Namdar Ahmadabad et al.

Parasites Vectors
[Link]
(2020) 13:363
Parasites & Vectors

RESEARCH Open Access

Cytokine profile and nitric oxide levels

in peritoneal macrophages of BALB/c
mice exposed to the fucose‑mannose
ligand of Leishmania infantum combined
with glycyrrhizin
Hasan Namdar Ahmadabad1, Reza Shafiei2*, Gholam Reza Hatam3, Reza Zolfaghari Emameh4
and Ashok Aspatwar5

Abstract 
Background:  The fucose-mannose ligand (FML) of Leishmania infantum is a complex glycoprotein which does not
elicit adequate immunogenicity in humans. In recent years, adjuvant compounds derived from plants have been
used for improving the immunogenicity of vaccines. Glycyrrhizin (GL) is a natural triterpenoid saponin that has known
immunomodulatory activities. In the present study, we investigated the effects of co-treatment with FML and GL on
the production of cytokines and nitric oxide (NO) by macrophages, in vitro.
Methods:  Lipopolysaccharide (LPS) stimulated murine peritoneal macrophages were treated with FML (5 μg/ml) of
L. infantum and various concentrations of GL (1 μg/ml, 10 μg/ml and 20 μg/ml). After 48 h of treatment, cell culture
supernatants were recovered and the levels of TNF-α, IL-10, IL-12p70 and IP-10 were measured by sandwich ELISA and
NO concentration by Griess reaction.
Results:  Our results indicate that the treatment of activated macrophages with FML plus GL leads to enhanced pro-
duction of NO, TNF-α and IL-12p70, and reduction of IL-10 levels in comparison with FML treatment alone.
Conclusions:  Therefore, we concluded that GL can improve the immunostimulatory effect of FML on macrophages
and leads to their polarization towards an M1-like phenotype.
Keywords:  Fucose-mannose ligand, Glycyrrhizin, Macrophage, Nitric oxide, Visceral leishmaniasis

Background and is transmitted by sand flies [1]. It is one of the most

Visceral leishmaniasis (VL) or kala-azar, a cosmopoli- important parasitic diseases among the many parasitic
tan vector-borne zoonotic disease, is the most severe diseases in Iran [2–5].
and fatal form of leishmaniasis if not diagnosed and In the recent past, the parasites have developed
treated in time. The disease is caused by parasitic pro- resistance to existing drugs; due to this and the lack
tozoan species of the Leishmania donovani complex of an effective human vaccine against VL, there has
been an increase in the incidence of VL [6–9]. Differ-
ent generations of vaccines against the different regions
*Correspondence: reza_shafi@[Link]; shafiei.r@[Link]
2
Vector‑borne Diseases Research Center, North Khorasan University of parasitic antigens have been examined in the hope
of Medical Sciences, Bojnurd, Iran of finding an appropriate treatment for leishmaniasis
Full list of author information is available at the end of the article

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Namdar Ahmadabad et al. Parasites Vectors (2020) 13:363 Page 2 of 7

or induce protection against the disease with long-term upregulation of costimulatory molecules on dendritic
immunity [9–12]. cells, increasing T cell proliferation, reduction of IL-4
Among the various antigens that serve as targets for production from T cells, and direction of the immune
VL vaccine design, fucose-mannose ligand (FML) has response towards Th1 [19].
attracted much attention owing to its excellent immu- With regard to improvements in the production of a
noprotective properties against experimental VL in vaccine against human VL by using purified FML, and
several animal models [13]. The FML is a glycopro- considering the importance of macrophages in protec-
tein antigen which is present both in amastigotes and tion and control against VL, it seems that characteriza-
in motile promastigotes of species in the L. donovani tion of the immunomodulatory effects of a combination

