally,oral treatment of imipramine clears organ parasites in hamsters(21). Imipramine is a potent inducer in macrophages (Mfs) ofTNF-a (22), which is an important cytokine for antileishmanialdefense (23) and is a competitive inhibitor of trypanothione re-ductase, an enzyme upregulated in SbR L. donovani (SbRLD) (8).Two important cytokines, IL-10 and IL-12, play a crucial role in

Leishmania infection. IL-10 promotes intracellular infection andpersistence (24) and increases the risk of relapse (25); IL-12 is thekey cytokine for IFN-g–producing Th1 cells (26, 27). EndogenousIL-12 is also required for the enhancement of the leishmanicidaleffect of conventional antileishmanial drugs (28). It acts as a potenthistone modulator in the brains of maternally deprived adult rats(29). Previously we have seen that the upregulation of MDR-1 inMfs by SbRLD is IL-10–dependent and requires recruitment of p50/c-Rel at the IL-10 promoter site (4). Imipramine has been shown toreverse multidrug resistance phenotype in acute myelogenous leu-kemia cell lines (30). There is a recent report that histone deace-tylase (HDAC)11, one of the newly added members of the HDACfamily, regulates IL-10 expression in mouse and human APCs (31).We showed that hamsters infected with SbRLD show a decreasedIL-12/IL-10 ratio that is reversed upon oral imipramine treatment(21). The mechanism by which imipramine activates the SbRLD-infected host to increase IL-12 levels with a concomitant decrease inIL-10 is a matter of considerable interest. In this study, we show thatimipramine inhibits IL-10 by overexpressing HDAC11, which inturn inhibits MDR-1 upregulation in SbRLD-infected Mfs. A de-crease in IL-10 favors IL-12 expression, which in turn favors ex-pansion of antileishmanial T cells, leading to parasite clearance.

Materials and MethodsAnimals

BALB/c mice (Mus musculus) and hamsters (Mesocricetus auratus) weremaintained and bred under pathogen-free conditions. Use of animals wasapproved by the Institutional Animal Ethics Committee of Indian Instituteof Chemical Biology. Il122/2 mice were obtained from, and experimentswere performed in, the animal facility of the National Center for CellScience (Pune, India). All animal experiments were performed accordingto the National Regulatory Guidelines issued by the Committee for thePurpose of Supervision of Experiments on Animals, Ministry of Envi-ronment and Forest, government of India.

Ethics statement

Use of human subjects was approved by the Ethical Committee of HumanSubjects of the Indian Institute of Chemical Biology. Blood samples were drawnfrom normal healthy individuals after obtaining their written informed consent.

Parasite cultures and maintenance

SbRLD (MHOM/IN/2009/BHU575/0 and MHOM/IN/2005/BHU138)and SbS L. donovani (SbSLD; MHOM/IN/83/AG83 and MHOM/NP/03/BPK206) (8) maintained in golden hamsters (32) were used for this study.

Preparation of soluble leishmanial Ag

Soluble leishmanial Ag (SLA) was prepared from stationary phaseL. donovani promastigotes (BHU 575 and Ag 83) following the published

protocol (33). The protein concentration was determined by the Bradfordprotein assay method (Bio-Rad, Hercules, CA). The prepared Ag wasstored at 270˚C until further use.

Preparation of macrophages from human PBMCs and infection

Peripheral blood was collected from three healthy donors by venipuncture,and mononuclear cells were separated by centrifugation over Ficoll-Hypaque as described previously (34). Cells were washed using PBSsupplemented with 0.5% FCS and harvested in tissue culture plates for 3 h.Nonadherent cells were discarded by washing, and adherent cells werecultured for 3 d and designated as Mfs. Cells were then incubated in thepresence or absence of L. donovani (103 the cell number) for 48 h incomplete RPMI 1640 media. Imipramine treatment was carried out for24 h more in selected sets. The supernatant was collected and stored frozenfor subsequent experiments.

Cell culture and infection

Peritoneal exudate cells (PECs), conveniently named PEC-Mfs, wereharvested from BALB/c mice and purified as described previously (8).PEC-Mfs were infected with stationary-phase L. donovani promastigotesat a ratio of 1:10 for 6 h, washed to remove free parasites, and incubatedfor another 24 h. Treatments were given where needed. Supernatants ob-tained from infected and treated PEC-Mfs were used for cytokine analysisby ELISA. In some experiments PEC-Mfs were infected with adenovirusfor overexpression of HDAC6 or HDAC11 or GFP (SignaGen Laborato-ries) following the manufacturer’s protocol. Transfection experiments wereperformed using the RAW264.7 cell line, defined as RAW-Mfs.

Infection of BALB/c mice

To infect BALB/c mice (6 wk old), amastigotes from SbSLD strain Ag83and SbRLD strain BHU 575 L. donovani were purified as described (35)and inoculated (107 parasites in 100 ml) via intracardiac routes as reportedpreviously (33).

Preparation of drug stocks for drug assays

Imipramine hydrochloride (Sigma-Aldrich, St. Louis, MO) and sodiumstibogluconate (SSG; provided by Albert David, Kolkata, India) solutionswere prepared at 1 mg/ml in PBS (Sigma-Aldrich), then subjected to sterilefiltration using 0.22-mm filters (Millipore) when required.

