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Modellingandsimulatinginterleukin-10 production
and regulationbymacrophagesafterstimulation with
an immunomodulatorofparasitic nematodes
Ana Sofia Figueiredo
1
, Thomas Ho
¨
fer
2
, Christian Klotz
3
, Christine Sers
4
, Susanne Hartmann
3
,
Richard Lucius
3
and Peter Hammerstein
1
1 Institute for Theoretical Biology, Humboldt University, Berlin, Germany
2 Research Group Modeling of Biological Systems, German Cancer Research Center and BioQuant Center, Heidelberg, Germany
3 Department of Parasitology, Berlin, Germany
4 Institute of Pathology, Universitaetsmedezin Charite
´
, Berlin, Germany
Parasitic nematodes are multicellular organisms occupy-
ing diverse niches within their hosts. The economically
and medically important nematodes reside in the intesti-
nal tract, skin, muscles, blood or connective tissue of
their hosts. In all of these sites, nematodes are
constantly exposed to various host immune responses.
Keywords
autocrine crosstalk; host–parasite
interaction; regulation; signalling cascades;
systems biology
Correspondence
A. S. Figueiredo, Institute for Theoretical
Biology, Humboldt University,
Invalidenstrasse 43, 10115 Berlin, Germany
Fax: +49 30 20938801
Tel: +49 30 20938450
E-mail: s.figueiredo@biologie.hu-berlin.de
Note
The mathematical models described here
have been submitted to the Online Cellular
Systems Modelling Database and can be
accessed at http://jjj.biochem.sun.ac.za/
database/figueiredo1/index.html, http://jjj.
biochem.sun.ac.za/database/figueiredo2/
index.html, http://jjj.biochem.sun.ac.za/
database/figueiredo3/index.html and http://
jjj.biochem.sun.ac.za/database/figueiredo4/
index.html free of charge
(Received 5 September 2008, revised 22
February 2009, accepted 21 April 2009)
doi:10.1111/j.1742-4658.2009.07068.x
Parasitic nematodes can downregulate the immune response of their hosts
through the induction of immunoregulatory cytokines such as interleukin-
10 (IL-10). To define the underlying mechanisms, we measured in vitro the
production of IL-10 in macrophages in response to cystatin from Acantho-
cheilonema viteae, an immunomodulatory protein of filarial nematodes, and
developed mathematical models of IL-10 regulation. IL-10 expression
requires stimulationof the mitogen-activated protein kinases extracellular
signal-regulated kinase (ERK) and p38, and we propose that a negative
feedback mechanism, acting at the signalling level, is responsible for tran-
sient IL-10 production that can be followed by a sustained plateau. Specifi-
cally, a model with negative feedback on the ERK pathway via secreted
IL-10 accounts for the experimental data. Accordingly, the model predicts
sustained phospho-p38 dynamics, whereas ERK activation changes from
transient to sustained when the concentration of immunomodulatory
protein of Acanthocheilonema viteae increases. We show that IL-10 can
regulate its own production in an autocrine fashion, and that ERK and
p38 control IL-10 amplitude, duration and steady state. We also show that
p38 affects ERK via secreted IL-10 (autocrine crosstalk). These findings
demonstrate how convergent signalling pathways may differentially control
kinetic properties of the IL-10 signal.
Abbreviations
AIC, Aikaike information criterion; AU, arbitrary units; Av17, cystatin from Acanthocheilonema viteae; ERK, extracellular signal-regulated
kinase; H3, histone 3; IL-10, interleukin-10; IL-10
e
, extracellular interleukin-10; LPS, lipopolysaccharide; MAPK, mitogen-activated protein
kinase; MKP, mitogen-activated protein kinase phosphatase; ODE, ordinary differential equation; rAV17, recombinant cystatin from
Acanthocheilonema viteae; RSS, residual sum of squares; SP1, Sp1 transcription factor; STAT3, signal transducer and activator of
transcription.
3454 FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS
Nevertheless, many parasitic nematode species live for
years, a fact that has recently been explained by sophisti-
cated immune evasion mechanisms deployed by the
worms. For example, the species Onchocerca volvulus is
the causative agent of the tropical disease river blindness
(which afflicts about 20 million people worldwide), and
can persist in its human host for more than 10 years. A
parasitic nematode of rodents, Acanthocheilonema
viteae, is used as an animal model to study basic ques-
tions of host–parasite interaction, e.g. host immune
responses and parasite immune evasion mechanisms.
