Differential expression of pro- and anti-inflammatory mediators in pulmonary macrophages upon Toll-like receptor activation

Toll-like receptor (TLR) activation plays a crucial role in both infectious as well as non-infectious lung disease. TLRs are capable of sensing different microbial and viral molecular patterns, and TLR engagement is a prerequisite for the initiation of macrophage responses to infections. Thus, aim of this work was to elucidate different aspects of TLR activation in human pulmonary macrophages. Interestingly, the activation profiles of the two macrophage populations examined, i.e. alveolar and interstitial macrophages, differed largely, indicating that alveolar macrophages are more effective as a non-specific first line of defence against inhaled pathogens, whereas interstitial show a more pronounced regulatory function. The glucocorticoid-induced leucine zipper (GILZ) is highly expressed in human alveolar macrophages and critically attenuates inflammatory signalling pathways. The mechanisms of GILZ regulation in inflammation, however, have as yet been completely unknown. Our investigations on the expression of GILZ in human alveolar macrophages upon TLR activation reveal that GILZ is downregulated by different post-transcriptional mechanisms. Both the TLR signalling pathways and GILZ have emerged as potential therapeutic targets for the treatment of inflammatory lung diseases. Our results support this concept and contribute to a better understanding of their role in pulmonary immune homeostasis.Die Aktivierung von Toll-like Rezeptoren (TLRs) ist von großer Bedeutung für den Verlauf infektiöser sowie nicht infektiöser Erkrankungen der Lunge. TLRs ermöglichen die Erkennung verschiedenster mikrobieller und viraler Muster, und die Aktivierung von TLRs stellt eine Vorraussetzung für die Einleitung von Abwehrmechanismen in Lungenmakrophagen dar. Im Rahmen dieser Arbeit wurde die Expression von pro- und anti-inflammatorischen Mediatoren nach TLR-Aktivierung in humanen Lungenmakrophagen untersucht. Dabei ließen sich große Unterschiede zwischen Alveolarmakrophagen und interstitiellen Makrophagen feststellen. Unsere Ergebnisse legen nahe, dass Alveolarmakrophagen eine effektive erste Abwehr von Pathogenen vermitteln, während interstitielle Makrophagen eher in regulatorische Prozesse involviert sind. Der anti-inflammatorische Faktor GILZ (glucocorticoid-induced leucine zipper) wird von Lungenmakrophagen stark exprimiert. Die Regulationsmechanismen, denen GILZ im Rahmen einer Entzündungsreaktion unterliegt, waren bisher jedoch unbekannt. Unsere Untersuchen zur Expression von GILZ in Alveolarmakrophagen zeigen erstmals, dass GILZ nach TLR-Aktivierung herabreguliert wird und liefern Einblicke in die zugrunde liegenden Mechanismen. Die Aufklärung der Mechanismen, die in die Regulation von GILZ und TLR-Signalwegen involviert sind, kann zu einem besseren pathophysiologischen Verständnis und somit zu einer besseren Therapierbarkeit von entzündlichen Lungenerkrankungen beitragen

Control of CAR-T cell activity in space and time: the next level of anti-tumor action

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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

M2 polarization enhances silica nanoparticle uptake by macrophages

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

Yields and Immunomodulatory Effects of Pneumococcal Membrane Vesicles Differ with the Bacterial Growth Phase

Streptococcus pneumoniae infections are a leading cause of death worldwide. Bacterial membrane vesicles (MVs) are promising vaccine candidates because of the antigenic components of their parent microorganisms. Pneumococcal MVs exhibit low toxicity towards several cell lines, but their clinical translation requires a high yield and strong immunogenic effects without compromising immune cell viability. MVs are isolated during either the stationary phase (24 h) or death phase (48 h), and their yields, immunogenicity and cytotoxicity in human primary macrophages and dendritic cells have been investigated. Death-phase vesicles showed higher yields than stationary-phase vesicles. Both vesicle types displayed acceptable compatibility with primary immune cells and several cell lines. Both vesicle types showed comparable uptake and enhanced release of the inflammatory cytokines, tumor necrosis factor and interleukin-6, from human primary immune cells. Proteomic analysis revealed similarities in vesicular immunogenic proteins such as pneumolysin, pneumococcal surface protein A, and IgA1 protease in both vesicle types, but stationary-phase MVs showed significantly lower autolysin levels than death-phase MVs. Although death-phase vesicles produced higher yields, they lacked superiority to stationary-phase vesicles as vaccine candidates owing to their similar antigenic protein cargo and comparable uptake into primary human immune cells

Endotoxin Tolerance Acquisition and Altered Hepatic Fatty Acid Profile in Aged Mice

(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

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

Cell-Derived Vesicles for Antibiotic Delivery-Understanding the Challenges of a Biogenic Carrier System

Recently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs’ technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro–in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073 are introduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVs is comprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae model is applied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesicles are successfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesicles are then tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics

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 mRNA-binding Protein TTP/ZFP36 in Hepatocarcinogenesis and Hepatocellular Carcinoma

Hepatic lipid deposition and inflammation represent risk factors for hepatocellular carcinoma (HCC). The mRNA-binding protein tristetraprolin (TTP, gene name ZFP36) has been suggested as a tumor suppressor in several malignancies, but it increases insulin resistance. The aim of this study was to elucidate the role of TTP in hepatocarcinogenesis and HCC progression. Employing liver-specific TTP-knockout (lsTtp-KO) mice in the diethylnitrosamine (DEN) hepatocarcinogenesis model, we observed a significantly reduced tumor burden compared to wild-type animals. Upon short-term DEN treatment, modelling early inflammatory processes in hepatocarcinogenesis, lsTtp-KO mice exhibited a reduced monocyte/macrophage ratio as compared to wild-type mice. While short-term DEN strongly induced an abundance of saturated and poly-unsaturated hepatic fatty acids, lsTtp-KO mice did not show these changes. These findings suggested anti-carcinogenic actions of TTP deletion due to effects on inflammation and metabolism. Interestingly, though, investigating effects of TTP on different hallmarks of cancer suggested tumor-suppressing actions: TTP inhibited proliferation, attenuated migration, and slightly increased chemosensitivity. In line with a tumor-suppressing activity, we observed a reduced expression of several oncogenes in TTP-overexpressing cells. Accordingly, ZFP36 expression was downregulated in tumor tissues in three large human data sets. Taken together, this study suggests that hepatocytic TTP promotes hepatocarcinogenesis, while it shows tumor-suppressive actions during hepatic tumor progression