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of June 17, 2017.
Imipramine Exploits Histone Deacetylase 11
To Increase the IL-12/IL-10 Ratio in
Macrophages Infected with
Antimony-Resistant Leishmania donovani
and Clears Organ Parasites in Experimental
Infection
Sandip Mukherjee, Budhaditya Mukherjee, Rupkatha
Mukhopadhyay, Kshudiram Naskar, Shyam Sundar,
Jean-Claude Dujardin and Syamal Roy
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2014; 193:4083-4094; Prepublished online 12
September 2014;
doi: 10.4049/jimmunol.1400710
http://www.jimmunol.org/content/193/8/4083
The Journal of Immunology
Imipramine Exploits Histone Deacetylase 11 To Increase
the IL-12/IL-10 Ratio in Macrophages Infected with
Antimony-Resistant Leishmania donovani and Clears
Organ Parasites in Experimental Infection
Sandip Mukherjee,*,1 Budhaditya Mukherjee,*,1 Rupkatha Mukhopadhyay,*
Kshudiram Naskar,* Shyam Sundar,† Jean-Claude Dujardin,‡ and Syamal Roy*
R
esistance to sodium stibogluconate (SSG) or pentavalent
antimonials in the treatment of visceral leishmaniasis
(VL) or kala-azar is a major problem in the Indian subcontinent (1). In Bihar, the epicenter of Indian kala-azar, traditional pentavalent antimonial treatment was abandoned not
because of toxicity but owing to high resistance (2). Pentavalent
antimonials remain the treatment of choice in Africa, South
America, Bangladesh, Nepal, and India (except north Bihar) (3).
*Department of Infectious Diseases and Immunology, Council of Scientific and
Industrial 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
1
S.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 (New
Delhi) Network Projects BSC 0114 and BSC 0120, as well as by European Commission–funded Kaladrug-R Project Grant Health-F3-2008-222895. S.M., B.M., and
R.M. are recipients of a fellowship from the Council of Scientific and Industrial
Research (New Delhi).
Address correspondence and reprint requests to Dr. Syamal Roy, Department of
Infectious Diseases and Immunology, Council of Scientific and Industrial Research–Indian Institute of Chemical Biology, Kolkata 700032, India. E-mail address:
[email protected]
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, rhodamine
123; SbR, antimony-resistant; SbRLD, antimony-resistant Leishmania donovani; SbS,
antimony-sensitive; SbSLD, antimony-sensitive L. donovani; SLA, soluble leishmanial
Ag; SSG, sodium stibogluconate; VL, visceral leishmaniasis.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400710
The resistance is due to upregulation of the multidrug resistance
protein (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 relapse 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–kalaazar dermal leishmaniasis after successful treatment with miltefosine (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 leishmaniasis 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 antileishmanial 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
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kalaazar 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 SbRLDMfs. 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.
4084
IMIPRAMINE ACTIVATES HDAC11 IN LEISHMANIA-INFECTED CELLS
Materials and Methods
Animals
BALB/c mice (Mus musculus) and hamsters (Mesocricetus auratus) were
maintained and bred under pathogen-free conditions. Use of animals was
approved by the Institutional Animal Ethics Committee of Indian Institute
of Chemical Biology. Il122/2 mice were obtained from, and experiments
were performed in, the animal facility of the National Center for Cell
Science (Pune, India). All animal experiments were performed according
to the National Regulatory Guidelines issued by the Committee for the
Purpose of Supervision of Experiments on Animals, Ministry of Environment and Forest, government of India.
protocol (33). The protein concentration was determined by the Bradford
protein assay method (Bio-Rad, Hercules, CA). The prepared Ag was
stored 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 FicollHypaque as described previously (34). Cells were washed using PBS
supplemented with 0.5% FCS and harvested in tissue culture plates for 3 h.
Nonadherent cells were discarded by washing, and adherent cells were
cultured for 3 d and designated as Mfs. Cells were then incubated in the
presence or absence of L. donovani (103 the cell number) for 48 h in
complete RPMI 1640 media. Imipramine treatment was carried out for
24 h more in selected sets. The supernatant was collected and stored frozen
for subsequent experiments.
