Abstract
Activation of toll-like receptors (TLRs) plays a pivotal role in the host defense against bacteria and results in the activation of NF-κB-mediated transcription of proinflammatory mediators. Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory mediator, which inhibits NF-κB activity in macrophages. Thus, we aimed to investigate the regulation and role of GILZ expression in primary human and murine macrophages upon TLR activation. Treatment with TLR agonists, e.g., Pam3CSK4 (TLR1/2) or LPS (TLR4) rapidly decreased GILZ mRNA and protein levels. In consequence, GILZ downregulation led to enhanced induction of pro-inflammatory mediators, increased phagocytic activity, and a higher capacity to kill intracellular bacteria (Salmonella enterica serovar typhimurium), as shown in GILZ knockout macrophages. Treatment with the TLR3 ligand polyinosinic: polycytidylic acid [Poly(I:C)] did not affect GILZ mRNA levels, although GILZ protein expression was decreased. This effect was paralleled by sensitization toward TLR1/2- and TLR4-agonists. A bioinformatics approach implicated more than 250 miRNAs as potential GILZ regulators. Microarray analysis revealed that the expression of several potentially GILZ-targeting miRNAs was increased after Poly(I:C) treatment in primary human macrophages. We tested the ability of 11 of these miRNAs to target GILZ by luciferase reporter gene assays. Within this small set, four miRNAs (hsa-miR-34b*,−222,−320d,−484) were confirmed as GILZ regulators, suggesting that GILZ downregulation upon TLR3 activation is a consequence of the synergistic actions of multiple miRNAs. In summary, our data show that GILZ downregulation promotes macrophage activation. GILZ downregulation occurs both via MyD88-dependent and -independent mechanisms and can involve decreased mRNA or protein stability and an attenuated translation.
Highlights
Macrophages are strategically distributed all over the body and represent the first line of defense to invading pathogens
As reported previously [21], Glucocorticoid-induced leucine zipper (GILZ) mRNA levels were highly reduced upon TLR1/2 and TLR4, but not TLR3 stimulation (Figure 1B)
GILZ protein levels decreased both after myeloid differentiation primary response gene 88 (MyD88)-dependent stimulation by Pam3CSK4 and TRIFdependent activation by Poly(I:C) (Figures 1C–E), implicating a dual regulation of GILZ
Summary
Macrophages are strategically distributed all over the body and represent the first line of defense to invading pathogens. In response to bacterial or viral infections, these phagocytic cells engulf and destroy the infectious agent. Macrophages recognize a variety of microbial products, such as bacterial cell wall components and nucleic acids, via pathogen recognition receptors (PRRs). These receptors include Toll-like receptors (TLRs), as well as cytosolic NOD-like receptors, RIG-I-like receptors, and DNA sensors [1, 2]. Once a pathogen is recognized and phagocytosed, macrophages generate a wide range of biologically active molecules, e.g., pro-inflammatory cytokines, chemokines, and growth factors. Cytokines released by macrophages, such as interferons (IFNs), tumor necrosis factor (TNF)-α, and interleukin (IL)-1α/β, stimulate the activity of both the innate and adaptive immune response. Pathogen uptake usually results in antimicrobial responses such as reactive oxygen species (ROS) and nitric oxide (NO) production, enabling a rapid response to infection [3, 4]
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