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27 May 2011 Mathematical Biosciences xxx (2011) xxx–xxx 1 Contents lists available at ScienceDirect Mathematical

Contents lists available at ScienceDirect

Mathematical Biosciences

journal homepage: www.el sevier.com/locate/mbs

journal homepage: www.el sevier.com/locate/mbs 2 A dual negative regulation model of Toll-like receptor 4

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A dual negative regulation model of Toll-like receptor 4 signaling

for endotoxin preconditioning in human endotoxemia Qian Yang a , Steven E. Calvano b ,
for endotoxin preconditioning in human endotoxemia
Qian Yang a , Steven E. Calvano b , Stephen F. Lowry b , Ioannis P. Androulakis a , b , c , ⇑
a Chemical Engineering, Rutgers University, 98 Brett Road, Piscataway, NJ 08854, USA
b Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA
c Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, USA
article info
abstract
Article history:
Accepted 16 May 2011
Available online xxxx
Keywords:
Mathematical modeling
Lipopolysaccharide
Endotoxin
We discuss a model illustrating how the outcome of repeated endotoxin administration experiments can
emerge as a natural consequence of the tightly regulated signaling pathways and also highlight the
importance of a dual negative feedback regulation including PI3K/Akt and IRAK-M (IRAK3). We identify
the relative time scales of the onset and the magnitude of the stimulus as key determinants of outcome in
repeated administration experiments. The results of our simulations involve potentiated response, toler-
ance, and protective tolerance. Moreover, the knockout of negative regulators shows that IRAK-M is a
necessary and sufficient factor for generation of endotoxin tolerance (ET). The effects of the knockout
of IRAK-M gene or administration of PI3K inhibitor do yield predictions that have been verified experi-
mentally. Finally, the pretreatment with PI3K inhibitor reveals the interaction between these two nega-
tive regulations.
Potentiation
Tolerance
2011 Published by Elsevier Inc.
Humans
1. Introduction
Endotoxin (LPS), a membrane glycolipid of Gram-negative bac-
teria, is a potent inducer of pro-inflammatory responses in mono-
cytes, macrophages, and neutrophils and is widely accepted model
for the study of inflammatory responses [1] . While immune cells
exposure to LPS induces the release of both pro- and anti-inflam-
matory cytokines (small proteins that are the principal mediators
of inflammation), repeated treatment with LPS can lead to
enhancement or desensitization of subsequent pro-inflammatory
cytokine responses [2] so-called potentiation or tolerance, respec-
tolerance was initially depicted when animals survived a lethal
dose of bacterial endotoxin if they had been previously treated
with sublethal stimulus [4] .
In an attempt to interpret LPS preconditioning, model-based ap-
proaches have been proposed to explore potential underlying
mechanisms and to establish relationships between the various
LPS preconditioning strategies and the alternative outcomes. A
number of excellent prior studies [3,9–13] have investigated pre-
conditioning phenomena while evaluating alternative computa-
tional models. The central signaling receptor for LPS is Toll-like
receptor 4 (TLR4), and all previous work address preconditioning
tively [3] . Potentiation is defined as the enhanced response to a
secondary LPS administration [4] whereas endotoxin tolerance
(ET) is defined as a diminished secondary response to LPS activa-
tion following a primary exposure. LPS tolerance has also been
termed hyporesponsiveness, refractoriness, adaptation, deactiva-
tion, desensitization, immunoparalysis or reprogramming [2,5] .
Studies of ET induced in vitro [6,7] and in vivo [8] have shown a de-
crease in the production of several cytokines by macrophages;
including IL-1 b , TNF-a , and IL-6. In the extreme, endotoxin

Corresponding author at: Biomedical Engineering, Rutgers University, 599

Taylor Road, Piscataway, NJ 08854, USA. Tel.: +1 (732) 445 0099; fax: +1 (732) 445

37534.

E-mail addresses: qiany@eden.rutgers.edu (Q. Yang), calvanst@umdnj.edu (S.E. Calvano), lowrysf@umdnj.edu (S.F. Lowry), yannis@rci.rutgers.edu (I.P. Androula- kis).

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14 Received in revised form 10 May 2011

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as it relates to TLR4 signaling. Day et al. [9] construct a four-dimen- sional model whose key feature is the presence of anti-inflamma- tory factors in the system suppressing the growth of inflammatory cytokines in response to the secondary stimulus. Similarly, Vasile- scu et al. [10] build a two differential equation model describing the dynamics of TNF- a concentration and the ‘brake system’, and assumed that the generation of ET is induced by the suppression effect by the ‘brake system’. More recently, Riviere’s work [3] sug- gests that preconditioning is controlled by the regeneration rate of TLR4 without invoking a specific signaling inhibition mechanism. Finally, negative feedback regulation by specific proteins consid- ered as mechanism is also used to induce ET in an agent based

model proposed by An and coworkers [11,12]. 81

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Our fundamental understanding of LPS signaling has improved

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dramatically over the recent years as new experimental evidence

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emerges. Thus it is believed that ET may not be solely induced by

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0025-5564/$ - see front matter 2011 Published by Elsevier Inc. doi: 10.1016/j.mbs.2011.05.005

Please cite this article in press as: Q. Yang et al., A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia, Math. Biosci. (2011), doi: 10.1016/j.mbs.2011.05.005

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Q. Yang et al. / Mathematical Biosciences xxx (2011) xxx–xxx

85 suppression of anti-inflammatory mediators [14] , nor through the

86 exclusive down-regulation of cell surface receptors [15] . Further-

87 more, simple feedback control mechanisms could not explain

88 why immune responses are not suppressed simultaneously under

89 ET condition [16] . Thus, the complexity of the response to LPS pre-

90 conditioning implies the possibility of multilevel regulation requir-

91 ing the development of more elaborate underlying mechanisms.