Parasitic nematode infections induce a Th2 response,
which has the potential to trigger immune effector
mechanisms that can efficiently kill parasitic worms.
However, the presence of these worms seems to blunt
this effect, and vigorous effector mechanisms do not
develop. One way of interfering with immune effector
mechanisms is to stimulate the productionof anti-
inflammatory cytokines such as interleukin-10 (IL-10).
As a consequence, the ability of the host to kill the para-
sites is compromised, and host pathology due to inflam-
matory reactions is minimized. This balance allows
survival of parasites and hosts. Blunted Th2 responses
may represent a benefit for the host, as the ensuing
downregulation of effector mechanisms decreases auto-
immune responses and allergies [1,2].
The search for molecules that modulate host immune
responses has led to the identification of A. viteae cysta-
tin (Av17), a filarial protein constantly secreted by the
nematode [3] that inhibits cysteine proteases with impor-
tant functions in immune processes such as antigen
processing and presentation [4,5]. Furthermore, recom-
binant Av17 (rAv17) has recently been shown to specifi-
cally inhibit allergic and inflammatory responses in mice
[6]. In this scenario, rAv17 induced macrophages to
produce the anti-inflammatory cytokine IL-10 as a key
element of immunomodulation. The fact that signalling
through the extracellular signal-regulated kinase (ERK)
and p38 mitogen-activated protein kinase (p38) induces
IL-10 production in macrophages [7,8] has prompted us
to model the respective signalling pathways.
IL-10 is a cytokine with immunoregulatory proper-
ties. It executes its functions on a wide range of cells,
macrophages being a major source of this cytokine.
One major function of IL-10 is to control and reduce
excessive immune responses during infections and auto-
immunity, mainly by inhibiting the productionof pro-
inflammatory cytokines in macrophagesand other cell
types. However, several studies show a regulatory role
of IL-10 on T-helper cell responses of different types,
e.g. Th1 responses, which can lead to autoimmune
pathologies, and Th2 responses, which can lead to aller-
gies. IL-10-deficient mice develop spontaneous colitis
under normal conditions and are more prone to immu-
nopathology in general, being able to clear infection by
intracellular pathogens more effectively than wild-type
mice [6,9]. This emphasizes the importance of IL-10 as
an immune regulator.
The promoter region of the il-10 gene in macrophages
contains binding sites for the transcription factors, e.g.
Sp1 transcription factor (SP1) [10–12] and signal trans-
ducer and activator of transcription (STAT3) [7,8], that
regulate gene expression and are controlled by ERK
and p38 [13]. In a sequential mechanism, the ERK
signalling cascade remodels the chromatin of the il-10
promoter region by phosphorylating its histone 3 (H3)
sites, and the p38 signalling pathway activates the tran-
scription factors SP1 and STAT3 [7,8]. These transcrip-
tion factors bind to the phosphorylated H3 sites and
thereby initiate il-10 gene expression [7,8,14]. Macro-
phages express the IL-10 receptor complex on their
surface [15], suggesting feedback regulationby IL-10. A
negative autoregulatory role for IL-10 is suggested for
lipopolysaccharide (LPS)-stimulated or lipoprotein-
stimulated IL-10 production in monocytes and mono-
cyte-derived macrophages [16–19]. On the basis of these
findings, we assume that Av17 activates the ERK and
p38 signalling pathways in macrophages, leading to
IL-10 production, and we hypothesize that IL-10 is reg-
ulated via a negative feedback mechanism of secreted
IL-10 that binds to the macrophagesand deactivates
the ERK signalling pathway either by kinase inhibition
or by phosphatase activation. Other ERK-induced
molecules, e.g. mitogen-activated protein kinase
phosphatases (MKPs) can deactivate ERK and thereby
regulate IL-10 induction (Fig. 1).
The ERK and p38 signalling cascades are two exam-
ples of MAPK cascades. They are central and highly
conserved, and are present in many cell types. A myr-
iad of stimuli can activate these kinases, which, in
turn, activate many other transcription factors and
regulators of transcription, controlling the expression
of many genes. Although the mechanisms that control
the different ERK activities are as yet unclear, diverse
activation modes lead to diverging outcomes, despite
the fact that the same cascade is in play [20–22].
In order to better understand the complexity of
MAPK signalling pathways, many mathematical mod-
els of these cascades have been developed. Heinrich
et al. [23] implemented mathematical models for
different topologies of the receptor-stimulated kinase ⁄
phosphatase signalling cascades and analysed key
parameters that characterize the signalling pathways
(signal amplitude, signalling time, and signal duration).
