Human Immunology 74 (2013) 28–31

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Identification and characterization of a human IL-10 receptor antagonist

Mohammed Mumtaz Naiyer a, Shipra Saha a, Vijayshree Hemke a, Somenath Roy b, Shailza Singh a,
Krishnasastry V Musti a, Bhaskar Saha a,⇑
a
National Centre for Cell Science, Ganeshkhind, Pune 411007, India
b
Department of Physiology, Vidyasagar University, Midnapore 721102, India

a r t i c l e i n f o a b s t r a c t

Article history: Interleukin-10 (IL-10) is an anti-inflammatory cytokine that works through IL-10 receptor alpha subunit-
Received 3 January 2012 and suppresses immune responses in many infectious diseases such as leishmaniasis as well as in cancer.
Accepted 10 September 2012 Therefore, in order to restore the host-protective immune responses in such diseases, an antagonist to this
Available online 19 September 2012
cytokine is a pressing need. Herein, using phage peptide library display, we have identified a dodecameric
peptide that functions as an antagonist to human IL-10 receptor in an IL-10-induced STAT3 phosphoryla-
tion assay. The peptide antagonist’s ability to restore anti-leishmanial function in CD40-activated macro-
phages was also tested. We observed that the peptide reduced IL-10-induced STAT-3 phosphorylation and
enhanced CD40-activated leishmanial functions in macrophages.
Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights
reserved.

1. Introduction
Interleukin-10 (IL-10) is found to be responsible for the progres-
sion of many infectious diseases and cancer [1–3]. Owing to its po-
tent immunosuppressive effects, IL-10 has been an attractive target
for therapy especially in visceral leishmaniasis (VL), caused by the
parasite Leishmania donovani (Ld). IL-10 neutralization or IL-10
receptor blockade by respective antibodies had promising host-
protective effects in Ld infection [4–5]. However, antibodies cannot
be used for long term in humans due to several limitations. In addi-
tion, the present drugs against VL are less effective due to drug
resistance and toxicity [6–8]. Therefore, it was imperative to formu-
late a new IL-10-targeted therapeutic principle that would be effec-
tive against Ld [9]. We adopted a novel approach to design small
peptide antagonist of IL-10R using phage peptide display library.
We have identified a novel dodecameric peptide that reduces IL-
10-induced STAT-3 phosphorylation and increases CD40-activated
anti-leishmanial activity in THP-1-derived macrophages.
2. Materials and methods
England Biolabs, MA) on a 96-well plate coated with the soluble
human interleukin-10 receptor alpha (shIL-10R; Catalogue no.
274-R1/CF; R&D, Minneapolis, MN) using 500 ng, 300 ng, &
200 ng in the first, second and third round of biopanning respec-
tively. Bound phages were eluted after thorough washing and
amplified by infection of host strain ER2738. Subsequent rounds
of binding and elution were done with 1  1011 pfu obtained from
the first round of screening. After 3 rounds, individual plaques
were characterized by DNA sequencing. All peptides were synthe-
sized (Sigma–Aldrich, St. Louis, MO) by f-MOC chemistry and con-
firmed by mass spectrometry.
2.2. Cell and parasite culture
THP-1 cells or, PMA (20 ng/ml for 18 h) induced THP-1-derived
macrophages and L. donovani were cultured in RPMI-1640 medium
(GIBCO BRL, Grand Island, NY) containing penicillin (70 lg/ml),
streptomycin (100 lg/ml), 2-mercaptoethanol (50 lM), sodium
pyruvate (1 lM), HEPES (20 lM) and 10% heat-inactivated FCS
(GIBCO BRL, Grand Island, NY) in 5% CO2 at 37 °C.

2.1. Screening dodecameric phage display library and bio-panning 2.3. Characterization of shIL-10Ra binding peptides by ELISA
experiments
In brief, multi-well plates (Immuno plate Maxisorp, Nunc) were
Biopanning was performed by incubating a library of phage coated overnight at 4 °C with the peptides or rhIL-10 (1 lg/well in
displayed peptides (4  1010 phages; Catalogue no. E8110S; New 0.1 M NaHCO3 (pH 8.6)), followed by blocking (BSA at 5 mg/ml in
bicarbonate buffer) at 4 °C for 3 h. After washing several times with
Abbreviation: Ld, Leishmania donovani. TBS/0.1% Tween-20 (TBST), shIL10Ra- diluted in 100 ll blocking
⇑ Corresponding author. Fax: + 91 20 2569 2259. buffer- was added to each well (100 ng/ well) and allowed to bind
E-mail address: sahab@nccs.res.in (B. Saha). for 1 h at room temperature. Plates were washed with TBST and

0198-8859/$36.00 - see front matter Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.humimm.2012.09.002
 M.M. Naiyer et al. / Human Immunology 74 (2013) 28–31 29

