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Mathematical Biosciences
journal homepage: www.elsevier.com/locate/mbs

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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, Stephen F. Lowry b, Ioannis P. Androulakis a,b,c,
a

Chemical Engineering, Rutgers University, 98 Brett Road, Piscataway, NJ 08854, USA 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
b

a r t i c l e

i n f o

a b s t r a c t
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, tolerance, and protective tolerance. Moreover, the knockout of negative regulators shows that IRAK-M is a necessary and sufcient 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 veried experimentally. Finally, the pretreatment with PI3K inhibitor reveals the interaction between these two negative regulations. 2011 Published by Elsevier Inc.
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Article history: Received 9 September 2010 Received in revised form 10 May 2011 Accepted 16 May 2011 Available online xxxx Keywords: Mathematical modeling Lipopolysaccharide Endotoxin Potentiation Tolerance Humans

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1. Introduction Endotoxin (LPS), a membrane glycolipid of Gram-negative bacteria, is a potent inducer of pro-inammatory responses in monocytes, macrophages, and neutrophils and is widely accepted model for the study of inammatory responses [1]. While immune cells exposure to LPS induces the release of both pro- and anti-inammatory cytokines (small proteins that are the principal mediators of inammation), repeated treatment with LPS can lead to enhancement or desensitization of subsequent pro-inammatory cytokine responses [2] so-called potentiation or tolerance, respectively [3]. Potentiation is dened as the enhanced response to a secondary LPS administration [4] whereas endotoxin tolerance (ET) is dened as a diminished secondary response to LPS activation following a primary exposure. LPS tolerance has also been termed hyporesponsiveness, refractoriness, adaptation, deactivation, desensitization, immunoparalysis or reprogramming [2,5]. Studies of ET induced in vitro [6,7] and in vivo [8] have shown a decrease in the production of several cytokines by macrophages; including IL-1b, 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. Androulakis). 0025-5564/$ - see front matter 2011 Published by Elsevier Inc. doi:10.1016/j.mbs.2011.05.005

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 approaches 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 [3,913] have investigated preconditioning phenomena while evaluating alternative computational models. The central signaling receptor for LPS is Toll-like receptor 4 (TLR4), and all previous work address preconditioning as it relates to TLR4 signaling. Day et al. [9] construct a four-dimensional model whose key feature is the presence of anti-inammatory factors in the system suppressing the growth of inammatory cytokines in response to the secondary stimulus. Similarly, Vasilescu 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, Rivieres work [3] suggests that preconditioning is controlled by the regeneration rate of TLR4 without invoking a specic signaling inhibition mechanism. Finally, negative feedback regulation by specic proteins considered as mechanism is also used to induce ET in an agent based model proposed by An and coworkers [11,12]. Our fundamental understanding of LPS signaling has improved dramatically over the recent years as new experimental evidence emerges. Thus it is believed that ET may not be solely induced by