Sasagawa et al. [24] constructed a mathematical model
of ERK signalling based on literature findings and
A. S. Figueiredo et al. Modellinginterleukin-10productionand regulation
FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS 3455
predicted ERK dynamics in response to increases in
the growth factors epidermal growth factor and nerve
growth factor. Both studies show that the same
MAPK pathway can undergo a sustained or transient
activation, as experimentally shown by Marshall [20]
and Santos et al. [22].
The aim of this study was to develop mathematical
models of IL-10 regulation on Av17 stimulation in
macrophages in order to understand quantitatively
whether the current qualitative knowledge of the mecha-
nism is compatible with available data. Moreover, the
feedback regulationof IL-10 in macrophages is not well
understood at the moment. Therefore, we implement
models distinguished by different types ofregulation of
IL-10 production to test how these different hypotheses
can explain the available experimental data. We select
the model that best represents the data, and analyse its
key features, such as the amplitude and duration of the
signal output. On the based of the results, we provide
insights about the potential regulatory modes of IL-10
production on macrophagesafter Av17 stimulation.
The mathematical models described here have been
submitted to the Online Cellular Systems Modelling
Database and can be accessed at http://jjj.biochem.sun.
ac.za/database/figueiredo1/index.html, http://jjj.biochem.
sun.ac.za/database/figueiredo2/index.html, http://jjj.
biochem.sun.ac.za/database/figueiredo3/index.html and
http://jjj.biochem.sun.a c.za/database/figueir edo4/index.
html free of charge.
Results
The model
On the basis of the experimental and literature
evidence described above, we developed mathematical
models of IL-10 regulation on Av17 stimulation in
macrophages. Model development was based on the
principle of parsimony. In order to keep the number
of parameters as small as possible, we included only
those components and processes that we considered
paramount to describe the systems dynamics and
where data were available (see Fig. 2 for all compo-
nents and reactions included in the models).
We propose two different methods of regulation, via
IL-10 (model 1 and model 2) or via an inhibitor
(model 3), and compare them to a model with no feed-
back (model 0). Model 1 assumes promotion of ERK
dephosphorylation via IL-10 (kinase deactivation).
Model 2 assumes inhibition of ERK phosphorylation
via IL-10 (phosphatase activation). Model 3 assumes
promotion of ERK dephosphorylation via an inhibitor
(kinase deactivation). The components and reactions
of these models are described in Table 1.
Fig. 1. A literature-based model of IL-10 induction andregulationby the helminthic immune modulator Av17. Av17 binds to the macrophage
and activates the p38 signalling pathway (which will activate the transcription factors SP1 and STAT3) and the ERK signalling pathway (which
will phosphorylate the H3 site of the il-10 promoter region). These transcription factors bind to this promoter site, inducing il-10 mRNA
expression [14]. IL-10 protein is subsequently produced and secreted. We assume that extracellular IL-10 binds to the IL-10 receptor of
macrophages and deactivates phospho-ERK, either by kinase inhibition or by phosphatase activation, hence regulating its own production in
a negative feedback loop. IL-10 regulation can also occur through a redundant negative feedback loop: the ERK signalling pathway induces
the expression of MKPs that can deactivate ERK.
Modelling interleukin-10productionandregulation A. S. Figueiredo et al.
3456 FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS
Fig. 2. Mathematical model of IL-10 productionand regulation. The model receives the input stimulation (Av17) as a step function (from 0 to 1),
which activates ERK and p38. Phospho-ERK phosphorylates the H3 sites of the il-10 promoter region. Phospho-p38 activates the set of tran-
scription factors (A) necessary to induce il-10 gene expression, and il-10 mRNA expression (il-10
m
) and translation take place. IL-10 is secreted
by the macrophage (IL-10
e
), and promotes the feedback regulation. We hypothesize that extracellular IL-10 (IL-10
e
) binds to the macrophage
and deactivates phospho-ERK, either by kinase deactivation (model 1) or by phosphatase activation (model 2). IL-10 regulation can also be
achieved byan IL-10-independent inhibitor, X (model 3). These three models have in common the regulationby negative feedback.
Table 1. Description of reactions and its equations for the models of IL-10 productionand regulation.