Cell culture and infection
Peritoneal exudate cells (PECs), conveniently named PEC-Mfs, were
harvested from BALB/c mice and purified as described previously (8).
PEC-Mfs were infected with stationary-phase L. donovani promastigotes
at a ratio of 1:10 for 6 h, washed to remove free parasites, and incubated
for another 24 h. Treatments were given where needed. Supernatants obtained from infected and treated PEC-Mfs were used for cytokine analysis
by ELISA. In some experiments PEC-Mfs were infected with adenovirus
for overexpression of HDAC6 or HDAC11 or GFP (SignaGen Laboratories) following the manufacturer’s protocol. Transfection experiments were
performed 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 Ag83
and SbRLD strain BHU 575 L. donovani were purified as described (35)
and inoculated (107 parasites in 100 ml) via intracardiac routes as reported
previously (33).
Preparation of drug stocks for drug assays
Imipramine hydrochloride (Sigma-Aldrich, St. Louis, MO) and sodium
stibogluconate (SSG; provided by Albert David, Kolkata, India) solutions
were prepared at 1 mg/ml in PBS (Sigma-Aldrich), then subjected to sterile
filtration 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-free
medium with 10 mM ERK1/2 inhibitor U0126 (36), 1 mM PI3K inhibitor
wortmannin (37), 20 mM ΙkΒ of the kinase inhibitor BAY 11-7082 (8), 25
mM 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), or
neutralizing IL-10 Ab (10 ng/ml; BD Pharmingen). In some experiments
whole splenocytes isolated from either infected or treated mice were
treated with SLA as described previously (4), and subsequently ELISA and
T cell proliferation assays were performed.
Real-time quantitative PCR to estimate expression of HDAC in
imipramine-treated SbRLD-infected PECs
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.
Total RNA was isolated from normal and SbRLD-infected PEC-Mfs with or
without imipramine treatment using a total RNA isolation kit (Roche Biochemicals) and following the manufacturer’s protocol. cDNA synthesis and
real-time quantitative PCR were done as described elsewhere (8). SbRLDinfected PEC-Mfs were defined as SbRLD-PEC-Mf for convenience. The
quantitative PCR contained 23 SYBR Green Supermix (Applied Biosystems, Foster City, CA) diluted twice and forward and reverse primers. The
sequences of the forward and reverse primers are presented in Table I.
Reactions were run on an Applied Biosystems 7500 Fast Real-Time PCR
System. Experiments on negative controls of cDNA synthesis (i.e., without
reverse transcriptase) and no-template controls (i.e., without cDNA template)
were also done for each gene. All reactions were done in duplicate, and their
arithmetic average threshold cycle (Ct) was used for data analysis. The fold
of gene expression compared with the control was calculated as 22DDCt by
7500 Fast System SDS software, version 1.4 (Applied Biosystems).
Preparation of soluble leishmanial Ag
Reporter assays
Soluble leishmanial Ag (SLA) was prepared from stationary phase
L. donovani promastigotes (BHU 575 and Ag 83) following the published
All of the transfection experiments were performed with the RAW264.7 cell
line. The murine IL-12 p35 promoters 26/21411 (1.4 kb; 59-GGT GTC
Ethics statement
Use of human subjects was approved by the Ethical Committee of Human
Subjects of the Indian Institute of Chemical Biology. Blood samples were drawn
from normal healthy individuals after obtaining their written informed consent.
