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Received: 16 March 2018 Revised: 26 July 2018 Accepted: 2 September 2018 DOI: 10.1111/pim.12586
ORIGINAL ARTICLE
Leishmania infection activates host mTOR for its survival by M2 macrophage polarization Ajay Kumar1 | Sushmita Das2 | Abhishek Mandal1 | Sudha Verma1 | Kumar Abhishek1 | Ashish Kumar1 | Vinod Kumar1 | Ayan Kumar Ghosh3 | Pradeep Das1 1 Division of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, Bihar, India 2
Department of Microbiology, All India Institute of Medical Sciences, Patna, Bihar, India 3
Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, Maryland
Summary Mammalian target of rapamycin (mTOR) is a central regulator of growth and immunity of host cells. It’s involvement in cancer and tuberculosis is well documented but least explored in Leishmania donovani invasion of host cells. Therefore, in the present study, we aimed to investigate the role of mTOR in M2 macrophage polarization for Leishmania survival. We observed that Leishmania infection activated host mTOR pathway characterized by phosphorylation of mTOR, 70S6K and 4-EBP1. Inhibition
Correspondence Pradeep Das, Division of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, Bihar, India. Email:
[email protected]
of mTOR resulted in decreased parasite load and percent infectivity. Moreover,
Funding information This work was carried out through the Intramural Project Program (INT-117- BAS/2015) of the Indian Council of Medical Research (I.C.M.R.) and funded by I.C.M.R. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
IL-12, IL-1β and TNF-α), and increased expression of M2 macrophage markers (eg,
Leishmania infection triggered cell proliferation as was evidenced by increased expression of cyclin A and p-RPS6. mTOR activation during Leishmania infection resulted in reduced expression of M1 macrophage markers (eg, ROS, NO, iNOS, NOX-1, arginase-1, IL-10, TGF-β, CD206 and CD163). Furthermore, we observed that in case of Leishmania infection, mTOR inhibition increased the translocation of NF-κB to nucleus and deactivation of STAT-3. Eventually, we observed that inhibition of M2 macrophage polarization reduced Leishmania survival inside macrophages. Therefore, our findings suggest that mTOR plays a crucial role in regulation of M2 macrophage polarization and direct the innate immune homeostasis towards parasite survival inside host. KEYWORDS
Leishmania, M1 macrophage, M2 macrophage, mTOR, rapamycin, visceral leishmaniasis
1 | I NTRO D U C TI O N
such as toll-like receptor (TLR) signalling and stress responses, it is
Visceral leishmaniasis (VL) or Kala-A zar is caused by the obligate in-
asite adaptations that target and modify various aspects of host cell
tracellular protozoan parasite, Leishmania donovani, with the charac-
signalling. 2 The macrophage is the primary host cells of Leishmania
teristic feature of hepato-splenomegaly. Globally, the incidence and
parasites and secretion of macrophage derived cytokines like TNF-α
prevalence of VL are 0.5 and 2.5 million per year, respectively. In
and IL-12 influence the treatment of Leishmaniasis.
becoming increasingly clear that they also reflect sophisticated par-
India, VL has been reported in parts of Bihar, West Bengal and Uttar
Macrophages, important immune cells of innate immunity, are
Pradesh. However, it poses a serious burden to health problem in
highly dynamic, plastic and heterogeneous in nature that plays an im-
the state of Bihar, accounting for nearly 90% of the total cases in
portant role in host defence against microbial infection. Host immune
1
India. Disease outcomes depend upon complex interplay between
responses are modulated by infecting pathogens through interfering
infecting Leishmania species and the host immune factors. While cel-
with macrophage polarization and differentiation.2 It is well known
lular responses to parasitism involve host defence-related functions,
that macrophages possess a wide range of immunological function
Parasite Immunology. 2018;40:e12586. https://doi.org/10.1111/pim.12586
wileyonlinelibrary.com/journal/pim
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such as regulators, inducers and effectors of both innate and acquired
Biotechnology Ltd, Dallas, TX (mTOR, p-mTOR, β-actin, GAPDH,
immunity and their roles in tissue repair as well as wound healing
iNOS, NADPH Oxidase 1, arginase, 4EBP1, p-4EBP1, 70S6K and
were well documented.3 It was previously reported that many cardinal
p70S6K) or Cell Signaling Technology Ltd., Danvers, MA (p50, p65,
genes like mannose receptor (CD206), arginase 1 (ARG1), heme recep-
Lamin B, Cyclin A, STAT3 and pSTAT3). mTOR ELISA kit used in our
tor (CD163) and inducible nitric oxide synthase (iNOS) are involved in
study were purchased from Cell Signaling Technology Ltd.