gen in rabbits and dogs (e.g. Leishmune®, a vaccine for

complex. Even though the FML is a potent immuno- of FML/GL on macrophages can be useful in finding a
way to increase the immunogenicity of FML and vaccine
canine VL consisting of FML and saponin) [14], it does development. Therefore, to the best of our knowledge,
not have adequate immunogenicity in humans [9, 15]. for the first time, we investigated the effects of FML/GL
Previous studies have demonstrated that mac- on production of cytokines and NO by macrophages, in
rophages play a pivotal role in the outcome of Leishma- vitro.
nia infection depending on the type of macrophages: With regard to the improvement in producing human
classically activated (M1) macrophages as efficient type VL vaccines by using purified FML, and considering the
against Leishmania parasites, or alternatively, activated importance of macrophages in the protection and control
(M2) macrophages as favoring survival and growth of of VL, it is important to characterize the immunomod-
Leishmania parasites [16, 17]. In response to different ulatory effects of a combination of FML/GL on mac-
microbial stimuli and immune status of the microen- rophages which can be useful in finding ways to increase
vironment, naive macrophages (M0) differentiate into the immunogenicity of FML and vaccine development.
either M1 or M2 subpopulations with different pat-
terns of cytokine production and distinct properties.
Concerning the stimuli that can affect shifting of mac- Methods
rophages towards M1 or M2 subpopulations, recently Animals
we evaluated the immunomodulatory effects of FML on For the experimental studies, a total of seven 6–8 week-
macrophages [18]. Our findings showed that although old female BALB/c mice were used from Razi Institute
the FML significantly increases nitric oxide (NO), IL- (Mashhad, Iran). The mice were fed standard mouse
12p70 and IP-10 production in the macrophages, but chow ad libitum throughout the study and maintained
cannot alter TNF-α production in them [18]. The most under the standard conditions according to the protocol.
surprising aspect of this study was that FML signifi-
cantly increased the production of IL-10, an immuno- Leishmania promastigote culture and FML extraction
suppressive cytokine, from macrophages [18]. Leishmania infantum promastigotes (MCAN/IR/07/
Glycyrrhizin (GL) is a natural triterpenoid saponin Moheb-gh) were grown at 26 °C in brain heart infusion
derived from the root of licorice (Glycyrrhiza glabra) broth (37 g/l; Himedia, Mumbai, India) supplemented
that has been associated with numerous pharmacologic with 10% of fetal bovine serum (Gibco, Paisley, UK),
effects, including anti-bacterial, anti-inflammatory, hemin (0.01 g/l) and folic acid (0.02 g/l; Sigma-Aldrich,
anti-oxidant, anti-viral, anti-tumor, hepatoprotective, St. Louis, MO, USA).
and immunomodulatory activities [19–22]. Stimulation The stationary phase growth medium was centrifuged
of IL-12 and NO production and suppression of IL-10 at 6000× g for 10 min to collect the promastigotes. The
production from macrophages [23, 24], augmentation pellet containing promastigotes was washed with cold
of NK cell activity [23], upregulation of costimulatory phosphate-buffered saline (PBS) and was stored at − 20
molecules on dendritic cells [19], increase in T cell pro- °C until further analysis. Aqueous extraction of FML
liferation, reduction of IL-4 production from T cells, was carried out as previously described by Foroughi-
and direction of the immune response towards Th1 Parvar et  al. [25]. Briefly, frozen pellets of the parasite
[19], are the most important known immunomodula- were mixed with cold distilled water and centrifuged
tory activities of GL. at 6000×g for 10 min to collect the supernatant. This
It is well known that the GL plays an important role step was repeated once again, and both the superna-
in immunomodulatory activities such as stimulation tants were combined and boiled for 15 min at 100 °C.
of IL-12 and NO production, and suppression of IL-10 The sample was then centrifuged at 6000 for 20 min,
production from macrophages [23, 24]. GL also plays and the supernatant was lyophilized and subjected to
a central role in the augmentation of NK cell activity, chromatography by loading 2 ml of lyophilized sample
 Namdar Ahmadabad et al. Parasites Vectors (2020) 13:363 Page 3 of 7

in cold deionized distilled water (10 mg/ml) on 100 × culture supernatants from each well of the 96-well plate
1.6 cm column of P10 Bio-Gel (Bio-Rad, Watford, UK) were collected and stored at -80 °C until further analy-
to purify the FML. The collected FML samples were sis. Each experiment was performed in triplicate. The
analyzed further for their carbohydrate content and different study groups are shown in Table  1. The MTT
for the presence of 10–96 kDa bands corresponding (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
to FML glycoprotein on 10% SDS-PAGE. The purified bromide (Merck, Darmstadt, Germany) assay was per-
FML samples were lyophilized and stored at − 20 °C formed to evaluate the macrophage viability after 48 h of
until further use. incubation at 37 °C with different concentrations of GL
(0.1, 1, 10, 20, 50 and 100 μg/ml).