Parasite cultures and maintenance
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development (13). Fleximidazole, a nitroimidazole recently tested
in a phase I clinical trial for treating African trypanosomiasis (14),
could potentially be used to treat VL (15). Thioridazole, belonging
to the class of antipsychotic drugs, shows promising activities
against multidrug-resistant tuberculosis and extensively drugresistant tuberculosis in a mouse model (16). Most importantly,
it shows unique activity against nonreplicating Mycobacterium
tuberculosis culture grown under hypoxic conditions (17). Similar
to phenothiazines, imipramine, another antipsychotic drug used
for treating depression (18) and pediatric nocturnal enuresis (19),
alters the proton motive force in Leishmania donovani promastigotes (20) and kills antimony-sensitive (SbS) and SbR intracellular
amastigotes 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) of
TNF-a (22), which is an important cytokine for antileishmanial
defense (23) and is a competitive inhibitor of trypanothione reductase, 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 and
persistence (24) and increases the risk of relapse (25); IL-12 is the
key cytokine for IFN-g–producing Th1 cells (26, 27). Endogenous
IL-12 is also required for the enhancement of the leishmanicidal
effect of conventional antileishmanial drugs (28). It acts as a potent
histone modulator in the brains of maternally deprived adult rats
(29). Previously we have seen that the upregulation of MDR-1 in
Mfs by SbRLD is IL-10–dependent and requires recruitment of p50/
c-Rel at the IL-10 promoter site (4). Imipramine has been shown to
reverse multidrug resistance phenotype in acute myelogenous leukemia cell lines (30). There is a recent report that histone deacetylase (HDAC)11, one of the newly added members of the HDAC
family, regulates IL-10 expression in mouse and human APCs (31).
We showed that hamsters infected with SbRLD show a decreased
IL-12/IL-10 ratio that is reversed upon oral imipramine treatment
(21). The mechanism by which imipramine activates the SbRLDinfected host to increase IL-12 levels with a concomitant decrease in
IL-10 is a matter of considerable interest. In this study, we show that
imipramine inhibits IL-10 by overexpressing HDAC11, which in
turn inhibits MDR-1 upregulation in SbRLD-infected Mfs. A decrease in IL-10 favors IL-12 expression, which in turn favors expansion of antileishmanial T cells, leading to parasite clearance.
The Journal of Immunology
CTT CTT ATT GGC TTG-39 and 59-CCG GCA CTG AGA GGA GCT
GC-39) and p40 promoters 2103/21165 (1.06 kb; 59-CAG GAC AGG
AAT GGA GAA GCG GC-39 and 59-GTT AGC GAC AGG GAA GAG
GAG AG-39) were PCR amplified and cloned into a pGL3-Basic Vector
(Promega). Using the IL-12p35 promoter construct (1.4 kb) and a QuickChange II PCR-based, site-directed mutagenesis kit (Stratagene), three
mutated IL-12p35 promoters constructs, containing a deletion at the NFkB binding site 2100/2110, or at the NF-kB binding site 2229/2240, or
a double deletion at NF-kB binding sites 2100/2110 and 2229/2240 of
IL-12p35 promoter were generated. A mutated IL-12p40 promoter construct containing a deletion at NF-kB binding site 2121/2132 was also
generated using the IL-12p40 promoter construct (1.06 kb). All of the
inserts were confirmed by sequencing. RAW-Mfs were transiently transfected with 2 mg of these IL-12 or IL-10 (1.5 kb) or MDR-1 promoter
constructs (4) using Lipofectamine 2000 (Invitrogen), rested for 12 h, and
treated with rIL-10, rIL-2, or imipramine for 24 h. In some experiments
luciferase activity was measured in imipramine-treated RAW-Mfs 24 h
after SbRLD infection. Luciferase activity in cell extracts was measured
using the Dual-Luciferase reporter kit (Promega) according to the manufacturer’s protocols and normalized to the level of the protein content.
Chromatin immunoprecipitation assay
Confocal microscopy
The levels of expression of MDR-1 in rIL-10– or imipramine-treated PECMfs were determined by immunostaining followed by confocal microscopy (LSM 510; Carl Zeiss), as described elsewhere (4).
Dye uptake and retention assay
After treatment with several concentrations of imipramine, SbRLD- and
SbSLD-infected as well as rIL-10–stimulated PEC-Mfs were washed
and resuspended (2 3 105 cells/ml) in serum-free RPMI 1640 incubated
with 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 finally
lysed in 0.1% Triton X-100. The intracellular dye concentrations were
determined by measuring the fluorescence intensity of the cell lysates.
Western blot
Cytoplasmic and nuclear proteins were prepared and Western blotting was
performed for p50, p-p50, c-Rel, RelB, HDAC6, HDAC11 (Santa Cruz
Biotechnology), 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). In
brief, blots were probed with specific Abs. Binding of secondary HRPlabeled goat anti-rabbit or goat anti-mouse Abs (Cell Signaling Technology) was analyzed using SuperSignal West Pico or West Dura
chemiluminescent substrate (Pierce).
