(699e) A Dual Negative Regulation Model of TLR4 Signaling for LPS Preconditioning in Human Endotoxemia

Lipopolysaccharide (endotoxin; LPS), a major cell wall component of Gram-negative bacteria, induces an acute inflammatory response comparable to bacterial infection. Interesting phenomena termed as preconditioning have been attracting researchers' attention, in which repeated treatment with LPS can lead to either enhancement (potentiation) or desensitization (tolerance) of subsequent pro-inflammatory cytokine responses [1]. However, the mechanism underlies these phenomena are still unclear.

Recently a number of studies focusing on quantifying proteins or enzymes in TLR4 signaling pathways, considered as the main LPS signal transduction process, have suggested that the different results following preconditioning lie in the complex and tightly regulated molecular mechanisms within this pathway. Various negative regulatory mechanisms have evolved to control this signaling in order to maintain immunological balance including the negative regulator IRAKM [2] and additional signaling pathways PI3K/Akt [3], where 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 [4].

The work to be discussed in this presentation aims to develop a model based on the molecular mechanisms of the TLR4 signaling pathway to explore the complex dynamics of the LPS-induced inflammation and investigate different scenarios of preconditioning. Our previous model [5] describes the interaction between the ligand (LPS) and the transmembrane signaling receptor (TLR4) coupled with the activation of transcriptional factor (NFêB) which triggers the stimulation of expression of essential transcriptional motifs identified as ?P', the pro-inflammatory response, ?A', the anti-inflammatory response and ?E', and the energetic response and activation of endocrine immune system. We explore the concept of indirect response modeling [6] to describe dual negative regulation of this signaling pathway through modeling the following processes: activation of kinase PI3K/Akt via LPSR, negative feedback regulation of TLR4 signaling pathway by PI3K/Akt pathway, passing by signal from LPSR to IKK through the kinase IRAK, stimulation of production of negative regulator IRAKM and Negative feedback regulation of IRAK by IRAKM. Thus, the indirect activation of another signaling pathway PI3K/Akt by (LPSR) which suppresses the NFkB activity develops a short feedback loop. Similarly, the suppression of kinase IRAK by its specific inhibitor IRAKM whose transcription is stimulated by the activation of PI3K/Akt signaling pathway creates a long feedback loop. Such dual negative inhibition regulated inflammation could potentially emerge as critical enablers towards understanding the connectivity and relationship of critical components in the innate immune system. Out model is evaluated by predicting alternative preconditioning experimental observations. The capability of describing both potentiation and tolerance by our model illustrates how the outcomes of endotoxin administration experiments can emerge as a natural consequence of the tightly regulated signaling pathway in acute inflammatory response. Moreover, it is identified by our model that the relative time scales of the onset as key determinants of outcome in repeated administration experiments, potentiation will quickly switch to tolerance as the time interval between two stimuli increases. In addition, the in silico experiment of knockout of negative regulator IRAKM gene leading to lack of endotoxin tolerance demonstrates that it is a necessary and sufficient factor for generation of endotoxin. And the effects of the knockout of irak-M genes or administration of PI3K inhibitor respectively do yield predictions that have been verified experimentally highlighting the importance of a dual negative feedback regulation in the model with both the PI3K/Akt and IRAKM. Finally, the pretreatment with PI3K inhibitor reveals the ?crosstalk' between these two negative regulations.

In conclusion, the proposed dual negative regulation controlled indirect response model of inflammation describes the dynamics of inflammatory events connecting extracellular signal and transcriptional activation. The successful predicting various experimental preconditioning results by our model offers support for the biological relevance of the reduced model.

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5. P.T. Foteinou, S.E. Calvano, S.F. Lowry, and I.P. Androulakis, In silico simulation of corticosteroids effect on an NFkB- dependent physicochemical model of systemic inflammation, PLoS One, 4 (2009) e4706.

6. W.J. Jusko and H.C. Ko, Physiologic indirect response models characterize diverse types of pharmacodynamic effects, Clin Pharmacol Ther, 56 (1994) 406-19.