regulation of M1 and M2 macrophages polarization.4,5 Previous studies showed that monocytes polarized into M2 macrophages enhance anti- inflammatory cytokine expression which is
2.2 | Animals
mediated by the activation of PI3K- Akt- mTOR pathway.6 The M2
Inbred BALB/c mice (Mus musculus) used in the study were main-
macrophages inhibit the protective immunity of host in protozoan in-
tained under pathogen-free conditions in the animal house facil-
fection. However, in cutaneous leishmaniasis (CL), alternative activa-
ity of Rajendra Memorial Research Institute of Medical Sciences
tion helps in disease establishment, whereas the impairment of these
(RMRIMS), Patna. Use of animals was approved by Institutional
macrophages significantly put off the disease progression.7 The mam-
Animal Ethics Committee of RMRIMS, Patna with Project ID INT-
malian target of rapamycin has a vital role in regulating the growth as
117-BAS/2015. All the experiments involving animal were per-
well as cellular metabolism in response to growth factor signalling and
formed according to the guidelines and protocols recommended by
nutritional stress.8 The key function of mTOR is the promotion of cellu-
the institute for Laboratory Animal Research in “The guide for the
lar growth and proliferation. Deregulation of mTOR has frequently oc-
care and use of laboratory animal” 8th edition.
curred in neoplasms, and excessive stimulation of this pathway leads to defective control of cell survival and proliferation.9 Conversely, rapamycin, a specific inhibitor of mTOR, prevents mitogenic responses in certain cell types10,11 and is an effective anti-tumour agent in certain settings.12 mTOR controls protein synthesis through direct phos-
2.3 | Culture of Leishmania donovani promastigotes and infection to PECs Leishmania donovani promastigotes (MHOM/IN/1983/AG83) were
phorylation and inactivation of the repressor of mRNA translation,
cultured in Medium 199 (Invitrogen, Carlsbad, CA) aided with 10%
eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and through
foetal bovine serum (Invitrogen), 50 units/mL penicillin and 50 μg/mL
phosphorylation and activation of the S6 kinase (S6K1).13 It has been
streptomycin. Regular maintenance of parasites was performed by the
reported previously that mTOR plays an important role in the differen-
method as described elsewhere.15 For peritoneal exudate cells (PECs)
tiation and polarization of immune cells which is crucial for host pro-
isolation from female BALB/c, 4% starch was introduced into the peri-
tective responses to the pathogen as well as survival and replication
toneal cavity of mice. After 24 hours, PECs were isolated and were kept
of pathogen. Furthermore, mTOR polarizes the macrophage and T cells
in sterile tissue culture petri plates and maintained at 37°C, 5% CO2
towards the destiny of the disease in certain cases of cancer and im-
in RPMI 1640 medium (Invitrogen) aided with penicillin (100 units/mL),
mune disorder.14
streptomycin (100 μg/mL) and 10% FBS till the end of each experiment.
The present study explored the role of mTOR and its downstream
Purity of PEC macrophages was checked prior to experiment by analys-
players in the regulation of macrophage (M1/M2) polarization in L. don-
ing macrophage specific cell surface marker CD11b by flow cytometry
ovani infection. We observed L. donovani infection activates mTOR-S6K
methods (Figure S2). The purity level of isolated PECs was ~98%. In vitro
pathway which polarizes M2 macrophage. We also shown that L. don-
infections were carried out in female BALB/c mice derived PECs using
ovani infection significantly increases p-mTOR expression and leads to
stationary phase promastigotes at a 10:1 parasite/macrophage ratio.16
the phosphorylation of S6K followed by increased phosphorylation of
After 6 hours of infection, unbound parasites were washed out with
ribosomal protein S6 (RPS6). Furthermore, we identified that phos-
RPMI and the cells were kept in complete RPMI medium for definite
phorylation of mTOR leads to increased activity of STAT3 and reduced
time period as per experimental time setup. The parasite load was meas-
activity of NF-kB. Interestingly, rapamycin treatment significantly re-
ured using a May-Grunwald Giemsa staining protocol.17
duces the activity of mTOR-S6K pathway molecules thereby promoting
The viability of PEC macrophages (1 × 106 cells/well) in normal
M2 macrophage polarization. Moreover, our in vivo study showed that
condition, in case of L. donovani infection and in case of rapamycin
mTOR inhibitors along with conventional anti-leishmanial drug might
treatment prior to infection, was performed using trypan blue dye ex-
be used as an effective combinational drug therapy.