Reaction Description Equation
v
1
, v
2
ERK phosphorylation on Av17 stimulation
and constitutive dephosphorylation
v
1
¼ k
1
Á ERK tðÞÁ2
j
Á stðÞ
v
1model1
¼ k
1
Á ERK tðÞÁ2
j
Á stðÞ= 1 þ k
f
Á IL10
e
tðÞ
h
hi
v
1model3
¼ k
1
Á ERK tðÞÁ2
j
Á stðÞ= 1 þ k
f
Á XtðÞ
h
hi
v
2
¼ k
2
Á ERK
p
tðÞ
v
2model2
¼ k
2
Á ERK
p
tðÞÁk
f
Á IL10
e
tðÞ
v
3
, v
4
P38 phosphorylation on Av17
stimulation and constitutive
dephosphorylation
v
3
¼ k
3
Á 2
j
Á stðÞÁp38 tðÞ
v
4
¼ k
4
Á p38
p
tðÞ
v
5
, v
6
Transcription factor activation
and constitutive deactivation
v
5
¼ k
5
Á A Á p38
p
tðÞ
v
6
¼ k
6
Á A
p
tðÞ
v
7
, v
8
Histone phosphorylation
and dephosphorylation
v
7
¼ k
7
Á ERK
p
tðÞÁH3 tðÞ
v
8
¼ k
8
Á H3
p
tðÞ
v
9
, v
10
Complex formation, constituting
the transcription factor bound to the
phosphorylated H3 site and
constitutive disaggregation
v
9
¼ k
9
Á H3A
p
tðÞ
v
10
¼ k
10
Á H3
p
tðÞÁA
p
tðÞ
v
11
Induction of il-10 mRNA expression v
11
¼ k
11
Á H3A
p
tðÞ
v
12
Degradation of il-10 mRNA v
12
¼ k
12
Á IL10
m
tðÞ
v
13
Transcription and translation to
IL-10 intracellular protein
v
13
¼ k
13
Á IL10
m
tðÞ
v
14
Degradation of IL-10 extracellular protein v
14
¼ k
14
Á IL10
e
tðÞ
v
15
Production of X v
15
¼ k
15
Á ERK
p
tðÞ
A. S. Figueiredo et al. Modellinginterleukin-10productionand regulation
FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS 3457
We have implemented these models using ordinary
differential equations (ODEs) (Eqns 1–10), and fitted
them to experimental data on il-10 mRNA and IL-10
protein time series, and il-10 mRNA half-life. ODEs
are an effective way of mathematically describing the
dynamics of a biochemical reaction network through
its components and reactions [25,26]. These equations
allow the in silico representation of qualitative com-
plex systems and the quantification of their parame-
ters, providing insights into their emergent properties.
The models are also available in the SBML format,
which is a widely accepted standard of ODE models in
systems biology [27].
dERK
p
dt
¼ v
2
À v
1
(reversible) ð1Þ
dp38
p
dt
¼ v
4
À v
3
(reversible) ð2Þ
dA
p
dt
¼ v
5
À v
6
þ v
9
À v
10
ð3Þ
dA
dt
¼ v
6
À v
5
ð4Þ
dH3
dt
¼ v
8
À v
7
ð5Þ
dH3
p
dt
¼ v
7
À v
8
þ v
9
À v
10
ð6Þ
dH3A
dt
¼ v
10
À v
9
ð7Þ
dIL10
m
dt
¼ v
11
À v
12
ð8Þ
dIL10
e
dt
¼ v
13
À v
14
ð9Þ
dX
dt
¼ v
15
ð10Þ
Model fitting to the data
The different models were fitted to experimental data
on IL-10 protein and il-10 mRNA time series and il-10
mRNA half-life (for the half-life values, see Experi-
mental procedures). IL-10 protein and il-10 mRNA
time series were obtained by exposing murine macro-
phages to Av17 or NaCl ⁄ P
i
(as control experiment),
respectively. IL-10 protein and mRNA levels were
determined after several time points by ELISA and
quantitative real-time PCR, respectively (Fig. 3). For
experimental details, see Experimental procedures.
The maximum relative il-10 mRNA expression was
measured at 2 h after stimulation. After 4 h, the
mRNA levels reached background levels again. IL-10
protein in the cell supernatant was detectable after
2–3 h, showed a steady increase over time until 8 h,
and declined again after 14–24 h. We observed a
damped oscillation of the IL-10 mRNA between 4
and 8 h after stimulation. To determine whether this
was a biological effect, we repeated the same experi-
ment and obtained results similar to those expected
for the oscillatory effect (Fig. S1). We conclude that
the oscillation of the mRNA in Fig. 3 represents a
technical variation and is not an effect of the biolog-
ical system.