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suppression of anti-inammatory mediators [14], nor through the exclusive down-regulation of cell surface receptors [15]. Furthermore, simple feedback control mechanisms could not explain why immune responses are not suppressed simultaneously under ET condition [16]. Thus, the complexity of the response to LPS preconditioning implies the possibility of multilevel regulation requiring the development of more elaborate underlying mechanisms. Recently a number of studies focusing on quantifying proteins or enzymes in TLR4 signaling pathways have suggested that the different results following preconditioning are related to the complex and tightly regulated molecular mechanisms within this signaling pathway. TLR4 is involved in host defense against invading pathogens, functioning as the primary sensor of microbial products and activating signaling pathways that induce the expression of immune and pro-inammatory genes [17]. Due to this highly signicant biological role, the TLR4 signaling pathway is tightly regulated [18]. Thus, it is not surprising to nd that various negative regulatory mechanisms have evolved to control TLR4 signaling in order to maintain immunological balance. Several molecules which are identied as potential negative regulators of the LPS-induced TLR4 signaling pathway are highly likely to play a role in the signal transduction alterations associated with endotoxin tolerance based on recent in vitro and murine in vivo studies. Negative regulators such as intracellular molecules myeloid-differentiation-88-short (MyD88s) [19,20], IL-1R-associated-kinase-M (IRAK-M) [21], Toll interacting protein (TOLLP) [22] and suppressor-of-cytokine signaling 1 (SOCS1) [23,24] have been shown to play a vital role in endotoxin tolerance. Moreover, additional signaling pathways which are triggered by LPS are found to be able to negatively regulate TLR4 signaling. Phosphatidylinositol 3-kinase (PI3K), a family of intracellular signal transducer enzymes, has been linked to an extraordinarily diverse group of cellular functions including cell growth proliferation, differentiation, motility, survival and intracellular trafcking [25] which could be activated in many ways, directly by integrins [26], by growth factors [27], by G-protein coupled receptors [28]. Many of these functions relate to the ability of PI3K to activate protein kinase B (Akt). Recent data indicate that these molecules are also integral players in coordinating defense mechanisms in the innate immune system which could also be stimulated in diverse manners, by cytokines via JAK1 [29], by antigen via BCAP [30]. It has been reported that the LPS-induced activation of PI3K/Akt limits lipopolysaccharide activation of TLR4 signaling pathways and expression of inammatory mediators in human monocytic cells [31]. These mechanisms point to the possibility of a dual-phase mechanism of negative regulation associated with innate immune response. Though both PI3K/Akt and IRAK-M have roles in the gate-keeping system, preventing excessive innate immune response [32], there is a critical difference between PI3K/Akt- and IRAK-M-dependent negative regulatory mechanisms. Unlike IRAK-M that is induced by TLR signaling and functions during the second or continuous exposure to stimulation, PI3K/Akt acts at the rst phase of TLR signaling and modulate the magnitude of the primary activation. Therefore, PI3K/Akt functions as a negative controller in the early (or primary) phase of the innate immune response by inhibiting some of the shared signaling pathways downstream of TLR4, whereas IRAK-M acts in the late (or second) phase of the innate immune response [32]. The work to be discussed in this paper aims to develop a model based on the molecular mechanisms of the TLR4 signaling pathway exploring the synergies between these two negative feedback regulations in order to describe the complex dynamics of the LPS-induced inammation and investigate different scenarios of preconditioning. Our model describes the interaction between the ligand (LPS) and the transmembrane signaling receptor (TLR4) coupled with the recruitment of kinase (IRAK) and the

activation of transcriptional factor (NF-jB) which triggers the stimulation of expression of essential leukocyte-specic transcriptional dynamics. Simultaneously, the indirect activation of the PI3K/Akt signaling pathway by TLR4 which suppresses the NF-jB activity develops a short loop. On the other hand, the suppression of kinase IRAK by its specic inhibitor IRAK-M whose transcription is stimulated by the activation of PI3K/Akt signaling pathway creates a longer loop. Such dual negative inhibition can potentially emerge as a critical enabler towards understanding the connectivity 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 administration experiments can emerge as a natural consequence of the tightly regulated signaling pathway in acute inammatory response. 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 tolerance and demonstrates that IRAK-M is a necessary and sufcient factor for this complex behavior. In silico knockout of irak-M or administration of PI3K inhibitors respectively predicts experimentally veried responses highlighting the importance of a dual negative feedback regulation in the model with both the PI3K/Akt and IRAK-M. Finally, the pretreatment with PI3K inhibitor reveals the crosstalk between these two negative regulations. 2. Materials and methods 2.1. Human endotoxin model Gene expression data used herein were obtained from the Inammation and Host Response to Injury Large Scale Collaborative 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 testing the expression level of genes whose expression can be altered in response to endotoxin. A set of 5093 probe sets were characterized by signicant variation (corresponding to 0.2% false discovery rate) across the time course of the experiment using the SAM software [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 cortisol and epinephrine [36,37]. Specically, 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 was 0, 2, 4 and 6 h following endotoxin administration [37]. 2.2. An LPS-Induced acute inammation model We have previously [38], proposed a quantitative model of an endotoxin induced inammatory response. The activation process involves the induction of a signal transduction cascade that triggers transcriptional initiation of inammatory 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 degradation of IjBa and then the release of the transcriptional factor (NF-jB). NF-jB translocates into the nucleus and initiates the