Treatment

PEC-Mfs were treated with imipramine at several concentrations (25, 50,and 75 mM). In some experiments PEC-Mfs were treated in serum-freemedium with 10 mM ERK1/2 inhibitor U0126 (36), 1 mM PI3K inhibitorwortmannin (37), 20 mM ΙkΒ of the kinase inhibitor BAY 11-7082 (8), 25mM JNK inhibitor SP 600125 (38), or 10 mM p38 inhibitor SB 203580(39) for 45 min before infection. PEC-Mfs were also treated with rIL-12(100 and 200 pg/ml), rIL-10 (2, 20, and 200 pg/ml; BD Pharmingen), orneutralizing IL-10 Ab (10 ng/ml; BD Pharmingen). In some experimentswhole splenocytes isolated from either infected or treated mice weretreated with SLA as described previously (4), and subsequently ELISA andT cell proliferation assays were performed.

Real-time quantitative PCR to estimate expression of HDAC inimipramine-treated SbRLD-infected PECs

Total RNAwas isolated from normal and SbRLD-infected PEC-Mfs with orwithout imipramine treatment using a total RNA isolation kit (Roche Bio-chemicals) and following the manufacturer’s protocol. cDNA synthesis andreal-time quantitative PCR were done as described elsewhere (8). SbRLD-infected PEC-Mfs were defined as SbRLD-PEC-Mf for convenience. Thequantitative PCR contained 23 SYBR Green Supermix (Applied Bio-systems, Foster City, CA) diluted twice and forward and reverse primers. Thesequences of the forward and reverse primers are presented in Table I.Reactions were run on an Applied Biosystems 7500 Fast Real-Time PCRSystem. Experiments on negative controls of cDNA synthesis (i.e., withoutreverse transcriptase) and no-template controls (i.e., without cDNA template)were also done for each gene. All reactions were done in duplicate, and theirarithmetic average threshold cycle (Ct) was used for data analysis. The foldof gene expression compared with the control was calculated as 22DDCt by7500 Fast System SDS software, version 1.4 (Applied Biosystems).

Reporter assays

All of the transfection experiments were performed with the RAW264.7 cellline. The murine IL-12 p35 promoters 26/21411 (1.4 kb; 59-GGT GTC

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CTT CTT ATT GGC TTG-39 and 59-CCG GCA CTG AGA GGA GCTGC-39) and p40 promoters 2103/21165 (1.06 kb; 59-CAG GAC AGGAAT GGA GAA GCG GC-39 and 59-GTT AGC GAC AGG GAA GAGGAG AG-39) were PCR amplified and cloned into a pGL3-Basic Vector(Promega). Using the IL-12p35 promoter construct (1.4 kb) and a Quick-Change II PCR-based, site-directed mutagenesis kit (Stratagene), threemutated IL-12p35 promoters constructs, containing a deletion at the NF-kB binding site 2100/2110, or at the NF-kB binding site 2229/2240, ora double deletion at NF-kB binding sites 2100/2110 and 2229/2240 ofIL-12p35 promoter were generated. A mutated IL-12p40 promoter con-struct containing a deletion at NF-kB binding site 2121/2132 was alsogenerated using the IL-12p40 promoter construct (1.06 kb). All of theinserts were confirmed by sequencing. RAW-Mfs were transiently trans-fected with 2 mg of these IL-12 or IL-10 (1.5 kb) or MDR-1 promoterconstructs (4) using Lipofectamine 2000 (Invitrogen), rested for 12 h, andtreated with rIL-10, rIL-2, or imipramine for 24 h. In some experimentsluciferase activity was measured in imipramine-treated RAW-Mfs 24 hafter SbRLD infection. Luciferase activity in cell extracts was measuredusing the Dual-Luciferase reporter kit (Promega) according to the manu-facturer’s protocols and normalized to the level of the protein content.

Chromatin immunoprecipitation assay

Using a commercially available kit (Upstate Biotechnology), a chromatinimmunoprecipitation (ChIP) assay was performed as recommended by themanufacturer with minor modifications. Chromatin was immunoprecipi-tated at 4˚C overnight with Abs to rabbit IgG or NF-kB Abs such as anti-p50, anti-p65, RelB, and c-Rel, and DNAwas extracted. A PCR assay wasperformed to amplify the region of the IL-10 promoter (using primers 59-GCC CCA CAG CAC ACATAT CC-39 and 59-CCT GGG TTG AAC GTCCG-39), the regions of the IL-12 p35 promoter (using primers 59-CGGGAC AAG AGT GGC TAC TCG C-39 and 59-CTC GGA GTG CCC GTTTGA GG-39 or 59-GCG CCA CCA GCC TTG GG-39 and 59-GGA ACGCTG ACC TTG GGA GAC-39), or the region of the IL-12p40 promoter(using primers 59-CCT CTG TAT GATAGATGC AC-39 and 59-GTT TTGACA CTA GTT TTC-39).

Confocal microscopy

The levels of expression of MDR-1 in rIL-10– or imipramine-treated PEC-Mfs were determined by immunostaining followed by confocal micros-copy (LSM 510; Carl Zeiss), as described elsewhere (4).

Dye uptake and retention assay

After treatment with several concentrations of imipramine, SbRLD- andSbSLD-infected as well as rIL-10–stimulated PEC-Mfs were washedand resuspended (2 3 105 cells/ml) in serum-free RPMI 1640 incubatedwith an optimum concentration (250 ng/ml) of free rhodamine-123(Rh123) for 32 h, washed, and further incubated in media free of Rh123.At indicated time points, cells were washed three times in PBS and finallylysed in 0.1% Triton X-100. The intracellular dye concentrations weredetermined by measuring the fluorescence intensity of the cell lysates.

Western blot

Cytoplasmic and nuclear proteins were prepared and Western blotting wasperformed for p50, p-p50, c-Rel, RelB, HDAC6, HDAC11 (Santa CruzBiotechnology), p65, p-p38/p-38, p–c-Fos/c-Fos, p–c-Jun/c-Jun, b-actin,and histone (Cell Signaling Technology) as described elsewhere (4). Inbrief, blots were probed with specific Abs. Binding of secondary HRP-labeled goat anti-rabbit or goat anti-mouse Abs (Cell Signaling Tech-nology) was analyzed using SuperSignal West Pico or West Durachemiluminescent substrate (Pierce).