92 Recently a number of studies focusing on quantifying proteins

93 or enzymes in TLR4 signaling pathways have suggested that the

94 different results following preconditioning are related to the

95 complex and tightly regulated molecular mechanisms within this

96 signaling pathway. TLR4 is involved in host defense against invad-

97 ing pathogens, functioning as the primary sensor of microbial

activation of transcriptional factor (NF- j B) which triggers the stimulation of expression of essential leukocyte-specific transcrip- tional dynamics. Simultaneously, the indirect activation of the PI3K/Akt signaling pathway by TLR4 which suppresses the NF-j B activity develops a short loop. On the other hand, the suppression of kinase IRAK by its specific inhibitor IRAK-M whose transcription is stimulated by the activation of PI3K/Akt signaling pathway cre- ates a longer loop. Such dual negative inhibition can potentially emerge as a critical enabler towards understanding the connectiv- ity and relationship of critical components in the innate immune system. In addition, our model offers opportunity for unraveling the multiple outcomes associated with endotoxin preconditioning. The capability of describing both potentiation and tolerance using a single model illustrates how the outcomes of endotoxin adminis- tration experiments can emerge as a natural consequence of the tightly regulated signaling pathway in acute inflammatory re- sponse. Moreover, our model predicts that the relative time scales of the onset are key determinants of the outcome in repeated administration experiments. In addition, the in silico knockout of the negative regulator IRAK-M induces a lack of endotoxin toler- ance and demonstrates that IRAK-M is a necessary and sufficient factor for this complex behavior. In silico knockout of irak -M or administration of PI3K inhibitors respectively predicts experimen- tally verified responses highlighting the importance of a dual neg- ative feedback regulation in the model with both the PI3K/Akt and IRAK-M. Finally, the pretreatment with PI3K inhibitor reveals the

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2. Materials and methods 2.1. Human endotoxin model
2. Materials and methods
2.1. Human endotoxin model

98 products and activating signaling pathways that induce the expres-

99 sion of immune and pro-inflammatory genes [17] . Due to this

100 highly significant biological role, the TLR4 signaling pathway is

101 tightly regulated [18] . Thus, it is not surprising to find that various

102 negative regulatory mechanisms have evolved to control TLR4 sig-

103 naling in order to maintain immunological balance. Several mole-

104 cules which are identified as potential negative regulators of the

105 LPS-induced TLR4 signaling pathway are highly likely to play a role

106 in the signal transduction alterations associated with endotoxin

107 tolerance based on recent in vitro and murine in vivo studies. Neg-

108 ative regulators such as intracellular molecules myeloid-differenti-

109 ation-88-short (MyD88s) [19,20] , IL-1R-associated-kinase-M

110 (IRAK-M) [21] , Toll interacting protein (TOLLP) [22] and suppres-

111 sor-of-cytokine signaling 1 (SOCS1) [23,24] have been shown to

‘crosstalk’ between these two negative regulations. 177

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112 play a vital role in endotoxin tolerance. Moreover, additional sig-

113 naling pathways which are triggered by LPS are found to be able

114 to negatively regulate TLR4 signaling. Phosphatidylinositol 3-ki-

115 nase (PI3K), a family of intracellular signal transducer enzymes,

116 has been linked to an extraordinarily diverse group of cellular

117 functions including cell growth proliferation, differentiation,

118 motility, survival and intracellular trafficking [25] which could be

Gene expression data used herein were obtained from the Inflammation and Host Response to Injury Large Scale Collabora- tive Project funded by the USPHS, U54 GM621119 [33] . Human subjects were treated by intravenous injection with endotoxin (CC-RE, lot 2) at a dose of 2-ng/kg body weight (endotoxin treated subjects) or 0.9% sodium chloride (placebo treated subjects). After the lysis of erythrocytes and isolation of total RNA from leukocyte pellets [34] , biotin-labeled cRNA was hybridized to the HU133A and HU133B arrays which contain a total of 44,924 probes for test- ing the expression level of genes whose expression can be altered in response to endotoxin. A set of 5093 probe sets were character- ized by significant variation (corresponding to 0.2% false discovery rate) across the time course of the experiment using the SAM soft- ware [35] . The data are publicly available with accession number GSE3284 at the Gene Omnibus Database ( http://www.ncbi.nlm.- nih.gov/geo/ ). Blood samples were also extracted and analyzed to determine the plasma concentration of stress hormones including

119 activated in many ways, directly by integrins [26] , by growth fac-

120 tors [27] , by G-protein coupled receptors [28] . Many of these func-

121 tions relate to the ability of PI3K to activate protein kinase B (Akt).

122 Recent data indicate that these molecules are also integral players

123 in coordinating defense mechanisms in the innate immune system

124 which could also be stimulated in diverse manners, by cytokines

125 via JAK1 [29] , by antigen via BCAP [30]. It has been reported that

126 the LPS-induced activation of PI3K/Akt limits lipopolysaccharide

127 activation of TLR4 signaling pathways and expression of inflamma-

128 tory mediators in human monocytic cells [31] . These mechanisms

129 point to the possibility of a dual-phase mechanism of negative reg-

130 ulation associated with innate immune response. Though both

131 PI3K/Akt and IRAK-M have roles in the gate-keeping system, pre-

132 venting excessive innate immune response [32] , there is a critical

133 difference between PI3K/Akt- and IRAK-M-dependent negative

134 regulatory mechanisms. Unlike IRAK-M that is induced by TLR sig-

135 naling and functions during the second or continuous exposure to

cortisol and epinephrine [36,37] . Specifically, cortisol levels were tested at 0, 0.5, 1, 1.5, 2, 3, 4, 6, and 24 h in response to endotoxin administration [36] while the study period for epinephrine levels

136 stimulation, PI3K/Akt acts at the first phase of TLR signaling and

137 modulate the magnitude of the primary activation. Therefore,

138 PI3K/Akt functions as a negative controller in the ‘early’ (or pri-

was 0, 2, 4 and 6 h following endotoxin administration [37] . 200

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139 mary) phase of the innate immune response by inhibiting some

140 of the ‘shared’ signaling pathways downstream of TLR4, whereas

2.2. An LPS-Induced acute inflammation model

141 IRAK-M acts in the ‘late’ (or second) phase of the innate immune

142 response [32] .

143 The work to be discussed in this paper aims to develop a model

144 based on the molecular mechanisms of the TLR4 signaling pathway

145 exploring the synergies between these two negative feedback

146 regulations in order to describe the complex dynamics of the

147 LPS-induced inflammation and investigate different scenarios of

148 preconditioning. Our model describes the interaction between

149 the ligand (LPS) and the transmembrane signaling receptor

150 (TLR4) coupled with the recruitment of kinase (IRAK) and the

We have previously [38] , proposed a quantitative model of an endotoxin induced inflammatory response. The activation process involves the induction of a signal transduction cascade that trig- gers transcriptional initiation of inflammatory genes [39] . The model describes the kinetic interaction between the ligand (LPS) and its signaling receptor (TLR4) coupled with their activation of kinase activity (IKK) which induces the phosphorylation and deg- radation of I j B a and then the release of the transcriptional factor (NF-j B). NF- j B translocates into the nucleus and initiates the

Please cite this article in press as: Q. Yang et al., A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia, Math. Biosci. (2011), doi: 10.1016/j.mbs.2011.05.005