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expression of inammatory response related genes including P, the pro-inammatory component, A, the anti-inammatory component, and E, the energetic component. Moreover, the critical aspects of the neuro-endocrine immune crosstalk connecting the cellular response level were also described. These responses were integrated into a mathematical model using the basic principles of an Indirect Response Model (IDR) [40] that bridges the extracellular signal (LPS) with the downstream activation of the major transcriptional responses activation and neuro-endocrine system interaction. The model is succinctly presented in Eqs. (1)(5) and is described in great detail in [38]

translocation of NF-jB [17] resulting in the expression of inammation related genes. The inammatory dynamics are the manifestation of the complex interaction between activating and inhibitory interactions in order to constantly strike a balance between activation and inhibition and to drive the immune system back to homeostasis [46]. Numerous cytokines are responsible for amplifying the inammatory reaction, through the critical IKK node [47], while negative proteins inhibit IRAK which will nally suppress the release of inammatory mediators [18]. We will focus specically on four putative modes of regulation: (i) IRAK-M is one of the most important negative regulators of IRAK and has been shown to prevent dissociation of IRAK from MyD88 and the formation of the IRAK-TRAF6 complex

 dLPS klps;1 LPS 1 LPS klps;2 LPS LPS kinetics dt

8 > dR ksyn;mRNA;R mRNA; R k2 LPSR k1 LPS R ksyn R > dt < ligand receptor interactions dLPSR k1 LPS R k3 LPSR k2 LPSR > dt > dmRNA;R : kin;mRNA;R 1 kmRNA;R;P P kout;mRNA;R R dt 8   > dIKK k3 IKK=1 IkBa k4 IKK P IKK2 2 > dt > 1IKK > > > < dNFjBn kNFjB;1 IKK1NFjBn kNFjB;2 NFjBn IkBa dt 1IkBa NFjB signaling dynamics > dmRNAIkBa > > kin;IkBa 1 kIkBa;1 NFjBn kout;IkBa mRNA; IkBa > dt > > dIkBa : kI;1 mRNA; IkBa kI;2 1 IKK 1 NFjBn IkBa kI;1 dt 8 > dP kin;P 1 kP;NFjBn NFjBn 1 kP;E E=A kout;P P > dt > > dA < k 1 k A;cAMP cAMP 1 kA;E E 1 kA;FRN FRN in;A Intrinsic transcriptional responses dt > > kout;A A > > dE : kin;E 1 kE;P P=A kout;E E dt 8 dF > dt wF ex Rin;F kin;F 1 kF;P P kout;F F > > > dRm > FRN > > > dt ksyn Rm 1 IC50 Rm FRN kdeg Rm > > dRF > > > dt ksyn R Rm r f kre FRN kon F RF kdgr R RF > > > dFR > > dt kon F RF kT FR > > > dFRN > > > dt kT FR kre FRN > > > dEPI > > < dt wEPI;ex Rin;EPI kin;EPI 1 kEPI;P P kout;EPI EPI 0 neuro-endocrine immune system interactions dREPI > dt kREPI k1;REPI 1 kREPI EPI k2;REPI REPI > > > dEPIR > > dt k1;REPI 1 kREPI EPI REPI k3;EPIR EPIR > > > > dcAMP 1 n > > dt s EPIR cAMP > >  > > > > wF 1; exogenous hormone > > ex > > 0; elsewhere > >  > > > 1; exogenous hormone > > wEPIex : 0; otherwise

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3. Modeling the dual negative regulation LPS recognition mechanism 3.1. Putative structure of the regulation network LPS is recognized by its transmembrane signaling receptor, TLR4, and the accessory protein MD-2. The binding of LPS and TLR4 results in the formation of a complex, LPSR, and the recruitment of the adaptor molecules MyD88 [42] and TIRAP [43]. This will further result in recruiting and activating IRAK [44] subsequently activating TRAF6 [45]. Further intracellular events ultimately result in the activation of the IKK complex, involving phosphorylation and degradation of IjBa, enabling the nuclear

[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 specic negative regulators, MyD88s (the short form of MyD88) and A20 respectively [18], we consider a simplied feedback loop consisting only of IRAK and IRAK-M, since the redundancy of negative regulation of IRAK by several controllers, including IRAK-M, SOCS-1, and TOLLIP, implies the signicance 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