In vitro parasite clearance

To determine in vitro parasite clearance, PEC-Mfs were infected witheither SbSLD (AG83) or SbRLD (BHU 575) for 24 h and either left un-treated or treated. At the endpoints, the coverslips were washed with PBS,dried, fixed with 100% methanol (Merck), stained with 10% Giemsa(Sigma-Aldrich), and examined microscopically. One hundred Mfs percoverslip were scored and the amastigotes were enumerated.

In vivo parasite clearance

The 6-wk-infected BALB/c mice were randomly divided into four groups(groups I–IV). Group I received only saline, group II received only SSG atthe dose of 20 mg/kg/d once in a week for 4 wk (40), and group III re-ceived imipramine at the dose of 0.1 mg/kg/d for 4 wk by oral route usinga feeding needle as described by others (41). Group IV received imipra-mine (0.1 mg/kg/d) by oral route for 4 wk and, 6 h thereafter, i.p. SSG (20

mg/kg) once a week for 4 wk. Two days after the completion of treatment,mice were sacrificed to determine splenic and hepatic parasite burdens bythe stamp smear method as described elsewhere (42), as well as by theserial dilution method (33).

Cytokine measurement

Various cytokine levels (IL-10 and IL-12p70) in the splenocytes or peri-toneal Mfs were measured using a sandwich ELISA kit (BD Pharmingen,San Diego, CA) as per the manufacturer’s protocol. The level of IL-10from human Mfs was determined using commercially available mono-clonal anti-human IL-10, anti-human biotin, and rhIL-10 (BD Pharmingen)according to standard protocol. IL-12p70 in the same supernatant wasmeasured using sandwich ELISA kit (BD Pharmingen).

T cell proliferation assay

A T cell proliferation assay was performed as described elsewhere (33).Briefly, spleens were isolated from different experimental groups of mice.Single-cell suspensions of splenocytes prepared after Ficoll density gra-dient centrifugation were suspended in complete RPMI medium. Cellswere plated in triplicate in 96-well plates at a concentration of 105 cells perwell and allowed to proliferate for 3 d at 37˚C in a 5% CO2 incubatoreither in the presence or absence of SLA (5 mg/ml). At 4 h before harvest,cells were incubated with MTT and T cell proliferation was measured witha nonradioactive MTT cell proliferation assay using an ELISA plate reader(DTX 800 multimode detector, Beckman Coulter, Brea, CA).

Statistical analysis

Each experiment was performed three times and the data represent meansof three independent experiments (6SD) unless noted otherwise. Statis-tical significance between means of various groups was determined usinga two-tailed Student t test. Only p values ,0.05 were considered to bestatistically significant. Values were considered extremely significant(p , 0.001), very significant (p = 0.001–0.01), or significant (p = 0.01–0.05) as indicated. Error bars indicate means 6 SD. Data were ana-lyzed using Prism 5.0 (GraphPad Software, San Diego, CA).

ResultsImipramine abrogates SbRLD- or rIL-10–driven MDR-1overexpression

Imipramine treatment results in a significant dose-dependent in-crease in the accumulation of Rh123, a surrogate marker for an-timony, in SbRLD (BHU 575 or BHU 138)-infected or rIL-10–treated PEC-Mfs. However, in SbSLD (AG83 or BPK 206)-infected PEC-Mfs, Rh123 accumulation was found to be highwithout imipramine treatment (Fig. 1A). Because both BHU 575and BHU 138 showed similar results for the rest of the experi-ments, only BHU 575 has been used and defined as SbRLD forconvenience. Similarly, AG83 was selected as a representative ofSbSLD infection.The MDR-1 reporter assay showed a decrease in IL-10–driven

MDR-1 promoter activity as a function of imipramine concen-tration in RAW-Mfs (Fig. 1B). All transfection experiments wereperformed in the RAW264.7 cell line; for all other experiments,primary Mfs were used unless noted otherwise. The upregulationof MDR-1 is specific to IL-10 because rIL-2, an unrelated cyto-kine, failed to show the above effect. Furthermore, confocal mi-croscopy of rIL-10–treated PEC-Mfs stained with FITC-labeledAb to MDR-1 showed upregulation of MDR-1, which wasdownregulated in the presence of imipramine (Fig. 1C). Westernblot analysis showed that imipramine inhibited nuclear translo-cation of c-Fos/c-Jun, the subunits of AP-1 transcription factorsimportant in MDR-1 upregulation in SbRLD-PEC-Mfs (Fig. 1D).

Imipramine inhibits IL-10 promoter and histone acetylation

Because imipramine treatment abrogated IL-10 production in SbRLD-infected PEC-Mfs, we checked the status of IL-10 promoter ac-tivity. Imipramine treatment led to abrogation of SbRLD-mediatedhistone acetylation and binding of p50/c-Rel at the IL-10 promoter

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FIGURE 1. (A) Increase in Rh123 accumulation in SbRLD (BHU 575, BHU 138)-infected and rIL-10–treated normal Mfs as a function of imipramine

concentration. In the control set, SbSLD (Ag 83, BPK 206)-infected Mfs show accumulation of Rh123 regardless of absence or presence of imipramine. (B)

Reporter assay showing inhibition of MDR-1 promoter activity as a function of imipramine concentration. The RAW264.7 cell line was transfected with

