Influence of the glucocorticoid-induced leucine zipper (GILZ) on macrophage-associated inflammation and statin-mediated effects

The glucocorticoid-induced leucine zipper (GILZ) is an immunomodulatory protein playing a pivotal role in macrophages' immune response. The aim of the present studies was the investigation and role of GILZ in different settings: The first part of this study focused on the function and regulation of GILZ in aging and macrophage activation. The term “inflammaging” describes the acquired state of low- term chronic inflammation contributing to an age-related imbalanced immune system. The elucidation of impaired glucocorticoid metabolism and reduced levels of glucocorticoids (GC) in the myeloid compartment of aged mice resulting in a dysregulated immune network was part of this study. Second, this study focused on statins' effects on GILZ. Statins represent the most prescribed class of drugs for the treatment of hypercholesterolemia as the underlying condition in cardiovascular diseases (CVDs). Besides lipid-lowering actions, statins exert muscle-related side effects and are postulated to mediate anti-inflammatory actions by mechanisms not fully elucidated. Statins can induce GILZ in skeletal muscle and macrophages. The study revealed mechanistic aspects of Gilz-upregulation and the involvement of the mevalonate pathway in macrophages in vitro and ex vivo as well as the mediating role of GILZ in statin-induced muscle damage in vitro and in vivo.Der Glucocorticoid-induzierte Leucin Zipper (GILZ) ist ein Protein mit immunmodulierenden Eigenschaften, welches eine wichtige Rolle in der Immunantwort von Makrophagen spielt. Ziel der vorliegenden Studien war die Untersuchung von GILZ in unterschiedlichen Ansätzen: Die Rolle und Funktion von GILZ während des Alterungsprozesses und bei Makrophagenaktivierung war Bestandteil des ersten Studienteils. Mit zunehmendem Lebensalter verändert sich das Immunsystem hin zu einem entzündungsähnlichen Zustand. Die Untersuchung im myeloiden Kompartiment von gealterten Mäusen zeigte, dass reduzierte Glucocorticoid (GC) Spiegel und ein veränderter Glucocorticoidmetabolismus Teil dieses fehlregulierten Immunnetzwerkes sind. Ein weiteres Ziel dieser Studie war die Untersuchung des Einflusses von Statinen auf GILZ. Bei Statinen handelt es sich um die meistverordnete Gruppe von Arzneimitteln, die bei Hypercholesterinämie eingesetzt werden. Neben ihrer lipidsenkenden Wirkung werden Statinen auch muskelschädigende und anti-entzündliche Eigenschaften, deren Mechanismen nicht vollständig geklärt sind, zugeschrieben. Zunächst wurde gezeigt, dass Statine Gilz in Muskelzellen und Makrophagen induzieren kann. Es konnten mechanistische Aspekte der Gilz Induktion unter Beteiligung des Cholesterolsyntheseweges in Makrophagen in vitro und ex vivo, sowie die Beteiligung von GILZ an muskelschädigenden Eigenschaften in vivo und in vitro, gezeigt werden

Statins and Bempedoic Acid: Different Actions of Cholesterol Inhibitors on Macrophage Activation

Statins represent the most prescribed class of drugs for the treatment of hypercholes terolemia. 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 mat ter 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., simvas tatin and cerivastatin, impaired phagocytotic activity and, concurrently, enhanced pro-inflammatory responses upon short-term lipopolysaccharide challenge, as characterized by an induction of tu mor 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

Altered glucocorticoid metabolism represents a feature of macroph-aging

The aging process is characterized by a chronic, low-grade inflammatory state, termed "inflammaging." It has been suggested that macrophage activation plays a key role in the induction and maintenance of this state. In the present study, we aimed to elucidate the mechanisms responsible for aging-associated changes in the myeloid compartment of mice. The aging phenotype, characterized by elevated cytokine production, was associated with a dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis and diminished serum corticosteroid levels. In particular, the concentration of corticosterone, the major active glucocorticoid in rodents, was decreased. This could be explained by an impaired expression and activity of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), an enzyme that determines the extent of cellular glucocorticoid responses by reducing the corticosteroids cortisone/11-dehydrocorticosterone to their active forms cortisol/corticosterone, in aged macrophages and peripheral leukocytes. These changes were accompanied by a downregulation of the glucocorticoid receptor target gene glucocorticoid-induced leucine zipper (GILZ) in vitro and in vivo. Since GILZ plays a central role in macrophage activation, we hypothesized that the loss of GILZ contributed to the process of macroph-aging. The phenotype of macrophages from aged mice was indeed mimicked in young GILZ knockout mice. In summary, the current study provides insight into the role of glucocorticoid metabolism and GILZ regulation during aging

Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide

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 Pam3CSK4. In addition, the presence of EVs reduced inflammatory responses in Pam3CSK4-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

the glucocorticoid induced leucine zipper mediates statin induced muscle damage

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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

Altered glucocorticoid metabolism represents a feature of macroph-aging

The aging process is characterized by a chronic, low‐grade inflammatory state, termed “inflammaging.” It has been suggested that macrophage activation plays a key role in the induction and maintenance of this state. In the present study, we aimed to elucidate the mechanisms responsible for aging‐associated changes in the myeloid compartment of mice. The aging phenotype, characterized by elevated cytokine production, was associated with a dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis and diminished serum corticosteroid levels. In particular, the concentration of corticosterone, the major active glucocorticoid in rodents, was decreased. This could be explained by an impaired expression and activity of 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1), an enzyme that determines the extent of cellular glucocorticoid responses by reducing the corticosteroids cortisone/11‐dehydrocorticosterone to their active forms cortisol/corticosterone, in aged macrophages and peripheral leukocytes. These changes were accompanied by a downregulation of the glucocorticoid receptor target gene glucocorticoid‐induced leucine zipper (GILZ) in vitro and in vivo. Since GILZ plays a central role in macrophage activation, we hypothesized that the loss of GILZ contributed to the process of macroph‐aging. The phenotype of macrophages from aged mice was indeed mimicked in young GILZ knockout mice. In summary, the current study provides insight into the role of glucocorticoid metabolism and GILZ regulation during aging