Induction of the glucocorticoid‐induced leucine zipper (GILZ) by glucocorticoids plays a role in their antiinflammatory action, whereas GILZ expression is reduced under inflammatory conditions. The ...mechanisms regulating GILZ expression during inflammation, however, have not yet been characterized. Here, we investigated GILZ expression in human alveolar macrophages (AMs) following Toll‐like receptor (TLR) activation. Macrophages were shown to predominantly express GILZ transcript variant 2. Lipopolysaccharide‐treated AMs, THP‐1 cells, and lungs of lipopolysaccharide‐exposed mice displayed decreased GILZ protein and mRNA levels. The effect was strictly dependent on the adapter molecule MyD88, as shown by using specific ligands or a knockdown strategy. Investigations on the functional significance of GILZ downregulation performed by GILZ knockdown revealed a proinflammatory response, as indicated by increased cytokine expression and NF‐κB activity. We found that TLR activation reduced GILZ mRNA stability, which was mediated via the GILZ 3′‐untranslated region. Finally, involvement of the mRNA‐binding protein tristetraprolin (TTP) is suggested, since TTP overexpression or knockdown modulated GILZ expression and TTP was induced in a MyD88‐dependent fashion. Taken together, our data show a MyD88‐ and TTP‐dependent GILZ downreg‐ulation in human macrophages upon TLR activation. Suppression of GILZ is mediated by mRNA destabilization, which might represent a regulatory mechanism in macrophage activation.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Statins represent the most prescribed class of drugs for the treatment of hypercholesterolemia. Effects that go beyond lipid-lowering actions have been suggested to contribute to their beneficial ...pharmacological properties. Whether and how statins act on macrophages has been a matter of debate. In the present study, we aimed at characterizing the impact of statins on macrophage polarization and comparing these to the effects of bempedoic acid, a recently registered drug for the treatment of hypercholesterolemia, which has been suggested to have a similar beneficial profile but fewer side effects. Treatment of primary murine macrophages with two different statins, i.e., simvastatin and cerivastatin, impaired phagocytotic activity and, concurrently, enhanced pro-inflammatory responses upon short-term lipopolysaccharide challenge, as characterized by an induction of tumor necrosis factor (TNF), interleukin (IL) 1β, and IL6. In contrast, no differences were observed under long-term inflammatory (M1) or anti-inflammatory (M2) conditions, and neither inducible NO synthase (iNOS) expression nor nitric oxide production was altered. Statin treatment led to extracellular-signal regulated kinase (ERK) activation, and the pro-inflammatory statin effects were abolished by ERK inhibition. Bempedoic acid only had a negligible impact on macrophage responses when compared with statins. Taken together, our data point toward an immunomodulatory effect of statins on macrophage polarization, which is absent upon bempedoic acid treatment.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The era of personalized medicine for cancer treatment has just begun opening up novel molecular targets improving cancer therapy. Members of the insulin-like growth factor 2 (IGF2) axis have been ...described to be altered and to have prognostic relevance in several different tumor identities. IGF2 belongs to the most complexly regulated growth factors known. As an imprinted gene it is controlled by epigenetic alterations. IGF2 mRNA binding proteins (IMPs/IGF2BPs) further regulate its translation. IGF2 activity is contained through IGF binding proteins (IGFBPs) and differential expression of the target receptors. The necessity of such a complex regulation implies pathophysiological effects of a deregulated expression of IGF2. This review attempts to summarize the different levels of IGF2 regulation, especially in the context of cancer. Members of the IGF2 axis are enlightened from the perspective of novel molecular targets for cancer therapy. Preclinical as well as experimental therapeutic interventions targeting IGF2 in cancer will be surveyed.