MDR-1 reporter construct, stimulated with rIL-10 (200 pg/ml) or IL-2 (200 pg/ml), and either treated with an increasing concentration of imipramine or left

untreated. A progressive decrease in luciferase activity was observed as a function of imipramine concentration as compared with untreated control. (C)

Confocal imaging of MDR-1 expression in rIL-10–treated PEC-Mfs in the absence and presence of imipramine. Murine PEC-Mfs were harvested in eight-

well chambered slides and adhered for 48 h. After rIL-10 treatment for 24 h, Mfs were kept with imipramine (25 mM) for another 24 h. The cells were

stained with FITC-conjugated anti–MDR-1 Ab and subjected to confocal imaging (original magnification 3100). Imipramine treatment abrogated rIL-10–

mediated MDR-1 overexpression. (D) Expression of subunits of AP-1 transcription factors in rIL-10–stimulated PEC-Mfs in the presence and absence of

imipramine. Mfs were stimulated with rIL-10 for 24 h and then treated with imipramine for another 24 h. Expression of p–c-Fos/c-Fos and p–c-Jun/c-Jun

in cytoplasmic as well as the nuclear extract was analyzed by Western blot analysis. Histone was used as nuclear loading controls. (E) ChIP analysis

showing imipramine treatment prevents p50/c-Rel binding to IL-10 promoters. Nuclear extract from PEC-Mfs infected with SbRLD (BHU 575) for 24 h

was assessed with Abs to hyperacetylated histone H3 (Ace H3), p65, p50, c-Rel, and RelB and then subjected to PCR amplification. Chromatin was

immunoprecipitated by whole-rabbit IgG. No Ab was used as a negative control, and input DNA (5%) was used as an internal control. Results in (A) and (B)

are presented as means6 SD, with statistical significance being determined with respect to imipramine untreated control. (C)–(E) are representative of three

independent experiments. ***p , 0.001.

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site (Fig. 1E). This observation prompted us to study the role, ifany, of HDAC in the above process.

HDAC status in SbRLD-PEC-Mfs before and after imipraminetreatment

The status of HDAC1 to HDAC11 was analyzed by real-time PCRusing primer sets mentioned in Table I. From this analysis it wasevident that only two members of the HDAC family, HDAC6 andHDAC11, showed altered expression in SbRLD-PEC-Mfs withrespect to uninfected PEC-Mfs, and this was further modulatedupon imipramine treatment (Fig. 2A). Upregulation of HDAC6,which occurred by 3.2-fold in SbRLD-PEC-Mfs, was reduced to1.8-fold after imipramine treatment. Conversely HDAC11, whichwas downregulated 0.4-fold in SbRLD-PEC-Mfs, was upregu-lated 5.6-fold after imipramine treatment. Western blot analysis

confirmed that imipramine treatment induced HDAC11 in normalas well as SbRLD-PEC-Mfs but not in untreated SbRLD-PEC-Mfs (Fig. 2B). HDAC6 was expressed in SbRLD-PEC-Mfs, butthis was reduced to some extent after imipramine treatment.

Functional analysis of the role of HDAC11 and HDAC6 inIL-10/IL-12 reciprocity

The relationship, if any, between the altered expressions ofHDAC11 and HDAC6 and the production of IL-10 and IL-12 wasevaluated. It was observed that overexpression of HDAC11 but notof HDAC6 resulted in an ∼2-fold decrease in IL-10 and an∼3-fold increase in IL-12 promoter activity in SbRLD-RAW-Mfs.Interestingly, imipramine treatment of SbRLD-RAW-Mfs over-expressing HDAC11 further decreased IL-10 promoter activity to∼3-fold with respect to infected control, whereas IL-12 promoteractivity was increased by ∼8-fold (Fig. 3A). The cytokine assayrevealed an ∼2-fold decrease in IL-10 and an ∼4-fold increase inIL-12 level in SbRLD-PEC-Mfs overexpressing HDAC11. Imip-ramine treatment of SbRLD-PEC-Mfs overexpressing HDAC11showed further reduction of IL-10 by ∼4-fold, whereas IL-12 levelwas increased by ∼9-fold (Fig. 3B). Interestingly, an IL-12 re-porter assay showed similar luciferase activity in both HDAC11overexpressed and non-overexpressed RAW-Mfs in the presenceof imipramine (Fig. 3C). Furthermore, we analyzed IL-12 levelin imipramine-treated SbRLD-PEC-Mfs in the presence andabsence of IL-10–neutralizing Ab to determine whether IL-10has any influence on imipramine-mediated IL-12 production(Fig. 3D). Note that the presence of IL-10–neutralizing Ab furtherincreased imipramine-mediated IL-12 generation in SbRLD-PEC-Mfs (Fig. 3D).

Imipramine recruits p65/RelB for IL-12 induction inSbRLD-Mfs

Using an array of pharmacological inhibitors, we observed thatimipramine-mediated IL-12 generation from SbRLD-PEC-Mfswas significantly inhibited (∼3.5-fold) only in the presence ofp38 and NF-kB inhibitors, whereas inhibitors of ERK, PI3K,and JNK did not show any such inhibitory effect (Fig. 4A).

FIGURE 2. (A) Quantitative real-time RT-PCR analysis of HDAC1–HDAC11 mRNA. PEC-Mfs were infected with SbRLD (BHU 575) for 24 h and

either left untreated or treated with imipramine for an additional 24 h before total RNA isolation. Results are normalized to b-actin expression and

presented relative to those of control cells. (B) Western blot analysis of HDAC11 and HDAC6 expression. Nuclear extract was isolated from normal or 24-h-

infected SbRLD (BHU 575) PEC-Mfs either in the presence or absence of imipramine (24 h) . Expression of HDAC6 and HDAC11 in nuclear extract was

analyzed by Western blot analysis. Histone was used as nuclear loading controls. Results in (A) are presented as means 6 SD, with statistical significance

being determined between means of imipramine untreated and treated groups. (B) is representative of three independent experiments. ***p , 0.001.