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

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MyD88 in response to LPS was reported. Ojaniemis study on LPS-induced PI3K activation [49] demonstrated that activation of TLR4 results in the formation of the PI3KMyD88 complex, implying that one mode of activation of the PI3K/Akt pathway following exposure to LPS is via LPSTLR4-MyD88 approach. It was also shown that inhibition of PI3K/Akt pathway enhances LPS-induced TNF-a gene expression via increased activation of NF-jB [31]. Thus, the PI3K/Akt pathway imposes a braking mechanism limiting the expression of TNF-a in LPS-stimulated monocytes and ensures transient expression of these inammatory mediators. (iii) Recent data demonstrate that the expression of IRAK-M depends on the activation of the PI3K/Akt signaling pathway. Zacharioudaki et al. [50] demonstrated that administration

of PI3K inhibitors abolished IRAK-M induction by LPS suggesting that LPS mediates its signal via PI3K/Akt pathway to promote IRAK-M gene expression. (iv) Finally, the work [51] demonstrated that LPS preconditioning resulted in lowered levels of proinammatory cytokines (indicative of tolerance) accompanied with increased levels of IRAK-M mRNA expression. Therefore, it is hypothesized that pro-inammatory cytokines exert an inhibitory effect on the expression of mRNAIRAKM. 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-jB. Thus we hypothesize the existence of a minimal dual

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regulation of LPS recognition signaling. All the aforementioned qualitative relations are depicted in the form of a network in Fig. 1. Here, we just focus on MyD88-dependent pathway. In fact, LPS-induced activation of TLR4 signaling have been divided into MyD88-dependent and MyD88-independent (TRIF-dependent) pathways [52]. The study [53] on induction of cross-tolerance to multiple TLR ligands by in vivo LPS exposure of human blood leukocytes proposes a strong support that LPS tolerance in MyD88dependent pathway is mediated by IRAK-M which has already been observed in many previous experiments [54,55]. However, the factor which induces the tolerance in MyD88-independent pathway is still unknown [53]. Moreover, the MyD88-dependent pathway has been shown to mediate the expression of the majority of pro-inammatory cytokines, while to date the MyD88-independent pathway is associated only with the induction of Type 1 interferon [56]. Since we focus on the LPS-induced inammatory mediators expression, the indicator of endotoxin tolerance, in this study, we just consider the MyD88-dependent pathway as LPSactivated TLR4 signaling pathway. 3.2. Quantifying the dual negative regulation model The hypotheses earlier described are quantied in the following way: Negative feedback regulation of IRAK by IRAK-M: We proposed to model IRAK as a transient signal as described in Eq. (6). The cellular surface complex LPSR induces the activation of kinase activity IRAK with a rate k3, while being eliminated with a rate kout,IRAK. Moreover, its increase is suppressed by the presence of its primary inhibitor IRAK-M which adversely affects the transmission of the signal to the downstream. The dynamics of the gene transcript of IRAK-M, mRNAIRAKM, are characterized by a zero order production rate kin,mRNA,IRAKM which is stimulated by Akt (per mechanism iii, see above) while inhibited by P (per mechanism iv, see above) and a rst order degradation rate kout,mRNA,IRAKM, Eq. (7). The dynamics of IRAK-M, the inhibitor of IRAK, is based on the translation of its corresponding transcript, mRNAIRAKM, with synthesis rate kin,IRAKM and a rst order degradation rate kout,IRAKM, Eq. (8). Negative feedback regulation of TLR4 signaling pathway by PI3K/ Akt pathway: The inhibition of NF-jB by PI3K/Akt is modeled via the indirect stimulation of the production of IjBa with rate kIkBa,Akt. The degradation of the IjBa is described as in the earlier model [38], Eq. (9). Activation of kinase PI3K/Akt via LPSR: PI3K, the kinase which is constitutively expressed in immune cells, is activated by LPSR indirectly with activation rate kin,PI3K and eliminated with a rate kout,PI3K, (per mechanism ii, see above) Eq. (10). The activation of the kinase Akt by PI3K is modeled using a transit compartment model [57] with transit time s, Eq. (11)