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211 expression of inflammatory response related genes including ‘ P ’,

translocation of NF- j B [17] resulting in the expression of inflam-

236

212 the pro-inflammatory component, ‘ A ’, the anti-inflammatory com-

mation related genes. The inflammatory dynamics are the manifes-

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213 ponent, and ‘E ’, the energetic component. Moreover, the critical as-

tation of the complex interaction between activating and

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214 pects of the neuro-endocrine immune crosstalk connecting the

inhibitory interactions in order to constantly strike a balance be-

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215 cellular response level were also described. These responses were

tween activation and inhibition and to drive the immune system

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216 integrated into a mathematical model using the basic principles

back to homeostasis [46] . Numerous cytokines are responsible

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217 of an Indirect Response Model (IDR) [40] that bridges the extracel-

for amplifying the inflammatory reaction, through the critical IKK

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218 lular signal (LPS) with the downstream activation of the major

node [47] , while negative proteins inhibit IRAK which will finally

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219 transcriptional responses activation and neuro-endocrine system

suppress the release of inflammatory mediators [18] . We will focus

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220 interaction. The model is succinctly presented in Eqs. (1)–(5) and

specifically on four putative modes of regulation: 245

221 is described in great detail in [38]

222

224224

LPS kinetics

dLPS

(i) IRAK-M is one of the most important negative regulators of

dt ¼ k lps ;1 LPS ð 1 LPSÞ k lps; 2 LPS

ð 1Þ 8 dR > > k syn; mRNA; R mRNA; R þ k 2
ð 1Þ
8
dR
> > k syn; mRNA; R mRNA; R þ k 2 ð LPSR Þ k 1 LPS R k syn R
¼
<
dðLPSR Þ
dt
¼ k 1 LPS R k 3 ð LPSR Þ k 2 ðLPSR Þ
dt
> > :
dðmRNA; RÞ
¼ k in; mRNA; R ð 1 þ k mRNA; R; P P Þ k out ;mRNA; R R
dt
8
>
2
dIKK
IKK
>
¼
k 3 ð IKKÞ = ð1 þ IkBaÞ k 4 IKK þ P
>
dt
2
>
1þIKK
>
>
<
¼ k NFjB; 1 IKK ð 1 NFjBn Þ
dNFjBn
k NFjB; 2 NFjBn IkBa
dt
ð1þIkBaÞ
>
>
dmRNA IkBa
>
>
¼ k in ;IkBa ð 1 þ k IkBa; 1 NFjBnÞ k out; IkBa mRNA; IkBa
>
dt
>
:
dIkBa
¼ k I ; 1 mRNA; IkBa k I ; 2 ð 1 þ IKKÞ ð1 NFjBnÞ IkBa k I ; 1
dt
8
> > k in ; P ð 1 þ k P ;NF jBn NFjBnÞ ð 1 þ k P ; E EÞ= A k out; P P
dP
¼
dt
<
> > k out ; A A
> >
k in ; A ð 1 þ k A;cAMP cAMPÞ ð1 þ k A; E EÞ ð1 þ k A; FRN FR ð NÞÞ
dA
¼
dt
> >
k in ; E ð 1 þ k E; P P Þ =A k out ;E E
: dE
¼
dt
8
> ¼ w F ex R in; F þ k in ; F ð 1 þ k F ; P P Þ k out; F F
dF
> > >
>
dRm
> dt
FR ðNÞ
>
¼ k syn Rm ð1
>
dt
þFR ðNÞ Þ k deg Rm
IC 50 Rm
> >
>
dRF
> >
¼ k syn R R m þ r f k re FR ð NÞ k on F R F k dgr R R F
>
>
dt
> >
>
dFR
¼ k on F R F k T FR
> >
dt
>
>
dFR ðNÞ
> >
¼ k T FR k re FR ð NÞ
>
dt
> >
> >
dEPI
<
þ k in; EPI ð 1 þ k EPI; P P Þ k out ;EPI EPI
¼ w EPI; ex R in; EPI
dt
0
dREPI
>
¼
k R EPI ½ k 1; R EPI ð1 þ k R EPI EPIÞ þ k 2; R EPI REPI
>
dt
>
> > >
dEPIR
>
¼ k 1; R EPI ð1 þ k R EPI EPIÞ R EPI k 3;EPIR EPIR
>
dt
>
>
>
dcAMP
¼ 1 s ð EPIR n cAMPÞ
> >
dt
> >
>
>
1;
exogenous hormone
> >
> wF ex ¼
> >
0; elsewhere
>
>
> >
>
1;
exogenous hormone
>
>
:
w EPI ex ¼
0; otherwise

IRAK and has been shown to prevent dissociation of IRAK from MyD88 and the formation of the IRAK-TRAF6 complex

ð 2Þ

ligand receptor interactions

NFjB signaling dynamics

ð 3Þ

Intrinsic transcriptional responses

ð 4Þ

neuro-endocrine immune system interactions

ð 5Þ

225 3. Modeling the dual negative regulation LPS recognition

226 mechanism

[21] . Though other proteins, such as MyD88 and TRAF6, which play critical role in the recruitment and activation of downstream enzymes in the TLR4 signaling pathway are also tightly regulated by their specific negative regulators, MyD88s (the short form of MyD88) and A20 respectively [18] , we consider a simplified feedback loop consisting only of IRAK and IRAK-M, since the redundancy of negative regu- lation of IRAK by several controllers, including IRAK-M, SOCS-1, and TOLLIP, implies the significance of this node [48] . (ii) A critical pathway in the inhibition of TLR4 signaling is the PI3K/Akt kinase signaling pathway which is triggered by LPS stimulation [31] . Recently, the interaction of PI3K and

227 3.1. Putative structure of the regulation network

228 LPS is recognized by its transmembrane signaling receptor,

229 TLR4, and the accessory protein MD-2. The binding of LPS and

230 TLR4 results in the formation of a complex, LPSR, and the recruit-

231 ment of the adaptor molecules MyD88 [42] and TIRAP [43] . This

232 will further result in recruiting and activating IRAK [44]

233 subsequently activating TRAF6 [45] . Further intracellular events

234 ultimately result in the activation of the IKK complex, involving

235 phosphorylation and degradation of I j B a , enabling the nuclear

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Yang et al. / Mathematical Biosciences xxx (2011) xxx–xxx Fig. 1. Basic topological interactions composing the
Yang et al. / Mathematical Biosciences xxx (2011) xxx–xxx Fig. 1. Basic topological interactions composing the

Fig. 1. Basic topological interactions composing the multi-level model of endotoxin induced human inflammation with dual negative regulation.