In vitro parasite clearance
To determine in vitro parasite clearance, PEC-Mfs were infected with
either SbSLD (AG83) or SbRLD (BHU 575) for 24 h and either left untreated 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 per
coverslip 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 at
the dose of 20 mg/kg/d once in a week for 4 wk (40), and group III received imipramine at the dose of 0.1 mg/kg/d for 4 wk by oral route using
a feeding needle as described by others (41). Group IV received imipramine (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 by
the stamp smear method as described elsewhere (42), as well as by the
serial dilution method (33).
Cytokine measurement
Various cytokine levels (IL-10 and IL-12p70) in the splenocytes or peritoneal Mfs were measured using a sandwich ELISA kit (BD Pharmingen,
San Diego, CA) as per the manufacturer’s protocol. The level of IL-10
from human Mfs was determined using commercially available monoclonal anti-human IL-10, anti-human biotin, and rhIL-10 (BD Pharmingen)
according to standard protocol. IL-12p70 in the same supernatant was
measured 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 gradient centrifugation were suspended in complete RPMI medium. Cells
were plated in triplicate in 96-well plates at a concentration of 105 cells per
well and allowed to proliferate for 3 d at 37˚C in a 5% CO2 incubator
either 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 with
a 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 means
of three independent experiments (6SD) unless noted otherwise. Statistical significance between means of various groups was determined using
a two-tailed Student t test. Only p values ,0.05 were considered to be
statistically 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 analyzed using Prism 5.0 (GraphPad Software, San Diego, CA).
Results
Imipramine abrogates SbRLD- or rIL-10–driven MDR-1
overexpression
Imipramine treatment results in a significant dose-dependent increase in the accumulation of Rh123, a surrogate marker for antimony, 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 high
without imipramine treatment (Fig. 1A). Because both BHU 575
and BHU 138 showed similar results for the rest of the experiments, only BHU 575 has been used and defined as SbRLD for
convenience. Similarly, AG83 was selected as a representative of
SbSLD infection.
The MDR-1 reporter assay showed a decrease in IL-10–driven
MDR-1 promoter activity as a function of imipramine concentration in RAW-Mfs (Fig. 1B). All transfection experiments were
performed in the RAW264.7 cell line; for all other experiments,
primary Mfs were used unless noted otherwise. The upregulation
of MDR-1 is specific to IL-10 because rIL-2, an unrelated cytokine, failed to show the above effect. Furthermore, confocal microscopy of rIL-10–treated PEC-Mfs stained with FITC-labeled
Ab to MDR-1 showed upregulation of MDR-1, which was
downregulated in the presence of imipramine (Fig. 1C). Western
blot analysis showed that imipramine inhibited nuclear translocation of c-Fos/c-Jun, the subunits of AP-1 transcription factors
important 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 SbRLDinfected PEC-Mfs, we checked the status of IL-10 promoter activity. Imipramine treatment led to abrogation of SbRLD-mediated
histone acetylation and binding of p50/c-Rel at the IL-10 promoter
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Using a commercially available kit (Upstate Biotechnology), a chromatin
immunoprecipitation (ChIP) assay was performed as recommended by the
manufacturer with minor modifications. Chromatin was immunoprecipitated at 4˚C overnight with Abs to rabbit IgG or NF-kB Abs such as antip50, anti-p65, RelB, and c-Rel, and DNA was extracted. A PCR assay was
performed to amplify the region of the IL-10 promoter (using primers 59GCC CCA CAG CAC ACA TAT CC-39 and 59-CCT GGG TTG AAC GTC
CG-39), the regions of the IL-12 p35 promoter (using primers 59-CGG
GAC AAG AGT GGC TAC TCG C-39 and 59-CTC GGA GTG CCC GTT
TGA GG-39 or 59-GCG CCA CCA GCC TTG GG-39 and 59-GGA ACG
CTG ACC TTG GGA GAC-39), or the region of the IL-12p40 promoter
(using primers 59-CCT CTG TAT GAT AGA TGC AC-39 and 59-GTT TTG
ACA CTA GTT TTC-39).