dPI3K kin;PI3K LPSR kout;PI3K PI3K dt dAkt 1 PI3K-Akt dt s

10

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11

356
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Of note, induction of IRAK-M mRNA and IRAK-M in macrophages, following 10 ng/ml LPS stimulation and measured using Northern and Western blot respectively, in the study by Kobayashi et al. [3] were found to be correlated and dependent on LPS thus 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). 4. Results and discussion 4.1. Estimation of relevant model parameter 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 conjunction with least squares is explored [60]. Estimates of the parameter values and associated condence interval are evaluated. The bootstrap sampling with replacement is based on the 4 replicates of one representative gene in each essential motif as well as the corresponding measurements of mRNAR, mRNAIkBa, mRNAIRAKM. 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 specic genes representative of each essential response in order to reproduce the experimental data. IL-1b is selected to serve as the representative biomarker of P, the pro-inammatory response. The gene transcript of IL10RB is considered to be indicative of the immune-regulatory signal of A, the anti-inammatory 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

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Table 1 Values of the parameters involved in the propagation of LPS signaling on the transcriptional response level neuro-endocrine immune axis. Parameter kLPS,1 kLPS,2 ksyn K1 K2 K3 K4 Kin,mRNA,R Kout,mRNA,R kNFjB,1 KNFjB,2 Kin,IKBa Kout,IkBa kIkBa,1 ns Value 4.500 6.790 0.020 3.000 0.040 5.000 2.240 0.090 0.250 16.290 1.180 0.460 0.4634 13.270 1.185 Parameter KI,1 kI,2 Kin,P Kout,P Kin,A Kout,A Kin,E Kout,E KmRNA,P,R kP,NFjBn kP,2 kA,E kA,cAMP k1,REPI Value 1.400 0.870 0.030 0.330 0.461 0.809 0.080 0.280 1.740 29.75 9.050 0.534 0.145 2.657 Parameter k3,REPI Kout,EPI kR,EPI kin,Fen kA,FRN kFen,P kout,F kA,FRN kin,EPI kREPI Rin,F(WFex = 1) Rin,F(WFex = 0) k2,REPI Ts
0

dIRAK k3 LPSR kout;IRAK IRAK dt 1 IRAKM dmRNAIRAKM 1 kmRNA;IRAKM;Akt Akt kin;mRNA;IRAKM 1 kmRNA;IRAKM;P P dt

Value 2.500 7.286 0.649 0.842 0.401 0.256 1.058 0.401 5.921 6.594 2.922 0 2.213 0.723

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kout;mRNA;IRAKM mRNAIRKAM dIRAKM kin;IRAKM mRNA; IRAKM kout;IRAKM IRAKM kin;IRAKM dt

347 348

8 dIkBa kI;1 mRNAIkBa 1 kIkBa;Akt Akt dt

350

kI;2 1 IKK1 NFjBIkBa kI;1

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Table 2 Estimated value of parameters involved in the dual negative regulation of TLR4 signaling. Parameter KIkBa, Akt Kout,IRAK Kin,mRNA,IRAKM Kout,mRNA,IRAKM kmRNA,IRAKM,P kmRNA,IRAKM,Akt Kin,IRAKM Kout,IRAKM Kin,PI3K Kout,PI3K Average 0.4030 3.1568 0.9010 0.7089 0.2709 27.9706 1.1571 0.0499 16.8220 0.6506 0.6475 Min 0.0001 1.3031 0.5248 0.0459 10.2058 0.7179 0.0467 9.2529 0.5361 0.5096 Max 2.149 6.4827 1.6999 1.2129 46.0046 1.8030 0.0559 40.0392 1.4178 1.0464 5% Quantile 0.0001 1.8426 0.69529 0.1018 22.4290 0.8311 0.0483 12.787 0.6233 0.6181 95% Quantile 1.22175 4.2462 1.1375 0.5261 31.4328 1.4910 0.0515 21.3832 0.6769 0.6671

val. The 100(a/2) and 100 (1 a/2) percentile values of the bootstrap distribution are used as the upper and lower condence limits for a parameter. The value of a (0 < a < 1) indicates a 100a% condence that b 2 [bl(a), bu(a)]. In this study, a is chosen as 0.05, then 95% condence limits for b based on 2000 bootstrap replications are given by bl 50th and bu 1950th largest estimates of b [62]. The condence 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 condence limits are included with each histogram. All histograms are roughly Gaussian in shape, suggesting that the condence interval evaluation based on bootstrap percentile is reasonable. All the simulation and perditions in this study are done by using Matlab. 4.2. Timing of the secondary stimulus