100 ll/well of mouse anti-human anti-IL-10Ra primary antibody
(1 lg/ml; #MAB274, clone 37612, R&D systems) was added except
the controls. Thereafter, goat anti-mouse IgG-HRP conjugated anti-
body (1/5000 dilution) was added excepting the controls. After
incubation for 1 h at room temperature, plates were then washed
and developed by adding 100 ll substrate TMB substrate and incu-
bated at 37 °C in dark for 15 min. Optical density was measured in
an ELISA reader at 450 nm.
MN), washed twice with ice-cold PBS and lyzed in ice-cold lysis
buffer for 30 min at 4 °C. Cell lysates were clarified by centrifuga-
tion at 12,000xg (15 min, 4 °C). The supernatant was processed
for immunoblotting for STAT-3 phosphorylation using anti-phos-
pho-STAT3 antibody (Cell Signaling, catalogue no. 9131; San Diego,
CA) [10]. For equal protein loading, membranes were stripped and
re-probed with anti-STAT3 antibody (Cell Signaling; catalogue no.
9132, San Diego, CA).

2.4. In vitro parasite load experiment and Giemsa staining 2.6. Peptide and soluble human IL-10R modeling

Ld promastigotes (strain AG83) were used for infecting the
PMA-induced THP-1-derived macrophages at a 1:10 ratio for 8 h
in 16-chamber slides (Nunc, Catalogue no. 178599, Rochester,
NY). The extracellular parasites were washed out. Macrophages-
uninfected or Ld-infected- were stimulated with anti-CD40 anti-
body (3 lg/ml, Catalogue no. 555586, Clone 5C3; BD Biosciences,
San Diego, CA) along with peptide-04 (700 nM) and anti-IL10
antibody (5 lg/ml, Catalogue no. 554495, Clone JES3-97D, BD Bio-
sciences, San Diego, CA) for 48 h. The cells were washed with PBS,
fixed with ice-cold methanol, dried overnight and stained with
Giemsa stain (1:10 diluted with de-ionized water) for 15 min, fol-
lowed by mounting using DPX mountant. All cultures were set in
triplicates. Student’s t-test was employed to analyze the signifi-
cance of the effects of the treatments on parasite load.
2.5. Western blot analysis of STAT3 phosphorylation
The THP-1-derived macrophages were treated with recombi-
nant human IL-10 (Catalogue no. 217-IL/CF; R&D, Minneapolis,
For the ligand-binding studies, the single conformation closest
to the average conformation in the production run was minimized
and selected as the starting receptor structure for the docking
studies. Peptide-03 and peptide-04 were docked into the putative
binding site of the receptor model. After docking, the conforma-
tional space of the ligand in the pocket was explored by a simu-
lated annealing. Each simulated annealing run consisted of 50 ps
MD simulation of the receptor–ligand complex in a vacuum with
harmonic restraints of 1 kcal/mol/[A0]2 on the Ca atoms of the
receptor. This set up allows small conformational changes in the
Ca positions during simulated annealing without loss of the overall
receptor topology. The simulation was divided into the following
periods: 5 ps heating up to 1500 K, 20 ps hot phase, 25 ps slowly
cooling phase to 300 K followed by minimization of the complex
to an rms gradient of <0.05A0. Restraints used during the simulated
annealing were varied in order to find optimal ligand–receptor
interactions with respect to experimentally determined contact
points as well as with respect to interaction energies and optimal
ligand backbone conformations. For each peptide, three to six ser-

Fig. 1. A. Identification and characterization of IL-10R antagonist: (A) Phage display screening was carried out as described in methods section, where the bound phages were
amplified three times to obtain the specific binders to shIL-10Ra. (B) List of peptides that were identified by method described in A. (C) Binding of peptide-04 to shIL-10Ra by
ELISA. ELISA plates were coated with suboptimal concentrations of either Pep-04 or rIL-10 or both. After blocking the unoccupied space, the wells were treated with shIL-
10Ra, followed by purified primary antibody against hIL-10Ra and HRP-conjugated secondary antibody. The color was developed and read as described in materials and
methods. (D) Surface Plasmon Resonance (SPR) trace for determination of association and dissociation rates of binding of peptide-04 to shIL-10Ra as described in methods
section. The SPR analysis was performed thrice of which one representative data are shown.
30 M.M. Naiyer et al. / Human Immunology 74 (2013) 28–31