4085
4086
IMIPRAMINE ACTIVATES HDAC11 IN LEISHMANIA-INFECTED CELLS
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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 eightwell 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 means 6 SD, with statistical significance being determined with respect to imipramine untreated control. (C)–(E) are representative of three
independent experiments. ***p , 0.001.
The Journal of Immunology
4087
Table I. Sequences of primers used for real-time PCR
Primer
Product (bp)
HDAC1
59-CGCATGACTCACAATTTGCT-39
59-AAACACCGGACAGTCCTCAC-39
59-AGACTGCAGTTGCCCTTGAT-39
59-TTTGAACACCAGGTGCATGT-39
59-CACCTTTTCCAGCCAGTCAT-39
59-GGACAGTGTAGCCACCACCT-39
59-CAGACAGCAAGCCCTCCTAC-39
59-AGACCTGTGGTGAACCTTGG-39
59-AGGTAGCCGAGAGGAGAAGC-39
59-CTGAGCCAGTAAAGCCGTTC-39
59-AAGTGGAAGAAGCCGTGCTA-39
59-CTCCAGGTGACACATGATGC-39
59-TGAAGAATGGCTTTGCTGTG-39
59-CACTGGGGTCCTGGTAGAAA-39
59-CGACGGAAATTTGACCGTAT-39
59-TGAATGGGCACATTGACACT-39
59-GCGGTCCAGGTTAAAACAGA-39
59-GAGCTGAAGCCTCATTTTCG-39
59-CAGAGGAAGAGTTGGGCTTG-39
59-GCACAGCTCCTGTTAGCACA-39
59-TTCCTTGTGCAGAGGAAGGT-39
59-ACGCGTTCAAACAGGAACTT-39
204
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
HDAC7
HDAC8
HDAC9
HDAC10
HDAC11
198
202
198
198
200
201
200
203
201
197
site (Fig. 1E). This observation prompted us to study the role, if
any, of HDAC in the above process.
HDAC status in SbRLD-PEC-Mfs before and after imipramine
treatment
The status of HDAC1 to HDAC11 was analyzed by real-time PCR
using primer sets mentioned in Table I. From this analysis it was
evident that only two members of the HDAC family, HDAC6 and
HDAC11, showed altered expression in SbRLD-PEC-Mfs with
respect to uninfected PEC-Mfs, and this was further modulated
upon imipramine treatment (Fig. 2A). Upregulation of HDAC6,
which occurred by 3.2-fold in SbRLD-PEC-Mfs, was reduced to
1.8-fold after imipramine treatment. Conversely HDAC11, which
was downregulated 0.4-fold in SbRLD-PEC-Mfs, was upregulated 5.6-fold after imipramine treatment. Western blot analysis
Functional analysis of the role of HDAC11 and HDAC6 in
IL-10/IL-12 reciprocity
The relationship, if any, between the altered expressions of
HDAC11 and HDAC6 and the production of IL-10 and IL-12 was
evaluated. It was observed that overexpression of HDAC11 but not
of 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 overexpressing HDAC11 further decreased IL-10 promoter activity to
∼3-fold with respect to infected control, whereas IL-12 promoter
activity was increased by ∼8-fold (Fig. 3A). The cytokine assay
revealed an ∼2-fold decrease in IL-10 and an ∼4-fold increase in
IL-12 level in SbRLD-PEC-Mfs overexpressing HDAC11. Imipramine treatment of SbRLD-PEC-Mfs overexpressing HDAC11
showed further reduction of IL-10 by ∼4-fold, whereas IL-12 level
was increased by ∼9-fold (Fig. 3B). Interestingly, an IL-12 reporter assay showed similar luciferase activity in both HDAC11
overexpressed and non-overexpressed RAW-Mfs in the presence
of imipramine (Fig. 3C). Furthermore, we analyzed IL-12 level
in imipramine-treated SbRLD-PEC-Mfs in the presence and
absence of IL-10–neutralizing Ab to determine whether IL-10
has any influence on imipramine-mediated IL-12 production
(Fig. 3D). Note that the presence of IL-10–neutralizing Ab further
increased imipramine-mediated IL-12 generation in SbRLD-PECMfs (Fig. 3D).