262 MyD88 in response to LPS was reported. Ojaniemi’s study on

263 LPS-induced PI3K activation [49] demonstrated that

264 activation of TLR4 results in the formation of the PI3K-

265 MyD88 complex, implying that one mode of activation of

266 the PI3K/Akt pathway following exposure to LPS is via LPS-

267 TLR4-MyD88 approach. It was also shown that inhibition

268 of PI3K/Akt pathway enhances LPS-induced TNF- a gene

269 expression via increased activation of NF- j B [31] . Thus, the

270 PI3K/Akt pathway imposes a ‘‘braking’’ mechanism limiting

271 the expression of TNF- a in LPS-stimulated monocytes and

272 ensures transient expression of these inflammatory

273 mediators.

274 (iii) Recent data demonstrate that the expression of IRAK-M

275 depends on the activation of the PI3K/Akt signaling pathway.

276 Zacharioudaki et al. [50] demonstrated that administration

of PI3K inhibitors abolished IRAK-M induction by LPS sug- gesting that LPS mediates its signal via PI3K/Akt pathway to promote IRAK-M gene expression.

(iv) Finally, the work [51] demonstrated that LPS precondition- ing resulted in lowered levels of proinflammatory cytokines (indicative of tolerance) accompanied with increased levels of IRAK-M mRNA expression. Therefore, it is hypothesized that pro-inflammatory cytokines exert an inhibitory effect

on the expression of mRNA

IRAKM .

Thus, we hypothesize that the LPS-induced activation of TLR signaling pathway is tightly regulated by different mechanisms at multiple levels. Routes (i), (iii) and (iv) eventually control the activity of IRAK whereas route (ii) affects signaling through NF-j B. Thus we hypothesize the existence of a minimal dual

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Please cite this article in press as: Q. Yang et al., A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia, Math. Biosci. (2011), doi: 10.1016/j.mbs.2011.05.005

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292 regulation of LPS recognition signaling. All the aforementioned

293 qualitative relations are depicted in the form of a network in

294 Fig. 1 . Here, we just focus on MyD88-dependent pathway. In fact,

dPI3K

dt

¼ k in; PI3K LPSR k out ;PI3K PI3K

5

ð 10Þ

351

353353

354

295 LPS-induced activation of TLR4 signaling have been divided into dAkt ¼ 1 s ð
295 LPS-induced activation of TLR4 signaling have been divided into
dAkt
¼ 1 s ð PI3K-AktÞ
ð
11Þ
296 MyD88-dependent and MyD88-independent (TRIF-dependent)
356356
dt
297 pathways [52] . The study [53] on induction of cross-tolerance to
Of note, induction of IRAK-M mRNA and IRAK-M in macro-
phages, following 10 ng/ml LPS stimulation and measured using
Northern and Western blot respectively, in the study by Kobayashi
357
298 multiple TLR ligands by in vivo LPS exposure of human blood leu-
358
299 kocytes proposes a strong support that LPS tolerance in MyD88-
359
300 dependent pathway is mediated by IRAK-M which has already
et al. [3] were found to be correlated and dependent on LPS thus 360
301 been observed in many previous experiments [54,55]. However,
IRAK-M is not constitutively expressed. Therefore, in our model,
equation (7) describes the expression of IRAK-M whereas equation
(8) describes the dynamics of protein synthesis which is assumed
to correlate with the activated kinase. The constitutive expression
of IRAK was demonstrated in the Kobayashi study, whereas both
PI3K [4] and Akt [5] are also constitutively expressed in most cells.
Thus gene expression and protein activities are not expected to be
correlated; therefore, in our work we model the dynamics of the
activated kinase (see model Eqs. (6), (10) and (11) for IRAK, PI3K
and Akt, respectively).
361
302 the factor which induces the tolerance in MyD88-independent
362
303 pathway is still unknown [53] . Moreover, the MyD88-dependent
363
304 pathway has been shown to mediate the expression of the majority
364
305 of pro-inflammatory cytokines, while to date the MyD88-indepen-
365
306 dent pathway is associated only with the induction of Type 1 inter-
366
307 feron [56] . Since we focus on the LPS-induced inflammatory
367
308 mediators’ expression, the indicator of endotoxin tolerance, in this
368
309 study, we just consider the MyD88-dependent pathway as LPS-
369
310 activated TLR4 signaling pathway.
370
311 3.2. Quantifying the dual negative regulation model
4. Results and discussion
371
312 The hypotheses earlier described are quantified in the following
4.1. Estimation of relevant model parameter
372
313 way:
314 Negative feedback regulation of IRAK by IRAK-M: We proposed to
373
315 model IRAK as a transient signal as described in Eq. (6). The cellular
374
316 surface complex LPSR induces the activation of kinase activity IRAK
375
317 with a rate k 3 , while being eliminated with a rate k out,IRAK . More-
377
319 inhibitor IRAK-M which adversely affects the transmission of the
378
320 signal to the downstream. The dynamics of the gene transcript of
The dual negative regulation model components as described in
Eqs. (6)–(11) introduce 10 new parameters. In order to robustly
estimate their appropriate values a variant of bootstrap in conjunc-
tion with least squares is explored [60] . Estimates of the parameter
values and associated confidence interval are evaluated. The boot-
strap sampling with replacement is based on the 4 replicates of one
representative gene in each essential motif as well as the corre-
sponding measurements of mRNA R , mRNA IkBa , mRNA IRAKM . The
three responses P , A , E following LPS stimulation are obtained by
using the slingshot clustering method which include 343, 502
and 2919 coexpressed probe sets, respectively [61] . In previous
[61] and current study, we select the transcriptional signature of
specific genes representative of each essential response in order
to reproduce the experimental data. IL-1 b is selected to serve as
the representative ‘‘biomarker’’ of ‘ P ’, the pro-inflammatory re-
sponse. The gene transcript of IL10RB is considered to be indicative
of the immune-regulatory signal of ‘ A ’, the anti-inflammatory
response. Finally, a subunit of NADH ubiquinone dehydrogenase
complex (mitochondrial component) NDUFC2 is considered as
the proxy for the energetic component. Of note, the purpose for
us to use the ‘ P ’ component in our modeling methodology is to
376
318 over, its increase is suppressed by the presence of its primary
379
321 IRAK-M, mRNA IRAKM , are characterized by a zero order production
380
322 rate k in,mRNA,IRAKM which is stimulated by Akt (per mechanism iii,
381
323 see above) while inhibited by ‘ P ’ (per mechanism iv, see above)
382
324 and a first order degradation rate k out,mRNA,IRAKM , Eq. (7) . The
383
325 dynamics of IRAK-M, the inhibitor of IRAK, is based on the transla-
384
326 tion of its corresponding transcript, mRNA IRAKM , with synthesis
385
327 rate k in,IRAKM and a first order degradation rate k out,IRAKM , Eq. (8).
386
328 Negative feedback regulation of TLR4 signaling pathway by PI3K/
387
329 Akt pathway: The inhibition of NF-j B by PI3K/Akt is modeled via
388
330 the indirect stimulation of the production of I j B a with rate k Ik-
389
Ba,Akt . The degradation of the I j B a is described as in the earlier
331
390
332 model [38] , Eq. (9) .
391
333 Activation of kinase PI3K/Akt via LPSR: PI3K, the kinase which is
392
334 constitutively expressed in immune cells, is activated by LPSR indi-
393
335 rectly with activation rate k in,PI3K and eliminated with a rate k out,-
PI3K , (per mechanism ii, see above) Eq. (10) . The activation of the
336
337 kinase Akt by PI3K is modeled using a transit compartment model
338 [57] with transit time s , Eq. (11)
339
Table 1
Values of the parameters involved in the propagation of LPS signaling on the
transcriptional response level neuro-endocrine immune axis.
dIRAK
k 3
LPSR
¼
IRAKM k out; IRAK IRAK
ð
Parameter
Value
Parameter
Value
Parameter
Value
341341 dt
1 þ
k
4.500
1.400
2.500
342
LPS,1
K I,1
k
6.790
0.870
7.286
LPS,2
k I,2
dmRNA IRAKM
k in; mRNA; IRAKM 1 þ k mRNA; IRAKM;Akt Akt
¼
k
0.020
0.030
0.649
syn
K in,P
dt
1
þ k mRNA; IRAKM; P P
K 1
3.000
0.330
0.842
K out,P
K 2
0.040
0.461
k 3,REPI
K out,EPI
k R ,EPI
k in,Fen
k A ,FRN
0.401
344344
k
mRNA
ð
K in,A
out; mRNA; IRAKM
IRKAM
K 3
5.000
0.809
k
0.256
K out,A
Fen, P
345
K 4
2.240
0.080
k
1.058
K in,E
out, F
dIRAKM
0.090
0.280
k
0.401
¼
K in,mRNA, R
K out,mRNA, R
K out,E
A,FRN
k in ; IRAKM mRNA; IRAKM k out; IRAKM IRAKM k in; IRAKM
d t
0.250
1.740
k
5.921
K mRNA,P,R
in,EPI
16.290
k
29.75
0
6.594
347347
ð 8Þ
k NF j B,1
P,NF j Bn
k
REPI
1.180
k
9.050
2.922
348
K NF j B,2
K in,IKBa
K out,IkBa
P,2
0.460
k
0.534
A,E
0.4634
k
0.145
A,cAMP
R in, F ( W Fex = 1)
R in, F ( W Fex = 0)
k 2,REPI
0
dIkBa
¼
k I ; 1 mRNA IkBa ð 1 þ
2.213
k IkBa; Akt AktÞ
dt
k
13.270
k
2.657
T s
0.723
IkBa,1
1,REPI
n
1.185
ð 9Þ
s
350350
k I ; 2 ð 1 þ IKKÞð1 NFjBÞ IkBa k I ; 1
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Table 2 Estimated value of parameters involved in the dual negative regulation of TLR4 signaling.