ies of simulated annealing runs using different restraints were per-
formed, each series covering 20–40 separate runs. In total, for each
ligand, up to 220 runs were carried out. The simulations were per-
formed using the GROMACS software [11] and the force-field by
Cornell et al. [12]. The calculations were performed on 64bit Linux
cluster machine.
3. Results and discussion
3.1. Identification and characterization of peptides binding to soluble
human IL-10Ra
Because IL-10Ra binds IL-10 and hetero-dimerizes with IL-10Rb
leading to tyrosine phosphorylation and activation of DNA binding
of STAT3 and STAT1 [13–16], we tried biopanning of phage peptides
on shIL-10Ra. We identified 47 phages after 3 rounds of screening
(Fig. 1A). We confirmed the binding of these phages to the purified
shIL-10Ra by ELISA, which showed the varied affinity of different
phages towards shIL10Ra (results not shown). Based on their ELISA
results, we selected 14 different peptides (Fig. 1B) from low affinity
to high affinity for functional characterization. All the peptides
were further ascertained for binding to shIL-10Ra by ELISA
(Fig. 1C showing the representative data for peptide-04), of which
peptide-04 showed slowest dissociation, as observed in the surface
plasmon resonance (SPR) studies (Fig. 1D). SPR analyses suggested
that peptide-04 had high Ka and low Kd values, features required for
efficient binding to the receptor. Therefore, the peptide was tested
for the antagonist functions.
3.2. Peptide-04 inhibits IL-10 induced STAT3 phosphorylation
In THP-1-derived macrophages, IL-10 induces STAT3 phosphor-
ylation in a time- and dose-dependent manner, where maximal
phosphorylation of STAT3 was found to occur with 20 ng/ml
rhIL-10 by 30 min (Fig. 2A). To examine if peptide-04 was an IL-
10R antagonist, THP-1-derived macrophages were pre-incubated
with 43.75–700 nM of purified peptide before IL-10 treatment. At
350 nM, peptide-04 completely inhibited IL-10-induced STAT3
phosphorylation (Fig. 2B), identifying it as a strong IL-10R antago-
nist. Thus, we show for the first time that a phage peptide worked
as IL-10Ra antagonist possibly by binding to the receptor and inhi-
bition of its signaling.
3.3. Peptide-04 enhances the CD40-activated anti-leishmanial
function
THP-1-derived, Ld-infected macrophages were treated with
anti-CD40 in presence of the indicated peptides (700 nM). Only
peptide-04 but not peptide-03 enhanced CD40-activated anti-
leishmanial function (Fig. 3A). Peptide-04 alone substantially
reduced parasite burden in macrophages (Inset, Fig. 3A) corrobo-
rating with the inhibition of the IL-10-induced STAT3 phosphoryla-
tion and suggesting for the first time that an IL-10R-targeting
peptide acts as an IL-10R antagonist with host-protective effects.
3.4. Peptide-03 and peptide-04 shows different pattern of binding with
soluble human IL-10R
Next, we examined peptide-03 and peptide-04 binding with
shIL-10Ra. The presented model is the result of an iterative process
of molecular modeling, which relies on homology modeling of the
IL10R. The core binding pocket side chains are fully conserved in
peptide-03 whereas, in IL-10R modeled peptide-04, there are few
side chains in the binding pocket with all of them located in the
extracellular loop region (Fig. 3B) suggesting a different site and
mode of binding for these two peptides and explaining the ob-

Fig. 2. IL-10 induced STAT3 phosphorylation in THP-1 cell derived macrophages: (A) Kinetics of IL-10 induced stat3 phosphorylation, the highest phosphorylation was obtained
at 15–30 min with rhIL-10 (20 ng/ml). Western blot (left panel) and the densitometric data (right panel) are shown. (B) Dose-response for IL-10 induced STAT3
phosphorylation, the highest phosphorylation was obtained at 15–30 min with rhIL-10 (20 ng/ml). Western blot (left panel) and the densitometric data (right panel) are
shown. (C) Inhibition of the IL-10-induced STAT3 phosphorylation at different doses of peptide-04. (D) None of the peptides alone induced STAT3 phosphorylation. The
experiments were performed at least thrice, of which one representative data are shown.
 M.M. Naiyer et al. / Human Immunology 74 (2013) 28–31 31

Fig. 3. Parasite load assay and peptide-IL-10R model: THP-1 derived macrophages were infected with Ld at 1:10 ratio for 8 h, followed by treatment with different peptides.
After 72 h of infection, macrophages were fixed with methanol and stained with Giemsa. The numbers of intracellular parasites counted per 100 macrophages are plotted. (A)
The parasite load was lower in peptide-04 treated samples (p < 0.001) than peptide-03. In another set, THP-1 derived macrophages were infected with Ld, followed by
treatment with anti-CD40 antibody and anti-IL10 antibody or anti-IL-10R antibody in presence or absence of the peptides. Compared to peptide-03, peptide-04 significantly
(p < 0.01) decreases the parasite burden in anti-CD40 treated Ld infected macrophages. (B) Docking of peptide-03 and peptide-04 to IL-10R model shows distinct site of
interaction.

served differences between the activities of these peptides in the
reduction of parasite load. No major differences in the backbone
orientation between peptide-03 and peptide-04 could be detected
(Fig. 3B). Although contacts between receptor and ligands were dif-
ferent, the overall fold was similar, peptide-03 and peptide-04 C-
termini adopts similar positions.
In conclusion, we identify a phage peptide that can function as a
hIL-10R antagonist by virtue of its strong binding to the receptor
that leads to the inhibition of IL-10-induced STAT3 phosphoryla-
tion. This study also exemplifies a model for constructing 3D
pharmacophores.
Acknowledgments
The work was supported by the Department of Biotechnology,
New Delhi, Govt. of India. The fellowship of M.M.N is from the
University Grants Commission, New Delhi, Govt. of India.
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