Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages

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.

The glucocorticoid‐induced leucine zipper mediates statin‐induced muscle damage

“…Cells were harvested either with RIPA buffer (50 mM Tris‐HCl, 1% Triton X‐100, 0.1% SDS, 0.5% sodium deoxycholate, 150 mM NaCl) supplemented with a protease/phosphatase inhibitor mix (P8340, P0044, Sigma‐Aldrich, St. Louis, MO, USA) or with SB lysis buffer (50 mM Tris‐HCl, 1% SDS, 10% glycerol, 5% β‐mercaptoethanol, 0.004% bromophenol blue, in water) supplemented with a protease inhibitor cocktail (cOmplete Mini, Roche, Basel, Switzerland). Western blots were performed as described previously, and signals were either detected by an HRP‐based method or with the LI‐COR Odyssey imaging system (LI‐COR Biosciences, Lincoln, NE, USA) . Incubation with primary antibody dilutions was performed at 4°C overnight, and with secondary antibody dilutions at room temperature for 1‐1.5 hours.…”

“…Western blots were performed as described previously, and signals were either detected by an HRPbased method 27 or with the LI-COR Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA) . 28,29 Incubation with primary antibody dilutions was performed at 4°C overnight, and with secondary antibody dilutions at room temperature for 1-1.5 hours. The antibodies and dilutions used are listed in Table 2.…”

Altered glucocorticoid metabolism represents a feature of macroph‐aging

“…GILZ also interferes with mitogen‐activated protein kinase (MAPK) signaling, for example, by binding to Ras/Raf, resulting in the inhibition of downstream MAP kinases, such as ERK (Hoppstädter et al., 2015; Ricci et al., 2019). In this manner, GILZ plays a vital role in macrophage activation (Hoppstädter et al., 2012, 2015; Hoppstädter, Diesel, et al., 2019; Hoppstädter & Kiemer, 2015). Under inflammatory conditions, repression of GILZ expression represents a regulatory mechanism that prolongs and/or increases pro‐inflammatory responses in human and murine macrophages (Hoppstädter et al., 2012, 2015).…”

“…Murine TNF (TNF‐α/β) levels in cell culture supernatants were quantified by bioassay as previously described (Hoppstädter, Diesel, et al., 2019; Hoppstädter et al., 2015).…”

“…Quantitative RT–PCR (qPCR) was performed as described previously (Dembek et al., 2017; Hoppstädter, Dembek, et al., 2019; Hoppstädter, Diesel, et al., 2019; Hoppstädter et al., 2015, 2016; Kessler et al., 2017). Total RNA from cells was isolated using the High Pure RNA Isolation Kit (#11828665001; Roche), following the manufacturer's instructions.…”