Parameter

Average Min

Max

5% Quantile 95% Quantile

K IkBa, Akt K out,IRAK K in,mRNA,IRAKM

0.4030

0.0001

2.149

0.0001

1.22175

3.1568

1.3031

6.4827

1.8426

4.2462

0.9010

0.5248

1.6999

0.69529

1.1375

K out,mRNA,IRAKM 0.7089

 

k

mRNA,IRAKM,P

0.2709

0.0459

1.2129

0.1018

0.5261

k mRNA,IRAKM,Akt 27.9706 10.2058 46.0046 22.4290

31.4328

K in,IRAKM

1.1571

0.7179

1.8030

0.8311

1.4910

K out,IRAKM

0.0499

0.0467

0.0559 0.0483

0.0515

K in,PI3K

16.8220

9.2529 40.0392 12.787

21.3832

K out,PI3K

0.6506

0.5361

1.4178

0.6233

0.6769

val. The 100( a /2) and 100 (1 a /2) percentile values of the boot- strap distribution are used as the upper and lower confidence

value of a (0 < a < 1) indicates a

100 a % confidence that b 2 [ b l ( a ), b u ( a )]. In this study, a is chosen

as 0.05, then 95% confidence limits for b based on 2000 bootstrap replications are given by b l ¼ 50th and b u ¼ 1950th largest esti- mates of b [62] . The confidence intervals for parameter are also shown in Table 2 . Typical histograms of 2000 bootstraps are shown in Fig. 3 . A dashed line drawn at the parameter estimate and dotted lines drawn at the two confidence limits are included with each histogram. All histograms are roughly Gaussian in shape, suggest- ing that the confidence interval evaluation based on bootstrap per- centile is reasonable. All the simulation and perditions in this study are done by using Matlab.

limits for a parameter. The

s 0.6475

0.5096 1.0464 0.6181 0.6671 4.2. Timing of the secondary stimulus n i ¼ 1 b
0.5096
1.0464
0.6181
0.6671
4.2. Timing of the secondary stimulus
n
i ¼ 1 b i
, where i denotes bootstrap iteration.
n
l and u respectively denote
;
P ratio ¼ P 2
P
c
P c ¼ f P c; max ; LPSð0Þ ¼ 0; LPSð xÞ ¼ 1g

394 explore the broader concept of pro-inflammatory mediator activa-

395 tion. Therefore, the use of the IL-1 b data was used only for quanti-

It is believed that the timing of the secondary stimulus, i.e. LPS administration, plays a vital role in affecting the outcome be it either potentiation or tolerance [3] . The model allows us to explore the alternative effects by varying the injection time of the second- ary LPS stimulus. We assume that two equal, non-lethal doses are administered the first at time t = 0 and the second x times units la- ter, i.e., LPS(0) = LPS( x ) = 1. In order to evaluate the implication of the time delay between the two injections we evaluate the ratio of P 2 to P c, where P 2 is the peak value of the pro-inflammatory re- sponse ‘P ’ following the second injection where P c is the corre- sponding peak in the pro-inflammatory response following a single injection of LPS, namely:

396 fication purposes. This is also valid for ‘ A ’ and ‘E ’. For each

397 bootstrap sample a vector of model parameters is estimated using

398 a least squares method. The mean of the multiple bootstrap esti-

399 mates (2000 runs in our case) is reported as the most likely param-

400 eter value [62] , b ¼ P

^

401 Parameters associated with the prior model, which are considered

402 fixed, are presented in Table 1 , while the estimated parameter val-

403 ues associate with the dual-regulation model are depicted in Table

404 2 . The performance of the model in reproducing the self-limited re-

405 sponses is shown in Fig. 2 .