Model fitting was done using copasi [28] (see Exper-
imental procedures for the methods used). The differ-
ent regulation models fit the experimental data for
il-10 mRNA and IL-10 secreted protein (Fig. 4).
Model 0 fits the data with larger error. Figure 4 shows
the fitting of the model of Fig. 2 to IL-10 secreted
protein and il-10 mRNA. These data show that model
0 is not able to fit the decrease of IL-10 production
observed experimentally, keeping it at a sustained
level, whereas the models withregulation (model 1,
model 2, and model 3) can fit the increase and decrease
in IL-10 levels. For a complete listing of the best-
fitting parameters, constraints and initial conditions
for each model, see Doc. S1.
Fig. 3. IL-10 protein and IL-10 mRNA kinetics afterstimulation of
macrophages with the helminthic immune modulator Av17. Thiogly-
collate-elicited peritoneal macrophages from BALB ⁄ c mice were
stimulated with 0.25 l
M recombinant Av17 or with NaCl ⁄ P
i
for the
indicated times. The level of IL-10 protein in cell supernatants was
quantified by ELISA. Levels of il-10 mRNA were determined by
real-time PCR, and are presented as expression relative to the
endogenous control GAPDH. All data points represent triplicates.
We show one representative experiment out of two that gave
similar results.
Modelling interleukin-10productionandregulation A. S. Figueiredo et al.
3458 FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS
Model selection
The fitting results allow to select models. The experi-
mental data (Fig. 3) show that IL-10 production in
macrophages after Av17 stimulation is transient. This
decrease in IL-10 production is evidence for its regula-
tion by negative feedback. Hence, we discard model 0,
which includes no regulationand which presents sus-
tained IL-10 production. Moreover, the disagreement
between fitted and experimental values is very high
(Fig. 4A). We calculated the Aikaike information crite-
rion (AIC) and the residual sum of squares (RSS)
between the estimated values and the experimental
data for the four models, and model 0 yielded the
highest value (compare Table 2).
It is experimentally observed that LPS-induced
IL-10 does not reach zero in the macrophage, after IL-
10 stimulation [18]. Because IL-10 production in model
3 (model ofregulation via an inhibitor) approximates
zero, we discard model 3 and focus on the models of
regulation via IL-10 (model 1 and model 2).
Simulation predicts transient phosphorylation of
ERK and sustained phosphorylation of p38 after
Av17 stimulation
On the basis of the estimated parameters, we predict
the kinetics of phospho-ERK and phospho-p38 for
model 1 (Fig. 5A) and model 2 (Fig. 5B). These
Fig. 4. Fitted (lines) and experimental values (dots) for IL-10
secreted protein (maximum value at 8 h) and il-10 mRNA (maxi-
mum value at 2 h). (A) Model 0: no feedback. (B) Model 1: inhibi-
tion of ERK phosphorylation via IL-10. (C) Model 2: activation of
ERK dephosphorylation via IL-10. (D) Model 3: inhibition of ERK
phosphorylation via another molecule; the models fit the data for
the three regulation hypotheses. Model 0 follows the production of
IL-10, but cannot follow the decrease, because there is no regula-
tion, leading to cumulative productionof IL-10, which reaches a
steady state.
Table 2. AIC and RSS for the four models. Model 0 yields the
highest value and model 3 the lowest value. AIC presents a relative
value that scores the model, the lowest value being the best score.
A low RSS value indicates that the difference between estimated
and experimental values is low, and a high value indicates the
reverse.
Model AIC RSS
0 )13.1693 0.370
1 )31.4408 0.157
2 )41.6533 0.114
3 )48.3893 0.076
A. S. Figueiredo et al. Modellinginterleukin-10productionand regulation
FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS 3459
models show transient phospho-ERK and sustained
phospho-p38 dynamics. These predictions are qualita-
tively in accordance with experimental evidence [7,8].
These authors show that activation ofmacrophages by
immune complexes leads to IL-10 production through
the activation of ERK (transient) and p38 (sustained).
Both models present weak and transient ERK and
strong and sustained p38 activation.
Changing the Av17 stimulus shows that both
ERK and p38 control the amplitude of IL-10
The density of the parasite population, and hence the
concentration of secreted Av17, can vary. To under-
stand how this affects the system behavior, we changed
the concentration of Av17 (increasing and decreasing
it) and examined how this affects the dynamics of key
elements of each model. We implemented one-step
exponential increases (from 2 to 2
30
) and decreases
(from 2
)10
to 2
0
), and compared their impacts on the
dynamics of phospho-ERK (Fig. 6), phospho-p38 and
IL-10 (Figs 7 and 8) for kinase deactivation (model 1)
and phosphatase activation (model 2).