Table I. Sequences of primers used for real-time PCR

Gene Primer Product (bp)

HDAC1 59-CGCATGACTCACAATTTGCT-39 20459-AAACACCGGACAGTCCTCAC-39

HDAC2 59-AGACTGCAGTTGCCCTTGAT-39 19859-TTTGAACACCAGGTGCATGT-39

HDAC3 59-CACCTTTTCCAGCCAGTCAT-39 20259-GGACAGTGTAGCCACCACCT-39

HDAC4 59-CAGACAGCAAGCCCTCCTAC-39 19859-AGACCTGTGGTGAACCTTGG-39

HDAC5 59-AGGTAGCCGAGAGGAGAAGC-39 19859-CTGAGCCAGTAAAGCCGTTC-39

HDAC6 59-AAGTGGAAGAAGCCGTGCTA-39 20059-CTCCAGGTGACACATGATGC-39

HDAC7 59-TGAAGAATGGCTTTGCTGTG-39 20159-CACTGGGGTCCTGGTAGAAA-39

HDAC8 59-CGACGGAAATTTGACCGTAT-39 20059-TGAATGGGCACATTGACACT-39

HDAC9 59-GCGGTCCAGGTTAAAACAGA-39 20359-GAGCTGAAGCCTCATTTTCG-39

HDAC10 59-CAGAGGAAGAGTTGGGCTTG-39 20159-GCACAGCTCCTGTTAGCACA-39

HDAC11 59-TTCCTTGTGCAGAGGAAGGT-39 19759-ACGCGTTCAAACAGGAACTT-39

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Promoter scan analysis revealed two NF-kB binding sites atpositions 2100/2110 and 2240/2229 of IL-12 p35 promoterdesignated as sites A and B, respectively, and one NF-kB halfsite at position 2121/2132 of the IL-12p40 promoter desig-nated as site C (Fig. 4B). To determine the specific promoter siteinvolved in imipramine-mediated IL-12 generation, RAW-Mfswere transfected with IL-12p35 or IL-12p40 or their combina-tion, and luciferase activity was determined after imipraminetreatment (Fig. 4C). Deletion of either site A or site B of theIL-12p35 promoter results in an ∼45% reduction of luciferaseactivity, whereas deletion of both the sites (A and B) caused an

∼80% reduction. Interestingly, although the IL-12p40 promoterfailed to show significant luciferase activity alone, presence ofit resulted in a significant increase in luciferase activity of theIL-12p35 promoter. Similar experiments in the presence of SbRLDinfection also revealed that the presence of all three sites (A–C) isrequired for optimal imipramine-mediated IL-12 generation(Fig. 4D). To determine the NF-kB subunits critical for imipramine-mediated IL-12 generation in SbRLD-PEC-Mfs, ChIP of theIL-12 promoter was performed using Abs for acetylated histone,p65, p50, c-Rel, or RelB. The results revealed that histone acet-ylation and binding of p65/RelB at all three sites of the IL-12

FIGURE 3. (A) Overexpression of HDAC11 abrogates IL-10 and induces IL-12 production in SbRLD (BHU 575)-infected Mfs after treatment with

imipramine. RAW-Mfs were transfected with either IL-10 or IL-12 reporter construct, infected with SbRLD (BHU 575), and then either transfected with

adenovirus encoding HDAC6 or HDAC11 or GFP for 24 h, or left untreated or treated with imipramine for another 24 h. Finally, cells were lysed and

luciferase activity was measured. Results are normalized to protein concentrations. (B) IL-10 and IL-12 levels were measured in the culture supernatant of

PEC-Mfs under the same experimental condition as above but without transfecting IL-10 or IL-12 reporter constructs and in the presence of an uninfected

and untreated control. (C) Luciferase activity in IL-12 reporter construct transfected RAW-Mfs infected with adenoviral encoding either HDAC11 or GFP

for 24 h and the cells were either left unstimulated or stimulated with imipramine for another 24 h. Results are normalized to protein concentrations. (D) IL-

12 level in the culture supernatant of Mfs infected with SbRLD (BHU 575) and then either left untreated or treated with imipramine for 24 h in the presence

and absence of IL-10–neutralizing Ab. Results in (A)–(D) are presented as means6 SD. For (A) and (B) statistical significance was determined between the

infected control group and the HDAC11 overexpressed group and also between the HDAC11 overexpressed group and the imipramine-treated HDAC11

overexpressed group. For (D), statistical significance was determined between the infected control group and the imipramine-treated group and also be-

tween the imipramine-treated group and the anti–IL-10 Ab treated group. **p = 0.001–0.01, ***p , 0.001.

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promoter occurred upon imipramine treatment in SbRLD-PEC-Mfs (Fig. 4E).