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explore the broader concept of pro-inammatory mediator activation. Therefore, the use of the IL-1b data was used only for quantication purposes. This is also valid for A and E. For each bootstrap sample a vector of model parameters is estimated using a least squares method. The mean of the multiple bootstrap estimates (2000 runs in our case) is reported as the most likely paramPn bi ^ eter value [62], b i1 , where i denotes bootstrap iteration. n Parameters associated with the prior model, which are considered xed, are presented in Table 1, while the estimated parameter values associate with the dual-regulation model are depicted in Table 2. The performance of the model in reproducing the self-limited responses is shown in Fig. 2. The estimated condence intervals for each parameter are de^ ^ noted by bl a; bu a, where subscript l and u respectively denote the lower and the upper limits of the vector of model parameters b and percentiles are estimated using the a central condence inter-

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 secondary LPS stimulus. We assume that two equal, non-lethal doses are administered the rst at time t = 0 and the second x times units later, 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 P2 to Pc, where P2 is the peak value of the pro-inammatory response P following the second injection where Pc is the corresponding peak in the pro-inammatory response following a single injection of LPS, namely:

437

Pratio

P2 ; Pc

P2 fP 2;max ; LPS0 1; LPSx 1g;

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Pc fPc;max ; LPS0 0; LPSx 1g

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

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^ 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 condence limits bl (0.05), bu (0.05) are represented by dotted lines respectively.

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The denitions of P2 and Pc are graphically depicted in Fig. 4 (top panel). The in silico experiments depicted in Fig. 4 (bottom panel) indicate that signicant potentiation of the inammatory response is observed when the interval between the two successive injections is within a critical time window. It should also be noted that observed potentiation is not a simple additive effect by two successive injection of LPS since levels of P2 are much larger than the maximum value of the peak of P following a single injection of LPS with dose equal to 2. The robustness of the inammatory response is diminished as the interval x between the two successive injection increases until a maximum tolerance, quantied as a relative suppression of the pro-inammatory response, occurs when the interval between the injections is reached. From that point on, the extent of tolerance diminishes and eventually the effects of preconditioning slowly dissipate and the memory effects completely disappear (bottom panel of Fig. 4). The predicted trends related to the effect of timing of the secondary stimulation are qualitatively consistent with the result in vant Veers studies [54], in which the release of TNF-a per monocytes in whole blood was drastically diminished in the period 3 8 h after LPS injection. TNF-a measurement [54] is considered as a prototypical inammatory response, which corresponds to P in our model. Furthermore, it has been documented that the endotoxin tolerance is preserved over long periods of time as in several studies of the induction of endotoxin tolerance in animals, normal responsiveness resumes after 8 days of tolerance [2]. 4.3. Lethal potentiation When the time between successive administrations is short, our model predicts potentiation of the response consistent with experimental evidence [4]. Part of the internal system dynamics, 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 preconditioning will further activate the NF-jB which has already been stimulated and cause it to be persistently active which leads to the signicantly increased and lasting pro-inammatory and anti-inammatory 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. 4.4. Endotoxin tolerance As the interval between the successive administrations is increased rather than a persistent production of proinammatory cytokines, a much less vigorous inammatory response is observed. 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. In Fig. 6, a deduced activation of IRAK, NF-jB could be observed which will nally lead to the suppressed P, the pro-inammatory response. Our results are in agreement with experiment observations that some components in the signaling pathways are downregulated such as IRAK [16], NF-jB [63]. And studies of ET induced in vivo [8] have shown a decrease in the production of several cytokines by macrophages including IL-1b, TNF-a, and IL-6 which are biomarkers of proinammatory response. It is worth noting that the concentration of IRAK-M induced by preconditioning is relatively 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 inammatory process. The stimulation of IRAK-M transcripts expression was reected at the protein level, and signicant quantities of this

