Phosphoinositide-Dependent Kinase 1 Provides Negative Feedback Inhibition to Toll-Like Receptor-Mediated NF-κB Activation in Macrophages

Abstract

Phosphoinositide-dependent kinase 1 (PDK-1) represents an important signaling component in the phosphatidylinositol 3-kinase (PI3K) pathway, which plays an essential role in controlling a coordinated innate immune response. Here, we show that mice with conditional disruption of PDK-1 specifically in myeloid lineage cells (PDK-1^Δmyel mice) show enhanced susceptibility to lipopolysaccharide (LPS)-induced septic shock accompanied by exaggerated liver failure. Furthermore, primary macrophages derived from PDK-1^Δmyel mice lack LPS- and Pam3CSK4-stimulated AKT activity but exhibit increased mRNA expression and release of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Moreover, LPS- and Pam3CSK4-stimulated primary macrophages exhibit enhanced phosphorylation and degradation of IκBα. While immediate upstream Toll-like receptor 4 (TLR-4)-induced signaling, including IL-1 receptor (IL-1R)-associated protein kinase (IRAK) phosphorylation, is unaltered in the absence of PDK-1, macrophages from PDK-1^Δmyel mice exhibit prolonged ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF-6) in response to LPS stimulation. These experiments reveal a novel PDK-1-dependent negative feedback inhibition of TLR-induced NF-κB activation in macrophages in vivo.

We thank Gisela Schmall for secretarial assistance, I. Förster for providing LysMCre mice, and Dario Alessi for providing conditional PDK-1 knockout mice. We also thank Brigitte Hampel for her excellent technical assistance and F. Thomas Wunderlich and Christoph Goettlinger for their expertise in FACS.

This work was supported by grants SFB 670 and DFG Br 1492-7/4 to J.C.B. and a graduate fellowship to B.C. from the International Graduate School of Genomics and Functional Genomics, Cologne, Germany.