Isolation of peritoneal macrophages Nitric oxide and cytokine assay

Seven female BALB/c mice were sacrificed by ­CO2 eutha- The culture supernatants were evaluated for stable end-
nasia. Isolation of peritoneal macrophages was per- products of NO, nitrates, and nitrites, using Standard
formed based on the procedure described by Bibak et al Griess Reagent according to the instructions provided in
[26]. Briefly, murine peritoneal cells were harvested by the manual (Cayman Chemical, Michigan, USA). Levels
lavage of the peritoneal cavity with 10 ml of RPMI 1640 of NO in different treatment groups were determined by
medium (Invitrogen, Darmstadt, Germany). The cells reading the absorbances at 540 nm in a microplate reader
were centrifuged at 200×g for 10 min and washed in PBS/ (BioTek, Winooski, Vermont, USA). The mean optical
cold ­ddH2O. The cells were then cultured in RPMI 1640 density (OD) values of the blank were subtracted from
medium in petri dishes at 37 °C for 4 h. In this experi- the mean OD values of the test groups. The concentra-
ment, we considered three control groups: (i) PBS group: tion of the nitrite was calculated from the standard curve
activated macrophages were cultured in the presence obtained with serial dilutions of sodium nitrite as the
of PBS alone at the same volume as the other additions standard. Presence of TNF-α, IL-10 and IL-12p70 were
(10 μl/ml); (ii) GL group: activated macrophages treated measured in the cell culture supernatants using sandwich
with GL (5 μg/ml); and (iii) FML group: activated mac- ELISA kits according to the instructions of the manufac-
rophages treated with FML (5 μg/ml). Petri dishes were turer (eBioscience, San Diego, CA, USA). The minimum
carefully washed using Hanks’ solution to remove the detectable concentration was 5 pg/ml for both IL-12p70
non-adherent cells. The cells adhered to the Petri dishes and IL-10, and 1 pg/ml for TNF-α.
were trypsinized and the cell concentration was adjusted
to 1 × ­106 cells/ml in RPMI medium (RPMI medium
Statistical analysis
with 10% FCS (Invitrogen) containing 50 IU/ml penicil-
GraphPad Prism software version 5.0 (GraphPad Soft-
lin/streptomycin (Sigma-Aldrich).
ware, San Diego, USA) was used for statistical analysis of
the data. Data distribution was analyzed by a Kolmogo-
Treatment of peritoneal macrophages with a combination
rov-Smirnov test. According to the results of the normal-
of FML and GL
ity test, a one-way ANOVA followed by Dunn’s or Tukey’s
Isolated peritoneal macrophages were stimulated with 10
post-hoc test or a non-parametric Kruskal-Wallis test
μg/ml of LPS at 37 °C and 5% ­CO2 for 4 h. The activated
were used for statistical comparisons. Data are shown as
macrophages were treated with FML together with vary-
the mean ± SD of three independent experiments. P-val-
ing concentrations of GL to assess the immunomodu-
ues < 0.05 were considered statistically significant.
latory effects of the combination of FML and GL. To
prepare activated macrophages, 2 × ­105 macrophage cell
suspensions (200 μl/well in 96-well flat-bottom plates) in Results
each well were treated with 10 μg/ml of LPS from Escher- After the treatment of LPS stimulated macrophages with
ichia coli O111: B4 (Sigma-Aldrich) in complete RPMI FML and different concentrations of GL, we determined
1640 medium. Thereafter, 5 μg/ml of FML and 1, 10 and the viability of cells using an MTT reduction assay.
20 μg/ml of GL (Sigma-Aldrich) mixtures were added to Similarly, we measured the levels of TNF-α, IL-10 and
the activate cells in the wells in triplicate, as previously IL-12p70 using a sandwich ELISA method and NO con-
described [25, 27]. The cells were then cultured at 37 °C centration using a Griess reaction.
and 5% ­CO2 for 48 h. In the control or PBS group, acti- Results of the MTT assay indicated that co-treatment
vated macrophages were cultured in the presence of PBS of macrophages with FML (5 μg/ml) and GL (1, 10 and
alone using the same volume as the other additions (10 20 μg/ml) had no cytotoxic effect on the activated mac-
μl/ml). For the NO assay, we used only complete RPMI rophages, thus these concentrations were used for the
1640 medium as the blank control group. After 48 h, NO and cytokine assay. Since the activated macrophages
Namdar Ahmadabad et al. Parasites Vectors (2020) 13:363 Page 4 of 7