406 The estimated confidence intervals for each parameter are de-

^ ^

407 noted by ½ b l ð a Þ ; b u ð a Þ , where subscript

P 2 ¼ fP 2; max ; LPSð 0Þ ¼ 1; LPSð xÞ ¼ 1g ;

408 the lower and the upper limits of the vector of model parameters b

409 and percentiles are estimated using the a central confidence inter-

are estimated using the a central confidence inter- 410 411 412 413 414 415 416 417

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

439439

Fig. 2. Model building results: dynamic profiles of the elements that constitute the mechanistic model of endotoxin-induced inflammation. Experimentally [33] measured normalized mRNA transcript levels are denoted by symbols ( ), solid lines (–) are the model predictions.

Please cite this article in press as: Q. Yang et al., A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia, Math. Biosci. (2011), doi: 10.1016/j.mbs.2011.05.005

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et al. / Mathematical Biosciences xxx (2011) xxx–xxx 7 4.4. Endotoxin tolerance ^ Fig. 3. Histograms
4.4. Endotoxin tolerance
4.4. Endotoxin tolerance

^

Fig. 3. Histograms of 2000 bootstrap estimates of four parameters. The bars represent frequency. The average bootstrap estimator values of parameters b are indicated by a

dashed line and its lower and upper confidence limits b l (0.05), b u (0.05) are represented by dotted lines respectively.

440 The definitions of P 2 and P c are graphically depicted in Fig. 4 (top pa-

441 nel). The in silico experiments depicted in Fig. 4 (bottom panel) indi-

442 cate that significant potentiation of the inflammatory response is

443 observed when the interval between the two successive injections

444 is within a critical time window. It should also be noted that ob-

445 served potentiation is not a simple additive effect by two successive

446 injection of LPS since levels of P 2 are much larger than the maxi-

447 mum value of the peak of ‘ P ’ following a single injection of LPS with

448 dose equal to 2. The robustness of the inflammatory response is

449 diminished as the interval x between the two successive injection

450 increases until a maximum tolerance, quantified as a relative sup-

451 pression of the pro-inflammatory response, occurs when the inter-

452 val between the injections is reached. From that point on, the extent

453 of tolerance diminishes and eventually the effects of precondition-

454 ing slowly dissipate and the memory effects completely disappear

455 (bottom panel of Fig. 4 ).

456 The predicted trends related to the effect of timing of the sec-

457 ondary stimulation are qualitatively consistent with the result in

458 van’t Veer’s studies [54] , in which the release of TNF-a per mono-

459 cytes in whole blood was drastically diminished in the period 3–

460 8 h after LPS injection. TNF- a measurement [54] is considered as

461 a prototypical inflammatory response, which corresponds to ‘ P ’ in

462 our model. Furthermore, it has been documented that the endo-

463 toxin tolerance is preserved over long periods of time as in several

464 studies of the induction of endotoxin tolerance in animals, normal

465 responsiveness resumes after 8 days of tolerance [2] .

466 4.3. Lethal potentiation

467 When the time between successive administrations is short, our

468 model predicts potentiation of the response consistent with

469 experimental evidence [4] . Part of the internal system dynamics,

470 and the associated dysregulation of the responses, are depicted

in Fig. 5 . This phenomenon is feasible since the preconditioning has already changed the state in which the system lies when the second stimulus is given. The main stimulus 0.5 h following pre- conditioning will further activate the NF- j B which has already been stimulated and cause it to be persistently active which leads to the significantly increased and lasting pro-inflammatory and anti-inflammatory responses. Thus, an extra abrupt stimulus might dysregulate the dynamics of the host response to infection which may, in turn, have a lethal effect in the physiological state of the system.

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

As the interval between the successive administrations is in- creased rather than a persistent production of proinflammatory cytokines, a much less vigorous inflammatory response is ob- served. Our model predicts this kind of response when the system is pre-exposed to a non-lethal stimulus 24 h prior to the second

endotoxin injection and system dynamics are depicted in Fig. 6 . 487

In Fig. 6 , a deduced activation of IRAK, NF-j B could be observed which will finally lead to the suppressed ‘ P ’, the pro-inflammatory response. Our results are in agreement with experiment observa- tions that some components in the signaling pathways are down- regulated such as IRAK [16] , NF-j B [63] . And studies of ET induced in vivo [8] have shown a decrease in the production of several cyto- kines by macrophages including IL-1 b , TNF- a , and IL-6 which are biomarkers of proinflammatory response. It is worth noting that the concentration of IRAK-M induced by preconditioning is rela- tively high when the main stimulus is given at the 24th hour in Fig. 6 . In our model, the presence of IRAK-M is the key in induction of ET and to inhibit the signaling pathways required for the inflam- matory process. The stimulation of IRAK-M transcripts expression was reflected at the protein level, and significant quantities of this

488

489

490

491

492

493

494

495

496

497

498

499

500

501

Please cite this article in press as: Q. Yang et al., A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia, Math. Biosci. (2011), doi: 10.1016/j.mbs.2011.05.005

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Yang et al. / Mathematical Biosciences xxx (2011) xxx–xxx Fig. 4. The effect of the timing
Yang et al. / Mathematical Biosciences xxx (2011) xxx–xxx Fig. 4. The effect of the timing

Fig. 4. The effect of the timing of the secondary pro-inflammatory stimulus on the tolerance. The doses for two injections are equal and nonlethal to the subjec ts with the initial condition LPS(0) = LPS( x ) = 1 where x , the time point at which the secondary stimulus is introduced, varies from 0 h to 192 h. The outcome is monitored by the ratio of P 2 to P c , where P 2 is the peak value of the ‘P ’ of the second response. P c , the control, is the ‘ P ’, proinflammatory response under only one stimulus with the initial condition LPS(0) = 0, LPS( x ) = 1. P 2 and P c are intuitively depicted in top panel as well as the result in bottom panel.

502 kinase were detected 24 h after incubation with LPS [51] . The

of LPS tolerance [64] . This hypothesis implies that the endotoxin tolerance is not simultaneous suppression of all the immune re- sponse in the immune cells but an expression of a simultaneous upregulation of some components and downregulation of other components in the pathway. In other words, ET may not be generated from global kinases activity suppression in signaling pathways which finally leads to reduced production of proinflam- matory cytokines. The variety of responses to LPS after precondi- tioning implies extremely sophisticated mechanisms to support the proper magnitude of the immune response and to protect the host from its harmful edge in multiple levels and various phases [32] .

4.5. ‘‘Protective’’ tolerance

The extreme case of endotoxin tolerance as initially described was that animals survived a lethal dose challenge if they had been previously treated with a sublethal stimulus [4] . The pre-exposure

503 build-up of the IRAK-M at 24 h, induced by preconditioning, results

504 in a reduction of the inflammatory response through strong inhibi-

505 tion of IRAK activation.