Imipramine recruits p65/RelB for IL-12 induction in
SbRLD-Mfs
Using an array of pharmacological inhibitors, we observed that
imipramine-mediated IL-12 generation from SbRLD-PEC-Mfs
was significantly inhibited (∼3.5-fold) only in the presence of
p38 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-hinfected 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.
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Gene
confirmed that imipramine treatment induced HDAC11 in normal
as well as SbRLD-PEC-Mfs but not in untreated SbRLD-PECMfs (Fig. 2B). HDAC6 was expressed in SbRLD-PEC-Mfs, but
this was reduced to some extent after imipramine treatment.
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IMIPRAMINE ACTIVATES HDAC11 IN LEISHMANIA-INFECTED CELLS
Promoter scan analysis revealed two NF-kB binding sites at
positions 2100/2110 and 2240/2229 of IL-12 p35 promoter
designated as sites A and B, respectively, and one NF-kB half
site at position 2121/2132 of the IL-12p40 promoter designated as site C (Fig. 4B). To determine the specific promoter site
involved in imipramine-mediated IL-12 generation, RAW-Mfs
were transfected with IL-12p35 or IL-12p40 or their combination, and luciferase activity was determined after imipramine
treatment (Fig. 4C). Deletion of either site A or site B of the
IL-12p35 promoter results in an ∼45% reduction of luciferase
activity, whereas deletion of both the sites (A and B) caused an
∼80% reduction. Interestingly, although the IL-12p40 promoter
failed to show significant luciferase activity alone, presence of
it resulted in a significant increase in luciferase activity of the
IL-12p35 promoter. Similar experiments in the presence of SbRLD
infection also revealed that the presence of all three sites (A–C) is
required for optimal imipramine-mediated IL-12 generation
(Fig. 4D). To determine the NF-kB subunits critical for imipraminemediated IL-12 generation in SbRLD-PEC-Mfs, ChIP of the
IL-12 promoter was performed using Abs for acetylated histone,
p65, p50, c-Rel, or RelB. The results revealed that histone acetylation and binding of p65/RelB at all three sites of the IL-12
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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) IL12 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 means 6 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 between the imipramine-treated group and the anti–IL-10 Ab treated group. **p = 0.001–0.01, ***p , 0.001.
The Journal of Immunology
4089
promoter occurred upon imipramine treatment in SbRLD-PECMfs (Fig. 4E).
Reciprocity between IL-10 and IL-12 generation
We studied the influence of IL-10 over imipramine-activated IL-12
promoter activity. Low doses of rIL-10 (2 or 20 pg/ml) failed to alter
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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 production 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 inhibitor). (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 overexpression is dependent on both p35 and p40 of the
IL-12 promoter. Luciferase activity of the lysate of RAWMfs transfected with either whole-length construct of
pGL3-IL-12p35 promoter or with pGL3-IL-12p40 promoter or with both was measured. In some cases transfections 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 upregulation 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 imipramine 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 reporter 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.
the status of imipramine-mediated p65/RelB binding to the IL-12
promoter. However, at 200 pg/ml rIL-10 there was a partial abrogation of p65/RelB binding with the IL-12 promoter (Fig. 5A). We
also studied the influence of IL-12 or of neutralizing IL-10 Ab
on Sb RLD-mediated IL-10 generation from PEC-Mfs. Preincubation of PEC-Mfs with rIL-12 or neutralizing IL-10 Ab
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IMIPRAMINE ACTIVATES HDAC11 IN LEISHMANIA-INFECTED CELLS
resulted in reduced IL-10 production (Fig. 5B). We observed
that 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 compared with infected wild-type Mfs upon imipramine treatment
(Fig. 5D).
Cotreatment with SSG and imipramine eliminates organ
parasites of BALB/c mice infected with SbRLD
Having seen that imipramine is a potent inhibitor of MDR-1, we
asked 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
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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 neutralizing 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 means 6 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.