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Fig. 4. The effect of the timing of the secondary pro-inammatory stimulus on the tolerance. The doses for two injections are equal and nonlethal to the subjects 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 P2 to Pc, where P2 is the peak value of the P of the second response. Pc, the control, is the P, proinammatory response under only one stimulus with the initial condition LPS(0) = 0, LPS(x) = 1. P2 and Pc are intuitively depicted in top panel as well as the result in bottom panel. 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519

kinase were detected 24 h after incubation with LPS [51]. The build-up of the IRAK-M at 24 h, induced by preconditioning, results in a reduction of the inammatory response through strong inhibition of IRAK activation. However, recent experiments reveal that not all kinases in the TLR4 signaling pathway will be suppressed when the endotoxin tolerance occurs. When pretreated cells were again stimulated with LPS 24 after rst stimulus, the levels of IRAK-M mRNA and proteins were twofold greater than the maximal levels produced by the rst induction [51]. This observation indicates that endotoxin tolerance is no longer to be considered as a global downregulation of the immune response as before [16]. An increased production of IRAK-M is predicted by our model in Fig. 6. The augmentation of IRAK-M is due to the signicantly decreased inhibition of mRNAIRAKM by P. Therefore, the most important feature of current model is that it offers the opportunity to explore the leukocyte reprogramming which referrers to the alterations in signaling pathways and chromatin remodeling with the induction

of LPS tolerance [64]. This hypothesis implies that the endotoxin tolerance is not simultaneous suppression of all the immune response 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 nally leads to reduced production of proinammatory cytokines. The variety of responses to LPS after preconditioning 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

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Fig. 5. Lethal potentiation: successive administration of small doses of endotoxin can lead to an unresolved inammatory 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.

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to a lower nonlethal dose of LPS could modulate its intracellular dynamics by reversing the lethal outcome of the main much higher one which is responsible for an overwhelming inammatory response in Fig. 7. The physiological signicance of LPS tolerance 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

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Fig. 6. Tolerance: pre-exposure the system with a smaller inammatory insult results in a reduction in the cell capacity to produce pro-inammatory cytokines 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.

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Fig. 7. Protective tolerance: pre-existing infection might cause a profound hypo-responsiveness in systems response to a lethal LPS challenge. Solid line: LPS (t = 0 h) = 1 and LPS (t = 24 h) = 4. Dashed line: LPS (t = 0 h) = 0 and LPS (t = 24 h) = 4. 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563

changed the state in which the system lies when the lethal dose is encountered. Specically, the active IRAK-M rises enough to inhibit the activation of IRAK so that when the previously lethal endotoxin stimulus is given, the system will be driven back to the healthy state, rather than that of the unhealthy state. We conclude that by preconditioning the system with a low dose of LPS, one can reduce the response obtained with a larger dose of LPS. Interestingly, the induction of LPS tolerance during clinical conditions may in the short term be benecial by preventing excessive inammation, but in the longer term be deleterious by hampering an adequate defense response to opportunistic infections [53]. This may be demonstrated by the recent clinical observation that the Q2 severe immunosppression demonstrated by signicant endotoxin tolerance in sepsis patients have high correlation with mortality [55]. The signicant decrease of IRAK and elevation IRAK-M mRNA expression in mononuclear cells are the notable characters of these severe sepsis patients. Thus, endotoxin tolerance is just like a double-edged sword. The transient induction of IRAK-M in healthy

animals after preconditioning will protect the host from overactivation of another wave of proinammation following the secondary exposure to LPS. However, the long lasting high level IRAK-M expression in sepsis patient is an indicator of poor outcome and mortality [55]. 4.6. The effect of IRAK-M on Tolerance 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 claried. Thus, we are interested in exploring the effect of IRAK-M by knocking out this gene to check if it is required for tolerance. Our model allows us to test the effect of the knockout of 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 nd that the disruption of the gene encoding the negative regulator IRAK-M for the signaling pathway results

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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.