506 However, recent experiments reveal that not all kinases in the

507 TLR4 signaling pathway will be suppressed when the endotoxin

508 tolerance occurs. When pretreated cells were again stimulated

509 with LPS 24 after first stimulus, the levels of IRAK-M mRNA and

510 proteins were twofold greater than the maximal levels produced

511 by the first induction [51] . This observation indicates that endo-

512 toxin tolerance is no longer to be considered as a global downreg-

513 ulation of the immune response as before [16] . An increased

514 production of IRAK-M is predicted by our model in Fig. 6 . The

515 augmentation of IRAK-M is due to the significantly decreased inhi-

516 bition of mRNA IRAKM by ‘ P ’. Therefore, the most important feature

517 of current model is that it offers the opportunity to explore the

518 ‘leukocyte reprogramming’ which referrers to the alterations in

519 signaling pathways and chromatin remodeling with the induction

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

Please cite this article in press as: Q. Yang et al., A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia, Math. Biosci. (2011), doi: 10.1016/j.mbs.2011.05.005

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xxx (2011) xxx–xxx

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et al. / Mathematical Biosciences xxx (2011) xxx–xxx 9 Fig. 5. Lethal potentiation: successive administration of
et al. / Mathematical Biosciences xxx (2011) xxx–xxx 9 Fig. 5. Lethal potentiation: successive administration of

Fig. 5. Lethal potentiation: successive administration of small doses of endotoxin can lead to an unresolved inflammatory response. Solid line: LPS ( t = 0 h) = 1 and LPS

( t = 0.5 h) = 1.

Dashed line: LPS ( t = 0 h) = 0 and LPS ( t = 0.5 h) = 1.

536 to a lower nonlethal dose of LPS could modulate its intracellular

537 dynamics by reversing the lethal outcome of the main much higher

538 one which is responsible for an overwhelming inflammatory re-

539 sponse in Fig. 7 . The physiological significance of LPS tolerance

540 can be best demonstrated through the protection by a nonlethal

LPS against the lethal outcomes of a secondary high-dose LPS in animals [65] . In this experiment it was shown that wild type mice primed with a sublethal dose of LPS and then challenged with a lethal dose of LPS remained healthy. It is important to note that such a rescue is possible because the preconditioning has already

a rescue is possible because the preconditioning has already 541 542 543 544 545 Fig. 6.

541

542

543

544

545

Fig. 6. Tolerance: pre-exposure the system with a smaller inflammatory insult results in a reduction in the cell capacity to produce pro-inflammatory cytokin es which is characterized as an attenuation scenario. Solid line: LPS ( t = 0) = 1 and LPS ( t = 24) = 1. Dashed line: LPS ( t = 0 h) = 0 and LPS ( t = 24 h) = 1.

Please cite this article in press as: Q. Yang et al., A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia, Math. Biosci. (2011), doi: 10.1016/j.mbs.2011.05.005

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Q. Yang et al. / Mathematical Biosciences xxx (2011) xxx–xxx

Yang et al. / Mathematical Biosciences xxx (2011) xxx–xxx line: LPS ( t = 0 h)
line: LPS ( t = 0 h) = 0 and LPS ( t = 24
line: LPS ( t = 0 h) = 0 and LPS ( t = 24 h) = 4.
4.6. The effect of IRAK-M on Tolerance

Fig. 7. Protective tolerance: pre-existing infection might cause a profound hypo-responsiveness in system’s response to a lethal LPS challenge. Solid lin e: LPS ( t = 0 h) = 1 and

LPS ( t = 24 h) = 4. Dashed

546 changed the state in which the system lies when the lethal dose is

547 encountered. Specifically, the active IRAK-M rises enough to inhibit

548 the activation of IRAK so that when the previously lethal endotoxin

549 stimulus is given, the system will be driven back to the healthy

550 state, rather than that of the unhealthy state. We conclude that

551 by preconditioning the system with a low dose of LPS, one can re-

552 duce the response obtained with a larger dose of LPS.

553 Interestingly, the induction of LPS tolerance during clinical con-

554 ditions may in the short term be beneficial by preventing excessive

555 inflammation, but in the longer term be deleterious by hampering

556 an adequate defense response to opportunistic infections [53] . This

557 may be demonstrated by the recent clinical observation that the

558 Q2 severe immunosppression demonstrated by significant endotoxin

559 tolerance in sepsis patients have high correlation with mortality

560 [55] . The significant decrease of IRAK and elevation IRAK-M mRNA

561 expression in mononuclear cells are the notable characters of these

562 severe sepsis patients. Thus, endotoxin tolerance is just like a

563 double-edged sword. The transient induction of IRAK-M in healthy

animals after preconditioning will protect the host from overacti- vation of another wave of proinflammation following the second- ary exposure to LPS. However, the long lasting high level IRAK-M expression in sepsis patient is an indicator of poor outcome and mortality [55] .

As discussed in previous section, the suppression of IRAK by IRAK-M may play a role in the endotoxin tolerance. However, whether IRAK-M is the factor which actually induces ET is still not clarified. Thus, we are interested in exploring the effect of IRAK-M by knocking out this gene to check if it is required for tol-

564

565

566

567

568

569

570

571

572

573

574

erance. Our model allows us to test the effect of the knockout of 575

IRAK-M gene on LPS tolerance in the system which is pre-exposed to a stimulus for about 24 h before the main endotoxin challenge. It is not surprising to find that the disruption of the gene encoding the negative regulator IRAK-M for the signaling pathway results

576

577

578

579

IRAK-M for the signaling pathway results 576 577 578 579 Fig. 8. The lack of tolerance

Fig. 8. The lack of tolerance in in silico experiment with IRAK-M knockout animal . Knocking out of mRNA leads to the lack of endotoxin tolerance. Left plot: solid line: LPS ( t = 0) = 1 and LPS ( t = 24) = 1, IRAK-M knockout animal; Dashed line: LPS ( t = 0) = 1 and LPS ( t = 24) = 1, wild animal. Right plot: solid line: LPS ( t = 0) = 1 and LPS ( t = 24) = 4, IRAK-M knockout animal. Dashed line: LPS ( t = 0) = 1 and LPS ( t = 24) = 4, wild animal.

Please cite this article in press as: Q. Yang et al., A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia, Math. Biosci. (2011), doi: 10.1016/j.mbs.2011.05.005

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580 in the lack of the endotoxin tolerance as seen in left plot in Fig. 8 .