treated with GL at 50 and 100 μg/ml exhibited low via- from activated macrophages compared with the acti-
bility (< 90%), they were not included in further analysis vated macrophages treated with FML alone (749.3
(Fig. 1). ± 47.5 pg/ml vs 991.6 ± 79.1 pg/ml, Tukey’s post-hoc
The concentrations of NO in the supernatants of the test, q = 5.13, P < 0.001, and 964.6 ± 83 pg/ml, Tukey’s
activated macrophages treated with FML were signifi- post-hoc test, q = 5.78, P < 0.0004) (Fig. 5). Also, there
cantly higher than the activated macrophages treated was a significant difference between the concentration
with PBS (11.20 ± 1.80 μM/ml vs 7.84 ± 1.54 μM/ml, of IL-12p70 produced from the activated macrophages
Tukey’s post-hoc test, q = 4.32, P = 0.013). As shown in treated with FML and the activated macrophages
Fig. 2, treatment of activated macrophages with FML (5 treated with PBS (749.3 ± 47.5 pg/ml vs 514.6 ± 48.37,
μg/ml) plus GL (at concentration of 10 and 20 μg/ml) Tukey’s post-hoc test, q = 5.60, P = 0.002) (Fig. 5).
significantly increased the NO production in compari-
son with FML treatment alone (11.20 ± 1.80 μM/ml vs Discussion
14.40 ± 1.90 μM/ml, Tukey’s post-hoc test, q = 4.12, P Previous studies have shown that FML does not provide
= 0.026 and 15.60 ± 1.66 μM/ml, Tukey’s post-hoc test, adequate immunogenicity and cannot efficiently stimu-
q = 5.67, P = 0.003) (Fig. 2). late the immune response of macrophages [9, 15]. GL is a
The measurement of TNF-α in the culture superna- well-known immunomodulatory component that stimu-
tant of activated macrophages, showed no significant lates immune response of infected macrophages [28].
differences between the FML-treated macrophages and Therefore, we assumed that treatment with a combina-
PBS treated macrophages (577.6 ± 73.4 pg/ml vs 526 ± tion of FML and GL can improve the efficiency of mac-
45.6 pg/ml, Tukey’s post-hoc test, q = 1.75, P = 0.223). rophages against VL infection through the induction of
However, macrophages that were treated with FML protective cytokines and reactive nitrogen species (RNS).
in combination with 20 μg/ml GL showed a signifi- To evaluate this hypothesis, we studied the effects of FML
cant increase in the production of TNF-α in activated in combination with GL on the production of TNF-α,
macrophages compared to the activated macrophages IL-12p70, IL-10 and NO in murine peritoneal activated
treated with FML alone (782.0 ± 69.49 vs 577.6 ± 73.4 macrophages in vitro.
pg/ml, Tukey’s post-hoc test, q = 7.00, P = 0.0019) The results of our study indicated that the treatment
(Fig. 3). of activated macrophages with FML plus GL leads to
FML significantly increased IL-10 production from enhanced production of NO, TNF-α and IL-12p70 in
activated macrophages compared to the activated mac- comparison with FML treatment alone. Surprisingly, we
rophages treated with PBS (1601 ± 54.11 pg/ml vs 1242 found that co-treatment of macrophages with FML and
± 79.68 pg/ml, Tukey’s post-hoc test, q = 6.80, P = GL markedly inhibits the production of IL-10 compared
0.005). The concentration of IL-10 in the supernatant of to FML treatment alone. The cytokine profile and NO
the activated macrophages treated with a combination levels in macrophages treated with a combination of FML
of FML and GL (at concentrations of 10 and 20 μg/ml) and GL were similar to the patterns described in earlier
were significantly lower than the concentration of IL-10 studies on M1 macrophages [16].
in the supernatant of only FML-treated activated mac- NO has been demonstrated to be a principal effector
rophages (1601 ± 54.11 pg/ml vs 1028 ± 46.2 pg/ml, molecule responsible for mediating the intracellular kill-
Tukey’s post-hoc test, q = 10.87, P < 0.001, and 722.2 ± ing of Leishmania parasites, particularly the L. donovani
147.8 pg/ml, Tukey’s post-hoc test, q = 16.68, P < 0.001) complex [29]. The present study has shown that GL helps
(Fig. 4). in enhancing the production of NO from FML-treated
The results of our study showed that the co-treatment activated macrophages (Fig.  2). In accordance with the
with FML and GL (at concentrations of 10 and 20 μg/ present results, previous studies have demonstrated that
ml) significantly increase the production of IL-12p70