Reciprocity between IL-10 and IL-12 generation

We studied the influence of IL-10 over imipramine-activated IL-12promoter activity. Low doses of rIL-10 (2 or 20 pg/ml) failed to alter

the status of imipramine-mediated p65/RelB binding to the IL-12promoter. However, at 200 pg/ml rIL-10 there was a partial abro-gation of p65/RelB binding with the IL-12 promoter (Fig. 5A). Wealso studied the influence of IL-12 or of neutralizing IL-10 Abon SbRLD-mediated IL-10 generation from PEC-Mfs. Pre-incubation of PEC-Mfs with rIL-12 or neutralizing IL-10 Ab

FIGURE 4. Imipramine-mediated IL-12 generation in

SbRLD-infected PEC-Mfs in the absence and presence

of pharmacological inhibitors. PEC-Mfs were incubated

with an array of pharmacological inhibitors, infected with

SbRLD (BHU 575) for 24 h, and treated with imipramine

for another 24 h. (A) Imipramine-mediated IL-12 pro-

duction in the SbRLD-infected host was inhibited in the

presence of BAY110782 (NF-kB inhibitor) or SB203580

(p38 inhibitor) but not by wortmannin (PI3K inhibitor),

U0126 (ERK1/2 inhibitor), or SP600125 (JNK1/2 inhibi-

tor). (B) Graphical representation of IL-12 promoter with

NF-kB binding sites. There are two sites for NF-kB

binding in p35 subunit of IL-12 promoter, designated as

site A (2229/2240) and site B (2100/2110), whereas a single

NF-kB binding site, designated as site C (2121/2132),

was present in the IL-12p40 subunit. (C) IL-12 over-

expression is dependent on both p35 and p40 of the

IL-12 promoter. Luciferase activity of the lysate of RAW-

Mfs transfected with either whole-length construct of

pGL3-IL-12p35 promoter or with pGL3-IL-12p40 pro-

moter or with both was measured. In some cases trans-

fections are done with deletion construct (D; trun) of

pGL3-IL-12 promoter (lacking site A of the p35 promoter,

or site B of the same or half site of p40) followed by

imipramine treatment. (D) Reporter assay showing up-

regulation of IL-12. Luciferase activity was assessed for

the lysate of RAW-Mfs transfected with p35, p40, or p35

and p40 followed by infection with SbRLD and imipra-

mine treatment. (E) Imipramine treatment activates p65/

RelB to bind with IL-12 promoter regions. ChIP analysis

of IL-12 p35 promoter and p40 promoter with nuclear

extract derived from Mfs infected with SbRLD (BHU

575) and assessed with Abs to hyperacetylated histone H3

(Ace H3), p65, p50, c-Rel, and RelB followed by PCR

amplification. Chromatin was immunoprecipitated by

whole-rabbit IgG. No Ab was used as a negative control,

and input DNA (5%) was used as an internal control.

Results in (A), (C), and (D) are presented as means 6 SD,

and (E) is representative of three independent experiments.

Statistical significance for (A) was determined with respect

to infected control group, for (C) with respect to p35 re-

porter group, and for (D) between p35 reporter–infected

and imipramine-treated group and also between p35

imipramine–treated and cotransfected imipramine-treated

group. *p = 0.01–0.05, **p = 0.001–0.01, ***p , 0.001.

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resulted in reduced IL-10 production (Fig. 5B). We observedthat the number of amastigotes per 100 PEC-Mfs in the case of

SbRLD infection was significantly less when PEC-Mfs were

pretreated with rIL-12 or neutralizing IL-10 Ab as compared

with untreated control (Fig. 5C). Furthermore, the IL-10 level

was significantly higher in SbRLD-infected Il122/2 Mfs as com-

pared with infected wild-type Mfs upon imipramine treatment

(Fig. 5D).

Cotreatment with SSG and imipramine eliminates organparasites of BALB/c mice infected with SbRLD

Having seen that imipramine is a potent inhibitor of MDR-1, weasked whether a combination of imipramine with SSG would be

effective in clearing SbRLD from infected BALB/c mice. Thus,

SbRLD-infected BALB/c mice were treated with SSG (20 mg/kg)

in the absence or presence of imipramine (0.1 mg/kg; Fig. 6).

Although, SSG alone could clear ∼43% of splenic as well as

FIGURE 5. Reciprocal regulation between IL-10 and IL-12 induction. (A) ChIP analysis of IL-12 promoters p35 and p40 with nuclear extract derived

from PEC-Mfs infected with SbRLD (BHU 575), treated with rIL-10 in a dose-dependent manner (2, 20, or 200 pg/ml), and assessed with Abs to

hyperacetylated histone H3 (Ace H3), p65, p50, c-Rel, and RelB followed by PCR amplification. ChIP by whole-rabbit IgG. No Ab was used as a negative

control, and input DNA (5%) was used as an internal control. (B) IL-10 production in the culture supernatant of PEC-Mfs, preincubated with either rIL-12 or

rIL-2 and infected with SbRLD, by ELISA. (C) Number of intracellular amastigotes per 100 Mfs in PEC-Mfs preincubated with either rIL-12 or neu-

tralizing IL-10 or rIL-2 and infected with SbRLD. (D) IL-10 production in the culture supernatant of wild-type or Il122/2 PEC-Mfs either left uninfected or

infected with BHU 575 in the presence or absence of imipramine. (A) is representative of three independent experiments, and (B)–(D) are presented as means6 SD.

For (B) and (C), statistical significance was determined with respect to infected control. For (D), statistical significance was determined between infected IL122/2Mfs

in the presence and absence of imipramine and also between wild-type and the Il122/2 imipramine-treated group. ***p , 0.001.