Phospho-ERK
Changing the stimulus amplitude can switch ERK acti-
vation from transient to sustained. Phospho-ERK of
model 1 reaches maximal activation instantaneously,
and the input changes affect the signal duration as well
as the amplitude (Fig. 6A). The response to these input
variations in model 2 is different: first, they reach
maximal activation with a certain delay, which
decreases as the input increases; and second, they
affect only the phospho-ERK amplitude until ERK
Fig. 5. Phospho-ERK (transient curve) and phospho-p38 (sustained
curve) kinetics for model 1 (A) and model 2 (B). x-axis, time (h);
y-axis, concentration (AU). On the basis of the fitted parameters for
this model, we predict the dynamic behaviour of phospho-ERK
(black) and phospho-p38 (grey). (A) Model 1: ERK activation is weak
and fast; it lasts for 1 h, and is followed byan ERK decrease. At
2 h, the ERK concentration is zero, whereas the phospho-p38 con-
centration is sustained. (B) Model 2: ERK activation is weak and
fast; it has its peak at 1 h, and this is followed byan ERK decrease.
At 2 h, the ERK concentration is zero; the phospho-p38 increase is
slow and sustained, reaching steady state at 40 h.
Fig. 6. Phospho-ERK kinetics for different input amplitudes (2
)10
to
2
30
). (A) Model 1. (B) Model 2. Red line: input amplitude = 1.
Modelling interleukin-10productionandregulation A. S. Figueiredo et al.
3460 FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS
saturation, andafter this level, ERK duration increases
(Fig. 6B).
The mechanism of kinase inhibition (model 1) was
assumed to have a cooperative behavior (Hill coeffi-
cient of 2.5). Therefore, the inhibition has a switch-like
behavior and becomes effective only with a certain
delay, during which the phospho-ERK is maximally
active. When the feedback ‘kicks in’, there is a rapid
deactivation of phospho-ERK following the plateau of
maximal activity (Fig. 6A). In model 2, the inhibition
by the increase in phosphate activity is assumed to be
a linear function of IL-10, and the inhibition therefore
constantly increases, causing direct deactivation after
reaching the maximum (Fig. 6B).
Phospho-p38
P38 activation is sustained for both models after con-
stant increases in the concentration of Av17. The
amplitude of phospho-p38 of both models increases
until saturation is reached [2 arbitrary units (AU)], but
as phospho-p38 activation is faster for model 1 than
for model 2, the former reaches saturation faster than
the later.
Extracellular IL-10 (IL-10
e
)
Figures 7 and 8 show how the different Av17 concen-
trations affect IL-10
e
amplitude, duration, and steady
state. By comparing Fig. 7A with Fig. 7B, we observe
a shift in IL-10
e
behaviour. In terms of signal ampli-
tude, for j =15 (v
1
and v
2
in Table 1), model 1 and
model 2 yield very similar maximal amplitudes. For
both models, a decrease in Av17 concentration shows
that the macrophage produces IL-10 after a certain
threshold of Av17 concentration is attained (compare
Fig. 8). IL-10 production overshoots and goes down
to a steady-state level. As the input concentration
increases, so does the maximum value of IL-10.
In terms of duration, although there is no significant
difference between model 1 and model 2 for IL-10 rise
time (time to reach maximum production), IL-10
downregulation is faster for the former. Moreover, the
Fig. 7. IL-10
e
kinetics for different Av17 concentrations (1 to 2
15
).
(A) Model 1. (B) Model 2. Red line: input amplitude = 1.
Fig. 8. IL-10
e
kinetics for different input amplitudes (2
)10
to 2
0
). (A)
Model 1. (B) Model 2. The results show that there is an effective
Av17 concentration level needed to start the productionof IL-10.
Red line: input amplitude = 1.
A. S. Figueiredo et al. Modellinginterleukin-10productionand regulation
FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS 3461
difference between the maximum value and the steady-
state value is higher in model 1 than in model 2 (in this
model, the difference disappears for j = 13). These
observations correlate with phospho-ERK dynamics
(Fig. 5), which show a shift from transient to sustained
in both models. Model 1 shows faster activation of
ERK, followed by slower attainment of the sustained
level, which entails the same behaviour for IL-10 pro-
duction with respect to increasing Av17 concentration.