Table 1  Treatment of peritoneal macrophages with GL, FML and a combination of FML and different concentrations of GL in the
presence of LPS
Study group no. 1 2 3 4 5 6

LPS (10 μg/ml) + + + + + +

PBSa + – – – – –
GL – + (5 μg/ml) – + (1 μg/ml) + (10 μg/ml) + (20 μg/ml)
FML (5 μg/ml) – – + + + +
a
  Phosphate-buffered saline
 Namdar Ahmadabad et al. Parasites Vectors (2020) 13:363 Page 5 of 7

in activated macrophages [19]. In the present study, we

found that the treatment of activated macrophages with
FML plus GL significantly increases the production of
TNF-α, while treatment with FML or GL alone did not
increase the production of TNF-α significantly. This out-
come is contrary to that of Liu et al. [32] and Fu et al. [33]
who found that GL inhibits the secretion of TNF-α by
LPS-stimulated macrophages and mammary epithelial
cells, respectively. It is difficult to explain these discrep-
ancies, but it may be due to inhibition of LPS-induced
nuclear factor-ĸB by GL, without suppression of nuclear
Fig. 1  Peritoneal macrophage viability in the presence of factor-ĸB induced by MyD88-dependent downstream
different concentrations of glycyrrhizin (GL). The average values signaling [33].
of MTT reduction in activated macrophages treated with different
concentrations of GL were the same in all the groups except at
VL is characterized by the absence of cytokines such
concentrations of 50 and 100 μg/ml (*P < 0.05, ***P < 0.0001) as IFN-γ and IL-12 and cure of VL is associated with
restoration of these cytokines [34]. Several reports have
shown that IL-10 and IL-12 play opposite roles in control
of VL infections [35]. IL-10 is associated with progression
of VL, and IL-12 with the control of the disease caused
by species of the L. donovani complex [35]. In a recent
study, we demonstrated that FML increases the levels of
both IL-12p70 and IL-10 in activated macrophages [19].
In the present study, we have shown that the use of FML
in combination with GL exerts a strong booster effect
on IL-12 and a suppressive effect on IL-10 production
in activated macrophages. This finding is consistent with
that of Dai et al. [23] who demonstrated the ability of GL
to enhance LPS-induced IL-12 production in peritoneal
macrophages. The results of the present study are also

Fig. 2  The effect of the combination of fucose-mannose ligand

(FML) with glycyrrhizin (GL) on the production of nitric oxide (NO) by
activated macrophages. Co-treatment with FML and GL significantly
increased NO production in activated macrophages in comparison
with activated macrophages treated with PBS, FML or GL alone.
Results represent the mean (mean ± SD) of three independent
experiments with macrophages from seven mice per experiment (*P
< 0.05, **P < 0.01). Abbreviations: PBS, phosphate-buffered saline; ns,
not significant