581 This result is consistent with Kobayashi’s other experiment in the

582 same publication [21] . It is reported IRAK-M / macrophages

583 showed a loss of endotoxin tolerance demonstrated by the cyto-

584 kine levels produced upon LPS restimulation. Moreover, compared

585 with the scenario in Fig. 7 , under the same condition, it is further

586 expected that the preconditioning of smaller dose lost protective

587 effect for a subsequent lethal dose injection when IRAK-M gene is

588 knocked out as seen in the right plot in Fig. 8 . The model predicted

589 that the inhibition of stimulation of IRAK and the subsequent gene

590 expression is lost due to disappearance of IRAK-M. Therefore, we

591 can conclude that IRAK-M may be a key component of this impor-

592 tant control system since the development of tolerance upon re-

PI3K by five times. The increased production of the prototypical inflammatory response ‘ P ’ in our current model is reflected in the enhanced production of TNF-a [31] after in silico administration

of Wortmannin. Thus, both IRAK-M and PI3K negatively regulate

TLR4 signaling pathway though they function in different time per- iod. The PI3K will be activated immediately in response to stimulus

and lose its activity within 9 h which only inhibits the activation of NF-j B transiently. However, the IRAK-M will be stimulated a little bit latter but last for much longer time period. It appears that the different time periods directly decide their individual functions

in controlling the TLR4 signaling pathway which will be addressed

in detail in the discussion of ‘crosstalk’ between these two

proteins. M in in of of
proteins.
M
in
in
of
of

593 peated stimulation with LPS is dampened without IRAK-M.

4.8. The ‘crosstalk’ between PI3K and IRAKM

594 4.7. Increased cytokine production through IRAK-M gene knock-out or

595 administration of a PI3K inhibitor

It’s interesting to find a relationship between two negative reg- ulators, the induction of IRAK-M is induced by the other kinase

596 Both IRAK-M and PI3K are negative regulators of TLR4 signaling

PI3K. Thus, we expect there will be protein level change of IRAK-

597 pathway, so, it is expected that an enhanced proinflammatory re-

by pretreatment of the cells with specific inhibitor of PI3K. We

598 sponse will be observed in the absence of these regulators. As illus-

examine the influence of administrating Wortmannin against PI3K to the response by increasing the degradation rate of PI3K, k out,PI3K by 10 times which mimics the neutralization of active PI3K by its inhibitor. We discussed the role PI3K played by using

599 trated in the upper plots in Fig. 9 , we perform both an IRAK-M

600 knock-out experiment and an administration of a PI3K inhibitor

601 experiment respectively. The model is manipulated so that there

602 is no de novo transcriptional synthesis of IRAK-M which is respon-

pre-administration of its inhibitor Wortmannin. The roles it plays

603 sible for negative regulation of IRAK. After the disruption of IRAK-M

both single LPS stimulation as well as preconditioning are shown

604 gene, no expression of mRNA IRAKM and protein IRAK-M is observed.

Fig. 10 . As seen in Fig. 10 , 0–24 h shows the pre administration

605 The knockout causes an increased expression for ‘P’, pro-inflamma-

Wortmannin leads to the abolished expression of IRAK-M as

606 tion which is in agreement with the Kobayashi’s report that IRAK-

well as enhanced production of pro-inflammatory cytokines following single LPS stimulus. The result is in agreement with recent observation that LPS-derived immune cells pretreated with the PI3K inhibitor expressed significantly abolished expression of

607 M / macrophages revealed increased production of TNF a , IL-6

608 and IL-12 when compared to wild-type macrophages at 6 hr after

609 stimulation [21] . Similarly, in the lower plots in Fig. 9 , administra-

610 tion of Wortmannin, the PI3K inhibitor, shows enhanced TLR4 sig-

IRAK-M [7] . The 24–48 h responses show the effect of second LPS stimuli, we could see that the significantly decreased production

611 naling and enhanced production of TNF- a [31] . The neutralization

612 of active PI3K is mimicked by increasing the degradation rate of

IRAK-M following the first stimulus fails to inhibit the activation

the first stimulus fails to inhibit the activation 613 614 615 616 617 618 619 620

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

Fig. 9. The increased proinflammatory response in in silico experiment with IRAK-M knockout animal or pretreatment with PI3K inhibitor. Upper plots: solid line: LPS ( t = 0 h) = 1.5 with an IRAKM / animal; dashed line: LPS ( t = 0 h) = 1.5 with wild type animal. Lower plots: Solid line: LPS ( t = 0 h) = 1.5 pretreated with PI3K inhibitor; dashed line: LPS ( t = 0 h) = 1.5, pretreated with saline.

Please cite this article in press as: Q. Yang et al., A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia, Math. Biosci. (2011), doi: 10.1016/j.mbs.2011.05.005

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Q. Yang et al. / Mathematical Biosciences xxx (2011) xxx–xxx

Yang et al. / Mathematical Biosciences xxx (2011) xxx–xxx References [1] S64. Fig. 10. Implication of
References [1] S64.
References
[1]
S64.

Fig. 10. Implication of administration of PI3K inhibitor by decreasing concentration of active PI3K through increasing the degradation rate constant k out; PI3K by 10 times. Solid line: LPS ( t = 0 h) = 1and LPS ( t = 24 h) = 1pretreated with PI3K inhibitor; dashed line: LPS ( t = 0 h) = 1and LPS ( t = 24 h) = 1 pretreated with saline.

of IRAK and NFkB following the second LPS insult. This finally leads

to the strongly enhanced production of pro-inflammatory response

compared to the control. Thus, inhibiting the activation of the PI3K

with enough inhibitor will also lead to the loss of tolerance. This is

supported by the observation that ablation of Akt which blocks the

activation of IRAK-M by PI3K/Akt pathway inhibits the induction of

endotoxin tolerance [8] .

Model behavior implies that the presence of dual regulation

mechanisms leads to dual pattern of negative feedback, where

PI3K/Akt is an ‘early’ negative switch that attenuates the initial sig-

nal propagation, whereas IRAK-M represents the ‘memory’ compo-

nent of the system responsible for the tolerance behavior.

However, the question arises as to if IRAK-M alone can accomplish

the general response of induction of ET, or what additional role

does the PI3K/Akt pathway have? Actually, there is a ‘crosstalk’ be-

tween the two negative regulators; the induction of IRAK-M is due

to activation of PI3K/Akt signaling pathway which implies that

PI3K may function as a transient alarming signal to the system

which will further activate and intensify the magnitude of negative

regulation of TLR4 by activating other proteins in a long lasting

time interval for a sustained suppression. It is implied that IRAK-

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Please cite this article in press as: Q. Yang et al., A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia, Math. Biosci. (2011), doi: 10.1016/j.mbs.2011.05.005

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