Glucocorticoids (GCs) are widely prescribed therapeutics for the treatment of inflammatory diseases, and endogenous GCs play a key role in immune regulation. Toll-like receptors (TLRs) enable innate ...immune cells, such as macrophages, to recognize a wide variety of microbial ligands, thereby promoting inflammation. The interaction of GCs with macrophages in the immunosuppressive resolution phase upon prolonged TLR activation is widely unknown. Treatment of human alveolar macrophages (AMs) with the synthetic GC dexamethasone (Dex) did not alter the expression of TLRs -1, -4, and -6. In contrast, TLR2 was upregulated in a GC receptor-dependent manner, as shown by Western blot and qPCR. Furthermore, long-term lipopolysaccharide (LPS) exposure mimicking immunosuppression in the resolution phase of inflammation synergistically increased Dex-mediated TLR2 upregulation. Analyses of publicly available datasets suggested that TLR2 is induced during the resolution phase of inflammatory diseases, i.e., under conditions associated with high endogenous GC production. TLR2 induction did not enhance TLR2 signaling, as indicated by reduced cytokine production after treatment with TLR2 ligands in Dex- and/or LPS-primed AMs. Thus, we hypothesized that the upregulated membrane-bound TLR2 might serve as a precursor for soluble TLR2 (sTLR2), known to antagonize TLR2-dependent cell actions. Supernatants of LPS/Dex-primed macrophages contained sTLR2, as demonstrated by Western blot analysis. Activation of metalloproteinases resulted in enhanced sTLR2 shedding. Additionally, we detected full-length TLR2 and assumed that this might be due to the production of TLR2-containing extracellular vesicles (EVs). EVs from macrophage supernatants were isolated by sequential centrifugation. Both untreated and LPS/Dex-treated cells produced vesicles of various sizes and shapes, as shown by cryo-transmission electron microscopy. These vesicles were identified as the source of full-length TLR2 in macrophage supernatants by Western blot and mass spectrometry. Flow cytometric analysis indicated that TLR2-containing EVs were able to bind the TLR2 ligand Pam
CSK
. In addition, the presence of EVs reduced inflammatory responses in Pam
CSK
-treated endothelial cells and HEK Dual reporter cells, demonstrating that TLR2-EVs can act as decoy receptors. In summary, our data show that sTLR2 and full-length TLR2 are released by macrophages under anti-inflammatory conditions, which may contribute to GC-induced immunosuppression.
Intercellular communication is a fundamental process in the development and functioning of multicellular organisms. Recently, an essentially new type of intercellular communication, based on thin ...membrane channels between cells, has been reported. These structures, termed intercellular or tunnelling nanotubes (TNTs), permit the direct exchange of various components or signals (e.g., ions, proteins, or organelles) between non-adjacent cells at distances over 100 μm. Our studies revealed the presence of tunnelling nanotubes in microvascular endothelial cells (HMEC-1). The TNTs were studied with live cell imaging, environmental scanning electron microscopy (ESEM), and coherent anti-Stokes Raman scattering spectroscopy (CARS). Tunneling nanotubes showed marked persistence: the TNTs could connect cells over long distances (up to 150 μm) for several hours. Several cellular organelles were present in TNTs, such as lysosomes and mitochondria. Moreover, we could identify lipid droplets as a novel type of cargo in the TNTs. Under angiogenic conditions (VEGF treatment) the number of lipid droplets increased significantly. Arachidonic acid application not only increased the number of lipid droplets but also tripled the extent of TNT formation. Taken together, our results provide the first demonstration of lipid droplets as a cargo of TNTs and thereby open a new field in intercellular communication research.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for ...nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth. We employed different models of M1 and M2 polarization: granulocyte-macrophage colony-stimulating factor/lipopolysaccharide (LPS)/interferon (IFN)-γ was used to generate primary human M1 cells and macrophage colony-stimulating factor (M-CSF)/interleukin (IL)-10 to differentiate M2 monocyte-derived macrophages (MDM). PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-γ and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø26 and 41 nm) and microparticles (Ø1.75 μm) was quantified. At the concentration used (50 μg/ml), silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human MDM compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue. In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but might also open up therapeutic perspectives allowing to specifically target M2 polarized macrophages.
Zebrafish (ZF;
) larvae have become a popular in vivo model in drug metabolism studies. Here, we investigated the metabolism of methyl 2-1-(4-fluorobutyl)-1
...-indazole-3-carboxamido-3,3-dimethylbutanoate (4F-MDMB-BINACA) in ZF larvae after direct administration of the cannabinoid via microinjection, and we visualized the spatial distributions of the parent compound and its metabolites by mass spectrometry imaging (MSI). Furthermore, using genetically modified ZF larvae, the role of cannabinoid receptor type 1 (CB1) and type 2 (CB2) on drug metabolism was studied. Receptor-deficient ZF mutant larvae were created using morpholino oligonucleotides (MOs), and CB2-deficiency had a critical impact on liver development of ZF larva, leading to a significant reduction of liver size. A similar phenotype was observed when treating wild-type ZF larvae with 4F-MDMB-BINACA. Thus, we reasoned that the cannabinoid-induced impaired liver development might also influence its metabolic function. Studying the metabolism of two synthetic cannabinoids, 4F-MDMB-BINACA and methyl 2-(1-(5-fluoropentyl)-1
-pyrrolo2,3-bpyridine-3-carboxamido)-3,3-dimethylbutanoate (7'
-5F-ADB), revealed important insights into the in vivo metabolism of these compounds and the role of cannabinoid receptor binding.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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