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FIGURE 6. (A and B) Combination of imipramine and SSG clears organ parasites in BALB/c mice infected with SbRLD. Six-week-old mice were

infected with either SbRLD (BHU 575) or SbSLD (Ag83) parasites and infection was allowed to be established for the next 6 wk. Treatment was carried out

as described in Materials and Methods. Two days after the last treatment, mice were sacrificed and the hepatic (A) as well as the splenic (B) parasite loads

were determined by the stamp-smear method. Total parasite load in each organ is expressed in Leishman–Donovan unit (LDU) where 1 LDU = amastigote

per nucleated cell3 organ weight in milligrams. (C and D) Profile of IL-10 and IL-12 in BALB/c mice as in (A). Splenocytes isolated from different groups

of mice were harvested in six-well plates, kept for 48 h, stimulated with SLA (5 mg/ml) or left untreated for 24 h, and the resulting supernatant was assessed

for cytokine by ELISA. (E) Imipramine treatment along with SSG favors expansion of antileishmanial T cells. Splenocytes isolated from infected and drug-

treated animals were stimulated with SLA (5 mg/ml) or left untreated and the resulting T cell proliferation was measured using a nonradioactive MTT cell

proliferation assay. The mice received SSG alone (20 mg/kg), imipramine (0.1 mg/kg) alone, or a combination of both. All of the above experiments were

repeated three times with five animals per group, and data are represented for 15 animals per group.

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hepatic parasite load, imipramine alone at 0.1 mg/kg dose failed toclear any parasite. However, their combination cleared 98% of theorgan parasite burden (Fig. 6A, 6B).

Cotreatment favors expansion of antileishmanial T cellrepertoire with increased IL-12 and decreased IL-10production from splenocytes

Splenocytes were derived from SbRLD-infected BALB/c mice andstimulated with SLA ex vivo and the expansion of antileishmanialT cells was monitored. We observed that infected mice or infectedas well as imipramine (0.1 mg/kg)-treated mice failed to show anyanti-SLA–specific T cell expansion whereas the infected andcotreated group (antimony along with imipramine) showed ex-pansion of anti-SLA T cells (Fig. 6E). The resulting cytokine (IL-10 and IL-12) production in the culture supernatant was assayed.Cotreatment of imipramine and antimony was found to be effec-tive to reduce the IL-10 level (10-fold) as well as to increase theIL-12 level (8-fold) as compared with SbRLD infection alone(Fig. 6C, 6D).Similar experiments were performed in BALB/c mice infected

with SbSLD. In this case SSG alone could clear the hepatic andsplenic parasites essentially completely (Fig. 6A, 6B). There wasexpansion of the anti-SLA–specific repertoire (Fig. 6E) witha surge of IL-12 but not of IL-10 in the group receiving only SSG(Fig. 6C, 6D).

DiscussionThis study clearly identifies imipramine as a potent inhibitor ofMDR-1 pump (Fig. 1A–C) and favored accumulation of Rh123,a surrogate of antimonials in SbRLD-Mfs. Thus, imipraminewould favor accumulation of antimonials, which would kill in-tracellular parasites by generating superoxide and NO (43). It istempting to speculate that imipramine in combination with anti-monials may be useful for the treatment of SbR kala-azar cases.Pentavalent antimony remains the first line of treatment for VL insub-Saharan Africa and Brazil (44), but not in the Bihar statein India where continuous exposure to arsenic contamination indrinking water may have contributed to the lower efficacy ofantimonials because of cross-resistance (45, 46). Kala-azarpatients in the endemic zone of Bihar show overexpression ofmultidrug resistance–associated protein 1 and permeability gly-coprotein in monocytes (40).

Imipramine offers several advantages: it is a drug in use (18), itis orally active, it kills intracellular SbSLD and SbRLD parasites(21), and it is immunostimulatory (22). Previously, we showedthat infection of Mfs with SbRLD, but not with SbSLD, producesIL-10, which in turn upregulates MDR-1 (4). In the present studywe show that imipramine activates HDAC11 (Fig. 2A, 2B) whoseoverexpression inhibits IL-10 and upregulates IL-12 in SbRLD-PEC-Mfs (Fig. 3A, 3B). Our results also suggest that imipramineinhibits IL-10 and upregulates IL-12 in SbRLD-infected humanMfs (Supplemental Fig. 1), although further experiments are re-quired to determine whether a similar HDAC-dependent mecha-nism is in operation in the case of human Mfs. There is a previousreport that HDAC11 is a negative regulator of IL-10 (31). Wefound that imipramine treatment leads to deacetylation of the IL-10 promoter and prevents binding of NF-kB transcription factorsto the promoter site (Figs. 1, 2). Lack of acetylation of the IP-10promoter is already known to cause reduced binding of NF-kBtranscription factors (47).Interestingly, IL-12 promoter activity remains unaltered in

imipramine-treated, HDAC11 overexpressed RAW-Mfs (Fig. 3C),suggesting that HDAC11 may not have any direct influence on IL-12 promoter activity. It has been shown that overexpression ofHDAC11 does not affect IL-12 mRNA or IL-12 promoter activity(31). We show that p38 and NF-kB are important in imipramine-dependent upregulation of IL-12 in SbRLD-PEC-Mfs (Fig. 4). Itis also evident from our study that p65/RelB binds to three dif-ferent sites in the IL-12 promoter for optimal IL-12 generation(Fig. 4D, 4E). Although imipramine treatment resulted in p65/RelB binding with the IL-12p40 promoter, it failed to show anysignificant luciferase activity. It is therefore possible that althoughthe binding of transcription factor with the IL-12p40 promoterfails to cause its transcriptional activation, it might result in op-timal transcriptional activation of the IL-12p35 promoter throughfunctional interaction resulting in optimal transcriptional synergyas previously observed in the case of other transcription factors(48). However, further experiments are required to completelyaddress the role played by the IL-12p40 promoter in imipramine-mediated IL-12 production. Additionally, neutralizing Ab to IL-10enhances imipramine-mediated IL-12 generation from SbRLD-PEC-Mfs, suggesting the inhibitory role of IL-10 on IL-12 gen-eration (Fig. 3A, 3B, 3D). This is in agreement with other reports(49). We also show that imipramine-mediated recruitment of p65/

FIGURE 7. Schematic representation of molecular

mechanism of imipramine action in SbRLD-Mfs. Panel

left of the vertical line indicates SbRLD-infected Mfs,

and that right of the vertical line indicates SbRLD-

infected Mfs treated with imipramine. SbRLD infec-

tion leads to activation of NF-kB (p50/c-Rel) as indi-

cated by an A in the diagram. The B represents p50/c-Rel

binding to IL-10 promoter, resulting in IL-10 surge as

indicated by a C. SbRLD-induced IL-10–mediated

MDR-1 overexpression was indicated by D1, whereas

D2 represents suppression of IL-12 promoter activity.