The Journal of Immunology
4091
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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 cell 3 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 drugtreated 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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IMIPRAMINE ACTIVATES HDAC11 IN LEISHMANIA-INFECTED CELLS
hepatic parasite load, imipramine alone at 0.1 mg/kg dose failed to
clear any parasite. However, their combination cleared 98% of the
organ parasite burden (Fig. 6A, 6B).
Cotreatment favors expansion of antileishmanial T cell
repertoire with increased IL-12 and decreased IL-10
production from splenocytes
Discussion
This study clearly identifies imipramine as a potent inhibitor of
MDR-1 pump (Fig. 1A–C) and favored accumulation of Rh123,
a surrogate of antimonials in SbRLD-Mfs. Thus, imipramine
would favor accumulation of antimonials, which would kill intracellular parasites by generating superoxide and NO (43). It is
tempting to speculate that imipramine in combination with antimonials may be useful for the treatment of SbR kala-azar cases.
Pentavalent antimony remains the first line of treatment for VL in
sub-Saharan Africa and Brazil (44), but not in the Bihar state
in India where continuous exposure to arsenic contamination in
drinking water may have contributed to the lower efficacy of
antimonials because of cross-resistance (45, 46). Kala-azar
patients in the endemic zone of Bihar show overexpression of
multidrug resistance–associated protein 1 and permeability glycoprotein in monocytes (40).
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 SbRLDinfected Mfs treated with imipramine. SbRLD infection leads to activation of NF-kB (p50/c-Rel) as indicated 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. Imipramine 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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Splenocytes were derived from SbRLD-infected BALB/c mice and
stimulated with SLA ex vivo and the expansion of antileishmanial
T cells was monitored. We observed that infected mice or infected
as well as imipramine (0.1 mg/kg)-treated mice failed to show any
anti-SLA–specific T cell expansion whereas the infected and
cotreated group (antimony along with imipramine) showed expansion of anti-SLA T cells (Fig. 6E). The resulting cytokine (IL10 and IL-12) production in the culture supernatant was assayed.
Cotreatment of imipramine and antimony was found to be effective to reduce the IL-10 level (10-fold) as well as to increase the
IL-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 and
splenic parasites essentially completely (Fig. 6A, 6B). There was
expansion of the anti-SLA–specific repertoire (Fig. 6E) with
a surge of IL-12 but not of IL-10 in the group receiving only SSG
(Fig. 6C, 6D).
Imipramine offers several advantages: it is a drug in use (18), it
is orally active, it kills intracellular SbSLD and SbRLD parasites
(21), and it is immunostimulatory (22). Previously, we showed
that infection of Mfs with SbRLD, but not with SbSLD, produces
IL-10, which in turn upregulates MDR-1 (4). In the present study
we show that imipramine activates HDAC11 (Fig. 2A, 2B) whose
overexpression inhibits IL-10 and upregulates IL-12 in SbRLDPEC-Mfs (Fig. 3A, 3B). Our results also suggest that imipramine
inhibits IL-10 and upregulates IL-12 in SbRLD-infected human
Mfs (Supplemental Fig. 1), although further experiments are required to determine whether a similar HDAC-dependent mechanism is in operation in the case of human Mfs. There is a previous
report that HDAC11 is a negative regulator of IL-10 (31). We
found that imipramine treatment leads to deacetylation of the IL10 promoter and prevents binding of NF-kB transcription factors
to the promoter site (Figs. 1, 2). Lack of acetylation of the IP-10
promoter is already known to cause reduced binding of NF-kB
transcription 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 IL12 promoter activity. It has been shown that overexpression of
HDAC11 does not affect IL-12 mRNA or IL-12 promoter activity
(31). We show that p38 and NF-kB are important in imipraminedependent upregulation of IL-12 in SbRLD-PEC-Mfs (Fig. 4). It
is also evident from our study that p65/RelB binds to three different 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 any
significant luciferase activity. It is therefore possible that although
the binding of transcription factor with the IL-12p40 promoter
fails to cause its transcriptional activation, it might result in optimal transcriptional activation of the IL-12p35 promoter through
functional interaction resulting in optimal transcriptional synergy
as previously observed in the case of other transcription factors
(48). However, further experiments are required to completely
address the role played by the IL-12p40 promoter in imipraminemediated IL-12 production. Additionally, neutralizing Ab to IL-10
enhances imipramine-mediated IL-12 generation from SbRLDPEC-Mfs, suggesting the inhibitory role of IL-10 on IL-12 generation (Fig. 3A, 3B, 3D). This is in agreement with other reports
(49). We also show that imipramine-mediated recruitment of p65/
The Journal of Immunology
anism of imipramine-mediated inhibition of IL-10 production and
upregulation of IL-12 is presented schematically in Fig. 7.
