Quantitative characterization and analysis of the dynamic NF-κB response in microglia

Patrick W Sheppard,Xiaoyun Sun,Mustafa Khammash,John F Emery,Rona G Giffard

doi:10.1186/1471-2105-12-276

Patrick W Sheppard, Xiaoyun Sun + Show 3 more

Open Access

https://doi.org/10.1186/1471-2105-12-276

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Abstract

BackgroundActivation of the NF-κB transcription factor and its associated gene expression in microglia is a key component in the response to brain injury. Its activation is dynamic and is part of a network of biochemical species with multiple feedback regulatory mechanisms. Mathematical modeling, which has been instrumental for understanding the NF-κB response in other cell types, offers a valuable tool to investigate the regulation of NF-κB activation in microglia at a systems level.ResultsWe quantify the dynamic response of NF-κB activation and activation of the upstream kinase IKK using ELISA measurements of a microglial cell line following treatment with the pro-inflammatory cytokine TNFα. A new mathematical model is developed based on these data sets using a modular procedure that exploits the feedback structure of the network. We show that the new model requires previously unmodeled dynamics involved in the stimulus-induced degradation of the inhibitor IκBα in order to properly describe microglial NF-κB activation in a statistically consistent manner. This suggests a more prominent role for the ubiquitin-proteasome system in regulating the activation of NF-κB to inflammatory stimuli. We also find that the introduction of nonlinearities in the kinetics of IKK activation and inactivation is essential for proper characterization of transient IKK activity and corresponds to known biological mechanisms. Numerical analyses of the model highlight key regulators of the microglial NF-κB response, as well as those governing IKK activation. Results illustrate the dynamic regulatory mechanisms and the robust yet fragile nature of the negative feedback regulated network.ConclusionsWe have developed a new mathematical model that incorporates previously unmodeled dynamics to characterize the dynamic response of the NF-κB signaling network in microglia. This model is the first of its kind for microglia and provides a tool for the quantitative, systems level study the dynamic cellular response to inflammatory stimuli.

Highlights

Activation of the nuclear factor-B (NF-B) transcription factor and its associated gene expression in microglia is a key component in the response to brain injury
TNFa stimulates dynamic NF-B and IKK activation in BV2 microglia To characterize the dynamics of canonical NF-B activation in microglia, cells from the microglial cell line BV2 were cultured and treated with 10 ng/ml TNFa
At these parameters the test yielded a P-value of 0.038, implying that the null hypothesis could not be rejected with a high significance level. This result was corroborated by obtaining a large number of parameter estimates and finding that nearly 50% of the estimates with this model structure had P > 0.01 (Figure 3E). These results provide strong evidence that the addition of dynamics roughly corresponding to the steps involving phosphorylated IBa recognition and binding by the E3 ligase, polyubiquitination, and proteasomal degradation is sufficient to account for the slightly delayed NF-B activation observed in microglia

Summary

Introduction

Activation of the NF-B transcription factor and its associated gene expression in microglia is a key component in the response to brain injury. In the nervous system NF-B is known to play a key role in the immune and injury responses and in governing normal brain function [1]. The resident immune cells in the brain, are activated following ischemia and play a controversial role in this decision. Members of the NF-B family of transcription factors are found in their inactive state as dimers bound to their IkB inhibitor proteins. Upon stimulation by a diverse set of stimuli, NF-B is freed from its inhibitor to coordinate gene expression in a highly specific and tightly regulated manner. The IBa inhibitor and p65(RelA):p50 NF-B heterodimer are the most extensively studied members of their respective families, and their response to extracellular stimuli illustrates the canonical pathway of NF-B activation (Figure 1)

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