Imipramine treatment activates HDAC-11 as indicated

by E1, which remodels the IL-10 promoter and thereby

inhibits binding of p50/c-Rel, leading to transcriptional

inhibition of SbRLD-induced IL-10 production. Imip-

ramine blocks IL-10–mediated MDR-1 upregulation as

represented by E2. E3 represents imipramine-mediated

suppression of IL-10 resulting in binding of p65/RelB

with IL-12 promoter and activating IL-12 transcription,

which in turn may inhibit IL-10 as represented by E4.

Continuous line represents activation, whereas dotted

line represents inhibition.

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RelB is abrogated at 200 pg/ml IL-10 (Fig. 5A). Serum IL-10levels have been found to attain very high values (480 pg/ml)under some pathological conditions such as metastatic malignantmelanoma (50). Thus it seems that high IL-10 may influence IL-12 promoter activity. Interestingly, note that IL-10 has been shownto inhibit IL-12p40 gene transcription (51, 52). Naturally one mayask whether IL-12 may also have any inhibitory influence on IL-10 production. In rIL-12–pretreated SbRLD-PEC-Mfs, the IL-10level was found to decrease in culture supernatant, and a signifi-cant decrease was noted in the number of SbRLD amastigotes per100 Mfs as compared with untreated control (Fig. 5B, 5C),suggesting an inhibitory influence of IL-12 on IL-10. Our obser-vation suggests that infection of wild-type Mfs resulted in sig-nificantly higher IL-10 production as compared with Il122/2 Mfs(Fig. 5D). However, the situation was different upon imipraminetreatment of infected Mfs. This resulted in an ∼5-fold decrease inIL-10 production in infected wild-type Mfs as opposed to only an∼ 2-fold decrease in IL-122/2 Mfs. Our data thus suggest thatthere may be a role of IL-12 in inhibiting IL-10 production uponimipramine treatment. This is in agreement with other reports thatthere is a negative influence of IL-12 over IL-10 induction (53, 54).IL-12 is the key cytokine for the initiation and maintenance of

Th1 responses and IFN-g production from Th1 cells (26, 55).Endogenous IL-12 is also required for the expression of theleishmanicidal effect of conventional chemotherapy (28). Havingestablished that imipramine is a potent inhibitor of MDR-1, westudied its efficacy in combination with SSG to clear SbRLD fromthe organs of infected mice. Previously we showed that in SbRLD-infected hamsters, SSG is effective in combination with theinhibitors of multidrug resistance–associated protein 1 and per-meability glycoprotein (40). In the present study, we showed thatthe combination of SSG (20 mg/kg) (43) with a low dose ofimipramine (0.1 mg/kg/d) is capable of effecting excellent organparasite clearance, whereas SSG alone failed (Fig. 6A, 6B).Imipramine at a lower dose is free from noticeable side effects(56), with the common antidepressant dose being ∼150 mg inhumans. If we consider the average weight of humans to be 60 kg,then the dose becomes 2.5 mg/kg/d. According to the human doseequivalent formula, the dose in mice should be 7.2-fold higherthan the normal human dose (57). Thus, the mouse equivalentshould be 18 mg/kg/d. However, the dose that we used was only0.1 mg/kg/d, which is a very low dose.The success of antileishmanial chemotherapeutics depends

on effective T cell–based host immune response (58). Infectionwith SbRLD was associated with depressed Ag-specific T cell re-sponses, decreased IL-12, TNF-a, and IFN-g production, andupregulation of suppressive cytokines IL-10 and TGF-b in ananimal model of VL (24, 28). SSG with imipramine was found tobe effective to mount anti-SLA–specific T cell expansion, coupledwith generation of IL-12 and suppression of IL-10 (Fig. 6C–E).Thus, we think that imipramine, by inhibiting IL-10 and upregu-lating IL-12 in SbRLD-infected BALB/c mice, modulates theimmune repertoire in favor of the host. It is quite possible thatimipramine-mediated downregulation of MDR-1 leads to SSGaccumulation in the SbRLD-infected host, leading to parasiteclearance by producing superoxide and NO (12, 43).In summary, SbRLD infection leads to IL-10 surge from host

cells, which proceeds further to MDR-1 overexpression and sup-pression of IL-12 promoter activity. Imipramine treatment acti-vates HDAC11, which inhibits the binding of p50/c-Rel with IL-10promoter, leading to transcriptional inactivation of SbRLD-inducedIL-10 production. As a consequence, there is an inhibition ofIL-10–mediated MDR-1 upregulation. The compound activatesIL-12 transcription, which in turn may inhibit IL-10. The mech-

anism of imipramine-mediated inhibition of IL-10 production andupregulation of IL-12 is presented schematically in Fig. 7.

AcknowledgmentsWe thank Dr. Dan Zilberstein for critically reviewing the manuscript and

Drs. Bhaskar Saha and Sekhar C. Mande, Director of the National Center

for Cell Science (Pune, India), for providing Il122/2 mice from the animal

house facility.

DisclosuresThe authors have no financial conflicts of interest.

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