FML or GL alone enables an increase in NO production

from activated macrophages [19, 24].
TNF-α plays a critical role in the control of intracellu-
lar pathogens, especially those that infect macrophages
[30]. It has been demonstrated that TNF-α is required Fig. 3  The effect of treatment with a combination of
fucose-mannose ligand (FML) with glycyrrhizin (GL) on the
for the control of VL infection in humans through the
production of TNF-α by activated macrophages. The production of
stimulation of IFN-γ production [30]. Similarly, Tumang TNF-α was significantly higher in the macrophages treated with the
et  al. [31] have also shown that endogenous TNF-α combination of FML and GL using a high concentration (20 μg/ml)
appears to be critical for both the initial acquisition of compared to the activated macrophages treated with PBS, FML or GL
resistance to L. donovani and resolution of experimen- alone (P = 0.0019). Results represent the mean (mean ± SD) of the
three independent experiments with macrophages from seven mice
tal VL infection. We know from our previous study that
per experiment (**P < 0.01). Abbreviations: PBS, phosphate-buffered
FML is unable to significantly enhance TNF-α levels saline
Namdar Ahmadabad et al. Parasites Vectors (2020) 13:363 Page 6 of 7

phenotype, which is an efficient phenotype against Leish-

mania parasites. Further studies using animal models
of VL are needed to evaluate the protective effect of the
combined treatment with FML/GL.

Abbreviations
VL: visceral leishmaniasis; FML: fucose-mannose ligand; LPS: lipopolysaccha-
ride; NO: nitric oxide; GL: glycyrrhizin; PBS: phosphate buffered saline; MTT:
microwave theory and techniques; OD: optical density.

Acknowledgments
The authors thank the members of the Department of Pathobiology and
Medical Laboratory Sciences, North Khorasan University of Medical Sciences,
Fig. 4  The effect of a combination of fucose-mannose ligand Bojnurd, Iran for their technical assistance.
(FML) with glycyrrhizin (GL) on the production of IL-10 by activated
macrophages. Treatment with FML together with GL at 10 and 20 Authors’ contributions
μg/ml concentrations significantly reduced IL-10 production by HNA and RS performed the experiments and prepared the draft of the manu-
script. GRH helped with the preparation of FML. RZE and AA helped with the
activated macrophages compared to FML treatment alone. Results
scientific design of the study and writing of the manuscript. All authors read
are expressed as the mean ± SD of triplicate cultures. Results and approved the final manuscript.
represent the mean (mean ± SD) of three independent experiments
with macrophages from seven mice per experiment (**P < 0.01). Funding
Abbreviations: PBS, phosphate-buffered saline This study was supported by North Khorasan University of Medical Sciences,
Bojnurd, Iran (grant No. 95/P/914).

Availability of data and materials

Data supporting the conclusions of this article are included within the article.

Ethics approval and consent to participate

The experimental protocols used in the study were approved by the Animal
Ethics Committee of North Khorasan University of Medical Sciences, Bojnurd,
Iran (95-914/p). ([Link]. REC.1395.06).

Consent for publication

Not applicable.

Competing interests
The authors declare that they have no competing interests.

Author details
1
 Natural Products & Medicinal Plants Research Center, North Khorasan
University of Medical Sciences, Bojnurd, Iran. 2 Vector‑borne Diseases Research
Center, North Khorasan University of Medical Sciences, Bojnurd, Iran. 3 Depart-
ment of Parasitology and Mycology, Shiraz University of Medical Sciences, Shi-
raz, Iran. 4 Department of Energy and Environmental Biotechnology, National
Fig. 5  The effect of a combination of fucose-mannose ligand Institute of Genetic Engineering and Biotechnology (NIGEB), 14965/161, Teh-
(FML) with glycyrrhizin (GL) on the production of IL-12p70 by ran, Iran. 5 Faculty of Medicine and Health Technology, Tampere University,
macrophages. Treatment with FML and with 10 and 20 μg/ml 33014 Tampere, Finland.
concentrations of GL significantly increased IL-12p70 levels in the
activated macrophages. Results are expressed as the mean ± SD of Received: 7 April 2020 Accepted: 15 July 2020
measurements from triplicate cultures (**P ≤ 0.001). Abbreviations:
PBS, phosphate-buffered saline; ns, not significant

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