The different feedback mechanisms of model 1
(kinase inhibition) and model 2 (phosphatase activa-
tion) have implications for IL-10 dynamics. Model 2
shows more rapid and robust IL-10 dynamics as more
phosphatase accelerates dephosphorylation, whereas
the dephosphorylation rate of the kinase inhibition
mechanism is constant.
Sensitivity analysis
We performed a sensitivity analysis in order to under-
stand how perturbations in the system affect the out-
put (IL-10 production). Therefore, we perturbed, in a
systematic manner, all the parameters and checked
their influence on phospho-ERK, phospho-p38, il-10
mRNA, and IL-10 protein, in terms of amplitude and
steady state. The sensitivities were calculated using the
formula:
S ¼
DO
O
:
p
Dp
(O is the output and p is the perturbed parameter).
We imposed on these parameters perturbations of
10 and 0.1. The results show different behaviours for
model 1 and model 2. For model 1, the most sensitive
parameter is the Hill coefficient for the feedback inhi-
bition of ERK activation.
For model 2, k
9
and k
12
are the most sensitive
parameters, affecting the IL-10
e
steady state.The
parameter k
9
is associated with the production of
X2(t), which is the complex formed by the phosphory-
lated transcription factors bound to the phosphory-
lated chromatin of the il-10 promoter region. The
parameter k
12
is the parameter associated with il-10
mRNA production, and its value is the half-life of
il-10 mRNA. In terms of amplitude, the system is
insensitive for both models.
Fig. 9. Perturbations of phospho-ERK (model 1). Range of perturba-
tion: factor 10 and factor 0.1. Red line: no perturbation. Green line:
factor of perturbation is 0.1. Blue line: factor of perturbation is 10.
(A) Phospho-ERK: perturbing phospho-ERK affects its duration and
amplitude, but not steady state. (B) il-10 mRNA: perturbations
affect il-10
m
amplitude and the duration but not the steady state.
(C) IL-10 protein: perturbations affect IL-10
e
amplitude, duration and
steady state. (D) Phospho-p38 is not affected at all by phospho-
ERK perturbations.
Modelling interleukin-10productionandregulation A. S. Figueiredo et al.
3462 FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS
Perturbing phospho-ERK in model 1 affects IL-10
production but has no influence on phospho-p38
Figure 9 shows the variations of parameter k
1
of
model 1. In Fig. 9A, we can see the imposed
perturbations of phospho-ERK and observe that these
changes affect the duration and amplitude of phospho-
ERK. As the perturbation increases, the amplitude of
phospho-ERK increases and the duration decreases.
These same perturbations also affect the amplitude
and the duration of il-10 mRNA (Fig. 9B) and IL-10
protein (Fig. 9C). The steady state of IL-10 protein is
also affected, but not the steady state of il-10 mRNA.
These perturbations have no direct effect on p38, phos-
pho-p38 maintaining its curve over the whole perturba-
tion range (Fig. 9D).
Perturbing phospho-p38 in model 1 affects
phospho-ERK and IL-10 production
We perturbed the phosphorylation rate constant of
p38, k
3
(Fig. 10A) and observed that p38 activity,
although not directly affected by the negative feed-
back, has an indirect impact on the feedback mecha-
nism by influencing the productionof IL-10 and,
consequently, ERK activity. This reveals autocrine
feedback between the MAPKs.
In this model, secreted IL-10 binds to the macro-
phage and promotes the dephosphorylation of phos-
pho-ERK, establishing in this way a negative
feedback mechanism. Hence, the productionof IL-10
interferes with the ERK signalling pathway, higher
IL-10 production reflecting higher feedback strength
and lower duration of phospho-ERK (Fig. 11B–D).
By comparing both perturbations on parameters k
1
for phospho-ERK and k
3
for phospho-p38, we can
observe that phospho-ERK has a stronger influence
on il-10 mRNA and IL-10 protein amplitude and that
phospho-p38 exerts control over the feedback mecha-
nism strength.
Fig. 10. Perturbations of phospho-p38 (model 1). Range of pertur-
bation: factor 10 and factor 0.1. Red line: no perturbation. Green
line: factor of perturbation is 0.1. Blue line: factor of perturbation is
10. (A) Phospho-p38: perturbing phospho-p38 affects its amplitude.
(B) Phospho-ERK: phospho-ERK is sensitive to phospho-p38 pertur-
bations, owing to the feedback mechanism. Its amplitude maintains
a constant level, its duration increases as the perturbation
decreases, and for a perturbation factor of 0.1, its steady state
increases. (C) il-10 mRNA: amplitude and duration are sensitive to
perturbations of phospho-p38, but not the steady state. (D) IL-10
protein: amplitude, duration and steady state are sensitive to per-
turbations of phospho-p38.