clusion method as described previously.18 Thereafter, the cells were counted in haemocytometer under 40× compound light microscope
2 | M ATE R I A L S A N D M E TH O DS
(Olympus, Tokyo, Japan) chamber and primed to the required density in each assay. Cells that remained colourless were considered as viable and those that were stained blue were considered as non-viable.
2.1 | Reagents and antibodies All the reagents and chemicals used in this study were purchased
2.4 | Semi-quantitative RT PCR
from Sigma, St. Louis, MO unless indicated otherwise. The anti-
Total RNA from uninfected, Leishmania donovani infected, and ra-
bodies used in our study were purchased either from Santa Cruz
pamycin treated L. donovani-infected PEC was isolated using the
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KUMAR et al.
Trizol (Invitrogen) method according to the manufacturer’s instructions. One microgram of RNA was taken for a template to synthesize cDNA using the Superscript first strand synthesis system for
2.6 | mTOR Kinase assay by ELISA 1 × 106 PEC macrophages were seeded in six well sterile tissue cul-
the RT-P CR kit (Invitrogen). PCR was performed applying first dena-
ture petri plates. Cells were either left untreated or pre-treated with
turation for 5 minutes at 94°C and 22 amplification cycles (94°C for
rapamycin then infected with L. donovani. After 24 hours, cells were
30 seconds, 54°C or 56°C for 30 seconds, and 1 minutes at 72°C)
washed with 1× chilled PBS and 0.5 mL ice-cold 1× cell lysis buffer
followed by a final extension for 5 minutes at 72°C. The PCR prod-
(Cell Signaling Technology) plus 1 mmol/L PMSF were put into tissue
ucts were electrophoresed on 1.5% agarose gel and stained with
culture plate and incubated the plate for further 5 minutes on ice.
Ethidium Bromide (EtBr) prior to analysis. Primers used in Semi-Q
Cells were scraped off the plate and lysed by sonication (30 seconds
PCR were iNOS_F. 5ʹ-CCCTTCCGAAGTTTCTGGCAGCAGC-3ʹ; iNO
of pulse and 40 seconds of rest repeating five times) till the suspen-
S_R.5ʹGGCTGTCAGAGCCTCGTGGCTTTGG-3ʹ;19 GAPDH_F.5ʹCAA
sion become transparent. The supernatants were separated by spin-
GGCTGTGGGCAAGGTCA-3ʹ; GAPDH_R.5ʹAGGTGGAAGAGTGGG
ning at 3300 g for 5 minutes at 4°C. The supernatant was used for
AGTTGCTG-3ʹ
mTOR Kinase ELISA assays. The mTOR kinase ELISA was performed
GAPDH was used as an internal experimental control, and data
using mTOR Kinase ELISA kit according to manufacturer protocols
were normalized to GAPDH mRNA levels and expressed as a fold
(Cell Signaling and Technology). Briefly, 100 μL of cell lysates were
change compared to uninfected controls. Densitometry analyses for
added to micro-well strips and incubated at 37°C for 2 hours. After
all experiments were carried out using Quantity One software (Bio-
the incubation was over, the wells were washed three times using 1×
Rad, Hercules, CA). The ratios of band intensity of bands were showed
wash buffer thereafter detection antibody (100 μL) was added every
in bar graphs.
well and left for incubation at 37°C for 1 hour. After the incubation was over, the cells were washed four times followed by addition of
2.5 | Immunoblot
100 μL of reconstituted HRP-linked secondary antibody to each well and incubated for 30 minutes at 37°C. After incubation was over,
PEC macrophages were lysed in lysis buffer (Cell Signaling
washing process was repeated afterward TMB substrate was added
Technology) as per manufacturer protocol, and the protein
to each well and micro well-plate was left for incubation at 37°C
concentrations were estimated using Bradford method. 20 The
for 10 minutes. Before measuring the absorbance at 450nm in multi-
nuclear extracts were prepared from uninfected and L. dono-
plate ELISA reader (Thermo Fisher Scientific, Waltham, MA), 100 μL
vani-infected PEC macrophages in the presence or absence of
of STOP solution was added to each well. Each experiment was re-
rapamycin and SN50 (inhibitor of NF-κ B) as described previ-
peated three times, and results were expressed as mean ± SEM of all
ously. 21,22 Briefly, cells were first suspended in hypotonic buffer
the experiments.
