RESUMO
BACKGROUND: Tumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization. METHODS: EVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting. RESULTS: EVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells. CONCLUSION: Our results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype.
Assuntos
Vesículas Extracelulares , Proteínas de Ligação a RNA , Macrófagos Associados a Tumor , Peixe-Zebra , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Macrófagos Associados a Tumor/metabolismo , Células HCT116 , MicroRNAs/genética , MicroRNAs/metabolismo , Movimento Celular/genética , Macrófagos/metabolismoRESUMO
Glucocorticoids, which have long served as fundamental therapeutics for diverse inflammatory conditions, are still widely used, despite associated side effects limiting their long-term use. Among their key mediators is glucocorticoid-induced leucine zipper (GILZ), recognized for its anti-inflammatory and immunosuppressive properties. Here, we explore the immunomodulatory effects of GILZ in macrophages through transcriptomic analysis and functional assays. Bulk RNA sequencing of GILZ knockout and GILZ-overexpressing macrophages revealed significant alterations in gene expression profiles, particularly impacting pathways associated with the inflammatory response, phagocytosis, cell death, mitochondrial function, and extracellular structure organization activity. GILZ-overexpression enhances phagocytic and antibacterial activity against Salmonella typhimurium and Escherichia coli, potentially mediated by increased nitric oxide production. In addition, GILZ protects macrophages from pyroptotic cell death, as indicated by a reduced production of reactive oxygen species (ROS) in GILZ transgenic macrophages. In contrast, GILZ KO macrophages produced more ROS, suggesting a regulatory role of GILZ in ROS-dependent pathways. Additionally, GILZ overexpression leads to decreased mitochondrial respiration and heightened matrix metalloproteinase activity, suggesting its involvement in tissue remodeling processes. These findings underscore the multifaceted role of GILZ in modulating macrophage functions and its potential as a therapeutic target for inflammatory disorders, offering insights into the development of novel therapeutic strategies aimed at optimizing the benefits of glucocorticoid therapy while minimizing adverse effects.
Assuntos
Macrófagos , Mitocôndrias , Fagocitose , Piroptose , Fatores de Transcrição , Animais , Mitocôndrias/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Imunomodulação , Espécies Reativas de Oxigênio/metabolismo , Camundongos Knockout , Glucocorticoides/farmacologia , Camundongos Endogâmicos C57BL , Salmonella typhimurium/imunologia , Escherichia coli/imunologiaRESUMO
(1) Background: Aging is linked to an altered immune response and metabolism. Inflammatory conditions, such as sepsis, COVID-19, and steatohepatitis are more prevalent in the elderly and steatosis is linked both to severe COVID-19 and sepsis. We hypothesized that aging is linked to a loss of endotoxin tolerance, which normally protects the host from excessive inflammation, and that this is accompanied by elevated levels of hepatic lipids. (2) Methods: An in vivo lipopolysaccharide (LPS) tolerance model in young and old mice was used and the cytokine serum levels were measured by ELISA. Cytokine and toll-like receptor gene expression was determined by qPCR in the lungs and the liver; hepatic fatty acid composition was assessed by GC-MS. (3) Results: The old mice showed a distinct potential for endotoxin tolerance as suggested by the serum cytokine levels and gene expression in the lung tissue. Endotoxin tolerance was less pronounced in the livers of the aged mice. However, the fatty acid composition strongly differed in the liver tissues of the young and old mice with a distinct change in the ratio of C18 to C16 fatty acids. (4) Conclusions: Endotoxin tolerance is maintained in advanced age, but changes in the metabolic tissue homeostasis may lead to an altered immune response in old individuals.
RESUMO
Statins, the most prescribed class of drugs for the treatment of hypercholesterolemia, can cause muscle-related adverse effects. It has been shown that the glucocorticoid-induced leucine zipper (GILZ) plays a key role in the anti-myogenic action of dexamethasone. In the present study, we aimed to evaluate the role of GILZ in statin-induced myopathy. Statins induced GILZ expression in C2C12 cells, primary murine myoblasts/myotubes, primary human myoblasts, and in vivo in zebrafish embryos and human quadriceps femoris muscle. Gilz induction was mediated by FOXO3 activation and binding to the Gilz promoter, and could be reversed by the addition of geranylgeranyl, but not farnesyl, pyrophosphate. Atorvastatin decreased Akt phosphorylation and increased cleaved caspase-3 levels in myoblasts. This effect was reversed in myoblasts from GILZ knockout mice. Similarly, myofibers isolated from knockout animals were more resistant toward statin-induced cell death than their wild-type counterparts. Statins also impaired myoblast differentiation, and this effect was accompanied by GILZ induction. The in vivo relevance of our findings was supported by the observation that gilz overexpression in zebrafish embryos led to impaired embryonic muscle development. Taken together, our data point toward GILZ as an essential mediator of the molecular mechanisms leading to statin-induced muscle damage.