Acknowledgments
We 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.
Disclosures
The authors have no financial conflicts of interest.
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RelB is abrogated at 200 pg/ml IL-10 (Fig. 5A). Serum IL-10
levels have been found to attain very high values (480 pg/ml)
under some pathological conditions such as metastatic malignant
melanoma (50). Thus it seems that high IL-10 may influence IL12 promoter activity. Interestingly, note that IL-10 has been shown
to inhibit IL-12p40 gene transcription (51, 52). Naturally one may
ask whether IL-12 may also have any inhibitory influence on IL10 production. In rIL-12–pretreated SbRLD-PEC-Mfs, the IL-10
level was found to decrease in culture supernatant, and a significant decrease was noted in the number of SbRLD amastigotes per
100 Mfs as compared with untreated control (Fig. 5B, 5C),
suggesting an inhibitory influence of IL-12 on IL-10. Our observation suggests that infection of wild-type Mfs resulted in significantly higher IL-10 production as compared with Il122/2 Mfs
(Fig. 5D). However, the situation was different upon imipramine
treatment of infected Mfs. This resulted in an ∼5-fold decrease in
IL-10 production in infected wild-type Mfs as opposed to only an
∼ 2-fold decrease in IL-122/2 Mfs. Our data thus suggest that
there may be a role of IL-12 in inhibiting IL-10 production upon
imipramine treatment. This is in agreement with other reports that
there 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 the
leishmanicidal effect of conventional chemotherapy (28). Having
established that imipramine is a potent inhibitor of MDR-1, we
studied its efficacy in combination with SSG to clear SbRLD from
the organs of infected mice. Previously we showed that in SbRLDinfected hamsters, SSG is effective in combination with the
inhibitors of multidrug resistance–associated protein 1 and permeability glycoprotein (40). In the present study, we showed that
the combination of SSG (20 mg/kg) (43) with a low dose of
imipramine (0.1 mg/kg/d) is capable of effecting excellent organ
parasite 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 in
humans. 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 dose
equivalent formula, the dose in mice should be 7.2-fold higher
than the normal human dose (57). Thus, the mouse equivalent
should be 18 mg/kg/d. However, the dose that we used was only
0.1 mg/kg/d, which is a very low dose.
The success of antileishmanial chemotherapeutics depends
on effective T cell–based host immune response (58). Infection
with SbRLD was associated with depressed Ag-specific T cell responses, decreased IL-12, TNF-a, and IFN-g production, and
upregulation of suppressive cytokines IL-10 and TGF-b in an
animal model of VL (24, 28). SSG with imipramine was found to
be effective to mount anti-SLA–specific T cell expansion, coupled
with generation of IL-12 and suppression of IL-10 (Fig. 6C–E).
Thus, we think that imipramine, by inhibiting IL-10 and upregulating IL-12 in SbRLD-infected BALB/c mice, modulates the
immune repertoire in favor of the host. It is quite possible that
imipramine-mediated downregulation of MDR-1 leads to SSG
accumulation in the SbRLD-infected host, leading to parasite
clearance 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 suppression of IL-12 promoter activity. Imipramine treatment activates HDAC11, which inhibits the binding of p50/c-Rel with IL-10
promoter, leading to transcriptional inactivation of SbRLD-induced
IL-10 production. As a consequence, there is an inhibition of
IL-10–mediated MDR-1 upregulation. The compound activates
IL-12 transcription, which in turn may inhibit IL-10. The mech-
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29.
IMIPRAMINE ACTIVATES HDAC11 IN LEISHMANIA-INFECTED CELLS