(1.5 mmol/L MgCl2 , 0.2 mmol/L PMSF, 10 mmol/L HEPES, and 0.5 mmol/L DTT, 10 mmol/L KCl) and allowed to swell on the ice up to 10 minutes. Cells were lysed by sonication (30 seconds of pulse and 40 seconds of rest repeating five times) till the
2.7 | BrdU cell proliferation assay The rate of proliferation of normal PEC macrophages, Leishmania-
suspension become transparent. The nuclei were obtained by
infected macrophages, rapamycin pre-treated L. donovani-infected
spinning the solution at 3300 g at 4°C for 5 minutes. The super-
macrophages and PF-4708671 (160 nmol/L) (inhibitor of S6K)23
natant was used as the cytoplasmic extract. The nuclear pellet
pre-treated L. donovani-infected macrophages was analysed using
was extracted using nuclear extraction buffer (0.4 mol/L NaCl,
a Cell Proliferation ELISA BrdU Colorimetric kit (Roche, Basel,
0.2 mmol/L EDTA, 1.5 mmol/L MgCl2 , 0.5 mmol/L PMSF, 25%
Switzerland) as described previously. 24 Briefly, 1 × 105 cells of
glycerol, 20 mmol/L HEPES [pH 7.9], and 0.5 mmol/L DTT) for
macrophage/500 μL media were cultured in a 12 well sterile tissue
30 minutes on ice and centrifuged for 30 minutes at 12 000 g.
culture plate (Nunc, Roskilde, Denmark). After 12 hours, the mac-
The nuclear extract was found in the supernatant. ~50 μg of pro-
rophages from different experimental conditions were incubated
tein was run on SDS-PAGE and transferred to PVDF membrane
with 5 μL of BrdU labelling agent (final conc. 10 μmol/L) for 5 hours
(Merck Millipore, Burlington, MA). The membranes were blocked
and its uptake was measured at 450 nm on multi-scan ELISA reader
with 5% BSA in wash buffer (TBS, 0.1% Tween 20) for 1 hour at
(Thermo Fisher Scientific). Results were shown as mean ± SD for
room temperature and probed with primary antibodies (1:1000)
three independent experiments performed in triplicate set.
overnight at 4°C. Membranes were washed three times with wash buffer and then incubated with HRP-conjugated secondary antibody (1:10 000) and detected by enhanced chemilumi-
2.8 | Cell cycle analysis
nescence (ECL) substrates (GE healthcare, Chicago, IL) according
The number of cells in different phases of cell cycles was de-
to the manufacturer’s instructions. Densitometry analyses of all
tected by flow cytometry using propidium iodide (PI) staining
experiments were carried out using Quantity One software (Bio-
kit methods. Briefly, at the end of experiment that is normal
Rad). The intensity of immunoblot bands was quantitated, and
PEC macrophages, Leishmania-infected macrophages, rapamycin
the values were represented as arbitrary unit.
pre-t reated L. donovani-infected macrophages and PF- 4708671
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pre-t reated L. donovani-infected macrophages, the cells were dislodged with trypsin, washed twice with 1× PBS. The cells
2.12 | Cytokine ELISA
were then fixed with 80% chilled ethanol and kept at 4°C for
The culture supernatants after infection of PECs with L. donovani
1 hour. The cells were again washed with 1× PBS and resuspend
and rapamycin treated PECs prior to L. donovani infection were
in 500 μL DNase free RNase A (200 μg/mL) for 1 h at 37°C. The
collected assayed for measuring the concentration of cytokines
cells were then stained with PI (50 μg/mL) and incubated in the
IL-1β, IL-12, IL-10, TNF-α and TGF-β with the use of sandwich
dark for 20 minutes at 4°C. Data were acquired using BD FACS-
ELISA kit (R&D System, Minneapolis, MN). The assay was per-
ARIA-I I flow cytometer, and data were analysed using FACSDiva™
formed according to the manufacturer’s protocol. The detection
Software (Becton Dickinson, Franklin Lakes, SD).
limits of these assays were
