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

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 long non-coding RNA H19 suppresses carcinogenesis and chemoresistance in hepatocellular carcinoma.

The long non-coding RNA (lncRNA) H19 represents a maternally expressed and epigenetically regulated imprinted gene product and is discussed to have either tumor-promoting or tumor-suppressive actions. Recently, H19 was shown to be regulated under inflammatory conditions. Therefore, aim of this study was to determine the function of H19 in hepatocellular carcinoma (HCC), an inflammation-associated type of tumor. In four different human HCC patient cohorts H19 was distinctly downregulated in tumor tissue compared to normal or non-tumorous adjacent tissue. We therefore determined the action of H19 in three different human hepatoma cell lines (HepG2, Plc/Prf5, and Huh7). Clonogenicity and proliferation assays showed that H19 overexpression could suppress tumor cell survival and proliferation after treatment with either sorafenib or doxorubicin, suggesting chemosensitizing actions of H19. Since HCC displays a highly chemoresistant tumor entity, cell lines resistant to doxorubicin or sorafenib were established. In all six chemoresistant cell lines H19 expression was significantly downregulated. The promoter methylation of the H19 gene was significantly different in chemoresistant cell lines compared to their sensitive counterparts. Chemoresistant cells were sensitized after H19 overexpression by either increasing the cytotoxic action of doxorubicin or decreasing cell proliferation upon sorafenib treatment. An H19 knockout mouse model (H19Δ3) showed increased tumor development and tumor cell proliferation after treatment with the carcinogen diethylnitrosamine (DEN) independent of the reciprocally imprinted insulin-like growth factor 2 (IGF2). In conclusion, H19 suppresses hepatocarcinogenesis, hepatoma cell growth, and HCC chemoresistance. Thus, mimicking H19 action might be a potential target to overcome chemoresistance in future HCC therapy.

Induction of Glucocorticoid-induced Leucine Zipper (GILZ) Contributes to Anti-inflammatory Effects of the Natural Product Curcumin in Macrophages.

GILZ (glucocorticoid-induced leucine zipper) is inducible by glucocorticoids and plays a key role in their mode of action. GILZ attenuates inflammation mainly by inhibition of NF-κB and mitogen-activated protein kinase activation but does not seem to be involved in the severe side effects observed after glucocorticoid treatment. Therefore, GILZ might be a promising target for new therapeutic approaches. The present work focuses on the natural product curcumin, which has previously been reported to inhibit NF-κB. GILZ was inducible by curcumin in macrophage cell lines, primary human monocyte-derived macrophages, and murine bone marrow-derived macrophages. The up-regulation of GILZ was neither associated with glucocorticoid receptor activation nor with transcriptional induction or mRNA or protein stabilization but was a result of enhanced translation. Because the GILZ 3'-UTR contains AU-rich elements (AREs), we analyzed the role of the mRNA-binding protein HuR, which has been shown to promote the translation of ARE-containing mRNAs. Our results suggest that curcumin treatment induces HuR expression. An RNA immunoprecipitation assay confirmed that HuR can bind GILZ mRNA. In accordance, HuR overexpression led to increased GILZ protein levels but had no effect on GILZ mRNA expression. Our data employing siRNA in LPS-activated RAW264.7 macrophages show that curcumin facilitates its anti-inflammatory action by induction of GILZ in macrophages. Experiments with LPS-activated bone marrow-derived macrophages from wild-type and GILZ knock-out mice demonstrated that curcumin inhibits the activity of inflammatory regulators, such as NF-κB or ERK, and subsequent TNF-α production via GILZ. In summary, our data indicate that HuR-dependent GILZ induction contributes to the anti-inflammatory properties of curcumin.

Downregulation of the glucocorticoid-induced leucine zipper (GILZ) promotes vascular inflammation.

OBJECTIVE: Glucocorticoid-induced leucine zipper (GILZ) represents an anti-inflammatory mediator, whose downregulation has been described in various inflammatory processes. Aim of our study was to decipher the regulation of GILZ in vascular inflammation. APPROACH AND RESULTS: Degenerated aortocoronary saphenous vein bypass grafts (n = 15), which exhibited inflammatory cell activation as determined by enhanced monocyte chemoattractrant protein 1 (MCP-1, CCL2) and Toll-like receptor 2 (TLR2) expression, showed significantly diminished GILZ protein and mRNA levels compared to healthy veins (n = 23). GILZ was also downregulated in human umbilical vein endothelial cells (HUVEC) and macrophages upon treatment with the inflammatory cytokine TNF-α in a tristetraprolin (ZFP36, TTP)- and p38 MAPK-dependent manner. To assess the functional implications of decreased GILZ expression, we determined NF-κB activation after GILZ knockdown by siRNA and found that NF-κB activity and inflammatory gene expression were significantly enhanced. Importantly, ZFP36 is induced in TNF-α-activated HUVEC as well as in degenerated vein bypasses. When atheroprotective laminar shear stress was employed, GILZ levels in HUVEC increased on mRNA and protein level. Laminar flow also counteracted TNF-α-induced ZFP36 expression and GILZ downregulation. MAP kinase phosphatase 1 (MKP-1, DUSP1), a negative regulator of ZFP36 expression, was distinctly upregulated under laminar shear stress conditions and downregulated in degenerated vein bypasses. CONCLUSION: Our data show a diminished expression of the anti-inflammatory mediator GILZ in the inflamed vasculature and indicate that GILZ downregulation requires the mRNA binding protein ZFP36. We suggest that reduced GILZ levels play a role in cardiovascular disease.

Activation of Rac1 GTPase by nanoparticulate structures in human macrophages.

UNLABELLED: Inflammatory activation of alveolar macrophages by ambient particles can be facilitated via Toll-like receptors (TLR). The action of TLR agonists and antagonists has been reported to depend on the formation of nanoparticulate structures. Aim of the present study was to identify the signaling pathways induced by nanoparticulate structures in human macrophages, which might be critical for inflammatory cell activation. METHODS: Studies were performed in primary human alveolar macrophages or in differentiated THP-1 macrophages. Silica nanoparticles were prepared by Stöber synthesis and characterized by dynamic light scattering and scanning electron microscopy. Mycobacterial DNA was isolated from Mycobacterium bovis BCG, and nanoparticle formation was assessed by atomic force microscopy and dynamic light scattering. Actin polymerization was measured by phalloidin-TRITC staining, and cell activation was determined by reverse transcription quantitative PCR analysis, L929 cytotoxicity assay (cytokine induction), and pull-down assays (Rho GTPases). RESULTS: In contrast to immune stimulatory sequence ISS 1018, BCG DNA spontaneously formed nanoparticulate structures and induced actin polymerization as did synthetic silica nanoparticles. Co-incubation with silica nanoparticles amplified the responsiveness of macrophages toward the TLR9 ligand ISS 1018. The activation of Rac1 was induced by silica nanoparticles as well as BCG DNA and is suggested as the critical signaling event inducing both cytoskeleton changes as well as inflammatory cell activation. CONCLUSION: Nanoparticles can induce signaling pathways, which amplify an inflammatory response in macrophages.