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in the lack of the endotoxin tolerance as seen in left plot in Fig. 8. This result is consistent with Kobayashis other experiment in the same publication [21]. It is reported IRAK-M/ macrophages showed a loss of endotoxin tolerance demonstrated by the cytokine levels produced upon LPS restimulation. Moreover, compared with the scenario in Fig. 7, under the same condition, it is further expected that the preconditioning of smaller dose lost protective effect for a subsequent lethal dose injection when IRAK-M gene is knocked out as seen in the right plot in Fig. 8. The model predicted that the inhibition of stimulation of IRAK and the subsequent gene expression is lost due to disappearance of IRAK-M. Therefore, we can conclude that IRAK-M may be a key component of this important control system since the development of tolerance upon repeated stimulation with LPS is dampened without IRAK-M. 4.7. Increased cytokine production through IRAK-M gene knock-out or administration of a PI3K inhibitor Both IRAK-M and PI3K are negative regulators of TLR4 signaling pathway, so, it is expected that an enhanced proinammatory response will be observed in the absence of these regulators. As illustrated in the upper plots in Fig. 9, we perform both an IRAK-M knock-out experiment and an administration of a PI3K inhibitor experiment respectively. The model is manipulated so that there is no de novo transcriptional synthesis of IRAK-M which is responsible for negative regulation of IRAK. After the disruption of IRAK-M gene, no expression of mRNAIRAKM and protein IRAK-M is observed. The knockout causes an increased expression for P, pro-inammation which is in agreement with the Kobayashis report that IRAKM/ macrophages revealed increased production of TNFa, IL-6 and IL-12 when compared to wild-type macrophages at 6 hr after stimulation [21]. Similarly, in the lower plots in Fig. 9, administration of Wortmannin, the PI3K inhibitor, shows enhanced TLR4 signaling and enhanced production of TNF-a [31]. The neutralization of active PI3K is mimicked by increasing the degradation rate of

PI3K by ve times. The increased production of the prototypical inammatory response P in our current model is reected 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 period. The PI3K will be activated immediately in response to stimulus and lose its activity within 9 h which only inhibits the activation of NF-jB 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. 4.8. The crosstalk between PI3K and IRAKM

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Its interesting to nd a relationship between two negative regulators, the induction of IRAK-M is induced by the other kinase PI3K. Thus, we expect there will be protein level change of IRAKM by pretreatment of the cells with specic inhibitor of PI3K. We examine the inuence of administrating Wortmannin against PI3K to the response by increasing the degradation rate of PI3K, kout,PI3K by 10 times which mimics the neutralization of active PI3K by its inhibitor. We discussed the role PI3K played by using pre-administration of its inhibitor Wortmannin. The roles it plays in both single LPS stimulation as well as preconditioning are shown in Fig. 10. As seen in Fig. 10, 024 h shows the pre administration of Wortmannin leads to the abolished expression of IRAK-M as well as enhanced production of pro-inammatory cytokines following single LPS stimulus. The result is in agreement with recent observation that LPS-derived immune cells pretreated with the PI3K inhibitor expressed signicantly abolished expression of IRAK-M [7]. The 2448 h responses show the effect of second LPS stimuli, we could see that the signicantly decreased production of IRAK-M following the rst stimulus fails to inhibit the activation

Fig. 9. The increased proinammatory 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.

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Fig. 10. Implication of administration of PI3K inhibitor by decreasing concentration of active PI3K through increasing the degradation rate constant kout;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. 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670

of IRAK and NFkB following the second LPS insult. This nally leads to the strongly enhanced production of pro-inammatory 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 signal propagation, whereas IRAK-M represents the memory component 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 between 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 IRAKM is a direct regulator for induction of ET, while PI3K/Akt play the role behind the curtain. Thus, ablation of Akt which blocks the activation of IRAK-M by PI3K/Akt pathway inhibits the induction of endotoxin tolerance [66].

Injury Glue Grant program which is supported by the National Institute of General Medical Sciences.

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Acknowledgements Q.Y. and I.P.A. acknowledge support from NIH GM082974. S.E.C. and S.F.L. are supported, in part, from NIGMS Grant GM34695. The data used are part of the Inammation and the Host Response to

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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