A. S. Figueiredo et al. Modellinginterleukin-10productionand regulation
FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS 3463
[...]... to and activate Toll-like receptor 2, an event leading to the productionof IL-10 All of these receptors (CD36, FccR, and Toll-like receptor 2) signal through the ERK signalling cascade, which makes them attractive for studying the productionandregulationof this cytokine via Av17 Understanding what is the receptor addressed by Av17 could pave the way to understanding the dynamics of Av17 and macrophages. .. macrophagesand allow refining of the mathematical model that we currently have A mathematical model goes hand in hand with experimental models It is a caricature of reality, and its features depend on the question that the investigator wants to answer The aim of this work was to understand how IL-10 is produced and regulated in macrophagesafter Av17 stimulation We have shown that: (a) IL-10 is regulated in an. .. regulation First, we analysed the dynamics of ERK and p38 Both models suggest transient ERK and sustained p38 activation Bone marrow macrophages exposed to immune complexes and Leishmania mexicana present transient ERK and sustained p38 activation [8] Other authors have pointed out that transient dynamic behaviour of ERK could be due to internalization and degradation of the growth factor receptor [24], a... in accordance with our results Av17 increases change IL-10 dynamics IL-10 needs a certain minimum level of Av17 to be produced and, as we increased the concentration of Av17 in our models, the IL-10 concentration rose However, the downregulation of IL-10 production became less, and sustained productionof IL-10 was achieved This raises the question of what the effect of high levels of IL-10 production. .. IL-10 regulationand macrophage deactivation could open the door to understanding the role of ERK in macrophage fate Av17 and IL-10 deactivate macrophages This could be a consequence of the transient time course of ERK How IL-10 deactivates ERK is still an open question Staples et al [18] reported that IL-10 activates the JAK–STAT signalling pathway when bound to macrophages, and induces IL-10 protein and. .. faster with increasing perturbation factor Discussion We modelled the productionandregulationof IL-10 via ERK and p38 signalling bymacrophagesafter Av17 stimulation Quantification of IL-10 in the supernatant over a certain time course indicates that there is a regulatory mechanism that reduces IL-10 concentration until a constant basal level is reached This motivated us to design models with three... compilation ª 2009 FEBS 3465 Modellinginterleukin-10productionandregulation A S Figueiredo et al Second, we changed the input concentration and checked key features of ERK, p38, and IL-10, namely signal amplitude and signal duration The results show that activation of phosphatases (model 2) is a more efficient negative feedback mechanism for limiting signal duration than inhibition of kinases (model 1)... expression is controlled by the transcription factors Sp1 and Sp3 J Immunol 165, 286–291 Chanteux H, Guisset AC, Pilette C & Sibille Y (2007) LPS induces IL-10 productionby human alveolar macrophages via MAPKinases- and Sp1-dependent mechanisms Respir Res 8, 71–81 Zhang X, Edwards JP & Mosser DM (2006) Dynamic and transient remodeling of the macrophage IL-10 promoter during transcription J Immunol 177,... lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages J Immunol 164, 1940–1951 11 Gee K, Angel JB, Mishra S, Blahoianu MA & Kumar A (2007) IL-10 regulationby HIV-Tat in primary human monocytic cells: involvement of calmodulin ⁄ ELISA and real-time PCR Quantification of IL-10 protein was performed withan ELISA, according to the protocol of the manufacturer (BD Biosciences, Heidelberg, Germany) For real-time... feedback mechanisms and one without feedback, and to fit them to the observed time course for il-10 mRNA and IL-10 protein quantities IL-10 protein, secreted from the macrophage, binds to the same cell through the IL-10 receptor and deacti3464 FEBS Journal 276 (2009) 3454–3469 ª 2009 The Authors Journal compilation ª 2009 FEBS A S Figueiredo et al Modellinginterleukin-10productionandregulation Fig . Modelling and simulating interleukin-10 production and regulation by macrophages after stimulation with an immunomodulator of parasitic nematodes Ana Sofia Figueiredo 1 ,. weak and transient ERK and strong and sustained p38 activation. Changing the Av17 stimulus shows that both ERK and p38 control the amplitude of IL-10 The density of the parasite population, and. factor. Discussion We modelled the production and regulation of IL-10 via ERK and p38 signalling by macrophages after Av17 stimulation. Quantification of IL-10 in the super- natant over a certain time course